ميكرو ومضادات حيوية

Microbiology & Antibiotics
Third Year
Chem 353
Contents
No Topic Page
1 Food Spoilage 1
2 The role of microorganisms in soil 6

formation
3 Some diseases caused by microorganisms 13
4 Common Diseases (Infectious diseases) 66
5 Questions 26
6 Antibiotics 27
7 Practical part 622
8 References 167
Food Spoilage: Microorganisms and their prevention
Introduction
Food spoilage is a metabolic process that causes foods to be undesirable
or unacceptable for human consumption due to changes in sensory
characteristics. Spoiled foods may be safe to eat, i.e. they may not cause
illness because there are no pathogens or a toxin present, but changes in
texture, smell, taste, or appearance cause them to be rejected.
Food spoilage microorganisms
Chemical reactions that cause offensive sensory changes in foods are
mediated by a variety of microbes that use food as a carbon and energy
source. Spoilage microbes are often common inhabitants of soil, water, or
the intestinal tracts of animals and may be dispersed through the air and
water and by the activities of small animals, particularly insects.
Yeasts
Yeasts are a subset of a large group of organisms called fungi that also
includes molds and mushrooms. They are generally single-celled
organisms that are adapted for life in specialized, usually liquid,
environments and, unlike some molds and mushrooms, do not produce
toxic secondary metabolites. Yeasts can grow with or without oxygen
(facultative) and are well known for their beneficial fermentations that
produce bread and alcoholic drinks. They often colonize foods with a
high sugar or salt content and contribute to spoilage of sauerkraut. Fruits
and juices with a low pH are another target, and there are some yeasts
that grow on the surfaces of meat and cheese.
There are four main groups of spoilage yeasts: Zygosaccharomyces and
related genera tolerate high sugar and high salt concentrations and are the
usual spoilage organisms in foods such as honey, dried fruit, jams and soy
sauce. They usually grow slowly, producing off-odors and flavors and
carbon dioxide that may cause food containers to swell and burst.
Debaryomyces hansenii can grow at salt concentrations as high as 24%,
accounting for its frequent isolation from salt brines used for cured meats,
cheeses, and olives. This group also includes the most important spoilage
organisms in salad dressings. Saccharomyces spp. are best known for
their role in production of bread and wine but some strains also spoil
wines and other alcoholic beverages by producing gassiness, turbidity and
offflavors associated with hydrogen sulfide and acetic acid. Some species
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grow on fruits, including yogurt containing fruit, and some are resistant to
heat processing.
Candida and related genera are a heterogeneous group of yeasts, some of
which also cause human infections. They are involved in spoilage of
fruits, some vegetables and dairy products. Dekkera/Brettanomyces are
principally involved in spoilage of fermented foods, including alcoholic
beverages and some dairy products. They can produce volatile phenolic
compounds responsible for off-flavors.
Molds
Molds are filamentous fungi that do not produce large fruiting bodies like
mushrooms. Molds are very important for recycling dead plant and
animal remains in nature but also attack a wide variety of foods and other
materials useful to humans. They are well adapted for growth on and
through solid substrates, generally produce airborne spores, and require
oxygen for their metabolic processes.
Most molds grow at a pH range of 3 to 8 and some can grow at very low
water activity levels (0.7–0.8) on dried foods. Spores can tolerate harsh
environmental conditions but most are sensitive to heat treatment.
Different mold species have different optimal growth temperatures, with
some able to grow in refrigerators. They have a diverse secondary
metabolism producing a number of toxic and carcinogenic mycotoxins.
Some spoilage molds are toxigenic while others are not.
Penicillium and related genera are present in soils and plant debris.
Although they can be useful to humans in producing antibiotics and blue
cheese, many species are important spoilage organisms, and some
produce potent mycotoxins (patulin, ochratoxin, citreoviridin, penitrem).
Penicillium spp. cause visible rots on citrus, pear, and apple fruits and
cause enormous losses in these crops. They also spoil other fruits and
vegetables, including cereals. Some species can attack refrigerated and
processed foods such as jams and margarine.
Bacteria
Spore-forming bacteria are usually associated with spoilage of heat-
treated foods because their spores can survive high processing
temperatures. These Gram-positive bacteria may be strict anaerobes or
facultative (capable of growth with or without oxygen). Some spore-
formers are thermophilic, preferring growth at high temperatures (as high
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as 55ºC). Some anaerobic thermophiles produce hydrogen sulphide
(Desulfotomaculum) and others produce hydrogen and carbon dioxide
(Thermoanaerobacterium) during growth on canned/ hermetically sealed
foods kept at high temperatures.
Spoilage of fruits and vegetables
The main sources of microorganisms in vegetables are soil, water, air,
and other environmental sources, and can include some plant pathogens.
Fresh vegetables are fairly rich in carbohydrates (5% or more), low in
proteins (about 1 to 2%), and, except for tomatoes, have high pH.
Microorganisms grow more rapidly in damaged or cut vegetables.
The presence of air, high humidity, and higher temperature during storage
increases the chances of spoilage.
Vegetables are another tempting source of nutrients for spoilage
organisms because of their near neutral pH and high water activity.
Bacterial spoilage first causes softening of tissues as pectins are degraded
and the whole vegetable may eventually degenerate into a slimy mass.
Starches and sugars are metabolized next and unpleasant odors and
flavors develop along with lactic acid and ethanol.
Spoilage of dairy products
Milk is an excellent medium for growth for a variety of bacteria. Spoilage
bacteria may originate on the farm from the environment or milking
equipment or in processing plants from equipment, employees, or the air.
LAB are usually the predominant microbes in raw milk and proliferate if
milk is not cooled adequately. When populations reach about 106 cfu/ml,
off-flavors develop in milk due to production of lactic acid and other
compounds.
Refrigeration suppresses growth of LAB and within one day
psychrophilic bacteria (Pseudomonas, Enterobacter, Alcaligenes and
some spore-formers) grow and can eventually produce rancid odors
through the action of lipases and bitter peptides from protease action.
Pasteurization kills the psychrophiles and mesophilic bacteria (LAB), but
heat-tolerant species (Alcaligenes, Microbacterium, and the sporeformers
Bacillus and Clostridium) survive and may later cause spoilage in milk or
other dairy products. Immediately following pasteurization, bacterial
counts are usually <1000 cfu/ml. However, post-pasteurization
contamination of milk, particularly with Pseudomonas and some Gram-
positive psychrophiles does occur. Spoilage problems in cheese can
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sometimes be traced to low quality milk but may also result from
unhygienic conditions in the processing plant. Hard and semi-hard
cheeses have a low moisture content (<50%) and a pH ~5.0, which limits
the growth of some microbes.
Prevention from food spoilage microorganism
The development of food preservation processes has been driven by the
need to extend the shelf-life of foods. Food preservation is a continuous
fight against microorganisms spoiling the food or making it unsafe.
Several food preservation systems such as heating, refrigeration and
addition of antimicrobial compounds can be used to reduce the risk of
outbreaks of food poisoning; however, these techniques frequently have
associated adverse changes in organoleptic characteristics and loss of
nutrients.
The most common classical preservative agents are the weak organic
acids, for example acetic, lactic, benzoic and sorbic acid. These
molecules inhibit the outgrowth of both bacterial and fungal cells and
sorbic acid is also reported to inhibit the germination and outgrowth of
bacterial spores. In the production of food it is crucial that proper
measures are taken to ensure the safety and stability of the product during
its whole shelf-life. In particular, modern consumer trends and food
legislation have made the successful attainment of this objective much
more of a challenge to the food industry. Firstly, consumers require more
high quality, preservative-free, safe but mildly processed foods with
extended shelf-life.
Use of food additives
Food additives are substances or mixture of substances other than basic
foodstuffs, which are present in the foods as reagent of any aspects of
production, processing, storage, packaging etc. Food additives are (i)
sugar, (ii) salt, (iii) acids, (iv) spices. In case of sugar and salts, they
exerts osmotic pressure by water is diffuses from the product through a
semi-permeable membrane until the concentration reached equilibrium.
They kills the microorganisms or do not allow them to multiplication.
(i) Sugar: The concentration of 68-70% is used for preparation of jam,
jelly, marmalades etc. sugar act as a preservative by osmosis and not as a
true poison for micro organisms. It absorbs most of the available water,
so little water available for the growth of micro organisms.
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(ii) Salt: 15-20% concentration is used for the preparation such as pickles.
Salt inhibits enzymatic browning and discolouration and also acts as an
anti-oxidant. It exerts its preservative action by: a. Causing high osmotic
pressure resulting in the plasmolysis of microbial cells; b. Dehydrating
food and micro organisms by tying up the moisture; c. Ionizing to yield
the chloride ion which is harmful to micro organisms, and d. Reducing
the solubility of oxygen in water, sensitizing the cells against CO2.
(iii) Acids: Many processed foods and beverages needs the addition of
acids to impart their characteristic flavor and taste in the final product
because acids provides desired flavour and taste. They adjust the sugar
and acid ratio in the food. They give proper balance flavour of the food.
Acetic acid (Vinegar), Citric acid (Lime juice), Lactic acid (Lactose) etc.
Spices are plant products, are used in flavouring the foods and beverages
to enhance the food flavour, colour and palatability, act as antibacterial
and antifungal activity.
Weak carboxylic acids, such as acetic, sorbic and benzoicacids, are
generally regarded as safe anti-microbial additives, and have wide
application as preservatives in foods and beverages. However, many
yeasts are able to survive, adapt and even grow in the presence of the
maximum levels of these preservatives permitted for use in foods. When
compared with other fungi and bacteria, yeast are more resistant to weak
carboxylic acids.
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The role of microorganisms in soil formation
Soil Microbes overview

Soil looks lifeless but a quarter of a teaspoon of soil (1 gram) contains
many microbes - 100 million bacteria, 1 million actinomycetes and 5
metres of fungal filaments. Bacteria and fungi have similar ways of
feeding. Their main food is organic detritus in leaf litter, soil, dung and
carrion. In contrast to invertebrate animals which engulf food and digest
it within their bodies, microbes digest their food outside themselves by
secreting digestive enzymes over their food. Under the action of the
enzymes, organic matter breaks down into simpler molecules external to
the microbe, which then re-absorbs the simpler products of digestion
through its cell wall. Microbes have a vast number of enzymes to do this
job – 50 to 60 different ones have been discovered. Few invertebrates
possess this many digestive enzymes.
Fungi
Most of the time fungi hideaway in the murky depths of the soil and are
not visible. But their microscopic, mouldy strands are growing through
bits of organic matter and soil, through leaf litter, dead wood and dung
and into carcasses of dead animals.
Here, the fungi quietly go about their job of decomposing detritus and
recycling the nutrients back to the soil. Some soil fungi form a positive
relationship with plant roots. They live in roots, but also extend their
filament networks out into the soil where they absorb nutrients and water
which they bring back to the plant to use. What the fungus gets out of this
relationship is food in the form of sugars. This mutually beneficial
relationship (or symbiosis) between root and fungus is called a
mychorrizal association.
Bacteria
Bacteria form resistant spores when the climate turns harsh. They can
survive droughts in a spore stage and when favorable conditions return,
they can germinate and grow again. The main food of bacteria is organic
matter in leaf litter, dung, soil and carrion. They (along with fungi) are
the main decomposer groups in soil because of the huge number of
digestive enzymes they can secrete onto organic matter to break it down.
Some soil bacteria can fix atmospheric nitrogen, ultimately making it
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available to plants. Some of these live free in the soil e.g. Azotobacter,
while others fix nitrogen while living in nodules on the roots of legumes
e.g. Rhizobia. Here, these bacteria gain food in the form of sugars from
the plants and the plant benefits through increased nitrogen fixed by the
bacteria. This "win-win" relationship between bacteria and plant is called
"symbiosis" - or a relationship where both organisms gain a mutual
benefit from living together.
Other microbes
Other microbes, apart from bacteria and fungi, also live in soil. These are
actinomycetes, yeasts, viruses and algae. Not all of these other microbes
are decomposer organisms. Actinomycetes are bacteria-like microbes,
joined end to end in a filamentous strand to form a branched, fungus like
network. They seem to be half-way between a bacterium and a fungus.
They are good decomposers and can break down particularly resistant
forms of organic matter such as cellulose and chitin. Soil yeasts are
unicellular fungi. They are decomposers. Soil viruses prey on soil
bacteria but not a lot is known about these microorganisms. Soil algae are
also included in the microbes but these are different as they
photosynthesise like green plants do.
Roles of soil organisms
What do soil organisms do?
There are three main roles that soil organisms perform in soil.
 Decompose organic residues
 Re-cycle nutrients from organic residues
 Enhance soil structure
Soil organisms clear away and degrade organic debris such as dead
plants, animals and dung and use it as a source of food and nutrients, and
in the process release chemical elements (nutrients) into the soil solution
so that living plants may re-use them. This process sustains soil chemical
fertility. But soil organisms are also responsible for soil physical fertility
as well. They help the formation of good soil structure. In addition, soil
organisms degrade chemicals and pollutants that enter soil. Also, some
soil organisms live in mutually beneficial relationships with plants,
enhancing the plant‟s nutrition by increasing the nitrogen uptake from the
atmosphere (Rhizobium) or phosphorus uptake from the soil by soil
(mycorrhizae).
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Decomposition and Nutrient Cycling
Decomposer invertebrate animals and microbes have quite distinct roles
in breaking down organic detritus. Although invertebrates play only a
small part in chemically degrading organic detritus, they help the more
important microbes in many ways to do their job. During decomposition
of organic matter, nutrients are released.
Nitrogen cycling in a pasture soil. Springtails fragment the dead grass
while microbes chemically degrade it.
Enhancement of soil structure
Good soil structure helps with physical fertility of soil. Both the large soil
animals (e.g. earthworms) and the tiny microbes have roles in improving
soil structure. Soil with good structure has many beneficial effects
including enhanced water transmission into and through soil, lower bulk
density and lower potential for soil erosion. Large soil animals make
tunnels through the soil. These are often called macropores as they form
the larger pores in the soil.
Some earthworms, dung beetles, spiders, ants and cicadas (as they
emerge from the soil to change into adults), make vertical tunnels that
open to the soil surface and down which water can infiltrate. Other
tunnelers form macropores that don‟t open to the soil surface
(earthworms, termites). The formation of holes (or macropores) in the
soil, helps water transmission and soil hydrology. If water can easily
enter the soil, less runs off to cause erosion. Soil animals mix soil layers
together and also mix organic matter that they eat with mineral soil
layers. Ants, earthworms and termites bring organic matter into the soil
from the surface and deposit it, thus increasing the organic matter content
of soil. This helps water retention in soil. Earthworm activity lowers soil
bulk density and makes soil more friable - roots can penetrate this soil
more easily.
Diagram of a soil aggregate
Bacteria and fungi help in the formation of water-stable soil aggregates.
Fungal hyphae grow around and between soil mineral and organic
particles and physically bind them together. Both bacteria and fungi
secrete polysaccharide mucilages which are sticky and glue the soil
particles together into aggregates. These aggregates can be stable to the
action of water for several months and help prevent slaking and
dispersion of the soil.
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Fertilizers
Fertilizers are used to increase agricultural productivity and to replace
nutrients that are either removed in agricultural products (meat, wool,
crops) or eroded by water and wind. Agricultural fertilizers fall into
several groups. Inorganic chemical fertilizers include phosphate, nitrogen
and sculpture fertilizers while organic fertilizers include manures and
compost. Lime is used mainly as a soil ameliorant to reduce acidity.
Inorganic Fertilizers
Mostly, inorganic Fertilizers increase soil biological activity because they
increase plant production leaving organic residues of high quality as food
for soil biota. There may be some short-term adverse effects, possibly due
to pH change but, in the long-term, effects of inorganic Fertilizers on
overall biological activity are mostly stimulatory. However, species types
and diversity of soil biota may change with fertilizer use.
Organic Fertilizers
Organic amendments in the form of composts or manures, generally
increase biological activity in soil through an increased supply of energy
(food for soil biota) and nutrients to the detrital food web. Effects of
organic Fertilizers on soil biota
• Increase in earthworm numbers
• Increase in nematode, springtail and mite abundance
• Increase in microbial biomass
Pesticides
Chemicals used to control pests and diseases in plants and animals can
have undesirable toxic side-effects on the non-target soil biota. Pesticides
have variable effects on soil biota. The same pesticide may affect
different species of soil biota in different ways. Also, effects of pesticides
depend on the nature of the chemical, its dose, the method of application,
temperature and moisture conditions in soil, crop residue management,
the rate of decay of the chemical and the extent of leaching from the site.
For example, some herbicides are more persistent if sprayed onto mulch
than if sprayed onto bare soil as they may be quickly inactivated by
adsorption onto soil particles. However, some generalizations of effects
of pesticides on soil biota can be made. A general rule-of-thumb is that if
the toxic effect of a pesticide on a non-target organism lasts longer than
60 days, then the chemical can be regarded as persistent and its toxic
effects only slowly reversible.
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Another general rule is that toxicity of pesticides from the least to the
most toxic to soil biota, follows the order: herbicides < insecticides <
fungicides. The following points can be made about the eco-toxic effects
of pesticides.
Herbicides
• Newer herbicides affect enzyme pathways in plants which are not found
in soil biota
• Older herbicides can have some toxic effects on soil biota e.g.
springtails, mites
• Many fungi are tolerant to herbicides
• Earthworms can increase in numbers following herbicide application as
their food supply, in the form of dead plants, is increased.
Insecticides
Organ chlorines are more active in soil than organophosphates or
carbamates
Earthworms are not very susceptible to insecticides except carbamates.
Fungicides
Benomyl is toxic to earthworms
Some fungicides contain copper which is toxic to earthworms and
microbes – see the inhibition of the decay of cellulose by soil microbes in
cotton strips buried in soil containing copper. Staining of the strips are
areas where microbes have colonized.
Plant residue retention
In zero tillage cropping systems, a layer of mulch is retained on the soil
surface and is not cultivated into the soil. Under these systems soil biota
generally increase in abundance.
Effects of retention of organic surface residues on soil biota
include
• Increase in earthworm numbers (x6 times)
• Increase in springtails and mites
• Increase in microbial biomass
Crop rotations
Crop rotations occour when different crops are planted in succession, on
the same area of land. Benefits of this practice are that they provide a
disease break as diseases specific to one crop are denied a host for several
years.
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Crop rotations that include a legume phase (e.g. lucerne) increase the
nitrogen content of organic residues and soil. The quality of the diet for
decomposer organisms is increased.
Effects of legume in crop rotations on soil biota include:
• Increase in abundance of some small soil animals
• Increase in earthworm abundance
• Increase in soil microbial biomass
Irrigation
The increased soil moisture which follows irrigation favors many soil
biota. However, waterlogged soil conditions decreases the oxygen content
of soil and there are few soil macrofauna (e.g. earthworms) present under
these conditions.
Effects of irrigation on soil biota
 Irrigation allows earthworms to remain active in summer
 Increase in springtail and mite abundance
 Increase in Protozoa abundance
 Increase in soil microbial biomass
Fire
Burning of vegetation is used to remove stubble from cropping soils or in
grasslands to improve the quality of rough pastures such as savannah
grasslands where it eliminates woody plants and inedible dry vegetation.
In addition, many wildfires occur throughout Australia in the summer
months which burn whole forests. The adverse effects of fire are
generally restricted to litter dwellers and true soil species are little
affected. However, repeated burning of crop residues or forests depletes
the soil of organic matter and biological activity falls as the food supply
to soil biota is reduced.
Effects of fire on soil biota
• Litter dwelling organisms die
• True soil dwellers are little affected in uncultivated soils
• Some large invertebrates can survive fires by sheltering under
rocks
• Soil microbes increase after fire due to “liming effects” of wood
ash.
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The soil biota plays many fundamental roles in delivering key ecosystem
goods and services. Ecosystems goods provided by soil biota are
Food production;
Fiber production;
Fuel production;
Provision of clean water;
Provision of secondary compounds (e.g. pharmaceuticals
and agrochemicals).
Ecosystems services provided by soil biota
Driving nutrient cycling and regulation of water flow and storage;
Regulation of soil and sediment movement and biological regulation of
other biota (including pests and diseases); Soil structure maintenance;
Detoxification of xenobiotic and pollutants and regulation of atmospheric
composition. Soil structure holds a vital, but often overlooked role in
sustainable food production and the wellbeing of society. A more holistic
approach to land use and management is needed to cope with increased
pressure on soil resources for sustainable food and fiber production while
reducing the adverse off-site environmental impacts of agricultural
practices. The impact of soil structure ranges from a global to a highly
localized scale. Improved C sequestration in soil aggregates can reduce
the rate of increase in CO2 concentration in the atmosphere and
associated global warming. Improved soil structure enhances nutrient
recycling, water availability and biodiversity while reducing water and
wind erosion, and improving surface and ground water quality.
Processes and mechanisms involved in soil aggregation are complex with
intricate feedback mechanisms. Soil aggregation can be improved by
management practices that decrease agro-ecosystem disturbances,
improve soil fertility, increase organic inputs, increase plant cover . Soil
structure can be enhanced through the use of crops and crop management
practices that promote aggregation such as the use of crops with high CR
and high biomass production, the return of crop residues and
incorporation of cover crops. Aggregation also tends to increase with
increasing root length density; extensive fibrous roots produce highest
levels of macro aggregation. Enhancing the diversity and quantity of soil
flora and fauna are important in improving soil structure. Activity of soil
fauna is important in the formation of organomineral complexes and
aggregation.
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Some diseases caused by microorganisms
What is a disease?
Any condition which interferes with the normal functioning of the body is
called a disease. In other words, disease may be defined as a disorder in
the physical, physiological, psychological or social state of a person
caused due to nutritional deficiency, physiological disorder, genetic
disorder, pathogen or any other reason.
Types of Diseases
The diseases may be classified into two broad categories
A. Congenital disease: The disease which is present from birth (e.g. hole
in the heart in infants). They are caused by some genetic abnormality or
metabolic disorder or malfunctioning of an organ.
B. Acquired disease: The disease which may occur after birth during
one‟s lifetime.
Acquired diseases may generally be classified into:
(i) Infectious diseases: The diseases which can be transmitted from
person to person e.g. measles.
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(ii) Degenerative diseases: The diseases caused by the malfunction of
some vital organs of the body e.g. heart failure.
(iii) Deficiency diseases: These are caused due to nutritional deficiency
such as that of minerals or vitamins in the diet e.g. anaemia (Fe, Beri-
beri (vitamin B).
(iv) Cancer: This is an abnormal, uncontrolled and unwanted growth of
cells. e.g. breast cancer.
Acquired diseases are studied under two categories.
(i) Communicable diseases: The diseases which can be transmitted from
an infected person to a healthy person.
(ii) Non-communicable diseases: These diseases do not spread from an
affected person to a healthy person.
Modes of Spread of Communicable Diseases

Communicable diseases spread from the infected person to a healthy
person in the following ways.
Direct transmission
The pathogens of diseases infect a healthy person directly without an
intermediate agent. It can take place by various means such as,
(i) Direct contact between the infected person and the healthy person:
Diseases like small pox, chicken pox, syphilis, gonorrhoea spread through
direct contact.
(ii) Droplet infection: The infected person throws out tiny droplets of
mucus by coughing, sneezing or spitting. These droplets may contain the
pathogen. By inhaling the air containing the droplets, a healthy person
may get the infection.
Diseases like common cold, pneumonia, influenza, measles, tuberculosis
and whooping cough spread through droplet infection.
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(iii) Contact with soil contaminated with disease-causing viruses,
bacteria etc.
(iv)Animal bite: Viruses of rabies are introduced through the wound
caused by the bite of rabid animals, especially dogs. The virus is present
in the saliva of the rabid animals.
Indirect transmission
The pathogens of certain diseases reach the human body through some
intermediate agents. It can take place by various means, which are as
follows:
(i) By vectors such as houseflies, mosquitoes, and cockroaches.
Examples: Houseflies carry the causative organisms of cholera on their
legs and mouth parts from the faeces and sputum of infected persons to
food and drinks and contaminate them. When this contaminated food is
taken by a healthy person, he gets the infection. Similarly, mosquitoes
carry virus of dengue and malarial parasite which causes malaria.
(ii) Air-borne: The pathogens may reach humans with air and dust. The
epidemic typhus spreads by inhalation of dried faeces of infected fly.
(iii) Object borne (Fonite borne): Many diseases are transmitted
through the use of contaminated articles, such as clothes, utensils, toys,
door handles, taps, syringes and surgical instruments, etc.
(iv) Water borne: If potable water (drinking water) is contaminated with
pathogens of diseases such as cholera, diarhhoea, hepatitis or jaundice, it
reaches a healthy person upon consuming such water.
Some definitions:.2 SOME IMPORTANT TERMMEMBER
Pathogen: A living organism which causes a disease.
Parasite: An organism which gets food and shelter from host.
Host: The living body on or inside which the disease-producing organism
takes shelter.
Infestation: A large number of parasitic organisms present on the surface
of body of the host or on the clothings.
Vector: It is an organism which harbours a pathogen and may pass it on
to another person to cause a disease (Mosquitoes harbour malarial
parasite and transmits it to humans).
Carrier: It is an organism which itself does not harbour the pathogen but
physically transmits it to another person (Housefly is the carrier of
cholera germs).
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Reservoir: An organism which harbours pathogen in large numbers and
does not suffer itself.
Epidemic: Spreading of a disease among a large number of people in the
same place for some time e.g. plague.
Endemic: A disease which is regularly found among a particular group
of people e.g. goitre.
Pandemic: A disease which is found all over the world e.g. AIDS.
Interferon: Type of proteins produced by infected cells of the body when
attacked by a virus, which act to prevent the further development of the
virus.
Inoculation: Introduction of antigenic material inside the body to prevent
suffering from a disease.
Vaccination: Injection of a weak strain of a specific bacterium (Vaccine)
in order
to secure immunity against the corresponding disease. It is also called
immunisation.
Incubation period: The period between entry of pathogen inside a
healthy body and appearance of the symptoms of the disease.
Symptoms: Specific expressions which appear on the deseased and help
in the identification of the disease.
Common Diseases (Infectious diseases)
The diseases which spread from one person to another through
contaminated food, water or contact or through insecticides, animals etc.
are called the communicable diseases. These are caused by different
causative agents (pathogens).
28.3.1 Diseases caused by viruses
1. Chicken pox
Pathogen : Chicken pox virus (varicella)
Mode of transmission : By contact or through scabs
Incubation period : 12-20 days
Symptoms
(i) Fever, headache and loss of appetite
(ii) Dark red-coloured rash on the back and chest which spreads on the
whole body. Later, rashes change into vessicles.
(iii) After few days these vessicles start drying up and scabs (crusts) are
formed.
(v) These scabs start falling (infective stage)
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Prevention and cure
There is no vaccine against chicken pox as yet. But precautions must be
taken as follows:
(i) The patient should be kept in isolation.
(ii) Clothings, utencils, etc. used by the patient should be sterilised.
(iii) Fallen scabs should be collected and burnt. One attack of chicken
pox gives life long immunity to the person recovered from this disease.
2. Measles
Pathogen : Virus (Rubeola)
Mode of transmission : By air
Symptoms
(i) Common cold
(ii) Appearance of small white patches in mouth and throat.
(iii) Appearance of rashes on the body.
Prevention and cure
(i) The patient should be kept in isolation.
(ii) Cleanliness should be maintained.
(iii) Antibiotics check only the secondary infections which can easily
occur.
3. Poliomylitis
Pathogen : Polio Virus
Mode of transmissions : Virus enters inside the body through food or
water.
Symptoms
(i) The virus multiplies in intestinal cells and then reaches the brain
through blood.
(ii) It damages brain and nerves and causes infantile paralysis.
(iii) Stiffness of neck, fever, loss of head support.
Prevention and Cure
Polio vaccine drop (oral polio vaccine, OPV) are given to children at
certain intervals.
Pulse polio programme is organised in our country to give polio vaccine
to children.
4. Rabies (also called hydrophobia)
Pathogen: Rabies virus
17
Mode of Transmission: Bite by a rabid dog.
Incubation period: 10 days to 1-3 months depending upon the distance
of bite from Central Nervous System (CNS), that is the brain or spinal
cord.
Symptoms
(i) Severe headache and high fever.
(ii) Painful contraction of muscles of throat and chest.
(iii) Choking and fear of water leading to death.
Prevention and Cure
(i) Compulsory immunisation of dogs.
(ii) Killing of rabid animals.
(iii) Anti-rabies injections or oral doses are given to the person bitten by a
rabid animal.
5. Hepatitis
Pathogen: Hepatitis B virus.
Mode of Transmission: Mainly through contaminated water.
Incubation Period: Generally 15-160 days.
Symptoms
(i) Bodyache.
(ii) Loss of appetite and nausea.
(iii) Eyes and skin become yellowish, urine deep yellow in colour (due to
bile pigments).
(iv) Enlarged liver.
Prevention and Cure
(i) Hepatitis B vaccine is now available in India.
(ii) Proper hygeine is to be observed.
(iii) Avoid taking fat rich substances.
6. Influenza
Influenza, commonly known as „flu‟ is an illness caused by viruses that
infect the respiratory tract. Compared to common cold, influenza is a
more severe illness.
Causes
Influenza is caused by a virus which attacks our body‟s cells, resulting in
various effects depending on the strain of the virus.
18
There are many strains of influenza virus. The virus mutates all the time
and new variations (strains) arise. This constant changing enables the
virus to evade the immune system of its host. Unfortunately immunity
against one strain (which is conferred by exposure or immunisation) does
not protect against other strains. A person infected with influenza virus
develops antibodies against that virus; as the virus changes, the antibodies
against the virus do not recognize the changed virus,
and influenza can recur, caused by the changed or mutated virus.
Symptoms
Typical symptoms of influenza include:
(i) fever (Usually 100° F to 103° F in adults and often even higher in
children).
(ii) respiratory tract infection symptoms such as, cough, sore throat,
running nose, headache, pain in the muscles, and extreme fatigue.
Although nausea and vomiting and diarrhoea can sometimes accompany
Influenza infection, especially in children, gastrointestinal symptoms are
rarely prominent.
Most people who get flu, recover completely in 1 to 2 weeks, but some
people develop serious and potentially life-threatening complications,
such as pneumonia.
Treatment and Control
(i) Much of the illness and death caused by influenza can be prevented by
annual influenza vaccination. Influenza vaccine is specifically
recommended for those who arc at high risk for complications with
chronic diseases of the heart, lungs or kidneys, diabetes, or severe forms
of anemia.
(ii) The persons suffering from influenza should
 drink plenty of fluids
 take symptom relief with paracetamol, aspirin (not in children
under the age of 16) or ibuprofen etc. as recommended by the
doctor.
 Consult doctor immediately for treatment.
7. Dengue
Dengue is an acute fever caused by virus. It is of two types: (i) Dengue
fever, (ii) Dengue hemorrhagic fever.
Dengue fever is characterized by an onset of sudden high fever, severe
headache, pain behind the eyes and in the muscles and joints.
19
Dengue hemorrhagic fever is an acute infectious viral disease. It is an
advanced stage of dengue fever. It is characterized by fever during the
initial phase and other symptoms like headache, pain in the eye, joint pain
and muscle pain, followed by signs of bleeding, red tiny spots on the skin,
and bleeding from nose and gums.
How does Dengue spread?
Dengue spreads through the bite of an infected Aedes aegypti mosquito.
The transmission of the disease occurs when a mosquito bites an infected
person and subsequently bites a healthy person. In doing so, it transmits
blood containing the virus to the healthy person and the person becomes
infected with dengue. The first symptoms of the disease occur about 5 to
7 days after the infected bite. Aedes mosquito rests indoors, in closets and
other dark places, and is active during day time. Outside, it rests where it
is cool and shaded. The female mosquito lays her eggs in stagnant water
containers such as coolers, tyres, empty buckets etc., in and around
homes, and other areas in towns or villages. These eggs become adults
in about 10 days.
Incubation period
The time between the bite of a mosquito carrying dengue virus and the
start of symptoms averages 4 to 6 days, with a range of 3 to 14 days.
Diagnosis
Diagnosis is made through blood tests by scanning for antibodies against
dengue viruses. In addition the blood platelets counts also drastically
reduce in the infected person.
Symptoms
Symptoms of Dengue fever
(i) Sudden onset of high fever, generally 104-105 °F (40 °C), which may
last 4- 5 days.
(ii) Severe headache mostly in the forehead.
(iii) pain in the joints and muscles, body aches.
(iv) Pain behind the eyes which worsens with eye movement.
(v) Nausea or vomiting.
Symptoms of Dengue hemorrhagic fever
These include symptoms similar to dengue fever, plus other symptoms
such as:
(i) Severe and continuous pain in the abdomen.
21
(ii) Rashes on the skin.
(iii) Bleeding from the nose, mouth, or in the internal organs.
(iv) Frequent vomiting with or without blood.
(v) Black stools due to internal bleeding.
(vi) Excessive thirst (dry mouth).
(vii) Pale, cold skin, weakness.
Prevention
Following steps can be taken to prevent spread of dengue fever:
(i) Avoid water stagnation for more than 72 hours so that the mosquitoes
do not breed there.
(ii) Prevent mosquito breeding in stored water bodies, like ponds, wells
etc.
(iii) Destroy discarded objects like old tyres, bottles, etc. as they collect
and store rain water.
(iv) Use mosquito repellents and wear long sleeved clothes to curtail
exposure.
(v) Use mosquito nets, also during daytime.
(vi) Avoid outdoor activities during dawn or dusk when these mosquitoes
are most active.
(vii) Patients suffering from dengue fever must be isolated for at least 5
days.
(viii) Report to the nearest health centre for any suspected case of Dengue
fever.
Treatment for dengue and dengue hemorrhagic fever
There is no specific treatment for dengue fever. Persons with dengue
fever should rest and drink plenty of fluids. Dengue hemorrhagic fever is
treated by replacing lost fluids. Some patients need blood transfusions to
control bleeding.
Diseases caused.by Bacteria
1. Tuberculosis
Pathogen: A bacterium (Mycobacterium tuberculosis).
Mode of Transmission: airborne-discharged through sputum, cough,
sneeze, etc. of the infected person.
Incubation period: 2-10 weeks during which the bacteria produce a
toxin, tuberculin.
Symptoms
(i) Persistent fever and coughing.
21
(ii) Chest pain and blood comes out with the sputum.
(iii) General weakness.
Prevention and Cure
(i) Isolation of patient to avoid spread of infection.
(ii) BCG vaccination is given to children as a preventive measure.
(iii) Living rooms should be airy, neat and with clean sorroundings.
(iv) Antibiotics be administered as treatment.
2. Typhoid
Pathogen : A Bacillus rod-shaped bacterium (Salmonella typhi)
Mode of transmission : Through contaminated food and water
Incubation period : About 1-3 weeks
Symptoms
(i) Continuous fever, headache, slow pulse rate.
(ii) Reddish rashes appear on the belly.
(iii) In extreme cases, ulcers may rupture resulting in death of the patient.
Prevention and Cure
(i) Anti-typhoid inoculation should be given.
(ii) Avoid taking exposed food and drinks.
(iii) Proper sanitation and cleanliness should be maintained.
(iv) Proper disposal of excreta of the patient.
(v) Antibiotics should be administered.
3. Cholera
It often breaks out among crowded and areas with poor sanitary
conditions.
Pathogen : Comma shaped bacterium (Vibrio cholerae)
Mode of transmission : Contaminated food and water. House - fly is the
carrier.
Incubation period : 6 hours to 2-3 days.
Symptoms
(i) Acute diarrohoea, rice watery stool.
(ii) Muscular cramps.
(iii) Loss of minerals through urine.
(iv) Dehydration leads to death.
Prevention and cure
(i) Cholera vaccination should be given.
22
(ii) Electrolytes (Na, K, sugar, etc.) dissolved in water should be given to
the patient to check dehydration (In market it is available as ORS–oral
rehydration solution).
(iii) Proper washing and cooking of food.
(iv) Proper disposal of vomit and human excreta.
(v) Flies should not be allowed to sit on eatables and utensils.
4. Diphtheria
This disease generally occurs in children of 1-5 years of age.
Pathogen : Rod-shaped bacterium (Cornybacterium diphtherea)
Mode of Transmission : Through air (droplet infection)
Symptoms
(i) Slight fever, Sore throat and general indisposition.
(ii) Oozing semisolidmaterial in the throat which develops into a
toughmembrane.
The membrane may cause clogging (blocking) of air passage, resulting
into death.
Prevention and cure
(i) Immediate medical attention should be given.
(ii) Babies should be given DPT vaccine.
(iii) Sputum, oral and nasal discharges of the infected child should be
disposed off.
(iv) Antibiotics may be given under doctor‟s supervision.
(v) Isolation of the infected child.
5. Leprosy
Pathogen: A bacterium (Mycobacterium leprae)
Mode of transmission: Prolonged contact with the infected person.
Nasal secretions are the most likely infectious material for family
contacts.
Incubation period : 1-5 years
Symptoms
(i) Affects skin.
(ii) Formation of nodules and ulcer.
(iii) Scabs and deformities of fingers and toes.
(iv) Infected areas lose sensation.
Prevention and Cure
(i) The children should be kept away from parents suffering from leprosy.
23
(ii) Some medicine may arrest the disease and prevent from spreading.
Diseases caused by protozoans
1. Malaria
Pathogen: Malarial parasite (different species of Plasmodium)
Mode of transmission: By bite of female Anopheles mosquitoes
Incubation period: Approximately 12 days
Symptoms
(i) Headache, nausea and muscular pain.
(ii) Feeling of chilliness and shivering followed by fever which becomes
normal along with sweating after some time.
(iii) The patient becomes weak and anaemic.
(iv) If not treated properly secondary complications may lead to death.
Prevention and cure
(i) Fitting of double door and windows (with “Jali” i.e. wire mesh) in the
house to prevent entry of mosquitoes.
(ii) Use of mosquito net and mosquito repellents.
(iii) No water should be allowed to collect in ditches or other open spaces
to prevent mosquito breeding.
(iv) Sprinkling of kerosene oil in ditches or other open spaces where
water gets collected.
(v) Antimalarial drugs to be taken.
2. Amoebiasis (Amoebic dysentery)
Pathogen : Entamoeba histolytica
Mode of transmission: Contaminated food and water
Symptoms
(i) Formation of ulcers in intestine.
(ii) Feeling of abdominal pain and nausea.
(iii) Acute diarrhoea and mucus in stool.
Prevention and cure
(i) Proper sanitation should be maintained.
(ii) Vegetables and fruits must be properly washed before eating.
(iii) Antibiotics may be given to the patients.
Diseases caused by worms (helminths)
1. Filariasis
Pathogen: Filarial worm (Wuchereria bancrofti)
Mode of transmission: Bites of mosquitoes - Aedes and Culex.
Symptoms
24
(i) Fever
(ii) Collection of endothellial cells and metabolites in the wall of lymph
vessels.
(iii) Swelling takes place in certain parts of the body like legs, breasts,
scrotum, etc.
(iv) Swelling of legs which appear as legs of elephant, so this disease is
also called elephantiasis (Fig. 28.1)
Prevention and cure

(i) Mesh doors and windows in the house to check the entry of
mosquitoes.
(ii) The water collected in tanks or other articles should be properly
covered.
(iii) Sprinkling of kerosene in ditches, etc.
(iv) Drugs may be administered.
25
Questions
1. What is a disease? How does it differ from disorder?
2. Name the two categories of acquired diseases.
3. Explain the term (i) parasitism (ii) reservoir.
4. Give two symptoms of coronary diseases and of typhoid.
5. What precautions should be taken to prevent malaria?
6. Name the pathogen that causes diphtheria and cholera.
7. Mention the four types of acquired diseases.
8. Differentiate between:
(i) Communicable and non-communicable diseases
(ii) Pathogen and vector
(iii) Syphilis and gonorrhoea
(iv) HIV and AIDS
(v) Benign and malignant tumors
26
Antibiotics 3rd Year Special Biochemistry Students
(Biochem. 364)
Third Year Students (Biochemistry)
Contents
Topics Page no.

Basic terminology 2
Discovery of Antibiotics 5
Sources of antibiotics 10
How do antibiotics work? 11
Mechanism of antibiotics actions 13
Beta lactamase enzyme 30
Multidrug resistance organisms 31
Why resistance is concern? 37
Multidrug resistance factors 39
Mechanisms of Multidrug resistance 41
Strategies of containment 46
In vivo and in vitro delivery 50
Drug resistance in caner 54
Overcoming multidrug resistance 55
Practical Antibiotics 62
References 95
1
Antibiotics
The term antibiotics literally means “against life”; in this case, against
microbes. There are many types of antibiotics—antibacterials, antivirals,
antifungals, and antiparasitics. Some drugs are effective against many
organisms; these are called broad-spectrum antibiotics. Others are
effective against just a few organisms and are called narrow spectrum
antibiotics.
The term "antibiotic" was coined by Selman Waksman in 1942 to
describe any substance produced by a microorganism that is
antagonistic to or inhibit the growth of other microorganisms in high
dilution. This original definition excluded naturally occurring substances
that kill bacteria but are not produced by microorganisms (such as gastric
juice and hydrogen peroxide) and also excluded synthetic antibacterial
compounds such as the sulfonamides.
With advances in medicinal chemistry, most antibiotics are now semi
synthetic modified chemically from original compounds found in nature,
as is the case with beta-lactams (which include the penicillins, produced
by fungi in the genus Penicillium, the cephalosporins, and the
carbapenems). Some antibiotics are still produced and isolated from
living organisms, such as the aminoglycosides, and others have been
created through purely synthetic means: the sulfonamides, the
quinolones, and the oxazolidinones.
In addition to this origin-based classification into natural, semi synthetic,
and synthetic, antibiotics may be divided into two broad groups according
to their effect on microorganisms: Those that kill bacteria are
bactericidal agents, whereas those that only impair bacterial growth are
known as bacteriostatic agents.
2
Antibiotic is a chemical substance produced by a microorganism that

inhibits the growth of or kills other microorganisms. Antibiotics belong to
the broader group of antimicrobial compounds, used to treat infections
caused by microorganisms, including fungi and protozoa.
Antimicrobial agent is a chemical substance derived from a biological
source or produced by chemical synthesis that kills or inhibits the growth
of microorganisms. The two terms are usually used synonymously.
Antimicrobial agents are an essential component of the practice of
medicine
Basic Terminology of chemotherapy

o Chemotherapy: The use of drugs to treat a disease
o Antimicrobial drugs: Interfere with the growth of microbes within a
host
o Antibiotic: Substances produced by the natural metabolic processes of
some microorganisms that can inhibit or destroy other microorganisms
o Selective toxicity: A drug that kills harmful microbes without
damaging the host
o Semisynthetic Drugs
Drugs that are chemically modified in the laboratory after being isolated
from natural sources
o Synthetic Drugs
Drugs produced entirely by chemical reactions
o Narrow Spectrum activity (Limited Spectrum)
Antimicrobials effective against a limited array of microbial types for
example, a drug effective mainly on gram-positive bacteria
o Broad Spectrum activity (Extended Spectrum)
Antimicrobials effective against a wide variety of microbial types—for
3
example, a drug effective against both gram-positive and gram-negative

bacteria
Lethal dose, LD50 The value of LD50 for a substance is the dose required
to kill half the members of a tested population after specified test
duration.
The minimum inhibitory concentration (MIC) is the lowest
concentration of a chemical that prevents visible growth of a bacterium
(in other words, at which it has bacteriostatic activity)
The minimum bactericidal concentration (MBC) is the concentration
that results in microbial death (In other words, the concentration at which
it is bactericidal)
Characteristics of the Ideal Antimicrobial Drug

o Selectively toxic to the microbe but nontoxic to host cells
o Microbicidal rather than microbistatic
o Relatively soluble; functions even when highly diluted in body fluids
o Remains potent long enough to act and is not broken down or excreted
prematurely
o Doesn’t lead to the development of antimicrobial resistance
o Complements or assists the activities of the host’s defenses
o Remains active in tissues and body fluids
o Readily delivered to the site of infection
o Low priced
o Does not disrupt the host’s health by causing allergies or predisposing
the host to other infections
o Spectrum of Activity (Broad vs. Narrow) Coordinated with Diagnosis
o For example:
o a broad-spectrum antibiotic would be indicated against a
4
polymicrobial infection, e.g., an intrabdominal anaerobic infection

o a narrow spectrum antibiotic would be ideal for an infection caused
by a single pathogen, e.g., a staphylococcal skin infection.
o Lack of "Side Effects"
o Able to Cross Outer and Cytoplasmic Membranes
o No or Low Level of Antibiotic Resistance in Target Pathogen and
Lack of Cross-Resistance in Closely Related Strains
o Resistant to Inactivation by Microbial Enzymes
Discovery of Antibiotics
The great modern advances in chemotherapy have come from the chance
discovery that many microorganisms synthesize and excrete compounds
that are selectively toxic to other microorganisms. These compounds are
called antibiotics and have revolutionized medicine.
The period since World War II has seen the establishment and extremely
rapid growth of a major industry, using microorganisms for the synthesis
of, amongst other compounds, chemotherapeutic agents.
The development of this industry has had a dramatic and far-reaching
impact. Nearly all bacterial infectious diseases that were, prior to the
antibiotic era, major causes of human death have been brought under
control by the use of chemotherapeutic drugs, including antibiotics. In the
United States, bacterial infection is now a less frequent cause of death
than suicide or traffic accidents.
The first chemotherapeutically effective antibiotic was discovered in
1929 by Alexander Fleming, a British bacteriologist, who had long been
interested in the treatment of wound infections. On returning from a
vacation in the countryside, he noticed among a pile of petri dishes on his
5
bench one that had been streaked with a culture of Saphyloccocus aureus,
which was also contaminated, by a single colony of mold. As Fleming
observed the plate, he noted that the colonies immediately surrounding
the mold were transparent and appeared to be undergoing lysis. He
reasoned that the mould was excreting into the medium a chemical that
caused the surrounding colonies to lyse. Sensing the possible
chemotherapeutic significance of his observation, Fleming isolated the
mold, which proved to be a species of Penicillium, and established that
culture filtrates contained an antibacterial substance, which he called
penicillin
History of the Development of Antibiotics

Penicillin, the first natural antibiotic discovered by Alexander Fleming in
1928.
 The discovery of the first antibiotic was an accident.

Alexander Fleming accidentally contaminated a plate with a fungus.
He observed a clearly defined region of no bacterial growth where the
fungi had contaminated the plate.
The area around the fungus was eventually referred to as a zone of
inhibition.
6
F.g.1 The original plate made by Alexander Fleming showing Penicillium

inhibiting bacterial growth
Sir Alexander Fleming (1928)

Observed that colonies of the bacterium Staphylococcus aureus could be
destroyed by the mold Penicillium notatum, demonstrating antibacterial
properties.
Gerhard Domagk (1935)
Prontosil, the first sulfa drug, was discovered in 1935 by German chemist
Howard Florey and Ernst Chain (1942)
The manufacturing process for Penicillin G was invented by Howard
Florey and Ernst Chain. Penicillin could now be sold as a drug. Fleming,
Florey, and Chain shared the 1945 Nobel Prize for medicine for their
work on penicillin.
7
Selman Waksman (1943)

In 1943, American microbiologist Selman Waksman made the drug
streptomycin from soil bacteria, the first of a new class of drugs called
aminoglycosides.
The success of antibiotics has been impressive. At the same time,

however, excitement about them has been tempered by a phenomenon
called antibiotic resistance. This is a problem that surfaced not long after
the introduction of penicillin and now threatens the usefulness of these
important medicines.
8
9
Sources of Antibiotics
o Natural - mainly fungal and actinomycetes sources
o Semi-synthetic - chemically altered natural compound
o Synthetic - chemically designed in the lab
• Natural The original antibiotics were derived from microbial sources.
These can be referred to as “natural” antibiotics. Organisms develop
resistance faster to the natural antimicrobials because they have been
pre-exposed to these compounds in nature. Benzylpenicillin and
Gentamicin are natural antibiotics
• Semi-synthetic drugs were developed to decrease toxicity and increase
effectiveness • Ampicillin and Amikacin are semi-synthetic
antibiotics
• Synthetic drugs have an advantage that the bacteria are not exposed to
the compounds until they are released. They are also designed to have
even greater effectiveness and less toxicity.
Moxifloxacin and Norfloxacin are synthetic antibiotics
There is an inverse relationship between toxicity and effectiveness as you
move from natural to synthetic antibiotics
Properties of an ideal antibiotic

 Broad spectrum
 Stable--long shelf life
 Soluble in body fluids
 Stable toxicity
 Non allergenic
 Reasonable cost
 Selectively toxic (kills harmful microbes without damaging the host
 Not likely to induce bacterial resistance
10
How Do Antibiotics Work?

Various types of antibiotics work in either of the following two ways:
1. A Bacteriostatic antibiotic stops bacteria from multiplying by
interfering with bacterial protein production, DNA replication, or other
aspects of bacterial cellular metabolism.
Some Bacteriostatic antibiotics are tetracyclines, sulphonamides,
spectinomycin, trimethoprim, chloramphenicol, macrolides and
lincosamides.
2. A Bactericidal antibiotic kills the bacteria generally by either

interfering with the formation of the bacterium's cell wall or its cell
contents.
Penicillin, daptomycin, fluoroquinolones, metronidazole, nitrofurantoin
and co-trimoxazole are some example of Bactericidal antibiotics.
At which drug concentration is the bacterial population

inhibited?
1- Bacteriostatic effect.
To inhibit multiplication Antibiotics have a bacteriostatic effect.
At which drug concentration is the bacterial population inhibited?
• Minimal Inhibitory Concentration = MIC
Bacteriostatic = inhibits bacterial growth
11
Quantitative Measure
• MIC=lowest concentration of antibiotic that inhibits growth
(measured visually)
2- Bactericidal effect.
To destroy the bacterial population Antibiotics have a

bactericidal effect.
At which drug concentration is the bacterial
population killed?
• Minimal Bactericidal Concentration = MBC

Bactericidal = kills bacteria
12
Quantitative Measure MBC=lowest concentration of

antibiotic that kills bacteria
Mechanisms of Action of Antibiotics
The goal of antimicrobial drugs is either to disrupt the cell processes or

structures of bacteria, fungi, and protozoa or to inhibit virus replication.
Most of the drugs used in chemotherapy interfere with the function of
enzymes required to synthesize or assemble macromolecules, or they
destroy structures already formed in the cell. Above all, drugs should be
selectively toxic, which means they should kill or inhibit microbial cells
without simultaneously damaging host tissues. This concept of selective
toxicity is central to antibiotic treatment, and the best drugs are those that
block the actions or synthesis of molecules in microorganisms but not in
13
vertebrate cells. Examples of drugs with excellent selective toxicity are

those that block the synthesis of the cell wall in bacteria (penicillins).
They have low toxicity and few direct effects on human cells because
human cells lack the chemical peptidoglycan and are thus unaffected by
this action of the antibiotic. Among the most toxic to human cells are
drugs that act upon a structure common to both the infective agent and
the host cell, such as the cell membrane (for example, amphotericin B,
used to treat fungal infections). As the characteristics of the infectious
agent become more and more similar to those of the host cell, selective
toxicity becomes more difficult to achieve, and undesirable side effects
are more likely to occur. The previous example briefly illustrates this
concept.
Antibiotic Targets and Mechanisms of Action

The most widely used antibiotics interfere with cellular processes and can
be grouped into 5 target categories
1. Bacterial cell wall biosynthesis

2. Bacterial cell membranes
3. Bacterial protein biosynthesis
4. Bacterial nucleic acid
5. Bacterial metabolites
CLASSIFICATION BY MECHANISM OF ACTION

a. Drugs that inhibit bacterial wall synthesis or activate
enzymes that disrupt the cell wall.
b. Drugs that increase cell membrane permeability (causing
leakage of intracellular material)
c. Drugs that cause lethal inhibition of bacterial protein
synthesis.
14
d. Drugs that cause nonlethal inhibition of protein synthesis

(bacteriostatics).
e. Drugs that inhibit bacterial synthesis of nucleic acids
15
Antibiotic Targets and Mechanisms of Action
Fig.2. Antibiotic target sites

16
Table 1 different class of antibiotics and their mode of action
Group Mode of action Examples

1) Beta-lactams Inhibition of Cell Wall Penicillins, Cephalosporins
Synthesis
Carbapenems, monobactams
2) Glycopeptides Inhibition of Cell Wall Vancomycin, Teichoplanin

Synthesis
3) Macrolides Inhibition of Protein Synthesis Azithromycin, Erythromycin
4) Aminoglycosides Inhibition of Protein Synthesis Gentamicin, Amikacin

streptomycin
5) Tetracyclines Inhibition of Protein Synthesis Tetracycline, Minocycline
Inhibition of Nucleic acid Ciprofloxacin, Norfloxacin,

6) Quinolones
synthesis Rifamycins
Alteration of Cell Metabolism Trimethoprim, Trimetrexate

7) Sulfonamides
Pyrimethamine
8) Polymyxins, Alteration of Cell Membrane Amphotericin B, Nystatin

Imidazole Function
17
1. Beta-lactams (Inhibition of Cell Wall Synthesis)

 Members of this class of antibiotics contain a 3-carbon and 1-nitrogen ring
that is highly reactive.
18
 They interfere with proteins essential for synthesis of bacterial cell wall,
and in the process either kills or inhibits their growth.
 More succinctly, certain bacterial enzymes termed penicillin-binding
protein (PBP) are responsible for cross-linking peptide units during
synthesis of peptidoglycan.
 Members of beta-lactam antibiotics are able to bind themselves to these
PBP enzymes, and in the process, they interfere with the synthesis of
peptidoglycan resulting to lysis and cell death.
 The most prominent representatives of the beta-lactam class include
Penicillins, Cephalosporins, Monobactams and Carbapenems.
Structure of the peptidoglycan
19
2- Glycopeptide antibiotics (Inhibition of Cell Wall Synthesis)

 A class of drugs of microbial origin that are composed of
glycosylated cyclic or polycyclic nonribosomal peptides.
 Significant glycopeptide antibiotics include the anti-infective
antibiotics vancomycin, teicoplanin, telavancin. Vancomycin is
used if infection with methicillin-resistant Staphylococcus aureus
(MRSA) is suspected.
 This class of drugs inhibits the synthesis of cell walls in
susceptible microbes by inhibiting peptidoglycan synthesis. They
bind to the amino acids within the cell wall preventing the addition
of new units to the peptidoglycan.
20
 In particular, they bind to acyl-D-alanyl-D-alanine in

peptidoglycan.
Mode of action of Glycopeptides

21
3-Macrolides, Aminoglycosides and Tetracycline (Inhibition

of protein synthesis):-
Macrolides bind to the 50S ribosomal subunit and interfere with the
elongation of nascent polypeptide chains.
Aminoglycosides inhibit initiation of protein synthesis and bind to the
30S ribosomal subunit. Chloramphenicol binds to the 50S ribosomal
subunit blocking peptidyltransferase reaction.
Tetracyclines inhibit protein synthesis by binding to 30S subunit of
ribosome, thereby weakening the ribosome-tRNA interaction.
22
30S Inhibitors
Aminoglycosides
Gentamicin, Neomycin, Kanamycin, Amikacin, Streptomycin
 Binds tightly to 30S ribosome causing misreading of mRNA. They can
also interfere with the initiation complex (binding of 30S & 50S
ribosomes with mRNA), and can cause the breakup of polysomes into
nonfunctional monosomes. The overall effect is irreversible, resulting in
bacterial cell death.
Tetracyclines
 Bind reversibly to 30S subunit, blocking the binding of the amino acid
containing tRNA (aminoacyl-tRNA) to the acceptor side on the mRNA-
ribosome complex.
50S Inhibitors
Macrolides
 Macrolides: Erythromycin, Clarithromycin & Azithromycin
23
 Macrolides bind to the peptide exit tunnel in the 50S subunit (near the
peptidyltransferase center) and prevent peptide chain elongation.
 Can also inhibit formation of the 50S ribosomal subunit.
 Bacteriostatic (usually), but can be bactericidal to some bacteria in high
concentrations.
6. Quinolones (Inhibition of nuclei acid synthesis)
Antibiotics interfere with nuclei acid synthesis by blocking replication or
stopping transcription.
DNA replication involves the unwinding of the traditional double helix
structure, a process facilitated by the helicase enzymes.
The quinolones group of antibiotics, for example, do interfere with the
functionality of the helicase enzyme thereby disrupts the enzyme from
playing its function of unwinding DNA.
This antibiotic action of the quinolones ultimately truncates the process
of DNA replication and repair amongst susceptible bacteria.
Antibiotics whose mode of action is inhibition of nucleic acid synthesis
also target topoisomerase II and topoisomerase IV of bacteria.
Disrupting the activities of these enzymes in bacteria adversely affects
RNA polymerase, which in turn prevents RNA synthesis.
Rifampicin interferes with a DNA-directed RNA polymerase.
Quinolones disrupt DNA synthesis by interference with type II
topoisomerases DNA gyrase and topoisomerase IV during replication and
by causing double strand breaks.
7- Sulfonamides (Inhibition of a metabolic pathway)
The sulfonamides (e.g. sulfamethoxazole) and trimethoprim each block
the key steps in folate synthesis, which is a cofactor in the biosynthesis of
nucleotides, the building blocks of DNA and RNA.
24
 Folic acid is vital in the metabolism of nucleic acid and amino acids;
for this reason, sulfonamides ultimately disrupt the production of
nucleic acids (DNA and RNA) and amino acids, as they mimic
substrates required for folic acid metabolism.
Antibiotic or Antimicrobial resistance (AMR)
Antibiotic resistance is the ability of a microbe to resist the effects of
antibiotic medication previously used to treat them.
Resistance arises through one of three ways: natural resistance in
certain types of bacteria, genetic mutation, or by one species acquiring
resistance from another.
Resistance can appear spontaneously because of random mutations; or
more commonly following gradual buildup over time, and because of
misuse of antibiotics or antimicrobials.
25
Resistant microbes are increasingly difficult to treat, requiring alternative

medications or higher doses, both of which may be more expensive or
more toxic.
Microbes resistant to multiple antimicrobials are called multidrug
resistant (MDR); or sometimes superbugs. Antimicrobial resistance is on
the rise with millions of deaths every year. All classes of microbes
develop resistance.
MDR has been identified as a major threat to the public health of human
being by the World Health Organization (WHO).
The overuse, underuse and general misuse of antibiotics are major factors
in the emergence and dissemination of resistance.
Antibiotic misuse, sometimes called antibiotic abuse or antibiotic
overuse, refers to the misuse or overuse of antibiotics, with potentially
serious effects on health. It is a contributing factor to the development of
antibiotic resistance, including the creation of multidrug-resistant
bacteria, informally called "super bugs": relatively harmless bacteria can
develop resistance to multiple antibiotics and cause life-threatening
infection
In summary, the main causes of antibiotic resistance have been linked

to:
 Over-prescription of antibiotics
 Patients not finishing the entire antibiotic course
 Overuse of antibiotics in livestock and fish farming
 Poor infection control in health care settings
 Poor hygiene and sanitation
 Absence of new antibiotics being discovered
26
 The overuse, underuse and general misuse of antibiotics are major

factors in the emergence and dissemination of resistance
Mechanisms of antibiotic resistance

The four main mechanisms by which microorganisms exhibit resistance
to antimicrobials are:
27
The four main mechanisms by which microorganisms exhibit

resistance to antibiotics
1. Drug inactivation or modification: for example, enzymatic deactivation
of penicillin in some penicillin-resistant bacteria through the production
of β-lactamases.
Most commonly, the protective enzymes produced by the bacterial cell
will add an acetyl or phosphate group to a specific site on the antibiotic,
which will reduce its ability to bind to the bacterial ribosomes and disrupt
protein synthesis.
2. Alteration of metabolic pathway: for example, some sulfonamide-
resistant bacteria do not require para-aminobenzoic acid (PABA), an
28
important precursor for the synthesis of folic acid and nucleic acids in
bacteria inhibited by sulfonamides, instead, like mammalian cells, they
turn to using preformed folic acid.
3. Reduced drug accumulation: by decreasing drug permeability or
increasing active efflux (pumping out) of the drugs across the cell
surface[133] These pumps within the cellular membrane of certain bacterial
species are used to pump antibiotics out of the cell before they are able to
do any damage. They are often activated by a specific substrate
associated with an antibiotic. as in fluoroquinolone resistance
4. Efflux pumps: are transport proteins involved in the extrusion of toxic
substrates (including virtually all classes of clinically relevant antibiotics)
from within cells into the external environment. These proteins are found
in both Gram-positive and -negative bacteria as well as in eukaryotic
organisms.
Pumps may be specific for one substrate or may transport a range of
structurally dissimilar compounds (including antibiotics of multiple
classes); such pumps can be associated with multiple drug resistance
(MDR).
29
Beta-lactamases (β-lactamases enzymes)

Beta-lactamases (β-lactamases) are enzymes produced by bacteria (also known as
penicillinase) that provide multi-resistance to β-lactam antibiotics such as penicillins,
cephamycins.
Beta-lactamase provides antibiotic resistance by breaking the antibiotics' structure.
These antibiotics all have a common element in their molecular structure: a four-atom
ring known as a β-lactam. Through hydrolysis, the lactamase enzyme breaks the β-
lactam ring open, deactivating the molecule's antibacterial properties.
Beta-lactam antibiotics are typically used to treat a broad spectrum of Gram-positive and
Gram-negative bacteria.
Beta-lactamases produced by Gram-negative organisms are usually secreted, especially
when antibiotics are present in the environment.
Methods for β lactamase detection
β lactamase production can be detected by three different methods. Chromogenic
method is based on the principle that hydrolysis of certain β lactam antibiotic leads to a
distinct color change from a light yellow to a deep red color.
Acidimetric method uses a pH indicator color change from purple pink to yellow to
detect the formation of at least one extra carboxyl group produced during the hydrolysis
of β lactam antibiotic by β lactamase.
Iodometric method detects the loss of blue color from a blue starch/iodine complex
caused by the removal of iodine from the complex by the reducing action of a β
lactamase hydrolysis product.
Iodometric method for detection of β-lactamase activity
This method is based on the fact that the Penicillin resistance gene product, β-lactamase,
can hydrolyze penicillin G and release a reducing product (penicilloic acid), which can
be visualized by the discoloration of a dark blue iodine starch complex.
30
Antimicrobial Therapy
Clinicians need to treat patients with these potentially life-threatening infections with an
appropriate initial antimicrobial regimen while also trying to minimize the emergence
of resistant pathogens.
MDRO (multidrug resistant organisms)
Definition
Microorganisms, predominantly bacteria, which are resistant to one or more classes of
antimicrobial agents.
Although the names of certain MDROs describe resistance to only one agent (e.G.,
MRSA, VRE), these pathogens are frequently resistant to most available antimicrobial
agents.
 Vancomycin-resistant Enterococcus (VRE) .
 Methicillin-resistant Staphylococcus aureus (MRSA)
31
In addition to MRSA and VRE, certain gram negative bacteria (GNB), including those
producing extended spectrum beta-lactamases (ESBLs) and others that are resistant to
multiple classes of antimicrobial agents, are of particular concern.
Drug-resistant pathogens are a growing threat to all people, especially in healthcare
settings.
Each year nearly 2 million patients in the United States get an infection in a hospital. Of
those patients, about 90,000 die as a result of their infection. More than 70% of the
bacteria that cause hospital-acquired infections are resistant to at least one of the drugs
most commonly used to treat them.
Persons infected with drug-resistant organisms are more likely to have longer hospital
stays and require treatment with second- or third-choice drugs that may be less
effective, more toxic, and/or more expensive.
Clinical importance of MDROs:
In most instances, MDRO infections have clinical manifestations that are similar to
infections caused by susceptible pathogens. However, options for treating patients with
these infections are often extremely limited. Although antimicrobials are now available
for treatment of MRSA and VRE infections, resistance to each new agent has already
emerged in clinical isolates.
Similarly, therapeutic options are limited for ESBL-producing isolates of gram-negative
bacilli.
These limitations may influence antibiotic usage patterns in ways that suppress normal
flora and create a favorable environment for development of colonization when exposed
to potential MDR pathogens (i.e., selective advantage).
Increased lengths of stay, costs, and mortality also have been associated with MDROs.
Gram-negative resistant Bacteria:
GNB resistant to ESBLs, fluoroquinolones, carbapenems, and aminoglycosides also
have increased in prevalence.
32
*For example, in 1997, the SENTRY Antimicrobial Surveillance Program found that
among K. pneumoniae strains isolated in the United States, resistance rates to
ceftazidime and other third-generation cephalosporins were 6.6%, 9.7%, 5.4%, and
3.6% for blood stream, pneumonia, wound, and urinary tract infections, respectively
*In 2003, 20.6% of all K. pneumoniae isolates from NNIS ICUs were resistant to these
drugs.
Resistance in microbes is a natural phenomenon
 Resistance is unresponsiveness to antimicrobial agents in standard doses
 A natural biological unstoppable phenomenon
 Resistance is generally slow to reverse or irreversible
 All antimicrobial agents have the potential to select drug-resistant subpopulations of
microorganisms.
Resistance is fallout of inappropriate use of antimicrobials in different settings:
 In animals and plants:
– Therapeutic and non-therapeutic (e.g. as growth promoters)
 In community acquired infections
 In hospital-associated infections
 Irrational use of antibiotics is the greatest driver of resistance
– 50% of antibiotics are prescribed inappropriately
– 50% of patients have poor compliance
– 50% of populations do not have access to essential antibiotics
Risk factors that promote antimicrobial resistance in healthcare settings include:

1) Extensive use of antimicrobials
2) Transmission of infection
3) Susceptible hosts
Key Prevention Strategies:
 “Prevent infection”
33
 Diagnose and treat infection effectively “

 Use antimicrobials wisely “
 Prevent transmission
 Once resistant strains of bacteria are present in a population, exposure to
antimicrobial drugs favors their survival.
 Reducing antimicrobial selection pressure is one key to preventing antimicrobial
resistance and preserving the utility of available drugs for as long as possible.
 Bacteria have evolved numerous mechanisms to evade antimicrobial drugs.
 Chromosomal mutations are an important source of resistance to some
antimicrobials.
 Acquisition of resistance genes or gene clusters, via conjugation, transposition, or
transformation, accounts for most antimicrobial resistance among bacterial pathogens.
 These mechanisms also enhance the possibility of multi-drug resistance.
Plasmids:
 Rings of extra chromosomal DNA
 Can be transferred between different species of bacteria
34
 Carry resistance genes

 Most common and effective mechanism of spreading resistance
from bacteria to bacteria (Bacterial Conjugation)
Beta-Lactamases: What are they?

 Enzymes produced by certain bacteria that provide resistance to certain antibiotics
 Produced by both gram positive and gram negative bacteria
 Found on both chromosomes and plasmids
Beta-lactam Antibiotics
Examples
•Penicillins:
–Penicillin, amoxicillin, ampicillin
•Cephalosporins:
–Cephalexin, Cefuroxime, Ceftriaxone
•Carbapenems:
–Imipenem, meropenem
 Resistance that is produced through the actions of beta lactamases.

 Extended spectrum cephalosporins, such as the third generation cephalosporins,
were originally thought to be resistant to hydrolysis by beta-lactamases!
35
 Multiple antimicrobial resistances are often a characteristic of ESBL producing

gram-negative bacteria.
The story is more complicated….
 •Ceftazidime
 •Cefotaxime
•Ceftriaxone
 •Aztreonam
 •Genes encoding for ESBLs are frequently located on plasmids that also
carry resistance genes for:
 •Aminoglycosides
 •Tetracycline
 •TMP-SULFA
 •Chloramphenicol
 •Fluoroquinolones
If an ESBL is detected, all penicillins, cephalosporins, and aztreonam should be

reported as ―resistant‖, regardless of in vitro susceptibility test results
However: ESBL producing organisms are still susceptible to:
•Cephamycins:
–Cefoxitin
–Cefotetan
•Carbapenems:
–Meropenem
–Imipenem
Carbapenems are becoming the therapeutic option of choice
ESBLs are harbingers of multi-drug resistance
36
Why resistance is a concern?

• Resistant organisms lead to treatment failure
• Increased mortality
• Resistant bacteria may spread in Community
• Low level resistance can go undetected
37
• Added burden on healthcare costs

• Threatens to return to pre-antibiotic era
• Selection pressure
Antibiotic Resistance
• The concentration of drug at the site of infection must inhibit the organism and
also remain below the level that is toxic to human cells.
• Defined as micro-organisms that are not inhibited by usually achievable
systemic concentration of an antimicrobial agent with normal dosage schedule and / or
fall in the minimum inhibitory concentration (MIC) range.
Drug resistance occurs in:
BACTERIA—ANTIBIOTIC RESISTANCE
• Endoparasites
• Viruses—Resistance to antiviral drugs
• Fungi
• Cancer cells
Myths of Antibiotic Resistance
• Antibiotics select out the resistant strain
• Faulty use of antibiotics or widespread use of antibiotics increases the probability
of such selection.
• Antibiotic resistant strains appear to be more virulent because we cannot kill them
or stop their growth.
38
Factors of Antibiotic Resistance:
1. Environmental Factors:
• Huge populations and overcrowding
• Rapid spread by better transport facility
• Poor sanitation
• Increases community acquired resistance
• Ineffective infection control program
• Widespread use of antibiotics in animal husbandry and agriculture and as
medicated cleansing products
2. Drug Related:
• Over the counter availability of antimicrobials
• Counterfeit and substandard drug causing sub-optimal blood concentration
• Irrational fixed dose combination of antimicrobials
• Soaring use of antibiotics
3. Patient Related:
• Poor adherence of dosage Regimens
• Poverty
• Lack of sanitation concept
• Lack of education
• Self-medication
39
• Misconception
4- Overuse of broad-spectrum antibiotics (cephalosporins) leads to the rise of
resistance.
• It permits the superinfection effect.
• Pathogens occupy areas where normal microbes have been killed.
• Antibiotics have essentially compromised the patient.
So,
The potential for global antibiotic resistance is real due to:
• Overuse of antibiotics
• Improper adherence to hospital infection control protocols
• Difficulty finding new antibiotics
• Ease of worldwide travel
• There are ways to lengthen the useful life of antibiotics.
Types of Antibiotics Resistance:

Natural resistance:
It occurs naturally.
1. Lack target :
• No cell wall; innately resistant to penicillin
2. Innate efflux pumps:
• Drug blocked from entering cell or ↑ export of drug (does not achieve adequate
internal concentration). Eg. E. coli, P. aeruginosa
3. Drug inactivation:
• Cephalosporinase in Klebsiella
Acquired resistance:
Mutations
• It refers to the change in DNA structure of the gene.
40
• Occurs at a frequency of one per ten million cells.

• Eg.Mycobacterium.tuberculosis,Mycobacterium lepra , MRSA.
• Often mutants have reduced susceptibility
Plasmids
• Extra chromosomal genetic elements can replicate independently and freely in
cytoplasm.
• Plasmids which carry genes resistant ( r-genes) are called R-plasmids.
• These r-genes can be readily transferred from one R-plasmid to another plasmid or to
chromosome.
• Much of the drug resistance encountered in clinical practice is plasmid mediated
Mechanisms of Resistance Gene Transfer
• Transfer of r-genes from one bacterium to another
 Conjugation
 Transduction
 Transformation
 Conjugation : Main mechanism for spread of resistance
The conjugative plasmids make a connecting tube between the 2 bacteria through
which plasmid itself can pass.
 Transduction : Less common method
The plasmid DNA enclosed in a bacteriophage is transferred to another bacterium of
same species. Seen in Staphylococci , Streptococci
 Transformation: least clinical problem.
Free DNA is picked up from the environment (i.e.. From a cell belonging to closely
related or same strain.
• Transfer of r-genes between plasmids within the bacterium
 By transposons
• Transposons are sequences of DNA that can move around different positions within
the genome of single cell.
41
• The donor plasmid containing the Transposons, co-integrate with acceptor plasmid.
They can replicate during cointegration
• Both plasmids then separate and each contains the r-gene carrying the transposon.
 By Integrons
The four main mechanisms of antibiotic resistance are:

(1) Enzymatic degradation of the drug,
(2) Modification of the drug's target,
(3) Reduced permeability of the drug,
(4) Active export of the drug.
• Most drug resistance is the result of a genetic change in the organism, caused either
by a chromosomal mutation or the acquisition of a plasmid or transposon.
Biochemical mechanisms of antibiotic resistance:
• Prevention of drug accumulation in the bacterium
• Modification/protection of the target site
• Use of alternative pathways for metabolic / growth requirements
• By producing an enzyme that inactivates the antibiotic
• Quorum sensing
42
Modification/Protection of the Target site

Resistance resulting from altered target sites:
43
By producing enzymes that inactivates antibiotic

a)Inactivation of b-lactam antibiotics
• S. aureus, N. gonorrohoea, H.influenza, Produce b-lactamase which cleaves -
lactam ring
b)Inactivation of Chloramphenicol
44
• Inactivated by chloramphenicol acetyltransferase.

• Gram-ve (enzyme present constitutively hence higher resistance) gram +ve
bacteria (enzyme is inducible )
c)Inactivation of Aminoglycosides
• Inactivated by acetyl, phospho & adenylyl transferases Present in gram +ve and
gram –ve .
Use of alternative pathways for metabolic / growth requirements
• Resistance can also occur by alternate pathway that bypasses the reaction inhibited
by the antibiotic.
• Sulfonamide resistance can occur from overproduction of PABA (para-
aminobenzoic acid: an intermediate in the synthesis of folate by bacteria, plants, and
fungi)
Quorum sensing:
• Microbes communicate with each other and exchange signaling chemicals
(Auto-inducers)
• These auto-inducers allow bacterial population to coordinate gene expression
for virulence, conjugation, apoptosis, mobility and resistance
45
• Poor clinical practices that fail to incorporate the pharmacological properties of

antimicrobials amplify the speed of development of drug resistance.
• Faulty Antibiotic Use: Antimicrobials are over prescribed, Available without
prescription.
• Over Prescribed Antibiotics: Clinician should first determine whether antimicrobial
therapy is warranted for a given patient.
• Empirical Microbial Selection: Is antimicrobial agents indicated on the basis of clinical
findings? Or is it prudent to wait until such clinical findings become apparent?
- Can some simple bed side test done to confirm your suspicion? Microscopy, Gram
staining.
- Have appropriate clinical specimens been obtained to establish a microbial diagnosis?
- What are the likely etiologic agents for the patient‘s illness?
- What measures should be taken to protect individuals exposed to the index case to
prevent secondary cases (1), and what measures should be implemented to prevent
further exposure (2)?
46
 Definitive Treatment:
- Can a narrower spectrum agent be substituted for initial empiric drug?
- Is one agent or combination of agents necessary?
- What are the: optimum dose, route of administration and duration of therapy?
- What specific test to identify patients who will not respond to treatment?
- What adjunctive measures can be undertaken to eradicate infection? Vaccination,
Steroid, Drainage of pus, Amputation, Removal of catheter
Multidrug resistance (MDR): is a major problem encountered in chemotherapy that

negatively impacts the treatment efficacy of chemotherapeutics. A number of
mechanisms have been reported for MDR, including increased efflux pumping of drugs
by the overexpressed ATP-binding cassette (ABC) transporters, reduced intracellular
accumulation of drugs by non-ABC drug transporters, and blocked apoptosis, repair of
drug-induced DNA damage, metabolic modification, and detoxification by drug-
metabolizing enzymes. Among these mechanisms, the overexpression of plasma
47
membrane P-glycoprotein (P-gp, or ABCB1), a member of the ABC superfamily, is one

of the most common causes of MDR.
The overexpression of other ABC transporters, such as MDR proteins and breast cancer
resistance protein (BCRP), has also been identified as a primary cause of MDR. To
suppress MDR of cancerous cells and maximize the cytotoxic efficacy of anticancer
drugs, a general strategy is to co-administrate one drug (e.g., a gene) to inhibit ABC
transporters and promote apoptosis together with another anticancer drug for the actual
treatment. To this end, nanoparticle carriers based on liposomes and polymers have been
utilized to encapsulate the dual components and at the same time ensure precise delivery
to the targeted sites.
Figure: Mechanisms of multiple-drug resistance in cancer cells: drug efflux caused by multidrug
resistance protein (MRP) (e.g., P-glycoprotein or P-gp), down-regulation of the sensitivity to drug by
the tumor suppressor protein p53, reduction in sensitivity to methotrexate and fluorouracil by
phosphorylation of the retinoblastoma protein (Rb), and production of resistance to camptothecins
through down-regulation expression or mutations in topoisomerases (Topo).
48
Cancer is an inherently biological disease, in which cell replication—one of the

hallmarks of life—fails to be regulated by the usual mechanisms. Chemotherapy—
treatment with cytotoxic chemicals—kills cancer cells. But most chemotherapeutics also
kill healthy cells. Cancer cells replicate rapidly, so they evolve rapidly and are
extraordinarily quick at developing drug resistance. Cancer nano-therapeutics are
progressing at a steady rate; research and development in the field has experienced an
exponential growth since early 2000's. All of chemothrapic drugs have different adverse
side effects such as fatigue, pain (headaches, muscle pain, Stomach pain, and pain from
nerve damage), mouth and throat sores, diarrhea, nausea and vomiting, constipation and
blood disorders.
Recent advances in cancer nanotechnology have raised exciting opportunities for
specific drug delivery by an emerging class of nano-therapeutics that may be targeted to
neoplastic cells, thereby offering a major advantage over conventional chemotherapeutic
agents. There are two ways by which targeting of nanoparticles may be achieved,
namely passive and active targeting nanotechnology is an active field of cancer- related
research. Early detection, accurate diagnosis, and individual treatment could be achieved
using different nano-materials. Nanotechnology holds enormous potential for
overcoming many of the problems associated with conventional methods, faces
difficulties in the detection, treatment, and diagnosis of cancer. By using
nanotechnology, nano-materials have been developed and evaluated for cancer
diagnostics.
Thus, nano-diagnostics, defined as the use of nanotechnology for clinical diagnostic
purposes were developed to meet the demands of clinical diagnostics for increased
sensitivity and earlier detection of disease. There is an increasing desire for the
developing of materials used in the diagnosis of cancer by nanotechnology such as,
nanoshells, carbon nanotubes, quantum dots, polymeric nanoparticles, dendrimers and
polynucleotide nanoparticles. The trend is also changed for designing the nano-carriers
49
which are fused with target molecules to reach specific site. These include
carbohydrates, antibodies, peptides, aptamers and other small molecules.
According to the National Nanotechnology Initiative nano-technologic materials should
be 1–100 nm in at least one dimension. This size requirement can be engineered through
various rational designs, including top-down and bottom-up methods.
In vitro and in vivo Delivery
Nanoparticles, when empowered with either passive or active targeting capability, can
enhance the concentration of drugs inside a tumor, while reducing systemic toxicity in
healthy tissues. The nanoparticles to serve as a drug carrier must be first evaluated in
vitro at the cellular level before they are further tested in vivo at the tissue, organ, and
body levels. On the other hand, only with sufficient knowledge of nanoparticle– cell
interactions can one start to engineer the properties of nanoparticles for optimal delivery
in vivo and effective cancer therapy.
Nanoparticles as a multi-drug-delivery system for the apoptosis are a modulator
ceramide and the chemotherapeutic drug paclitaxel. Their results indicate that the dual-
drug-delivery system could greatly improve chemo-sensitivity of ovarian cancer cells
exhibiting MDR by bypassing P-gp drug efflux.
Upon introduction into the body, the carrier needs to reach the target lesion and be
accumulated there before any treatment can take place. As a result, one has to deal with
many additional issues related to the transport of nanoparticles, as well as immune
response, selectivity and efficiency in targeting, bio-distribution, biodegradation,
clearance, and toxicity at the organ and system levels.
Ideally, the nanoparticles that serve as the carrier of a drug-delivery system should have
the following attributes: 1) a good targeting efficiency to ensure selective deposition of
drug in the target lesion while maintaining low concentrations in healthy tissues/organs;
2) consisting of biocompatible and/or biodegradable materials only; and 3) clearance
from the body within a predetermined time frame. In reality, however, it is almost
impossible to satisfy all these requirements.
50
The physicochemical properties of nanoparticles, including composition, size, shape,

morphology, surface charge, and surface coating, can all be tailored to improve their
performance in vivo. An interesting example can be found in the use of QDs for cancer
diagnostics and therapeutics.
However, through surface modifications such as PEGylation and conjugation with
targeting agents, it is feasible to reduce their accumulation in and toxicity to major
organs to an acceptable level by increasing their circulation half-life and reducing their
accumulation in organs in a less nonspecific manner.
Intravenous injection represents the most commonly used route for the administration of
nanoparticle-based therapeutics as it bypasses the barriers in the epithelial absorption
process by directly entering the circulatory system. During circulation, the size, shape,
and surface properties of the nanoparticles can all strongly affect their
behaviors/performance with respect to targeting and clearance.
Once a malignant tumor grows to >2 ‑3 mm3 in size, the delivery of oxygen and
nutrients becomes diffusion‑limited and the formation of new blood vessels becomes
essential to meet the ever increasing demands of the rapidly growing malignant cells.
This is accomplished through the release of angiogenic factors by the neoplastic tissue
aiming to increase the microvasculature within the tumor in order to sustain further
growth. The resultant imbalance of angiogenic factors and matrix metalloproteinases
(MMPs) within neoplastic tissues results in highly disorganized vessels, which are
dilated, with numerous pores and wide gap junctions between endothelial cells. The
perivascular cells and basement membrane are absent or defective.
Furthermore, tumor vessels frequently lack the smooth muscle layer that normally
surrounds endothelial cells. The normal vasculature is endowed with tight junctions that
are impermeable to molecules sized >2‑4 nm, thus keeping the nanoparticles within the
circulation; however, the leaky vasculature of neoplastic tissue allows macromolecules
with a diameter of ≥600 nm to extravasate into the neoplastic tissues. Since tumors do
51
not have a well‑developed lymphatic system, these extravasated nanoparticles tend to

stagnate within the neoplastic tissue. This phenomenon of leaky vasculature and
impaired lymphatic drainage has been referred to as the enhanced permeability and
retention (EPR) effect.
The EPR effect is one of the most important features and results of tumor angiogenesis.
The EPR effect is further enhanced by many pathophysiological factors involved in
enhancement of the extravasation of macromolecules in tumor tissues. This is controlled
by number of local mediators [For instance: bradykinin, nitric oxide (NO) /
peroxynitrite, vascular endothelial growth factor (VEGF), and others]. One factor that
lends to the increased retention is the lack of lymphatics around the tumor region which
would filter out such particles under normal conditions
The utilization of nanoparticles as drug carriers promises a significant improvement in
cancer treatment. Targeted delivery can reduce the systemic side effects that patients
must endure under traditional chemotherapy by ensuring that pronounced cytotoxic
levels of the drugs are only present at the tumor sites. Besides targeting, nanoparticles
have been designed to release their payloads in response to a variety of different stimuli,
either those specific to the tumor microenvironment, such as acidic pH values and
elevated secretion of certain enzymes (e.g., matrix metalloproteinases, MMPs), or
external ones, such as light exposure and heating, among others. Nanoparticles also offer
multi-functionality, combining both diagnostic (i.e., image contrast enhancement or
molecular recognition capability).
As conjugated to various nanoparticles to improve efficacy and increase binding affinity
to the cancer cells. The increased specificity results in a higher accumulation of drug
within the tumor rather than other vital organs, reducing toxicity and making the drug
better equipped to overcome MDR. Nano-conjugates or encapsulated into nanoparticles
evade the capture of ABC drug efflux pumps. Also, Nano-conjugates reduced
opsonization, promotion of the adsorption of proteins which may mask the particle
52
(dysopsonization), aggregation prevention, steric hindrance to block the binding of

reticuloendothelial system (RES) cells, which are responsible for the clearance of
nanoparticles, and stabilization of lipid layers.
The addition of antibodies and targeting ligands is aimed at increasing receptor-mediated
endocytosis. Biological activity of nanoparticles often relies heavily on the ability to
escape the endosome and enter the cytosol, making endosome escape units. However,
the intact Fc domain may also bind to the Fc receptors on normal cells causing an
activated signaling cascade that may result in increased immunogenicity. Nano-
medicines that have increased circulation time, precise multiple targeting mechanisms,
enhanced drug accumulation at the tumor site, delivered into the cytoplasm and/or nuclei
of cancer cells, and have the ability to carry combinations of therapeutic payloads are
attractive treatment options in overcoming MDR.
53
Drug Resistance in Cancer

Cancer drug resistance is a complex phenomenon that is influenced by drug inactivation,
drug target alteration, drug efflux, DNA damage repair, cell death inhibition, EMT,
inherent cell heterogeneity, epigenetic effects, or any combination of these mechanisms.
The current paradigm states that combination therapy should be the best treatment option
because it should prevent the development of drug resistance and be more effective than
any one drug on its own. Therefore, such treatment regimens should be considered and
developed to counteract the increasing prevalence of drug resistance in cancers.
Multidrug Resistance and Cancer Chemoprevention
P-glycoprotein P-glycoprotein is encoded by MDR1, also referred to as ABCB1. The
human MDR1 (ABCB1) gene is located on chromosome 7q21. It consists of 28 exons,
which encode a 1280-amino acid glycoprotein. The molecular structure of P-
glycoprotein consists of two bundles of six trans-membrane helices that form a drug-
binding cavity with two ATP-binding sites. Besides anticancer agents, various clinically
important drugs, including digoxin, verapamil, cyclosporin A, tacrolimus, quinidine,
talinolol, erythromycin, ivermectin, fexofenadine, progesterone and saquinavir, are
substrates of P-glycoprotein. P-glycoprotein is also expressed in normal tissues. It is
abundant in the apical membranes of many pharmacologically important epithelial
barriers, such as the intestinal epithelium, renal proximal tubular epithelium and the
blood-brain barrier. Therefore, it is considered that P-glycoprotein plays very important
roles in the absorption, distribution and elimination of many commonly used drugs, and
thus determines the efficacy and toxicity of drugs. For instance, P-glycoprotein at the
apical membrane of enterocytes acts as a biochemical barrier and restricts the absorption
of orally administered drugs. In addition to drug-drug interactions, food-drug
interactions can also occur. It is now established that foods can have pronounced impacts
on drug absorption, disposition and elimination.
Recently, attention has focused on phytochemicals, non-nutritive components of a plant-
based diet that possess cancer preventive properties. The signaling pathways that govern
54
cell proliferation, survival and oncogenesis are of prime interest in the biology of cancer.
Chemo-preventive phytochemicals, such as curcumin and capsaicin, are known to block
the NF-κB activation process.
Depiction of the primary mechanisms that enable cancer cells to become drug resistant.
These include drug inactivation, alteration of drug targets, drug efflux, DNA damage
repair, inhibition of cell death, epithelial to mesenchymal transition (EMT), and
epigenetic effects. In the case of EMT, stromal cells assist in this process and signal for
improved drug resistance in cancer cells. Cell adhesion molecules on stromal cells and
extracellular matrix proteins attach to the cell adhesion molecules on cancer cells.
Stromal cells and cancer cells also secrete factors that regulate EMT. The depiction
displays a simplified example of these cell interactions.
Overcoming multidrug resistance
Cancer chemotherapy is usually a marginal proposition in the sense that the maximum
dose tolerated by the patient is often barely sufficient to kill a useful percentage of the
cancer cells. Relatively small increases in drug resistance in cancer cells are thus
sufficient to render the drug ineffective. ABC transporters are expressed at cancer cell
membranes and can cause multidrug resistance. Therefore, ABC transporters seem to be
good targets for circumventing multidrug resistance. Since the discovery of P-
glycoprotein in the 1970s, various attempts and clinical trials have been carried out using
55
a P-glycoprotein inhibitor such as verapamil or cyclosporine to overcome multidrug

resistance. However, due to the side effects or ineffectiveness of these compounds, a
successful outcome has not been achieved. In general, natural dietary phytochemicals
from foods, herbs, and dietary supplements are thought to be less toxic to the body than
medical drugs. The quest for inhibitors of anticancer drug exporters has uncovered
natural compounds including quercetin, (-)-epigallocatechin gallate, curcumin,
capsaicin, and [6]-gingerol, as promising candidates. Figure 1 shows the chemical
structures and source of dietary chemopreventive phytochemicals that can inhibit P-
glycoprotein.
56
Four Core Actions to Prevent Antibiotic Resistance
Preventing Infections, Preventing Spread:

Preventing infections from developing reduces the amount of antibiotics used. This
reduction in antibiotic use, in turn, slows the pace of antibiotic resistance. Preventing
infections also prevents the spread of resistant bacteria. Antibiotic-resistant infections
can be prevented in many ways. This section focuses on Control Diseases Centers
(CDC‘s) works to prevent antibiotic-resistant infections in healthcare settings, in the
community, and in food. By preventing antibiotic resistance in healthcare settings,
57
patients‘ lives are better protected and their health can be better preserved. In addition,
healthcare facilities, systems, insurers and patients can save dollars that otherwise would
have been spent on more complex care and medications needed to manage antibiotic-
resistant infections. CDC works to prevent antibiotic resistance in healthcare settings by
providing a system to track resistance and prescribing patterns at national, regional, and
local levels; providing guidance to healthcare facilities interested in better antibiotic use;
and working to prevent all patient infections through infection control guidelines,
assistance implementing these guidelines, and laboratory expertise.
Tracking Antibiotic Resistance:
The National Antimicrobial Resistance Monitoring System (NARMS) and The CDC
reference laboratory conducts antibiotic susceptibility testing on isolates from sporadic
cases and outbreaks of illness.
The primary objectives of the NARMS program are to:
■Monitor trends in antibiotic resistance among enteric bacteria from humans, retail
meats, and food-producing animals.
■ Disseminate information on antibiotic resistance to promote interventions that reduce
antibiotic resistance among foodborne bacteria.
■ Conduct research to better understand the emergence, persistence, and spread of
antibiotic resistance.
■ Provide data that assist the FDA in making decisions about approving safe and
effective antibiotic drugs for animals.
Improving Antibiotic Use:
Antibiotics are widely used in food-producing animals, and according to data published
by FDA, there are more kilograms of antibiotics sold in the United States for food
producing animals than for people. This use contributes to the emergence of antibiotic-
resistant bacteria in food-producing animals.
58
Scientists around the world have provided strong evidence that antibiotic use in food
producing animals can harm public health through the following sequence of events:
■Use of antibiotics in food-producing animals allows antibiotic-resistant bacteria to
thrive while susceptible bacteria are suppressed or die.
■Resistant bacteria can be transmitted from food-producing animals to humans through
the food supply.
■Resistant bacteria can cause infections in humans.
■Infections caused by resistant bacteria can result in adverse health consequences for
humans.
Preventing Infections
Efforts to prevent foodborne and other enteric infections help to reduce both antibiotic
resistant infections and antibiotic-susceptible infections (those that can be treated
effectively with antibiotics). CDC activities that help prevent these infections include:
■estimating how much foodborne illness occurs.
■Monitoring trends in foodborne infections.
■Investigating outbreaks and sporadic cases of foodborne illness to stop outbreaks and
improve prevention.
■Attributing illnesses to specific foods and settings.
■Tracking and responding to changes in resistance.
■Determining the sources of antibiotic-resistant enteric infections.
■Educating consumers and food workers about safe food handling practices.
■Identifying and educating groups at high risk for infection.
■Promoting proper hand washing.
■Strengthening the capacity of state and local health departments to detect, respond to,
and report foodborne infections.
59
■ Developing better diagnostic tools to rapidly and accurately find sources of

contamination.
■ Providing recommendations for travelers on safe food and clean water.
Summary:
Chemotherapy is the most effective treatment for patients with cancer. The effectiveness
however is seriously limited by the phenomenon of MDR. Anticancer drugs can fail to
kill cancer cells for various reasons including variations in the absorption, metabolism
and delivery of drug to target tissues and tumor location in parts of the body into which
the drugs do not easily penetrate. Three major mechanisms have been proposed: first,
decreased uptake of water soluble drugs such as folate antagonists and cisplatin ,which
require transporters to enter the cells; second, various changes in cells that effects the
capacity of cytotoxic drugs to kill cells such as reduced apoptosis; and third, increased
energy dependent efflux of hydrophobic drugs where the intracellular drugs inside the
resistant cancer cells are kept at sub-lethal level. The most common of these mechanisms
is the efflux of hydrophobic drugs mediated by energy driven ABC transporters such as
P-glycoprotein, an integral membrane protein overexpressed in various malignancies.
The broad substrate specificity and the abundance of ABC transporter proteins have
been a major challenge towards attempts to circumvent ABC-mediated MDR in vivo.
Various generations of MDR modulators have represented novel and improved
interventions, although not to the perfection. The perfect reversing agent would be the
one which is efficient, devoid of unrelated pharmacological effects, shows no
pharmacokinetic interaction with other drugs and restores the treatment efficiency of the
anticancer drug to that observed in MDR negative phenotype. In this regard, recent
studies have shown that natural compounds found in vegetables, fruits, plant derived
beverages and herbal dietary supplements not only have anticancer properties, but may
also modulate P-gp activity. P-gp inhibitors found in natural products, especially those
found in traditional medicine and dietary supplements, have the potential to be
60
developed as MDR reversing agents which could lead to more successful chemotherapy.
Such elements from dietary sources possess the advantage of having least or no
pharmacokinetic interactions with the anticancer drugs concomitant to their MDR
modulatory activity. Furthermore, the likelihood of multiple alternative mechanisms for
MDR also exists, thereby warranting further investigations regarding the mechanistic
actions of novel modulators, for treatment as well as prevention of multidrug resistance
in different types of cancer cells.
61
62
Definition
Greek. anti, "against"; bios, "life") An antibiotic is a chemical substance produced by one
organism that is destructive to another. The word antibiotic (given by Waksman) came
from the word antibiosis a term coined in 1889 by Louis Pasteur's pupil Paul Vuillemin
which means a process by which life could be used to destroy life.
Current Definition: Antibiotics are products (metabolites) of various organisms
(bacteria, fungi, algae, higher plants, lower and higher animals) which at low
concentrations cause inhibition of life processes of any living object.
History of antibiotics
3000 years ago ancient Egyptians, Chinese and people of Central America used molds to
cure diseases.
19th century: Louis Pasteur & Robert Koch: Bacteria as causative agents & recognized
need to control them
Penicillin- the first antibiotic – 1928
 Alexander Fleming observed the killing of staphylococci by a fungus (Penicillium
notatum).
 Florey & Chain purified it by freeze drying (1940) - Nobel prize 1945 first use in a
patient: 1942 World War II: saved 12-15% of lives
Streptomycin
Selman Waksman - Streptomycin (1943) from soil bacteria
– Active against all Gram-negatives
– First antibiotic active against
Mycobacterium tuberculosis
Is the first of the new class of drugs called aminoglycosides?
– extracted from Streptomyces
– 20 other antibiotics, incl. neomycin, actinomycin
Other antibiotics
1955 Tetracyclin was patented by Lloyd Conover, which became the most prescribed
broad spectrum antibiotic in the United States.
63
1957 Nystatin was patented and used to cure many disfiguring and disabling fungal
infections.
1981SmithKline Beecham patented Amoxicillin or amoxicillin/clavulanate potassium
tablets, and first sold the antibiotic in 1998 under the tradenames of Amoxicillin,
Amoxil, and Trimox. Amoxicillin is a semisynthetic antibiotic.
Antibiotic/Antimicrobial
• Antibiotic: Chemical produced by a microorganism that kills or inhibits the growth of
another microorganism
• Antimicrobial agent: Chemical that kills or inhibits the growth of microorganisms
Sources of Antibiotics
1. Cyanobacteria
2. Bacteria
3. Actinomycetes
4. Fungi
5. Higher Organisms
Cyanobacteria
 Mostly produce toxins that kill higher organisms.
 Malingolide is an antibiotic produced by a cyanobacterium.
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Bacteria
 Out of the 19 different principal groups into which bacterias can be
divided (Bergey‘s Classification ) the following groups mostly are source
of antibiotics
Bacterial group number Bacterial type

2 Gliding bacteria
7 Gram –ve aerobic rods & cocci
8 Gram –ve facultatively
anaerobic rods
14 Gram +ve cocci
15 Bacillus
16 Gram +ve ,asporogenous rods
17 Actinomycetes & related
organisms
12 Mycoplasmas
Actinomycetes
 It is the largest source of antibiotics.
 90%-95% of which is produced by the genus
―Streptomyces‖ (soil bacteria).
 Another genus important for antibiotics production is
Micromonospora
Fungi
 Richest source is the genera ―penicillium‖ and ―aspergillus‖
65
Higher organisms
Such as algae, lichens, higher plants, protozoa, insects, molluscs, sponges, worms and
vertebrates
Microbial Sources of Antibiotics
66
Antibiotic Spectrum of Activity
• No antibiotic is effective against all microbes

Mechanisms of Antimicrobial Action
• Bacteria have their own enzymes for
– Cell wall formation
– Protein synthesis
– DNA replication
– RNA synthesis
– Synthesis of essential metabolites
67
MODE OF ACTIONS OF ANTIBIOTICS

Possible targets
•Inhibition of cell-wall synthesis
–inhibition of peptidoglycan cross-linking (beta-lactams)
–inhibition of peptidoglycan synthesis (vancomycin)
•Disruption of cell membrane
–polymyxins
•Inhibition of protein synthesis
–at 30S ribosomal subunit (aminoglycosides, tetracyclines)
–Blocks attachment of tRNA to A site
–Prevents peptide bond formation
–Binds the 30S ribosome
–Misreads mRNA
–at 50S ribosomal subunit (macrolides, chloramphenicol)
•Inhibition of nucleic acid
–inhibition of folic acid synthesis (sulphonamides, trimethoprim)
–inhibition of DNA gyrase (fluoroquinolones) binds and cross-links the double helix
block replication.
–inhibition of RNA synthesis (rifampin).
68
Antibiotic Producing Microorganisms:

study of bacterial isolates from soil of a selection of seven bacteria isolates from soil with
a broad spectrum against bacterial testing was diagnosed using a variety of morphological
tests and biochemical as well as have been diagnosed with isolates that Bacillus subtilius
and studied the effect of different temperatures and the effect of salinity on the growth.
A trial to find out a new antimicrobial agent producing bacteria from soil samples
screened for their antimicrobial activity against the pathogenic bacteria. This study
indicates that microorganism Bacillus polymyxa isolated from the soil could be an
interesting source of antimicrobial bioactive substances.
Bacillus polymyxa Bacillus subtilius

It was declared that strains of antibiotic producing fungi are present in the soil, which it is
possible to be harnessed by the pharmaceutical industries for the production of antibiotics
from local soil samples were collected from ten different locations in Pour plate method
involving serial dilution was used for the isolation of fungi.
69
The media used for the isolation were Malt Extract Agar (MEA), Potato Dextrose Agar
(PDA) and Plate Count Agar (PCA). The genera were Penicillium sp. , Aspergillus
fumigates , and Aspergillus niger. All the fungal isolates were found to inhibit the growth
of at least one of the pathogens which are: Candida albicans, Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus aureus.
Penicillium sp.
In regard to actinomycetes one hundred seventy five strains, with potential antibiotic
producing, were isolated from 38 different soil samples from different locations.
It showed broad-spectrum antibacterial and antifungal properties which can be further
exploited for industrial and biological applications.
Materials and Methods:
A- Isolation of Antimicrobial Agent Producing Microorganisms:
1- Fungi: soil samples were collected from ten different locations in Nigeria of 250 g
each was collected into sterile plastic containers and transported to microbiology
laboratory for isolate and defined.
Isolation of Fungi: put 10 gm of the soil samples were diluted in 90 ml of sterile distilled
water. Ten-fold serial dilution was carried out, 0.1 ml of 10-3 and 10-4 dilution were
planted out in duplicate unto Sabouraud Dextrose Agar and Malt Extract-Yeast Extract
Agar using a spread plate technique supplemented with 50 mg/ml of streptomycin to
inhibit the growth of bacteria, also the pH of the medium was adjusted to 5.8 to encourage
the growth of the fungi. The plates were incubated at room temperature (28 C) for 96
hours.
The temperature of the soil at the ten different sites was determined by the use of
70
thermometer. The thermometer was inserted into the soil up to depth of 5 cm and allowed
to stay for 10 minutes, after which the temperature reading was obtained. The average of
three consecutive readings was recorded for each site
The soil pH values were determined by digital pH meter using standard methods of
Watson and Brown. Using this method, 3g of soil sample was weighed into a beaker
containing 3 ml of distilled water, which was stirred for five seconds and allowed to stand
for 10 minutes. The electrode of the pH meter was then inserted into the slurry and swirled
carefully. The reading was taken thereof and the average of the consecutive readings was
recorded for each site
1- Actinomycetes: (Spread Plate Method): Single colonies of actinomycetes were
isolated by serial dilution and spread-plate method using Starch Casein Agar (SCA)
Medium. Soil samples (0.1 gram) were suspended in 9.9 ml normal saline (0.87% NaCl,
w/v) and serially diluted. Then, 0.1 mL of inoculums from desired dilution was spread
onto sterile SCA agar plates. After incubation at 35 ± 1°C for 3-4 days, Screening of
single colonies for antimicrobial activity was performed by cross streak method.
71
2- Bacteria: Soil samples were collected from different localities of Bhopal region in
India. Each 1 g of the sample was suspended in 9 ml sterile distilled water and shaken
vigorously for 2-3 minutes. The soil suspension was serially diluted in sterile normal
saline (0.85%) and the dilution from 10-3 and 10-10 were then plated on overlaid
Nutrient agar 0.8% with seeded test organisms and incubated at 37°C for 12 to 24 hours,
to screen for antagonistic bacteria. Colonies giving a clear zone of inhibition were
isolated and re-streaked over a fresh media plate.
72
73
Methods of Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods are divided into types based on the principle
applied in each system. They include:
Diffusion Dilution Diffusion & Dilution
Stokes method Minimum Inhibitory Concentration E-Test method
Kirby-Bauer method i) Broth dilution
ii)Agar Dilution
Disk Diffusion
Reagents for the Disk Diffusion Test
1. Müeller-Hinton Agar Medium
Of the many media available, Müeller-Hinton agar is considered to be the best for routine
susceptibility testing of non-fastidious bacteria for the following reasons:
* It shows acceptable batch-to-batch reproducibility for susceptibility testing.
* It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.
* It gives satisfactory growth of most nonfastidious pathogens.
* A large body of data and experience has been collected concerning susceptibility tests
performed with this medium.
Although Müeller-Hinton agar is reliable generally for susceptibility testing, results
obtained with some batches may, on occasion, vary significantly. If a batch of medium
does not support adequate growth of a test organism, zones obtained in a disk diffusion test
will usually be larger than expected and may exceed the acceptable quality control limits.
Only Müeller-Hinton medium formulations that have been tested according to, and that
meet the acceptance limits described in, NCCLS document M62-A7- Protocols for
Evaluating Dehydrated Müeller-Hinton Agar should be used.
Preparation of Müeller-Hinton Agar
Müeller-Hinton agar preparation includes the following steps.
1. Müeller-Hinton agar should be prepared from a commercially available dehydrated
base according to the manufacturer's instructions.
74
2. Immediately after autoclaving, allow it to cool in a 45 to 50C water bath.

3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed petri
dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm. This
corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30
ml for plates with a diameter of 100 mm.
4. The agar medium should be allowed to cool to room temperature and, unless the plate is
used the same day, stored in a refrigerator (2 to 8C).
5. Plates should be used within seven days after preparation unless adequate precautions,
such as wrapping in plastic, have been taken to minimize drying of the agar.
6. A representative sample of each batch of plates should be examined for sterility by
incubating at 30 to 35C for 24 hours or longer.
2. Preparation of antibiotic stock solutions
Antibitiotics may be received as powders or tablets. It is recommended to obtain pure
antibiotics from commercial sources, and not use injectable solutions. Powders must be
accurately weighed and dissolved in the appropriate diluents (Annexure III) to yield the
required concentration, using sterile glassware. Standard strains of stock cultures should be
used to evaluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted in 5
ml volumes and frozen at -20ºC or -60ºC.
Stock solutions are prepared using the formula (1000/P) X V X C=W, where P+potency of
the anitbiotic base, V=volume in ml required, C=final concentration of solution and
W=weight of the antimicrobial to be dissolved in V.
Preparation of dried filter paper discs
Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which
are placed in a Petri dish and sterilized in a hot air oven.
The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter of
2 mm. This delivers 0.005 ml of antibiotics to each disc.
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Storage of commercial antimicrobial discs

Cartridges containing commercially prepared paper disks specifically for susceptibility
testing are generally packaged to ensure appropriate anhydrous conditions. Discs should
be stored as follows:
* Refrigerate the containers at 8C or below, or freeze at -14C or below, in a nonfrost-
free freezer until needed. Sealed packages of disks that contain drugs from the ß-lactam
class should be stored frozen, except for a small working supply, which may be
refrigerated for at most one week. Some labile agents (e.g., imipenem, cefaclor, and
clavulanic acid combinations) may retain greater stability if stored frozen until the day of
use.
* The unopened disc containers should be removed from the refrigerator or freezer one to
two hours before use, so they may equilibrate to room temperature before opening. This
procedure minimizes the amount of condensation that occurs when warm air contacts cold
disks.
* Once a cartridge of discs has been removed from its sealed package, it should be placed
in a tightly sealed, desiccated container. When using a disc-dispensing apparatus, it should
be fitted with a tight cover and supplied with an adequate desiccant. The dispenser should
be allowed to warm to room temperature before opening. Excessive moisture should be
avoided by replacing the desiccant when the indicator changes color.
* When not in use, the dispensing apparatus containing the discs should always be
refrigerated.
* Only those discs that have not reached the manufacturer's expiration date stated on the
label may be used. Discs should be discarded on the expiration date.
Turbidity standard for inoculum preparation
To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity standard,
equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle
suspension), should be used. A BaSO4 0.5 McFarland standards may be prepared as
follows:
76
1. A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 . 2H2O) is added to 99.5 ml
of 0.18 mol/L H2SO4 (1% v/v) with constant stirring to maintain a suspension.
2. The correct density of the turbidity standard should be verified by using a
spectrophotometer with a 1-cm light path and matched cuvette to determine the
absorbance. The absorbance at 625 nm should be 0.008 to 0.10 for the 0.5 McFarland
standard.
3. The Barium Sulfate suspension should be transferred in 4 to 6 ml aliquots into screw-
cap tubes of the same size as those used in growing or diluting the bacterial inoculum.
4. These tubes should be tightly sealed and stored in the dark at room temperature.
5. The barium sulfate turbidity standard should be vigorously agitated on a mechanical
vortex mixer before each use and inspected for a uniformly turbid appearance. If large
particles appear, the standard should be replaced. Latex particle suspensions should be
mixed by inverting gently, not on a vortex mixer
6. The barium sulfate standards should be replaced or their densities verified monthly.
Disc diffusion methods
The Kirby-Bauer and Stokes' methods are usually used for antimicrobial susceptibility
testing, with the Kirby-Bauer method being recommended by the NCCLS. The accuracy
and reproducibility of this test are dependent on maintaining a standard set of procedures
as described here.
NCCLS is an international, interdisciplinary, non-profit, non-governmental organization
composed of medical professionals, government, industry, healthcare providers, educators
etc. It promotes accurate antimicrobial susceptibility testing (AST) and appropriate
reporting by developing standard reference methods, interpretative criteria for the results
of standard AST methods, establishing quality control parameters for standard test
methods, provides testing and reporting strategies that are clinically relevant and cost-
effective
Interpretative criteria of NCCLS are developed based on international collaborative
studies and well correlated with MIC‘s and the results have corroborated with clinical
77
data. Based on study results NCCLS interpretative criteria are revised frequently.
NCCLS is approved by FDA-USA and recommended by WHO.
Procedure for Performing the Disc Diffusion Test
Inoculum Preparation
Growth Method
The growth method is performed as follows
1. At least three to five well-isolated colonies of the same morphological type are selected
from an agar plate culture. The top of each colony is touched with a loop, and the growth
is transferred into a tube containing 4 to 5 ml of a suitable broth medium, such as tryptic
soy broth.
2. The broth culture is incubated at 35C until it achieves or exceeds the turbidity of the
0.5 McFarland standards (usually 2 to 6 hours)
3. The turbidity of the actively growing broth culture is adjusted with sterile saline or
broth to obtain turbidity optically comparable to that of the 0.5 McFarland standards. This
results in a suspension containing approximately 1 to 2 x 108 CFU/ml for E.coli ATCC
25922. To perform this step properly, either a photometric device can be used or, if done
visually, adequate light is needed to visually compare the inoculum tube and the 0.5
McFarland standard against a card with a white background and contrasting black lines.
Direct Colony Suspension Method
1. As a convenient alternative to the growth method, the inoculum can be prepared by
making a direct broth or saline suspension of isolated colonies selected from a 18- to 24-
hour agar plate (a nonselective medium, such as blood agar, should be used). The
suspension is adjusted to match the 0.5 McFarland turbidity standards, using saline and a
vortex mixer.
2. This approach is the recommended method for testing the fastidious organisms,
Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for
potential methicillin or oxacillin resistance.
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Inoculation of Test Plates

1. Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension,
a sterile cotton swab is dipped into the adjusted suspension. The swab should be rotated
several times and pressed firmly on the inside wall of the tube above the fluid level. This
will remove excess inoculum from the swab.
2. The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the swab
over the entire sterile agar surface. This procedure is repeated by streaking two more
times, rotating the plate approximately 60 each time to ensure an even distribution of
inoculum. As a final step, the rim of the agar is swabbed.
3. The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for
any excess surface moisture to be absorbed before applying the drug impregnated disks.
NOTE: Extremes in inoculum density must be avoided. Never use undiluted overnight
broth cultures or other unstandardized inocula for streaking plates.
Application of Discs to Inoculated Agar Plates
1. The predetermined battery of antimicrobial discs is dispensed onto the surface of the
inoculated agar plate. Each disc must be pressed down to ensure complete contact with
the agar surface. Whether the discs are placed individually or with a dispensing
apparatus, they must be distributed evenly so that they are no closer than 24 mm from
center to center. Ordinarily, no more than 12 discs should be placed on one 150 mm plate
or more than 5 discs on a 100 mm plate. Because some of the drug diffuses almost
instantaneously, a disc should not be relocated once it has come into contact with the agar
surface. Instead, place a new disc in another location on the agar.
2. The plates are inverted and placed in an incubator set to 35C within 15 minutes after
the discs are applied. With the exception of Haemophilus spp., streptococci and N.
gonorrhoeae, the plates should not be incubated in an increased CO2 atmosphere,
because the interpretive standards were developed by using ambient air incubation, and
CO2 will significantly alter the size of the inhibitory zones of some agents.
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Reading Plates and Interpreting Results

1. After 16 to 18 hours of incubation, each plate is examined. If the plate was satisfactorily
streaked, and the inoculum was correct, the resulting zones of inhibition will be uniformly
circular and there will be a confluent lawn of growth. If individual colonies are apparent, the
inoculum was too light and the test must be repeated. The diameters of the zones of complete
inhibition (as judged by the unaided eye) are measured, including the diameter of the disc. Zones
are measured to the nearest whole millimeter, using sliding calipers or a ruler, which is held on
the back of the inverted petri plate. The petri plate is held a few inches above a black,
nonreflecting background and illuminated with reflected light. If blood was added to the agar
base (as with streptococci), the zones are measured from the upper surface of the agar
illuminated with reflected light, with the cover removed. If the test organism is a
Staphylococcus or Enterococcus spp., 24 hours of incubation are required for vancomycin and
oxacillin, but other agents can be read at 16 to 18 hours. Transmitted light (plate held up to
light) is used to examine the oxacillin and vancomycin zones for light growth of methicillin- or
vancomycin- resistant colonies, respectively, within apparent zones of inhibition. Any
discernable growth within zone of inhibition is indicative of methicillin or vancomycin
resistance.
2. The zone margin should be taken as the area showing no obvious, visible growth that can be
detected with the unaided eye. Faint growth of tiny colonies, which can be detected only with a
magnifying lens at the edge of the zone of inhibited growth, is ignored. However, discrete
colonies growing within a clear zone of inhibition should be subcultured, re-identified, and
retested. Strains of Proteus spp. may swarm into areas of inhibited growth around certain
antimicrobial agents. With Proteus spp., the thin veil of swarming growth in an otherwise
obvious zone of inhibition should be ignored. When using blood-supplemented medium for
testing streptococci, the zone of growth inhibition should be measured, not the zone of inhibition
of hemolysis. With trimethoprim and the sulfonamides, antagonists in the medium may allow
some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth), and
measure the more obvious margin to determine the zone diameter.
3. The sizes of the zones of inhibition are interpreted by referring to Tables 2A through 2I (Zone
Diameter Interpretative Standards and equivalent Minimum Inhibitory Concentration
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Breakpoints) of the NCCLS M100-S12: Performance Standards for Antimicrobial Susceptibility

Testing: Twelfth Informational Supplement and the organisms are reported as either susceptible,
intermediate, or resistant to the agents that have been tested. Some agents may only be reported
as susceptible, since only susceptible breakpoints are given.
Dilution Methods
Dilution susceptibility testing methods are used to determine the minimal
concentration of antimicrobial to inhibit or kill the microorganism. This can be
achieved by dilution of antimicrobial in either agar or broth media. Antimicrobials are
tested in log2 serial dilutions (two fold).
Minimum Inhibitory Concentration (MIC)
Diffusion tests widely used to determine the susceptibility of organisms isolated from
clinical specimens have their limitations; when equivocal results are obtained or in
prolonged serious infection e.g. bacterial endocarditis, the quantitation of antibiotic
action vis-a-vis the pathogen needs to be more precise. Also the terms ‗Susceptible‘
and ‗Resistant‘ can have a realistic interpretation. Thus when in doubt, the way to a
precise assessment is to determine the MIC of the antibiotic to the organisms
concerned.
There are two methods of testing for MIC:
(a) Broth dilution method
(b) Agar dilution method.
(a) Broth Dilution Method:
The Broth Dilution method is a simple procedure for testing a small number of isolates,
even single isolate. It has the added advantage that the same tubes can be taken for
MBC tests also.
Materials
Sterile graduated pipettes of 10ml, 5ml, 2ml and 1ml Sterile capped 7.5 x 1.3 cm tubes /
small screw-capped bottles, Pasteur pipettes, overnight broth culture of test and control
organisms ( same as for disc diffusion tests), required antibiotic in powder form (either
81
from the manufacturer or standard laboratory accompanied by a statement of its activity

in mg/unit or per ml. Clinical preparations should not be used for reference technique.),
required solvent for the antibiotic, sterile Distilled Water - 500ml and suitable nutrient
broth medium.
Trimethoprim and sulphonamide testing requires thymidine free media or addition of
4% lysed horse blood to the media
A suitable rack to hold 22 tubes in two rows i-e 11 tubes in each row.
Stock solution
Stock solution can be prepared using the formula
1000
------- x V x C= W
P
Where P=Potency given by the manufacturer in relation to the base

V= Volume in ml required
C=Final concentration of solution (multiples of 1000)
W= Weight of the antimicrobial to be dissolved in the volume V
Example: For making 10 ml solution of the strength 10,000mg/l from powder base whose
potency is 980 mg per gram,the quantities of the antimicrobials required is
W= 1000
------- x 10 x 10=102.04mg
980
Note: the stock solutions are made in higher concentrations to maintain their keeping qualities
and stored in suitable aliquots at -20oC .Once taken out, they should not be refrozen or reused.
Suggested dilution ranges of some antimicrobials are shown in Annexure II.
Method
Prepare stock dilutions of the antibiotic of concentrations 1000 and 100 µg/L as
required from original stock solution (10,000mg/L). Arrange two rows of 12 sterile 7.5
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x1.3 cm capped tubes in the rack. In a sterile 30ml (universal) screw capped bottle,
prepare 8ml of broth containing the concentration of antibiotic required for the first
tube in each row from the appropriate stock solution already made. Mix the contents of
the universal bottle using a pipette and transfer 2ml to the first tube in each row. Using
a fresh pipette, add 4 ml of broth to the remaining 4 ml in the universal bottle mix and
transfer 2ml to the second tube in each row. Continue preparing dilutions in this way
but where as many as 10 or more are required the series should be started again half the
way down. Place 2ml of antibiotic free broth to the last tube in each row. Inoculate one
row with one drop of an overnight broth culture of the test organism diluted
approximately to 1 in 1000 in a suitable broth and the second row with the control
organism of known sensitivity similarly diluted. The result of the test is significantly
affected by the size of the inoculum. The test mixture should contain 106 organism/ml.
If the broth culture used has grown poorly, it may be necessary to use this undiluted.
Incubate tubes for 18 hours at 37oC. Inoculate a tube containing 2ml broth with the
organism and keep at +4oC in a refrigerator overnight to be used as standard for the
determination of complete inhibition.
Calculations for the preparation of the original dilution:
This often presents problems to those unaccustomed to performing these tests. The
following method advocated by Pamela M Waterworth is presented. Calculate the total
volume required for the first dilution. Two sets of dilution are being prepared (one for
the test and one for the control), each in 2ml volumes i-e a total of 4 ml for each
concentration as 4ml is required to make the second dilution, the total requirement is
8ml. Now calculate the total amount of the antibiotic required for 8ml. For 64 g/l
concentration, 8x64mg/l =512µg in 8 ml. Place a decimal point after the first figure
(5.12) and take this volume in ml (i.e 5.12 ml) of the dilution below 512mg/l and make
up to 8ml with broth. In this example given above, the series has to be started again
mid-way at 2 mg/l which would be obtained in the same way:
83
8ml of 2mg/l=16µg in 8ml.

1.6 ml of 10 mg/ l + 6.4 ml of broth.
Reading of result
MIC is expressed as the lowest dilution, which inhibited growth judged by lack of
turbidity in the tube.
Because very faint turbidity may be given by the inoculum itself, the inoculated tube
kept in the refrigerator overnight may be used as the standard for the determination of
complete inhibition.
Standard strain of known MIC value run with the test is used as the control to check
the reagents and conditions.
Minimum Bactericidal Concentrations (MBC)

The main advantage of the ‗Broth dilution‘ method for the MIC determination lies in
the fact that it can readily be converted to determine the MBC as well.
Method
Dilutions and inoculations are prepared in the same manner as described for the
determination of MIC. The control tube containing no antibiotic is immediately sub-
cultured (Before incubation) by spreading a loopful evenly over a quarter of the plate
on a medium suitable for the growth of the test organism and incubated at 37oC
overnight. The tubes are also incubated overnight at 37 oC. Read the MIC of the control
organism to check that the drug concentrations are correct. Note the lowest
concentration inhibiting growth of the organisms and record this as the MIC.
Subculture all tubes not showing visible growth in the same manner as the control tube
described above and incubate at 37oC overnight. Compare the amount of growth from
the control tube before incubation, which represents the original inoculum. The test
must include a second set of the same dilutions inoculated with an organism of known
sensitivity .These tubes are not sub-cultured; the purpose of the control is to confirm by
84
its MIC that the drug level is correct, whether or not this organism is killed is
immaterial.
Reading of result
These subcultures may show
 Similar number of colonies- indicating bacteriostasis only.
 A reduced number of colonies-indicating a partial or slow bactericidal activity.
 No growth- if the whole inoculum has been killed
 The highest dilution showing at least 99% inhibition is taken as MBC
Micro-broth dilution test
This test uses double-strength Müeller-Hinton broth, 4X strength antibiotic solutions
prepared as serial two-fold dilutions and the test organism at a concentration of 2x106/ml.
In a 96 well plate, 100 l of double-strength MHB, 50 l each of the antibiotic dilutions
and the organism suspension are mixed and incubated at 35C for 18-24 hours. The
lowest concentration showing inhibition of growth will be considered the MIC of the
organism.
Reading of result
MIC is expressed as the highest dilution which inhibited growth judged by lack of
turbidity in the tube. Because very faint turbidity may be given by the inoculum itself,
the inoculated tube kept in the refrigerator overnight may be used as the standard for
the determination of complete inhibition. Standard strain of known MIC, run with the
test is used as the control to check the reagents and conditions.
The Agar dilution Method
Agar dilutions are most often prepared in petri dishes and have advantage that it is
possible to test several organisms on each plate .If only one organism is to be tested e.g
M.tuberculosis,the dilutions can be prepared in agar slopes but it will then be necessary
to prepare a second identical set to be inoculated with the control organism. The
dilutions are made in a small volume of water and added to agar which has been
85
melted and cooled to not more than 60oC.Blood may be added and if ‗chocolate agar‘
is required, the medium must be heated before the antibiotic is added.
It would be convenient to use 90 mm diameter petri dishes and add one ml of desired
drug dilutions to 19 ml of broth. The factor of agar dilution must be allowed for in the
first calculation as follows.
final volume of medium in plate = 20 ml
Top antibiotic concentrations = 64mg/l
Total amount of drug = 1280µg to be added to
1 ml of water
2ml of 1280 µg /ml will be required to start the dilution = 2560µg in 2 ml
= 1.28ml of 2000µg /ml
± 0.72 ml of water.
1 ml of this will be added to 19 ml agar.
(Note stock dilution of 2000µg /ml is required for this range of MIC)
The quickest way to prepare a range of dilutions in agar is as follows:

Label a sterile petri dish on the base for each concentration required. Prepare the
dilutions in water placing 1 ml of each in the appropriate dish. One ml water is added
to a control plate. Pipette 19 ml melted agar, cooled to 55oC to each plate and mix
thoroughly. Adequate mixing is essential and if sufficient technical expertise is not
available for the skilled manipulation, it is strongly recommended that the agar is first
measured into stoppered tubes or universal containers and the drug dilution added to
these and mixed by inversion before pouring into petri dishes. After the plates have set
they should be well dried at 37oC with their lids tipped for 20 to 30 minutes in an
incubator. They are then inoculated either with a multiple inoculator as spots or with a
wire loop or a platinum loop calibrated to deliver 0.001ml spread over a small area. In
either case the culture should be diluted to contain 10 5 to 106 organisms per ml. With
ordinary fast growing organisms, this can be obtained approximately by adding 5 µl of
an overnight broth culture to 5ml broth or peptone water.
86
It is possible to test spreading organism such as P.mirabilis by this method either by

cutting ditches in the agar between the inocula, or by confining each with small glass
or porcelain cylinders pressed into the agar. Although swarming of P.mirabilis can be
prevented by the use of higher concentration of agar in the medium, this is not
recommended for determination of MIC because of the difficulty of ensuring adequate
mixing of the drug with this very viscous medium. Selective media should not be used
and electrolyte deficient media will give false results because of the effect of variation
in the salt content on the action of many antibiotics.
Reading of results
The antibiotic concentration of the first plate showing  99% inhibition is taken as the
MIC for the organism.
87
Dilution and Diffusion

E test also known as the epsilometer test is an ‗exponential gradient‘ testing
methodology where ‗E‘ in E test refers to the Greek symbol epsilon ().The E test(AB
Biodisk) which is a quantitative method for antimicrobial susceptibility testing applies
both the dilution of antibiotic and diffusion of antibiotic into the medium.. A
predefined stable antimicrobial gradient is present on a thin inert carrier strip. When
this E test strip is applied onto an inoculated agar plate, there is an immediate release
of the drug. Following incubation, a symmetrical inhibition ellipse is produced. The
intersection of the inhibitory zone edge and the calibrated carrier strip indicates the
MIC value over a wide concentration range (>10 dilutions) with inherent precision and
accuracy.
E test can be used to determine MIC for fastidious organisms like S.
pneumoniae,
ß-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also
be used for Nonfermenting Gram Negative bacilli (NFGNB) for eg-Pseudomonas sp.
and Burkholderia pseudomallei.
Resistance of major consequence may be detected for e.g., the test is very useful in
detecting glycopeptide resistant Enterococci (GRE) and glycopeptide intermediate
S.aureus (GISA) and slow growing pathogens such as Mycobacterium tuberculosis.
Further it can be used for detection of extended spectrum beta lactamases (ESBL). In
conclusion E test is a simple, accurate and reliable method to determine the MIC for a
wide spectrum of infectious agents.
Susceptibility of Fastidious Bacteria
DISC DIFFUSION FOR FASTIDIOUS ORGANISMS
Antibiotic susceptibility testing of S.pneumoniae
Media for disc diffusion: Müeller -Hinton Sheep blood agar
Standardization of inoculum:
88
The inocula for seeding the susceptibility media with S.pneumoniae is prepared
from fresh pure cultures (grown overnight on Chocolate agar). Cell suspensions of
the bacteria to be tested are prepared in sterile saline or Müeller-Hinton broth. The cell
suspension is prepared by transferring a portion of the fresh growth with a swab or
inoculating loop to the suspending medium, using caution when mixing the cells
with the suspending medium so as not to form bubbles. The suspension is then
compared to the McFarland standard by holding the suspension and McFarland
standard in front of a light against a white background with contrasting black lines
and comparing the turbidity. If the turbidity is too heavy, the suspension should be
diluted with additional suspending medium. If the turbidity is too light additional
cells should be added to the suspension.
For S.pneumoniae – Direct colony suspension is made in normal saline and turbidity
adjusted to 0.5 McFarland standard. Within 15 minutes after adjusting the turbidity of
the suspension the plate should be inoculated.
Inoculation of the susceptibility test media
After proper turbidity is achieved, a new sterile swab (cotton or dacron) is submerged
in the suspension, lifted out of the broth, and the excess fluid is removed by pressing
and rotating the swab against the wall of the tube. The swab is then used to inoculate
the entire surface of the supplemented Müeller Hinton agar plate three times, rotating
the plate 60 degrees between each inoculation. The inoculum is allowed to dry
(usually taking only a few minutes but no longer than 15 minutes) before the discs are
placed on the plates. The discs should be placed on the agar with sterile forceps and
tapped gently to ensure the adherence to the agar. The plates containing the disks are
incubated at 35oC for 16 to 18 h in an inverted position in a 5% CO2 incubator. A
candle extinction jar may be used if a CO2 incubator is not available.
Estimating the susceptibility of the strains
After overnight incubation, the diameter of each zone of inhibition is measured with a
ruler or calipers. The zones of inhibition on the media containing blood are measured
89
from the top surface of the plate with the top removed. It is convenient to use a ruler
with a handle attached for these measurements, holding the ruler over the surface of the
disk when measuring the inhibition zone. Care should be taken not to touch the disk or
surface of the agar. Sterilize the ruler occasionally to prevent transmission of bacteria.
In all measurements, the zones of inhibition are measured from the edges of the
last visible colony-forming growth. The ruler should be positioned across the center
of the disc to make these measurements. The results are recorded in millimeters (mm)
and interpretation of susceptibility is obtained by comparing the results to the standard
zone sizes. For S.pneumoniae the zone measurement is from top of plate with the lid
removed. Faint growth of tiny colonies that may appear to fade from the more obvious
zone should be ignored in the measurement.
Interpretation
Each zone size is interpreted by reference to the Table 2G (Zone Diameter Interpretative
Standards and equivalent Minimum Inhibitory Concentration Breakpoints for S.pneumoniae)
of the NCCLS M100-S12: Performance Standards for Antimicrobial Susceptibility Testing:
Twelfth Informational Supplement as susceptible, intermediate and resistant.
Errors in Interpretation and reporting results
Resistant Minor Error Very Major Error
MIC
Minor Error Intermediate Minor Error
(g/ml
Major Error Minor Error Susceptible
Disk Diffusion Diameter (mm)
90
Tests for detection of β-lactamases

Direct tests for β-lactamase activity
Direct β-lactamase tests are mostly used for Haemophilus influenzae, Moraxella
catarrhalis and Neisseria spp., where few different enzyme types occur, and where
enzyme production has clear implications for therapy. Direct tests can be applied to
other species, but are less useful, since the important question usually is not whether a
β-lactamase is produced but which β-lactamase.
Numerous β-lactamase detection tests have been devised1 but few are convenient for
routine use. Most use chromogenic cephalosporins, or link the hydrolysis of penicillin
to a color change mediated by iodine or a pH indicator. Chromogenic cephalosporins
are very specific, whereas acidification and the reduction of iodine can occur for
reasons other than β-lactamase action, potentially giving false-positive results. Positive
and negative controls should be run in parallel with all tests but, because of the risk of
false-positive results, are especially critical for the acidimetric and iodometric methods.
Nitrocefin test:
Nitrocefin is a chromogenic cephalosporin that changes from yellow to red on
hydrolysis. It provides the most sensitive test for most β-lactamases, exceptions being
staphylococcal penicillinase and ROB-1, an uncommon plasmid-mediated enzyme of
haemophili.
A 0.5 mM nitrocefin solution is prepared by dissolving 2.58 mg of powder in 0.5 mL of
dimethylsulphoxide (DMSO) then diluting with 9.5 mL of 0.1 M phosphate buffer, pH
7.0. This solution is stable for 10 days at 4°C in a foil-wrapped bottle. Glass containers
should be used, since DMSO degrades plastics. Colonies of the test isolates are scraped
from nutrient agar plates and are suspended in 20 μL volumes of 0.1 M phosphate
buffer pH 7.0, to produce a dense suspension on a glass slide, and 20 μL amounts of the
nitrocefin solution are added. β-Lactamase activity is indicated by a red color within 1-2
min. Weak activities may take longer to appear, but reactions taking >10 min should be
91
treated with scepticism, as they may reflect the secondary ß-lactamase activity of those
penicillin-binding proteins that form unstable acyl complexes.
The following assays describe methods in which nitrocefin can be used to detect beta-
lactamase enzymes using inexpensive materials and equipment.[5] Working solutions
of nitrocefin lie within 0.5 mg/mL to 1.0 mg/mL.
Slide Surface Assay
1. Add one drop of 0.5 mg/ml Nitrocefin to the surface of a clean glass slide.
2. Select a colony from an agar surface using a sterile loop and mix with the drop.
3. Appearance of red color within 20-30 min. indicates beta-lactamase activity.
Direct Contact Assay
1. Place one drop of 0.5 mg/ml Nitrocefin directly on the surface of an isolated colony.
Broth Suspension Assay
1. Add 3-5 drops of 0.5 mg/ml Nitrocefin to 1 ml of broth suspension.
Lysed Cell Assay
1. Lyse 1ml of cell suspension by sonication.
2. Add 3-5 drops of 0.5 mg/ml Nitrocefin to lysed cell suspension.
Filter Paper Assay
1. Place a small piece of filter paper (~3 x 3 cm) in a clean petri dish or another clean
isolated surface and saturate (3-5 ml) with 0.5 mg/ml Nitrocefin
2. Select an isolated colony and smear over the surface of the impregnated filter paper.
3. Appearance of red color within 20-30 min. indicates beta-lactamase activity
Iodometric tests:
Hydrolysis of penicillin yields penicilloic acid, which reduces iodine, decolourising
starch-iodine complex. This reaction can be exploited to detect β-lactamase activity in
tubes or on paper strips. These tests are particularly sensitive for staphylococcal
92
penicillinase, but are less sensitive than nitrocefin for most of the β-lactamases from
Gram-negative bacteria.
(i) Tube method:
Benzylpenicillin, 6 g/L in 0.1 M phosphate buffer pH 6.0, is distributed in 0.1 mL
quantities in tubes or a microtitre tray.5 Bacterial growth from agar (not broth) is
suspended in these solutions until they are heavily turbid (c. 109 cfu/mL). The
suspensions are held at room temperature for 30-60 min, then 20 μL volumes of 1%
(w/v) soluble starch in distilled water are added, followed by 20 μL of 2% (w/v) iodine
in 53% (w/v) aqueous potassium iodide. β-lactamase activity is indicated by
decolourisation of the iodine within 5 min. Positive and negative controls are vital, as
extraneous protein reduces iodine, and over-heavily inoculated tests may give false-
positive results.
(ii) Paper strip method:
To prepare iodometric paper strips, 0.2 g of soluble starch is added to 100 mL of
distilled water and dissolved by boiling.6 After cooling, 1 g of benzylpenicillin is
added. Filter papers (1 x 5 cm, Whatman No. 3; Whatman, Maidstone, UK, or similar)
are soaked in this solution, and then air-dried for 2 h. The strips, which are stable for 1
year at –20°C, are moistened with 2% (w/v) iodine in 53% (w/v) aqueous potassium
iodide before use. They are then smeared with colonies from an overnight culture plate.
Decolourisation within 5 min indicates β-lactamase activity. Positive and negative
controls are mandatory.
Acidimetric tests:
Hydrolysis of the β-lactam ring generates a carboxyl group, acidifying un-buffered
systems. The resulting acidity can be tested in tubes or on filter papers. The method is
useful for tests on H. influenzae and Neisseria gonorrhoeae.
(i) Tube method:
For the tube method, 2 mL of 0.5% (w/v) aqueous phenol red solution is diluted with
16.6 mL distilled water and 1.2 g of benzylpenicillin is added. The pH is adjusted to
93
8.5 with 1 M NaOH. The resulting solution, which should be violet in colour, can be
stored at –20°C. Before use, 100 μl portions are distributed into tubes or microtitre
wells and inoculated with bacteria from culture plates (not broth) to produce dense
suspensions. A yellow color within 5 min indicates β-lactamase activity. Positive and
negative controls must be run in parallel.
(ii) Paper strip method:
For the acidimetric paper method,7 filter paper (Whatman No. 1) is cut into 5 x 1 cm
strips and soaked in a freshly-prepared solution containing 125 g/L benzylpenicillin,
0.1% (w/v) bromocresol purple and 1.25 mM NaOH. The strips are dried and can be
stored at 4°C for 6 months with a silica gel desiccant. Such strips are available
commercially [e.g. ‗IntraLactam‘ (Mast Diagnostics, Bootle, UK) or ‗Beta-Test‘
(Medical Wire and Equipment, Corsham, UK)]. Before use the strips must be
moistened: distilled water is recommended but it is essential that the water is not
acidic, and calcareous tap water may be preferable.8 Bacteria from agar (not broth)
cultures are smeared on the strip and development of a yellow colour within 5 min
indicates β-lactamase activity. Controls must be run in parallel.
Microbiological tests of ß-lactamase activity:
β-Lactamase activity can be detected biologically by demonstrating the loss of activity
of a β-lactam agent against a susceptible indicator organism. There are several
variations, including the cloverleaf (Hodge) method, which is highly sensitive for
staphylococci, and the Masuda double disc method, which can be used with whole
cells or cell extracts of test strains. While the use of such methods has declined, they
remain very sensitive. A review was provided by Livermore & Williams.
94
References:
 Doern G.V. Susceptibility tests of fastidious bacteria. Manual of Clinical Microbiology, 6th
edition, Murray P.R, Baron E.J, Pfaller M.A, Tenover F.C, Yolken R, American Society for
Microbiology, Washington DC, 1995, P. 1342-1349.
 Ira R. Bacteriology, Standard Operative procedure manual for microbiology laboratories,

National Institute of Biologicals. 1995, P73-97
 John D.T and James H.J Antimicrobial Susceptibility testing: General Considerations.
Manual of Clinical Microbiology 7th edition, Murray P.R, Baron E.J, Pfaller M.A, Tenover
F.C, Yolken R, American Society for Microbiology, Washington DC, 1999, P. 1469-1473.
 National Committee for Clinical Laboratory Standards. Performance Standards for

antimicrobial susceptibility testing. 8th Informational Supplement. M100 S12. National
Committee for Clinical Laboratory Standards, 2002. Villanova, Pa.
95
Practical Biochemistry
122
Sterilization
Sterilization: refers to any process that effectively kills or eliminates transmissible

agents (such as fungi, bacteria, viruses, spore forms, etc.) from a surface, equipment,
article of food or medication, or biological culture medium. Sterilization does not,
however, remove prions. Sterilization can be achieved through application of heat,
chemicals, irradiation, high pressure or filtration.
A) Heat sterilization:
a) Dry heat sterilization of an article is one of the earliest forms of sterilization

practiced. Dry heat, as the name indicates, utilizes hot air that is either free from water
vapor, or has very little of it, and where this moisture plays a minimal or no role in the
process of sterilization.
b) Moist heat sterilization Methods used
1. Below 100°C:
1. Water bath - 56°C for 60 minutes
2. Vaccine bath - 60°C for 60 minutes
3. Pasteurization of milk
1. Holder method - peaks at 63°C for 30 minutes
2. Flash method - peaks at 72°C for 20 seconds
3. Ultra high temperature (UHT) method - peaks at 125°C for a few
seconds
4. Fractional sterilization - serum inspissator
123
1. At 100°C:
1. Boiling for 10 to 30 minutes
2. Tyndallizer - steaming for 3 successive days at 100°C to kill all
organisms in their vegetative forms by allowing the spores' time to hatch
in between the heating periods.
3. Steam sterilizer - Steam at atmospheric pressure for 90 minutes
1. Above 100°C:
1. Autoclave or Pressure cooking
Action on micro-organisms
Moist heat coagulates the proteins in any organism and this is aided by the water
vapour that has a very high penetrating property, leading to their death. It also causes
oxidative free radical damage .This can even, at high enough temperatures
(vide:Autoclave), kill prions.
Steam sterilization utensils (Autoclave)
Front-loading autoclaves
124
A widely-used method for heat sterilization is the autoclave, sometimes called a

converter. Autoclaves commonly use steam heated to 121–134 °C (250–273 °F). To
achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) or 3 minutes
at 134 °C (273 °F) is required. Additional sterilizing time is usually required for
liquids and instruments packed in layers of cloth, as they may take longer to reach the
required temperature (unnecessary in machines that grind the contents prior to
sterilization). Following sterilization, liquids in a pressurized autoclave must be
cooled slowly to avoid boiling over when the pressure is released. Modern converters
operate around this problem by gradually depressing the sterilization chamber and
allowing liquids to evaporate under a negative pressure, while cooling the contents.
Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial
spores, which can be quite resistant. It will not necessarily eliminate all prions.
To ensure the autoclaving process was able to cause sterilization, most autoclaves
have meters and chart that record or display pertinent information such as temperature
and pressure as a function of time. Indicator tape is often placed on packages of
products prior to autoclaving. A chemical in the tape will change color when the
appropriate conditions have been met. Some types of packaging have built-in
indicators on them.
125
B) Chemical sterilization:
Chemicals are also used for sterilization. Although heating provides the most reliable
way to rid objects of all transmissible agents, it is not always appropriate, because it
will damage heat-sensitive materials such as biological materials, fiber optics,
electronics, and many plastics. Low temperature gas sterilizers function by exposing the
articles to be sterilized to high concentrations (typically 5 - 10% v/v) of very reactive
gases (alkylating agents such as ethylene oxide, and oxidizing agents such as
hydrogen peroxide and ozone). Liquid sterilants and high disinfectants typically
include oxidizing agents such as hydrogen peroxide and peracetic acid and aldehydes
such as glutaraldehyde and more recently o-phthalaldehyde. While the use of gas and
liquid chemical sterilants/high level disinfectants avoids the problem of heat damage,
users must ensure that article to be sterilized is chemically compatible with the
sterilant being used. The manufacturer of the article can provide specific information
regarding compatible sterilants. In addition, the use of chemical sterilants poses new
challenges for workplace safety. The chemicals used as sterilants are designed to
destroy a wide range of pathogens and typically the same properties that make them
good sterilants make them harmful to humans. Employers have a duty to ensure a safe
work environment (Occupational Safety and Health Act of 1970, section 5 for United
States) and work practices, engineering controls and monitoring should be employed
appropriately
1) Ethylene Oxide:
Ethylene oxide (EO or EtO) gas is commonly used to sterilize objects sensitive to
temperatures greater than 60 °C such as plastics, optics and electrics. Ethylene oxide
treatment is generally carried out between 30 °C and 60 °C with relative humidity
126
above 30% and a gas concentration between 200 and 800 mg/L for at least three
hours. Ethylene oxide penetrates well, moving through paper, cloth, and some plastic
films and is highly effective. Ethylene oxide sterilizers are used to process sensitive
instruments which cannot be adequately sterilized by other methods. EtO can kill all
known viruses, bacteria and fungi, including bacterial spores and is satisfactory for
most medical materials, even with repeated use. However, it is highly flammable, and
requires a longer time to sterilize than any heat treatment. The process also requires a
period of post-sterilization aeration to remove toxic residues. Ethylene oxide is the
most common sterilization method, used for over 70% of total sterilizations, and for
50% of all disposable medical devices.
2) Bleach:
Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists
of 5.25% sodium hypochlorite. It is usually diluted to 1/10 immediately before use;

however to kill Mycobacterium tuberculosis it should be diluted only 1/5, and 1/2.5 (1 part
bleach and 1.5 parts water) to inactivate prions. The dilution factor must take into
account the volume of any liquid waste that it is being used to sterilize. Bleach will
kill many organisms immediately, but for full sterilization it should be allowed to
react for 20 minutes. Bleach will kill many, but not all spores. It is highly corrosive
and may corrode even stainless steel surgical instruments.
Bleach decomposes over time when exposed to air, so fresh solutions should be made
daily.
127
3) Glutaraldehyde and Formaldehyde:
Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid
sterilizing agents, provided that the immersion time is sufficiently long. To kill all
spores in a clear liquid can take up to 12 hours with glutaraldehyde and even longer
with formaldehyde. The presence of solid particles may lengthen the required period
or render the treatment ineffective. Sterilization of blocks of tissue can take much
longer, due to the time required for the fixative to penetrate. Glutaraldehyde and
formaldehyde are volatile, and toxic by both skin contact and inhalation.
Glutaraldehyde has a short shelf life (<2 weeks), and is expensive. Formaldehyde is
less expensive and has a much longer shelf life if some methanol is added to inhibit
polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also
used as a gaseous sterilizing agent; in this case, it is prepared on-site by
depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk
polio vaccine, are sterilized with formaldehyde.
4) Hydrogen Peroxide:
Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic

when diluted to low concentrations, such as the familiar 3% retail solutions although
hydrogen peroxide is a dangerous oxidizer at high concentrations (> 10% w/w).
Hydrogen peroxide is strong oxidant and these oxidizing properties allow it to destroy
a wide range of pathogens and it is used to sterilize heat or temperature sensitive
articles such as rigid endoscopes. In medical sterilization hydrogen peroxide is used at
higher concentrations, ranging from around 35% up to 90%. The biggest advantage of
hydrogen peroxide as a sterilant is the short cycle time. Whereas the cycle time for
ethylene oxide (discussed above) may be 10 to 15 hours, the use of very high
128
concentrations of hydrogen peroxide allows much shorter cycle times. Some hydrogen
peroxide modern sterilizers, such as the Sterrad NX have a cycle time as short as 28
minutes.
C) Radiation Sterilization:
Methods of sterilization are radiation such as electron beams, X-rays, gamma rays, or
subatomic particles.
Gamma rays are very penetrating and are commonly used for sterilization of
disposable medical equipment, such as syringes, needles, cannulas and IV sets.
Gamma radiation requires bulky shielding for the safety of the operators; they also
require storage of a radioisotope (usually Cobalt-60), which continuously emits
gamma rays (it cannot be turned off, and therefore always presents a hazard in the area
of the facility).
Electron beam processing is also commonly used for medical device sterilization.
Electron beams use an on-off technology and provide a much higher dosing rate than
gamma or x-rays. Due to the higher dose rate, less exposure time is needed and
thereby any potential degradation to polymers is reduced. A limitation is that electron
beams are less penetrating than either gamma or x-rays.
X-rays, High-energy X-rays (bremsstrahlung) are a form of ionizing energy allowing

irradiating large packages and pallet loads of medical devices. Their penetration is
sufficient to treat multiple pallet loads of low-density packages with very good dose
uniformity ratios. X-ray sterilization is an electricity based process not requiring
chemical nor radio-active material. High energy and high power X-rays are generated
by an X-ray machine that can be turned off for servicing and when not in use.
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Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of
surfaces and some transparent objects. Many objects that are transparent to visible
light absorb UV. UV irradiation is routinely used to sterilize the interiors of biological
safety cabinets between uses, but is ineffective in shaded areas, including areas under
dirt (which may become polymerized after prolonged irradiation, so that it is very
difficult to remove). It also damages many plastics, such as polystyrene foam.
Growth medium:
A growth medium or culture medium is a liquid or gel designed to support the

growth of microorganisms or cells, or small plants like the moss Physcomitrella
patens. There are different types of media for growing different types of cells.
An agar plate -- an example of a bacterial growth medium.

Specifically, it is a streak plate; the orange lines and dots are
formed by bacterial colonies.
Types of growth media:
1) Nutrient media:
 a source of amino acids and nitrogen (e.g., beef, yeast extract)
This is an undefined medium because the amino acid source contains a variety of
compounds with the exact composition being unknown. Nutrient media contain all the
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elements that most bacteria need for growth and are non-selective, so they are used for
the general cultivation and maintenance of bacteria kept in laboratory culture
collections.
An undefined medium (also known as a basal or complex medium) is a medium that

contains:
 a carbon source such as glucose for bacterial growth

 water
 various salts needed for bacterial growth
Defined media (also known as chemically defined media)
 all the chemicals used are known

 Does not contain any yeast, animal or plant tissue.
Differential medium
 Some sort of indicator, typically a dye, is added, that allows for the
differentiation of particular chemical reactions occurring during growth .
2) Minimal media :
Minimal media are those that contain the minimum nutrients possible for colony
growth, generally without the presence of amino acids, and are often used by
microbiologists and geneticists to grow "wild type" microorganisms. Minimal media
can also be used to select for or against recombinants or exconjugants.
Minimal medium typically contains:
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 a carbon source for bacterial growth, which may be a sugar such as glucose, or
a less energy-rich source like succinate
 various salts, which may vary among bacteria species and growing conditions;
these generally provide essential elements such as magnesium, nitrogen,
phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic
acid
 water
Supplementary minimal media are a type of minimal media that also contains a single
selected agent, usually an amino acid or a sugar. This supplementation allows for the
culturing of specific lines of auxotrophic recombinants.
3) Selective media
Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter.
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Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a
positive Staphylococcus culture.
Selective media are used for the growth of only select microorganisms. For example,
if a microorganism is resistant to a certain antibiotic, such as ampicillin or
tetracycline, then that antibiotic can be added to the medium in order to prevent other
cells, which do not possess the resistance, from growing. Media lacking an amino acid
such as proline in conjunction with E. coli unable to synthesize it were commonly
used by geneticists before the emergence of genomics to map bacterial chromosomes.
Selective growth media are also used in cell culture to ensure the survival or
proliferation of cells with certain properties, such as antibiotic resistance or the ability
to synthesize a certain metabolite. Normally, the presence of a specific gene or an
allele of a gene confers upon the cell the ability to grow in the selective medium. In
such cases, the gene is termed a marker.
Selective growth media for eukaryotic cells commonly contain neomycin to select
cells that have been successfully transfected with a plasmid carrying the neomycin
resistance gene as a marker. Gancyclovir is an exception to the rule as it is used to
specifically kill cells that carry its respective marker, the Herpes simplex virus
thymidine kinase (HSV TK).
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Four types of agar plates demonstrating differential growth depending on bacterial metabolism.
Some examples of selective media include:
 eosin-methylene blue agar (EMB) that contains methylene blue – toxic to

Gram-positive bacteria, allowing only the growth of Gram negative bacteria
 YM (yeast and mold) which has a low pH, deterring bacterial growth
 blood agar (used in strep tests), which contains bouvine heart blood that
becomes transparent in the presence of hemolytic Streptococcus
 MacConkey agar for Gram-negative bacteria
 Hektoen enteric agar (HE) which is selective for Gram-negative bacteria
 mannitol salt agar (MSA) which is selective for Gram-positive bacteria and
differential for mannitol
 Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of
Escherichia coli.
 xylose lysine desoxyscholate (XLD), which is selective for Gram-negative
bacteria
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 buffered charcoal yeast extract agar, which is selective for certain gram-
negative bacteria, especially Legionella pneumophila
4) Differential media
Differential media or indicator media distinguish one microorganism type from

another growing on the same media. This type of media uses the biochemical
characteristics of a microorganism growing in the presence of specific nutrients or
indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the
medium to visibly indicate the defining characteristics of a microorganism. This type
of media is used for the detection of microorganisms and by molecular biologists to
detect recombinant strains of bacteria.
Examples of differential media include:
 eosin methylene blue (EMB), which is differential for lactose and sucrose
fermentation
 MacConkey (MCK), which is differential for lactose fermentation
 mannitol salt agar (MSA), which is differential for mannitol fermentation
 X-gal plates, which are differential for lac operon mutants
5) Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of organisms,
including some of the more fastidious ones. They are commonly used to harvest as many different
types of microbes as are present in the specimen. Blood agar is an enriched medium in which
nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with
heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is
named.
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Nutrient agar medium
Nutrient agar is a microbiological growth medium commonly used for the routine
cultivation of non-fastidious bacteria.
Nutrient agar typically contains (w/v)
0.5 % peptone
0.3 % beef extract
1.5 % agar
pH adjusted to neutral at 25 °C.
Nutrient broth does not contain agar.
CZAPEK-DOX agar medium
Use:
Hardy Diagnostics Czapek-Dox Agar is recommended for use in cultivating fungi and
bacteria capable of using inorganic nitrogen.
Czapek-Dox Agar is a modification of the Czapek (1902-1903) and Dox (1910)

formula prepared according to Thom and Church. The medium contains sucrose as the
sole source of cabon and nitrate as the only inorganic source of nitrogen. The medium
is useful in a variety of microbiological procedures, including fungi and mildew
resistance tests and soil microbiology testing. Czapek-Dox Agar produces luxuriant
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growth of most saprophytic aspergilli causing the organisms to produce characteristic

mycelia and conidia.
Czapek-Dox Agar is recommended in Standard Methods for the Examination of

Water and Wastewater for the isolation of Aspergillus, Penicillium, Paecilomyces and
other types of fungi with similar physiological requirements .
Formula
Ingredients per liter of deionized water:*
Sucrose 30.0gm
Sodium Nitrate 2.0gm
Dipotassium Phosphate 1.0gm
Magnesium Sulfate 0.5gm
Potassium Chloride 0.5gm
Ferrous Sulfate 0.01gm
Agar 15.0gm
Final pH 7.3 +/- 0.3 at 25 degrees C.
* Adjusted and/or supplemented as required to meet performance criteria.
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Slant culture
A culture made on the slanting surface of a solidified medium in a test tube that has
been tilted to provide a greater area for growth. Also called slope culture.
Biochemical tests (Physiological pathways of

microorganisms)
1) Coagulase test
Coagulase is a protein produced by several microorganisms, which enables the

convertion of fibrinogen to fibrin. In the laboratory, it is used to distinguish between
different types of Staphylococcus isolates. Coagulase negativity excludes S. aureus.
(That is to say, S. aureus is coagulase-positive.)
It is also produced by Yersinia pestis. Coagulase reacts with prothrombin in the blood.
The resulting complex is called staphylothrombin, which enables the enzyme protease
to convert fibrinogen to fibrin. This results in clotting of the blood. Coagulase is
tightly bound to the surface of the bacteria S. aureus and can coat its surface with
fibrin upon contact with blood. It has been proposed that fibrin-coated staphylococci
resist phagocytosis making the bacteria more virulent. Bound coagulase is part of the
larger family of MSCRAMMS
The coagulase test is used to differentiate Staphylococcus aureus from coagulase-

negative staphylococci. The test uses rabbit plasma that has been inoculated with a
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staphylococcal colony. The tube is then incubated at 37 degrees Celsius for 1-1/2
hours. If negative then continue incubation up to 24 hours.
 If positive (i.e., the suspect colony is S. aureus), the serum will coagulate,
resulting in a clot (sometimes the clot is so pronounced that the liquid will
completely solidify).
 If negative, the plasma remains liquid. The negative result may be S.
epidermidis but only a more detailed identification test can confirm this. Using
biochemical tests as like in API tests and BBL CRYSTAL methods.
 List of coagulase-positive staphylococci:
Staphylococcus aureus subsp. anaerobius, Staphylococcus aureus subsp. aureus,

Staphylococcus delphini, Staphylococcus hyicus, Staphylococcus intermedius,
Staphylococcus lutrae, Staphylococcus schleiferi subsp. coagulans.
2 ) Nitrate Reduction (Nitrate reductase test )
The nitrate reductase test is a test to differentiate between bacteria based on their ability
or inability to reduce nitrate (NO3) to nitrite (NO2) using anaerobic respiration.
The identification of some bacteria is aided by determining if the organism can reduce
nitrate (NO3) to nitrite (NO2)or another nitrogenous compound such as ammonia (NH 3) or
nitrogen gas (N2). This reaction is expressed as:
NO3 ----> NO2 ----> NH3 or N2
In order to determine if bacteria can reduce nitrate, the test organism is inoculated into
nitrate reduction broth, an undefined medium that contains large amounts of nitrate
(KNO3). After incubation, alpha-napthylamine and sulfanilic acid are added. These two
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compounds react with nitrite and turn red in color, indicating a positive nitrate reduction
test. (Tube 2 in image below.)
If there is no color change at this step, nitrite is absent. (Tube 1 below.) If the nitrate is
unreduced and still in its original form, this would be a negative nitrate reduction result.
However, it is possible that the nitrate was reduced to nitrite but has been further reduced
to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result.
To distinguish between these two reactions, zinc dust must be added. Zinc reduces nitrate
to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the
nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic acid are
already present in the tube. (Tube 4 below.) Thus a red color after the zinc is added
indicates the zinc found the nitrate unchanged. The bacteria were unable to reduce nitrate.
This is recorded as a negative nitrate reduction test.
If however, the tube does not change color upon the addition of zinc, then the zinc did not
find any nitrate in the tube. (Tube 3 below.) That means the test organism converted the
nitrate to nitrite and then converted the nitrite to ammonia and/or nitrogen gas. Thus no
color change upon the addition of zinc is recorded as a positive nitrate reduction test.
In the image below, alpha-napthylamine and sulfanilic acid were added to all four tubes.
Subsequently, zinc dust was added to the third and fourth tubes.
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nitrate reduction test
Procedure
1. Inoculate nitrate broth with an isolate and incubate for 48 hours.

2. Add 10-15 drops each of sulfanilic acid and N,N-dimethyl-1-naphthylamine. If
the bacterium produces nitrate reductase, the broth will turn a deep red within 5
minutes at this step.
3. If no color change is observed, then the result is inconclusive. Add a small
amount of zinc to the broth. If the solution remains colorless, then both nitrate
and nitrite reductase is present. If the solution turns red, nitrate reductase is not
present.
3) Carbohydrate Utilization:
Bacteria produce acidic products when they ferment certain carbohydrates. The
carbohydrate utilization tests are designed to detect the change in pH which would
occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the
medium which will cause the pH indicator (phenol red) to turn yellow. If the bacteria
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do not ferment the carbohydrate then the media remains red. If gas is produced as a by
product of fermentation, then the Durham tube will have a bubble in it.
The carbohydrate tests we perform are the:
A. Glucose (Dextrose) test

B. Lactose test
C. Sucrose test
All carbohydrate test media should be inoculated with the transfer loop.
Fermentation results from left to right:
 Left tube shows less acid formation than far right tube, but gas is still made
 Center shows no carbohydrate utilization to produce acid or gas.
 Right tube shows acid was produced as evidenced by the yellow color, and gas
was made (look at the bubble in the Durham tube)
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5) Citrate Utilization test:
Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle)
into oxaloacetate (another intermediate of the Kreb’s cycle). In this media, citrate is
the only carbon source available to the bacteria. If it can not use citrate then it will not
grow. If it can use citrate, then the bacteria will grow and the media will turn a bright
blue as a result of an increase in the pH of the media. To inoculate this slant, use the
transfer loop.
Left tube is a negative result. Right tube is a positive result.
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6) Gelatin Utilization:
This media is used to test if bacteria can digest the protein gelatin. To digest gelatin,
the bacteria must make an enzyme called gelatinase. To inoculate this media, use a
transfer needle to stab the gelatin. After incubating the inoculated media for at least 48
hrs, transfer the tube into a refrigerator. The tube should be completely chilled prior to
observation. If the media is solid after refrigeration then the test is negative (the
bacteria did not digest gelatin). If the media is liquefied even after refrigeration, then
the test result is positive…the bacteria is able to digest gelatin.
The 'Serratia marcescens' on the left is positive for gelatinase production, as evidenced by the liquidation of the
media. The 'Salmonella typhimurium' on the right is negative, as evidenced by the solidity of the media.
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7) Starch hydrolysis test:
This test is used to detect the enzyme amylase, which breaks down starch. After
incubation the plate is treated with Gram’s iodine. If starch has been hydrolyzed
(broken down) then there is a reddish color or a clear zone around the bacterial
growth; if it has not been hydrolyzed then there is a black/blue area indicating the
presence of starch. Simply use inoculating loop to spread bacteria onto plate surface.
After the bacteria have grown, you add a few drops of Gram’s iodine to the plate and
look for the color immediately after adding the iodine.
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8) Indole production test:
This test is done to determine if bacteria can breakdown the amino acid tryptophan
into indole. SIM media or TSB (tryptic soy broth) is inoculated using a transfer
needle. After incubating the bacteria for at least 48 hours, Kovac’s reagent is added to
the media to detect if indole has been made by the bacteria. The development of a
red/pink layer on top of the media is a positive result (the bacteria can breakdown
tryptophan to form indole). Failure to see a red layer is a negative result (indole was
not formed from tryptophan).
The tube on the left with the red ring is positive for indole production while the tube on the right shows a negative result.
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The tube on the left is negative and the tube on the right is a positive result.
Biochemistry
Indole is generated by reductive deamination from tryptophan via the intermediate

molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction,
during which the amine (-NH2) group of the tryptophan molecule is removed. Final
products of the reaction are indole, pyruvic acid, ammonia (NH3) and energy.
Pyridoxal phosphate is required as a coenzyme.
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Performing a Test
Like many biochemical tests on bacteria, results of an indole test are indicated by a
change in color following a reaction with an added reagent.
Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48
hours before performing the test. Following incubation, add 5 drops of Kovac's
reagent (isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric
acid) to the culture broth.
A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl
alcohol, developed by Paul Ehrlich) is used when performing the test on
nonfermenters and anaerobes.
A positive result is shown by the presence of a red or red-violet color in the surface
alcohol layer of the broth. A negative result appears yellow. A variable result can also
occur, showing an orange color as a result. This is due to the presence of skatole, also
known as methyl indole or methylated indole, another possible product of tryptophan
degradation.
Indole-Positive Bacteria
Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas
hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter sp., Edwardsiella
sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, most Proteus sp.
(not P. mirabilis), Plesiomonas shigelloides, Pasturella multocida, Pasturella
pneumotropica, Streptococcus faecalis, and Vibrio sp.
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Indole-Negative Bacteria
Bacteria which give negative results for the indole test include: Actinobacillus spp.,
Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordtella sp.,
Enterobacter sp., Lactobasillus spp., most Haemophilus sp., most Klebsiella sp.,
Neisseria sp., Pasturella haemolytica, Pasturella ureae, Proteus mirabilis,
Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp.
9) MRVP (methyl red-Vogues Proskauer) test :
This test is used to determine two things. The MR portion (methyl red) is used to
determine if glucose can be converted to acidic products like lactate, acetate, and
formate. The VP portion is used to determine if glucose can be converted to acetoin.
These tests are performed by inoculating a single tube of MRVP media with a transfer
loop and then allowing the culture to grow for 3-5 days. After the culture is grown,
about half of the culture is transferred to a clean tube. One tube of culture will be used
to conduct the MR test, the second tube serves as the VP test.
A. MR (methyl red) test:
Methyl red is added to the MR tube. A red color indicates a positive result
(glucose can be converted into acidic end products such as lactate, acetate, and
formate. A yellow color indicates a negative result; glucose is converted into
neutral end products.
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B. VP (Vogues Proskauer) test:
First alpha-napthol (also called Barritt’s reagent A) and then potassium hydroxide
(also called Barritt’s reagent B) are added to the VP tube. The culture should be
allowed to sit for about 15 minutes for color development to occur. If acetoin was
produced then the culture turns a red color (positive result); if acetoin was not
produced then the culture appears yellowish to copper in color (a negative result).
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10) Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S) test:
Looks at fermentation of glucose, lactose, and sucrose and checks if hydrogen sulfide
is produced in the process. Basically a pH indicator will change the color of the media
in response to fermentation…where that color change occurs in the tube will indicate
what sugar or sugars were fermented. The presence of a black color indicates that H 2S
was produced. In this media, H2S reacts with the ferrous sulfate in the media to make
ferrous sulfide…which is black. To inoculate, use a needle to stab agar and then uses a
loop to streak the top slated region.
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In addition to TSI media, SIM media can be used to determine if H 2S is produced. A

black color in the SIM medium following inoculation and incubation indicates that
H2S is made by the bacteria.
SLANT COLOR: Interpretation

RED does not ferment either lactose or sucrose
YELLOW ferments lactose and/or sucrose
BUTT
Interpretation
COLOR/CONDITION
RED no fermentation of glucose
some fermentation of glucose has occurred, acid has been
YELLOW
produced
Seen as cracks in the agar, bubbles, or the entire slant may be
GAS FORMED pushed out of the tube. (Caution:these gassy fermenters may
have bacteria close to the opening.)
BLACK H2S has been produced
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From left to right:
A. Uninoculated control
B. Red slant and red butt, no black color= no fermentation of glucose, sucrose or
lactose. No Hydrogen sulfide produced
C. Red slant and black butt= no lactose or sucrose fermentation, H 2S has been
produced
D. Red slant with yellow butt= no lactose or sucrose fermentation, lactose is
fermented, no H2S has been produced
E. Yellow slant, yellow butt and black coloration= Lactose, sucrose and glucose
fermented, and H2S has been produced
F. Yellow slant, yellow butt and lifting and/or cracking of media, no black
coloration= Lactose, sucrose and glucose fermented, H2S has not been produced
but gas has been produced
G. Yellow slant, yellow butt and no lifting and/or cracking of media, no black
coloration= Lactose, sucrose and glucose fermented, H2S has not been produced
nor has gas been produced
11) Urea test:
This test is used to detect the enzyme urease, which breaks down urea into ammonia.
Ammonia is a base and thus will raise the pH of the media if it is present. This change
in pH is indicated by a pH indicator called phenol red which is present in the media. A
color change from yellow to bright pinkish-red is positive; lack of color change is a
negative result. Inoculate the liquid media with a transfer loop.
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The tube on the left is a positive reaction; the tube in the middle is a negative reaction and the tube on the
right in an un-inoculated control.
12) Catalase test:
This test is can be used to detect the enzyme catalase. This enzyme is responsible for
protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can occur
during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the
organism. Catalase breaks H2O2 down into water and O2. To perform the catalase test
simply smear a small amount of the test organism onto the lid of a Petri plate/culture
dish. Then add a drop of hydrogen peroxide to the smear. If bubbles become visible
(these would be the O2 bubbling up) then the test is positive and you can conclude that
the organism makes catalase. A lack of bubbles indicates the absence of catalase.
*Note, most aerobic organism make catalase.
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Bubbling upon the addition of hydrogen peroxide is indicative of the presence of catalase for this organism.
13) Oxidase test:
To perform this test simply swab some of your test culture into one of the boxes on an
oxidase dry slide. If a color change to purple or blue is evident at 30 seconds-1 minute
then the result is positive. It is important that the test is read by one minute to ensure
accurate results (avoid false negatives and false positives). This laboratory test is
based on detecting the production of the enzyme cytochrome oxidase by Gram-
negative bacteria. It is a hallmark test for the Neiserria. It is also used to discriminate
between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other
Enterobacteriaciae.
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Additional Non-Biochemical Tests:
1) Motility test:
The motility test is not a biochemical test since we are not looking at metabolic
properties of the bacteria. Rather, this test can be used to check for the ability of
bacteria to migrate away from a line of inoculation thanks to physical features like
flagella. To perform this test, the bacterial sample is inoculated into SIM or motility
media using a needle. Simply stab the media in as straight a line as possible and
withdraw the needle very carefully to avoid destroying the straight line. After
incubating the sample for 24-48 hours observations can be made. Check to see if the
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bacteria have migrated away from the original line of inoculation. If migration away
from the line of inoculation is evident then you can conclude that the test organism is
motile (positive test). Lack of migration away from the line of inoculation indicates a
lack of motility (negative test result).
Left tube is result for a non-motile bacterium. Right tube is the result for a motile organism.
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Staining
There are two types of staining: 1) simple staining
2) compound staining
Simple staining Compound staining
 Show morphological shape  Show fine structure

 Only one stain used  More than one stain used
 Unistep staining process  Multistep staining process
 Have the same reaction with  Have different reaction with
bacteria bacteria
 Enhance the contrast between  Determine the type of cell wall
bacteria and background
 Example: crystal violet, carbon
fuchsin, saphranin
Procedure of simple stain:
1- Prepare the bacterial film

 Take one drop from the microorganism suspension on clean slide.
 Perform heat fixation process through putting the slide at suitable distance far
from the flame (15:25 cm) until complete drying of water
2- take 2:3 ml from stain by pipette
3- leave the stain on the slide for two minutes
4- wash gently
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5- dry by air
6- put drop of seeder oil and observe under microscope
Procedure of compound stain:
1- make a bacterial film

2- stain with crystal violet for two minutes
3- wash gently with H2O
4- add I2 solution for 1 minutes
5- wash gently again
6- add alcohol for 20 seconds
7- wash gently
8- add saphranin stain for two minutes
9- wash gently again
10- dry by air
11- add drops of Seder oil and observe under microscope
Theory of staining:
 The cell wall of the bacteria is responsible for the characteristic shape of the
bacterial cell.
 It surrounds the underlying plasma membrane and protecting it and the initial
parts of the from changes in the surrounding environment
Composition of the bacterial cell wall:
Bacterial cell wall is composed of macromolecular net work known as peptidoglycan

and other materials.
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In gram +ve bacteria: violet color with stain
The cell wall consists of several layers of peptidoglycan.
The high amount of peptidoglycan prevent the alcohol from making de-colorization to
the high molecular weight complex (crystal violet+ I2) color and so the bacteria
appear with violet color because the bacteria can't absorb saphranin stain.
In gram –ve bacteria: red color with stain
The cell wall contain much smaller amount of peptidoglycan and so alcohol can make
de-colorization to the high molecular weight complex (crystal violet+ I2) color and so
the bacteria can absorb saphranin stain and appear with red color.
Spore staining
 Bacteria in the end of its life cycle from spores
 Spores formed under unfavorable conditions
 Spores are resistant to dryness, ultraviolet, temperature, and toxic chemicals
 The most common types of bacteria formed spores are Bacillus, Clostridium
 Types of spores according to its position inside the cell:
1- central
2- terminal
3- sub-terminal
Procedure of spore staining
1- prepare a bacterial film from old age bacteria culture on a clean slide
2- stain by using malachite green
3- heat the under side of the slide in which the slide not steam or dry for 20
minutes
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4- wash gently with water

5- add one drop for saphranin for 1-2 min
6- wash again with water
7- dry in air
8- put a drop of seeder oil and observe under microscope
Theory of staining
 Spores are collection of protoplasts surrounds by thick wall

 Malachite green has the ability to stain dehydrated proteins of spores and so
appeared as green in color
 Malachite green penetrates both thick walls of spores and vegetative cell but
after de-colorization, stain get out of vegetative cells and so spores remain
green colored but vegetative cell loss the green color and counter stain by red
color of saphranin
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Antibiotic
 Antibiotic: it is any substance produced by a microorganism that kills or harms
another microorganism
 About 90% of antibiotics are isolated from bacteria
 There are a few antibiotics that completely synthesized in laboratory
 Antibiotic is not fixed for any microorganism this depends on environmental
conditions and the type of media
 Antibiotic may inhibit or kill microorganisms through effecting on metabolism
of organism, cell wall, or protein synthesis.
Determination of Antibiotic Activity
Procedure:
1- prepare sub-culture of microorganism on solid media

2- add the antibiotic discs on the culture
3- Incubate according to the type of microorganisms.
Theory of the experiment
 Theory of this experiment depends on appearance of the inhibition zone

 If strong inhibition zone appears. this indicates that antibiotics is very effective
against microorganisms and so it inhibit the growth
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 If minimum inhibition zone appears. this indicates that antibiotic is not very
effective and it is partially inhibits the growth and this is due to the adaption of
microorganism to the antibiotic by time
 If there isn't any inhibition zone, this indicates that this antibiotic haven't any
effect on bacteria due to:
1- the bacteria may devoid of the target of antibiotic
2- the bacteria may change the pathway that can be affected by the antibiotic
3- The bacteria may produce substance that compete the antibiotic.
Determination of antibiotic MIC
MIC: minimum inhibitory concentration
The lowest concentration can inhibit the growth of microorganism
MBC: minimum bacteriocide concentration
The lowest concentration can kill the microorganism
163
Determination of MIC occurs by two methods:
1) DIFFUSION METHOD
Procedure:
1- Prepare serial dilution from the antibiotic stock

Tube 1st 2nd 3rd 4th 5th
Stock 1 ml 2 ml 3 ml 4 ml 5 ml
water 9 ml 8 ml 7 ml 6 ml 5 ml
2- put a group of sterile disks into each tube to obtain antibiotic discs
3- prepare the suitable media for the microorganism (solid media)
4- make inoculation on the media by the desired microorganism
5- take your antibiotic discs and distribute them on the surface of plate where each
plate contain certain concentration of the antibiotic
6- incubate according to the type of microorganism
Observation
There is graduation in the inhibition zone in the plates where:
 The first plate have the inhibition zone, shows the MIC
 The second plate shows MBC
164
2) DILUTION METHOD
Procedure:
1- Prepare serial dilution from the antibiotic stock
Tube 1st 2nd 3rd 4th 5th
Stock 1ml 2 ml 3 ml 4 ml 5 ml
water 9 ml 8 ml 7 ml 6 ml 5 ml
2- Prepare the suitable media for the microorganism (liquid media)
3-make inoculation on the media by the desired microorganism
4- Transfer different concentration of antibiotics to the different tubes of the liquid

media
5- Incubate according to the type of microorganism
6- After incubation period take the reading by spectrophotometer for different

tubes at 600 nm
165
Observation:
The 1st tube which not have turbidity is represent MIC
The 2nd tube which not have turbidity is represent MBC
Diffusion method Dilution method
Media used Solid media Liquid media
Antibiotic diffuses from the The antibiotic mixed

discs to the area around the with the microorganism
discs forming inhiobition culture
zone
166
References
D.E. Burkepile, J.D. Parker, C.B. Woodson, H.J. Mills, J. Kubanek,
P.A. Sobecky and M.E. Hay. Ecol., 2006, 87: 2821–2831.
L.S. Kantor, K. Upton, A. Manchester and V. Oliveira. Food Rev.,
1997, 20: 2–12.
P. Martorell, M.T. Fernandez-Espinar and A. Querol. Int. J. Food
Microbiol., 2005, 101: 293–302.
V.B. Fonnesbech, K. Venkateswaran, M. Satomi and L. Gram. Appl.
Environ. Microbiol., 2005, 71: 6689–6697.
T. Kutzemeier. Europ. Dairy Magzin., 2006, 7: 34–36.
P. Lempert. Progressive Grocer, 2004, 83: 18p.
L. Gram, L. Ravn, M. Rasch, J.B. Bruhn, A.B. Christensen and M.
Givskov. Int. J. Food Microbiol., 2002, 78: 79–97.
Biró B. (2005): A talaj mint a mikroszervezetek élettere. In:
Magyarország az ezredfordulón. Stratégiai
tanulmányok a Magyar Tudományos Akadémián. II. Az agrárium
helyzete és jövője. A talajok jelentősége a században. (Szerk. Stefanovits
P. és Micheli E.) Budapest, Társadalomkutató Központ. 141-169.
Biró B. (2002): Talaj és rhizobiológiai eszközökkel a fenntartható
növénytermesztés és környezetminőség szolgálatában. Acta Agronom.
Hung. 50. 77-85.
761

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Microbiology & Antibiotics

2 The role of microorganisms in soil 6

3 Some diseases caused by microorganisms 13

4 Common Diseases (Infectious diseases) 66

7 Practical part 622

Soil Microbes overview

Modes of Spread of Communicable Diseases

Prevention and cure

Topics Page no.

Antibiotic is a chemical substance produced by a microorganism that

Basic Terminology of chemotherapy

example, a drug effective against both gram-positive and gram-negative

Characteristics of the Ideal Antimicrobial Drug

polymicrobial infection, e.g., an intrabdominal anaerobic infection

History of the Development of Antibiotics

 The discovery of the first antibiotic was an accident.

F.g.1 The original plate made by Alexander Fleming showing Penicillium

Sir Alexander Fleming (1928)

Selman Waksman (1943)

The success of antibiotics has been impressive. At the same time,

Properties of an ideal antibiotic

How Do Antibiotics Work?

2. A Bactericidal antibiotic kills the bacteria generally by either

At which drug concentration is the bacterial population

To inhibit multiplication Antibiotics have a bacteriostatic effect.

At which drug concentration is the bacterial population inhibited?

• Minimal Inhibitory Concentration = MIC

Bacteriostatic = inhibits bacterial growth

To destroy the bacterial population Antibiotics have a

• Minimal Bactericidal Concentration = MBC

Quantitative Measure MBC=lowest concentration of

Mechanisms of Action of Antibiotics

The goal of antimicrobial drugs is either to disrupt the cell processes or

vertebrate cells. Examples of drugs with excellent selective toxicity are

Antibiotic Targets and Mechanisms of Action

1. Bacterial cell wall biosynthesis

CLASSIFICATION BY MECHANISM OF ACTION

d. Drugs that cause nonlethal inhibition of protein synthesis

Antibiotic Targets and Mechanisms of Action

Fig.2. Antibiotic target sites

Table 1 different class of antibiotics and their mode of action

Group Mode of action Examples

2) Glycopeptides Inhibition of Cell Wall Vancomycin, Teichoplanin

3) Macrolides Inhibition of Protein Synthesis Azithromycin, Erythromycin

4) Aminoglycosides Inhibition of Protein Synthesis Gentamicin, Amikacin

5) Tetracyclines Inhibition of Protein Synthesis Tetracycline, Minocycline

Inhibition of Nucleic acid Ciprofloxacin, Norfloxacin,

Alteration of Cell Metabolism Trimethoprim, Trimetrexate

8) Polymyxins, Alteration of Cell Membrane Amphotericin B, Nystatin

1. Beta-lactams (Inhibition of Cell Wall Synthesis)

Structure of the peptidoglycan

2- Glycopeptide antibiotics (Inhibition of Cell Wall Synthesis)

 In particular, they bind to acyl-D-alanyl-D-alanine in

Mode of action of Glycopeptides

3-Macrolides, Aminoglycosides and Tetracycline (Inhibition

Resistant microbes are increasingly difficult to treat, requiring alternative

In summary, the main causes of antibiotic resistance have been linked

 The overuse, underuse and general misuse of antibiotics are major

Mechanisms of antibiotic resistance

The four main mechanisms by which microorganisms exhibit