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 Microbiology

History of Microbiology
Microbiology is a science that deals with the study of living organisms that cannot be seen by the
naked eye.

HISTORY OF MICROBIOLOGY

Microbiology
Microbiology is a science that deals with the study of living organisms that cannot
be seen by the naked eye. These can be seen with the aid of microscopes, which
magnify objects. Many scientists contributed to the science of microbiology.

Louis Pasteur (1822-1895)

Louis Pasteur was a French chemist and a crystallographer.

His contribution to microbiology is so great that he is con-side red to be the

“Father of Microbiology”.

Contribution to science as a chemist

He was working with tartaric acid crystals. He could pick up the dextrose and levo
rotatory crystals by seeing the morphology of the crystals. Later he was called to
solve some of the problems in fermentation industry and turned his attention to
biological pro-cess of fermentation.

Contribution to wine industry

1. He discovered that alcohol production from grape juice was due to Yeast

2. He found out that large amounts of lactic acid production were due to the
presence or contamination of rod shaped bacteria.
3. He observed that the process of alcohol production i.e. FERMENTATION
took place in the absence of air.

4. He coined the terms obic to describe those organisms requiring air

and anaerobic to describe those organisms which do not require air for their
growth.

Contribution to modern microbiology

Pasteur disproved the theory of spontaneous generation. The theory proposed that
living organisms originated spontaneously, particularly from decaying organic
matter. He disproved it.

Pasteur’s swan neck flask

Pasteur poured meat infusions into flasks and then drew the top of each flask into a
long curved neck that would admit air but not dust. He found that if the infusions
were heated, they remained sterile (free from any growth) until they were exposed
to dust. He opened them on a dusty road and resealed them and demonstrated the
growth of microorganisms in all the flasks. The unopened flasks were sterile.

Thus he disproved the theory of spontaneous generation

Edward Jenner 1796

It was an ancient observation that persons, who had suffered from a specific
disease such as small pox or mumps, resisted the infection on subsequent
exposures. They rarely contracted it second time. Such acquired resistance is
specific. Edward Jenner a country doctor in England noted a pustular disease on
the hooves of horses called the grease. This was carried by farm workers to the
nipples of cows (cow pox). This was again carried by milkmaids. They got
inflamed spots on the hands and wrists. The people who got this cow pox were
protected from small pox. He reported that 16 farm workers who had recovered
from cow pox were resistant to small pox infection.

He took the material from the cow pox and inoculated into the cut of an 8 year
old boy on 14 May 1796. Two months later Jenner inoculated the same boy with
material taken from small pox patients.
 This was a dangerous but accepted procedure of that time and the procedure
was called variolation. The boy was protected against small pox. His exposure to
the mild disease cow pox had made him immune to the disease small pox.

In this manner Jenner began the science of Immunology, the study of the body’s
response to foreign substances.

Robert Koch (1843-1910)

Robert Koch was a German physician.

1. For the first time he showed the evidence that a specific germ (Anthrax
bacillus) was the cause of a specific disease (spleenic fever in sheep)

2. He established that a specific germ can cause a specific disease and introduced
scientific approach in Microbiology

3. He discovered Bacillus anthracis (Anthrax bacillus), Mycobacterium

tuberculosis, and Vibrio cholera.

4. He modified Ziehl-Neelsen acid fast staining procedure which was introduced

by Ehrlich.

5. He devised the solid medium to grow the microorganism to get single

colonies.

6. He introduced Koch’s thread method to find out the efficacy of disinfectants

7. He established certain rules that must be followed to establish a cause and

effect relationship between microor diseases. They are known as Postulates
8. He also described this Phenomenon

Koch’s Postulates
Robert Koch developed powerful method to isolate the or-ganisms in pure culture
from diseased tissue. He also perfected the techniques of identification of the
isolated bacteria.

The need for Koch’s postulates

In those days there were no perfect techniques to identify the organisms. Solid
media and staining techniques were not avail-able. So the etiological role of
organisms was not known.
To prove the etiology there were not strict criteria. So there was a need to establish
criteria.

Koch’s Postulates
1. The organism should be regularly seen in the lesions of the disease.

2. It should be isolated in pure culture on artificial media.

3. Inoculation of this culture should produce a similar dis-ease in

experimental animals.

4. The organism must be recomveredthe lesions in these animals.

Postulate 1

The organism should be found in lesions of the disease

All the causative agents of the disease are seen in the particular diseases. If we
take pneumococci as example, they are seen in all the pneumonia cases.

Postulate 2

It should be isolated and grown in solid media

Pneumococci are grown in solid media and are isolated fromthe diseases. Some
organisms do not grow on solid media or the solid media are not developed yet.
Example: Mycobacteriu leprae and Treponema pallidum

Postulate 3

The organisms should produce the exact disease in experimental animals

Almost all the pathogenic organisms produce the same dis-ease in experimental
animals. Usually rats, mice, rabbits or guinea pigs are used as experimental
animals.

Pneumococci produce pneumonia in animals. Salmonellaspecies do not produce

typhoid fever in rat, mice or rabbit. So chimpanzee is taken as experimental animal
and it produces fever in chimpanzee.

Postulate 4

It should be isolated from the diseased animal also

Pneumococci are isolated from the experimentalanimals also.

Modern addition to Koch’s Postulates

Today we recognize additional criteria of causal relation between a microorganism
and a disease.

The important one is the demonstration of abnormally high concentration of

specific circulating antibodies to the organism in the infected host

Or, the presence of abnormally high degree of specific im-munity or

hypersensitivity to the infecting agent in a recently re-covered host.

Limitations
Some organisms have not yet been grown in artificial cul-ture media

Example: Mycobacterium leprae and Treponema pallidum.

Usefulness of Koch’s Postulates

1. It is useful in determining pathogenic organisms
2. To differentiate the pathogenic and nonpathogenic microor-ganism

3. For the classification of organisms

4. To detect the susceptibility, resistance of the laboratory ani-mals.

Conclusions
Koch has done a valuable work in the field of Microbiology and has made
postulates, which have merits, demerits and limita-tions with modern omission and
addition.
Anton van Leeuwenhoek of Delft, Holland, constructed simple microscopes composed of double
convex glass lenses held between two silver plates. His microscopes could magnify around 50 to
300 times. Microbiologists currently use a variety of light microscopes.

The compound light microscope

THE COMPOUND LIGHT MICROSCOPE
Anton van Leeuwenhoek of Delft, Holland, constructed simple microscopes
composed of double convex glass lenses held between two silver plates. His
microscopes could magnify around 50 to 300 times. Microbiologists currently use
a variety of light microscopes.

Modern microscopes are all compound microscopes. The light microscopy refers
to the use of any kind of microscope that uses visible light to make the specimens
observable. The most commonly used light microscopes are:

l Bright field microscopes

l Dark-field microscopes

l Phase contrast microscopes

l Fluorescence microscopes

The parts of a modern microscope and its light path are shown in figure. 2.1.

Each type of microscope is adapted for certain type of ob-servations. The standard
ordinary light microscope is called a bright-field microscope, because it forms a
dark image against a brighter background. A compound microscope with a single
eye piece (ocular) is called monocular and with two eye pieces is called binocular.
l A mirror or an electric illuminator is a light source which is located in the base
of the microscope.

l There are two focusing knobs, the fine and the coarse adjust-ment knobs which
are located on the arm. These are used to move either the stage or the nosepiece to
focus the image.
l The mechanical stage is positioned about halfway up the arm, which allows
precise contact of moving the slide.

l The sub stage condenser is mounted within or beneath the stage and focuses a
cone of light on the slide. In simpler microscopes, its position is fixed whereas in
advanced microscopes it can be adjusted vertically.

The upper part of the microscope arm holds the body assem-bly. The nose
piece and one or more eyepieces or oculars are at-tached to it. The body assembly
contains a series of mirrors and prisms so that the barrel holding the eyepiece may
be tilted for viewing. Three or five objectives with different magnification power
are fixed to the nose piece and can be rotated to the posi-tion beneath the body
assembly. A microscope should always be par focal, i.e. the image should remain
in focus when objectives are changed. Light enters the microscope from the base
and passes through a blue filter which filters out the long wavelengths of light,
leaving the shorter wavelengths and improving the resolution. The light then goes
through the condenser which converges the light beams so that they pass through
the specimen. The iris diaphragm controls the amount of light that passes through
the specimen and into the objective lens. For higher magnification, greater the
amount of light needed to view the specimen clearly. The objective lens magnifies
the image before it passes through body tube to the ocu-lar lens in the eyepiece.
The ocular of light needed to view the specimen clearly. The objective lens
magnifies the image before it passes through body tube to the ocular lens in the
eyepiece. The ocular lens further magnifies the image. The total magnification of
the light microscope is calculated by multiplying the magnify-ing power of the
objective lens by the magnifying power of the ocular lens.

Representative magnification values for a 10 X ocular are:

Scanning (4X) x (10X) = 40X magnification

Low power (10X) x (10x) = 100X magnification

High dry (40X) x (10X) = 400X magnification

Oil Immersion (100X) x (10X) = 1000X magnification

Microscope Resolution
Objective is the important part in the microscope which is responsible to produce a
clear image. The resolution of the objec-tive is most important. Resolution is the
capacity of a lens to separate or distinguish between small objects that are close to-
gether. The major factor in the resolution is the wave length of light used. The
greatest resolution obtained with light of the short-est wavelength, that is the light
at the blue end of the visible spec-trum in the range of 450 to 500 nm. The highest
resolution pos-sible in a compound light microscope is about 0.2 m. That means,
the two objects closer together than 0.2 m are not resolvable as distinct and
separate. The light microscope is equipped with three or four objectives. The
working distance of an objective is the distance between the front surface of the
lens and the surface of the cover glass or the specimen. Objectives with large
numerical apertures and great resolving power have short working distances.

Numerical Aperture
The resolving power of a light microscope depends on the wavelength of light used
and the numerical aperture (NA) of the objective lenses.

The numerical aperture of a lens can be increased by

l increasing the size of the lens opening and/or

l increasing the refractive index of the material between the lens and the
specimen.

The larger the numerical aperture the better the resolving power. It is important to
illuminate the specimens properly to have higher resolution. The concave mirror in
the microscope creates a narrow cone of light and has a small numerical aperture.
How-ever, the resolution can be improved with a sub stage condenser. A wide cone
of light through the slide and into the objective lens increases the numerical
aperture there by improves the resolution of the microscope.

Oil immersion
Oil immersion lens is designed to be in direct contact with the oil placed on the
cover slip. An oil immersion lens has a short focal length and hence there is a short
working distance between the objective lens and the specimen. Immersion oil has a
refrac-tive index closer to that of glass than the refractive index of air, so the use of
oil increases the cone of light that enters the objective lens.

Because of refractive index the light passing from the glass into air makes the
light to bend. The light passing from glass through oil does not bend much because
the oil has similar refractive index to that of a glass.
The immersion oil with a refractive index of about 1.5 in-creases the
numerical aperture and increases the resolving power of the microscope.
Stains and staining reactions

Bacteria are semi-transparent and consist of a clear proto-plasmic matter that differs slightly in
refractive index from the medium in which they are growing.

STAINS AND STAINING REACTIONS

Bacteria are semi-transparent and consist of a clear proto-plasmic matter that
differs slightly in refractive index from the medium in which they are growing. It is
difficult to observe the bacteria in unstained state, except when special methods of
illu-mination are used, to see them in the unstained state.

Stains are useful for the following reasons.

l It makes the microscopic semi-transparent objects visible

l To study the shape and size

l To reveal the presence of various internal and external struc-tures

l To produce specific chemical and physical reaction

The term stain and dye are not the same. A colouring agent that is used for general
purposes is called a dye. The one that is used for biological purposes is called a
stain. Based on their chemi-cal behavior, the dyes are classified as acidic, basic and
neutral.

An acid (or anionic) dye has a negative charge. eg., Eosin, Rose Bengal and
Acid fuchsin. The negatively charged groups are carboxyls (-COOH) and Phenolic
hydroxyls (-OH). Since they are negatively charged, bind to positively charged cell
structures. pH plays an important role in the effectiveness of staining, because the
nature and the degree of the charge on cell components change with pH. The
anionic dyes stain better under acidic conditions, where the proteins and many
other molecules carry a positive charge.
A basic dye (or cationic) carries a positive charge. eg., Methylene Blue, basic
fuchsin, crystal violet, malachite green, safranin. Ba-sic dyes bind to negatively
charged molecules like nucleic acid and many proteins. Since the bacterial cells
surfaces are negatively charged, basic dyes are most often used in Bacteriology.
Basic dyes are normally available as chloride salts.

A neutral dye is a complex salt of a dye acid with a dye base.

The dyes used in bacteriology have two features in common.

l They have chromophore groups, groups with double bonds, that give the dye
its colour

l They can bind with cells by ionic, covalent or hydrophobic bonding.

Relationship between the type of the dye and its charge when dissociated is
summarized.

In positive staining procedure, a stain that has a positively gedchromophorear

(coloured portion of the stain molecule) is attracted to the negatively charged outer
surface of the microbial cell. A stain such as Methylene blue has a positively
charged blue portion of the molecule that stains the microorganism.

In negative staining procedures, a negatively charged chro-mophore is repelled

by the negatively charged microorganisms, resulting in negative or indirect
staining of the microbial cell. Ni-grosin and Indian ink are frequently used for
negative staining of microbial cells, and this type of staining is particularly useful
for viewing some structures such as capsules that surround some bac-terial cells.

Stains are generally prepared largely as aqueous solutions. However in some

cases stock solutions are prepared in alcohol, and are diluted with water as needed.
Since alcohol removes the stains, pure alcoholic solutions should not be used.
Staining solu-tions are prepared to contain low concentrations of stains rarely
exceeding 1%. A very dilute staining solution activity for a long period of time will
produce much better results then a more con-centrated solution acting for a shorter
interval. This procedure has to be followed to reveal internal structure in bacteria.
Figure:3.1 The interaction of a cell with negative and positive stain re-agents: The
outer layer of a cell is negatively charged and a positive stain is attracted to the
cell, whereas a negative stain is repelled.

Staining reactions-Interpretation notes

When the pH of the surroundings of the microbial cells is either neutral or alkaline,
all microbial cells have a negative charge on their surface, called the surface
charge. Many bacterial cultures produce acids, thereby adding hydrogen ions to a
culture medium and decreasing it pH. These hydrogen ions(H+) interact with the
surface of the negative charges on the surface. When this happens, the cell surface
no longer strongly attracts positively charged dye ions (basic dyes). Thus the
microbes from acidic environments stain poorly with basic dyes. For this reason,
the basic dyes are made up as alkaline solutions. For example, potassium
hydroxide (KOH) is added to solutions of methylene blue to form the called
Loeffler’s Methylene blue.
Some bacteria excrete alkaline materials during growth and this decreases the
number of available hydrogen ions in the cul-ture medium. Under such conditions,
the cell surface has a greater negative charge, which is more attractive to basic
dyes and there- fore, allows greater binding, penetration and internal staining of the
microbe. Basic dyes stain microorganisms better under neutral or alkaline
conditions.
If the dye base molecule has a negative charge, it is repelled by the cell’s
negativelyThusnegativelycharged charsurface. ged dyes neither bind to the cell’s
surface nor are they able to penetrate into the cell. These are called acid dyes.

The methodology for using acid dyes are different from ba-sic dyes. An acid dye is
mixed with a drop of culture smeared on a microscope slide and allowed to air dry.
The negatively charged cells are not stained by the negatively charged dye, and
they ap-pear as clear area surrounded by a coloured back ground. Nega-tively
charged dyes used in this way are called negative stains.
Under neutral or alkaline conditions, the negative stains (acidic dyes) work
better, because these conditions allow the sur-face charge to be more negative.
Negative stains are of limited usefulness for those using light microscopes, but
they can be used to avoid some of the disadvantages of staining with basic dyes.

Simple staining
A simple staining solution, contains only one stain, which is dissolved in a solvent.
It is applied to the microorganism in one application. The microorganisms give the
colour characteristic of the staining solution. The purpose of simple staining is to
reveal the size and shape of the microorganism. The simple stains that are
commonly used by the microbiologists for routine purposes are dilute solution of
carbol fuchsin, crystal violet and methylene blue.

Methylene blue is more frequently used than any other stain in bacteriology. It is
because of its strong nature and it stains nu-clei and nucleic acid granules very
intensively. Methylene blue is used for the rapid survey of bacterial population of
milk. It is also used for the diagnosis of Diphtheria. This stain is incorporated with
Eosin in Lactose agar to distinguish typicalinconE-.coli taminated water.

Differential Staining
In this procedure, more than one dye is employed. Differen-tial staining procedure
helps to divide the bacteria into separate groups based on staining characteristics.
The two most important differential stains used by bacteriologists are Gram stain
and Acid-fast stain.

Gram Staining
The simple staining procedure makes to visualize bacteria clearly, but it does not
distinguish between organisms of similar morphology. In 1884, a Danish Physician
named, Christian Gram discovered a new technique to differentiate the bacteria of
similar morphology. He used two dyes in sequence, each of a different
colourganisms.Thethatorretain the colour of the first dye are called Gram positive
and those that cannot retain the first dye when washed with a decolourizing
solution, but then take on the colour of the second dye are called Gram negative .
In this method, the fixed bacterial smear is subjected to the following staining
regents in the order of sequence listed below:

Crystal violet - - > Iodine solution - - > alcohol (decolourizing agent) - -

> Safranin.

Principle
The Gram-positive bacteria will retain the crystal violet and appear deep violet in
colour. The Gram-negative bacteria lose the crystal violet on decolorization and
are counter stained by the sa-franine and appear red in colour. Iodine solution is
used as a mor-dant that fixes the primary stain in or on a substrate by combining
with the dye to form an insoluble compound-mordant, for the first stain.
The exact mechanism of action of this staining technique is not clearly understood.
However, the most plausible explanations for the reactions are associated with the
structure and composition of the cell wall.

The cell walls of Gram-negative bacteria are thinner than that of Gram-positive
bacteria and contain a higher percentage of lipid content. During the staining of
Gram-negative bacteria, the alcohol treatment extracts the lipid. This results in
increased po-rosity or permeability of the cell wall. The crystal violet-iodine (CV-
I) complex, thus can be extracted and the Gram-negative bac-teria is decolorized.
The cells subsequently take up the colour of the counter stain safranin.

The cell walls of Gram-positive bacteria with lower lipid content become
dehydrated during alcohol treatment. The pore size decreased, permeability is
reduced and the CV-I complex can-not be extracted. Therefore, the Gram-positive
cells remain purple-violet.

Endospore Staining

Endospore formation is a distinguishing feature of the fam-ily Bacillaceae, which

includes members of the aerobic genus, and the Bacillusanaerobic
genus, Clostridium. Endospore resists adverse environmental conditions such as
dryness, heat and poor nutrient supply. The endospore is a highly retractile body
formed within the vegetative bacterial cell at a certain stage of growth.The size,
shape, and position of the spore are relatively constant characteristics of a given
species and are therefore, of some value in distinguishing the kind of bacillus from
another. The position of the spore in the cell may be central, sub terminal or
terminal. It may be the same diameter as the cell, smaller, or larger causing a
swelling of the cell.

Endospores strongly resist application of simple dyes, but once stained are quiet
resistant to decolorization. This character suggests one way to make the structure
visible. If simple stains are used, the body of the bacillus is deeply colored,
whereas the spore is unstained and appears as a clear area in the organism. By
vigor-ous staining procedures the dye can be introduced into the sub-stance of the
spore. When thus stained, the spore tends to retain the dye after treatment with
decolorizing agents.

To make the distinction clear between the spore and the veg-etative portion of the
cell, a contrasting counter stain is usually applied in the ordinary fashion and the
resulting picture shows the initial stain taken up by the spore and the second stain
appear in the cytoplasm. Thus, it makes for a very simple method of distin-
guishing the endospore from the vegetative cell.
Sterilization
Sterilization is the freeing of an article from all living organ-isms, including bacteria and their spores.

STERILIZATION

Definition
Sterilization is the freeing of an article from all living organ-isms, including
bacteria and their spores.

Sterilization of culture media, containers and instruments is essential in

microbiological work for isolation and maintenance of microbes.

In surgery and medicine, the sterilization of instruments, drugs and other supplies
is important for the prevention of infec-tion.
Sterilization can be effected in a variety of ways, which can be conveniently
categorized as follows:

I. PHYSICAL METHODS
1. Heat :

1. Dry heat

2. Moist heat

B. Radiations

1. Ultraviolet radiations

2. Ionizing radiations
C. Filtration

II. CHEMICAL METHODS

STERILIZATION BY HEAT
Heat can be applied in two forms.

1. The dry heat

2 Moist heat.

Mechanism of killing by dry heat

l Dry heat kills the organisms by destructive oxidation of essen-tial cell
constituents

l Killing of the most resistant spores by dry heat requires a tem-perature of about
160C for 60 minutes

l Dry heat is employed for glassware; syringes, metal instru-ments and paper
wrapped goods, which are not spoiled by high temperatures.

l It is also used for anhydrous fats, oils and powders that are impermeable to
moisture.

Mechanism of killing by moist heat

l Moist heat kills the organisms by coagulating and denaturing their enzymes
and structural protein.
l Sterilization by moist heat of the most resistant spores gener-ally requires
121C for 15-30 minutes.

l Moist heat is used for the sterilization of culture media, and all other materials
through which steam can penetrate

l Moist heat is more effective than dry heat

Sterilization can be done at lower temperatures in a given time at a shorter duration

at the same temperature.

FACTORS INFLUENZING STERILIZATION BY HEAT

1. The temperature and time: they are inversely related, shorter time is sufficient
at high temperatures.

2. Number of microorganisms and spores: The number of survi-vors diminished

exponentially with the duration of heating

3. Depends on the species, strains and spore forming ability of the microbes.

4. Thermal death point is the lowest temperature to give com-plete killing in

aqueous suspension within 10 minutes

5. Depends on the nature of material: a high content of organic substances

generally tends to protect spores and vegetative organisms against heat.

6. Presenceganicdisinfectantsoforganic orfacilitatesinor kill-ing by heat

7. pH also plays an important role in the killing of microorgan-isms

METHODS OF STERILIZATION BY DRY HEAT

1. RED HEAT
Inoculating wires, points of forceps and searing spatulas are sterilized by holding
them in the flame of Bunsen burner until they are seen to be red-hot.

2. FLAMING

This method is used for sterilizing scalpel, mouth of culture tubes, glass slides etc.

It involves passing of an article through Bunsen flame with-out allowing it to

become red-hot.

3. HOT AIR OVEN

This is the main means of sterilization by dry heat.

Exposure at a temperature of 160C° for 1 hour is generally employed.

4. INFRARED RADIATIONS
Source employed is an electrically heated element, the infra red rays are directed
on to the object to be sterilized and tempera-ture of 180C° can be obtained.

METHODS OF STERILIZATION BY MOIST HEAT Moist heat can be

employed at

1. C Temperature below 100

2. TemperatureC of 100

3. Temperature above 100C

MOIST HEAT BELOW 100 °C

EXAMPLES

1. Pasteurization of milk

In Pasteurization of milk the temperature employed is either 63 °C for 30 minutes

or 72 °C for 20 seconds. All nonspore-forming pathogens in milk like Salmonellae,
M.tuberculosis are killed.

MOIST HEAT ABOVE 100°C

1. Sterilization in an autoclave

Autoclaving is the most reliable method

It is the method most widely used for sterilization of culture media and
surgical supplies

When water is boiled within a closed vessel at an increased pressure, the

temperature at which it boils and the steam it forms will rise above 100 C

This principle is used in the autoclave

Normally autoclaving is done at 15 lbs. (pounds per sq. inch pressure) and
115 C for 15 minutes

STERILIZATION BY FILTRATION
When fluids are passed through bacteria stopping filters, they are made free from
bacteria.

l It is useful for making preparations of soluble products of bac-terial growth

such as toxins

l Liquids that would be damaged by heat such as serum and antibiotic solutions
can be sterilized by filtration

l Efficient filters should be able to retain Serratia marcescens

TYPES OF FILTERS

There are different kinds of filter

1. Earthenware candles - called Berkfield & Chamberland filters

2. Asbestos and asbestos-paper discs filters - called Seitz fil-ters

3. Sintered glass filters

4. Cellulose membrane filters

5. Fibre glass filters.

Berkfield Filters

Made from Kieselguhr, a fossil diatomaceous earth

Three grades of porosity are available:

a. Veil - coarsest one

b. N - normal one

c. W- wenig the finest one

Chamberland Filters
l Made from unglazed porcelain

l Four grades are available

a. L1- clarifying filters

b. L1a-Big

c. L2 - normal
d. L3- Finest

Seitz filter
l Made up of asbestos pads

l Three grades are available

a. K- clarifying filters

b. Normal

c. Special EK bacteria stopping filters

Sintered glass filters

l Made from sintered glass

l Different grades available

Grades 1 to 5

Grades 1-2 are for clarifying purpose

Grades3-5 is for sterilization purpose

Membrane filters
l Made up of nitro-cellulose membranes

l Made with different grades of porosity by adjusting the con-centration of

constituents

MERITS AND DEMERITS OF HEAT STERILIZATION

Advantages of heat sterilization

1. Sterilization is very effective

2. Instruments are standardized to deliver the required effective heat

3. Heat deliver system can be monitored effectively with variouscontrols like

pressure gauge, temperature meters etc

4. Established quality control methods available

Disadvantages

1. Steam impermeable materials like fats, oils and powders can not be sterilized
by autoclaving.

2. Heat sensitive materials can not be sterilized by heat

Examples:

1. Serum can not be sterilized

2. Antibiotics

3. Plastic materials

4. Vaccines

5. Rubbers

3. Presence of organic matters interfere with effective sterilization

4. Dangers of explosion when high pressure is used

Factors Influenzing Sterilization by Heat

The temperature and time: they are inversely related, shorter time is sufficient at high temperatures.

FACTORS INFLUENZING STERILIZATION BY HEAT

1. The temperature and time: they are inversely related, shorter time is sufficient
at high temperatures.
2. Number of microorganisms and spores: The number of survi-vors diminished
exponentially with the duration of heating

3. Depends on the species, strains and spore forming ability of the microbes.

4. Thermal death point is the lowest temperature to give com

plete killing in aqueous suspension within 10 minutes

5. Depends on the nature of material: a high content of organic substances

generally tends to protect spores and vegetative organisms against heat.

6. Presence of organic disinfectants or facilitates in or killing by heat

7. pH also plays an important role in the killing of microorgan-isms

Microbial nutrition and growth
‘An army marches on its stomach’ said Napolean Bonapart. This indicates that food is important for
any living or-ganism and so also for microbes.

MICROBIAL NUTRITION AND GROWTH

‘An army marches on its stomach’ said Napolean Bonapart. This indicates that
food is important for any living or-ganism and so also for microbes. Food is any
substrate that can be metabolized to provide assimilable material or energy for the
cell. Plants synthesize their own food requirements through photosyn-thesis.
Animals ingest the presynthesized food from plants or by devouring other animals.
All living organisms, from micro to macroorganisms require nutrients for growth
and normal function-ing. Animals ingest the food and digest them in their digestive
system (Holozoic nutrition) to simpler nutrients which are absorbed by cells for
synthesis of all cellular material and derive energy. Plants absorb the nutrients
from soil solution (Holophytic nutri-tion) released by mineralisation of organic
matter and grow. Mi-croorganisms particularly fungi derive their nutrients from the
ex-tra corporeal digestion by secreting extracellular enzymes. The nutrients are
absorbed and cellular materials are synthesized.

All organisms exhibit two universal requirements viz., water and elements. All
organisms require energy which they de-rive from the chemical compounds or
radiant energy like light. The elemental components are carbon, nitrogen,
phosphorus, sul-phur and potassium besides hydrogen and oxygen major ones for
synthesizing cellular components. Metal ions like K, Ca, Mg and Fe are required
for normal growth. Other metal ions like Zn, Cu, Mn, Mo , Ni, B, Co are often
required in low quantities hence known as trace elements. Fe, Mg, Zn, Mo, Mn and
Cu are cofac-tors/coenzymes or prosthetic group of various enzymes. Most bac-
teria do not require Na but certain marine bacteria, cyanobacteria and
photosynthetic bacteria require it. Red extreme halophiles can- not grow with less
than 12 to 15% NaCl which is required to maintain the integrity of cell walls and
for the stability and activity of certain enzymes. Silicon is required for the growth
of diatoms.

Vitamins and vitamin like compounds are also present in living cells. These
function either as coenzymes or as building blocks of coenzymes. Some bacteria
synthesize their entire requirements of vitamins but some cannot grow unless
supplied from external source.
 Microorganisms are divided into several types based on the energy source or
electron source and carbon assimilation. Those derive energy from the oxidation of
chemical compounds are known as ‘chemotrophs’ and others utilizing radiant
energy like light are known as ‘phototrophs’. Electrons are required for me-
tabolism and based on the source from which bacteria derive elec-tron they are
grouped. Some organisms use reduced inorganic com-pounds as electron donors
and are termed as ‘lithotrophs’ literally meaning rock eating. Others use organic
compounds are termed as ‘organotrophs’. Those organisms that derive energy from
the chemical compounds (Chemotrophs) and uses inorganic com-pounds as e-
donors (lithotrophs) are known as chemolithotrophs. Those that derive energy
from light (phototrophs) and e- from in-organic compounds are photolithotrophs.
Similarly those chemotrophs that use organic compounds, as e - donors are
chemoorganotrophs and the phototrophs that utilize organic compounds as e-
donors are photoorganotrophs

Chromatium okenii, a photosynthetic bacterium, uses radi-ant energy and H2S

as electron donor oxidizing it to elemental sulphur. Some phototrophs use organic
compounds such as fatty acids and alcohols as electron donors and hence called
photoorganotrophs.

Rhodospirillum rubrum another phototrophic bacterium uti-lizes succinate as e-

donor converting it to fumarate. A phototrophic bacterium can grow as
chemotroph. In the anoxygenic environ-ment (absence of O2) this bacterium grow
as photoor but in the presence of oxygen and dark (absence of light) it grows as a
chemoorganotroph. Among the chemotrophs some utilize inorganic

compounds like NH4 as e- donors and hence called

chemolithotrophs. Nitrosomonas use ammonia for electrons and derive energy by
oxidizing ammonia to nitrite. Certain chemotrophs use organic compounds like
sugars and amino acids as e- donor

and are called chemoorganotrophs. Some of the chemotrophs can grow either as
chemolithotrophs or chemoorganotrophs. Pseudomonas pseudofulvacan use
glucose an organic compound (chemoorganotrophs) or inorganic compound H2 as
e- source (chemolithotrophs)
Autotrophs and Heterotrophs

Based on the source of carbon microorganisms are grouped as autotrophs and

heterotrophs. Some can use CO2 as their sole source of carbon like plants and
algae are termed as autotrophs. Others like some bacteria, fungi and actinomycetes
utilize preformed organic compounds as carbon source and hence called
heterotrophs.
Most organisms that involve in decomposition of organic matter in soil are
heterotrophs Fungi are saprophytic and depend on dead organic matter. Some fungi
are parasitic on living plants and animals. The saprophytic and parasitic organisms
are heterotrophs. Such of these heterotrophs that have elaborate requirements of
specific nutrients like vitamins and growth promoting substances are called
fastidious heterotrophs as they are not easily pleased or satisfied by ordinary
nutrients available in nature.

The source of carbon for microbes is CO2 or carbohydrates. Autotrophs derive

their entire requirement of C from CO2 while heterotrophs derive the carbon
chiefly from carbohydrate. In nature, cellulose, hemicelluloses, starch, pectin,
lignin etc serve as

carbon sources. Amino acid, purine and pyrimidine bases, protein serve as a source
of nitrogen. Phosphorus is obtained from the nucleotides, phytin etc. For
cultivation of microorganisms in labo-ratory, media containing monosaccharides
like glucose and disac-charides like sucrose are used as C sources. Peptone,
Tryptone, inorganic salts like ammonium salts, potassium nitrate serve as nitrogen
sources. Potassium dihydrogen phosphate and dipotas-sium hydrogen phosphate
are commonly employed to serve as sources of phosphorus and also as a buffering
agent.

Autotrophic bacteria have the simplest nutritional require-ments as they can grow
and reproduce in a mixture of inorganic compounds. They also possess an
elaborate capacity to synthesize the carbohydrate, proteins, lipids, nucleic acids,
vitamins and other complex substances of living cells. Photosynthesis is a normal
autotrophic way of life and this occurs in plants, algae, photosyn-thetic bacteria
and cyanobacteria. In this process, CO2 is reduced and converted to carbohydrate
utilizing light. However, photosyn-thesis of plants, algae and cyanobacteria
perform oxygen evolving photosynthesis by absorbing the reducing power from
the pho-tolysis of water. On the other hand, photosynthetic bacteria green and
purple bacteria obtain the reducing power from a compound similar to water (H 20)
viz., H2S i.e. available in anoxygenic envi-ronment. The pigments and the light
absorption also differ in these organisms.

Over all reaction of photosynthesis is,

2H20 + CO2−−> (C2H0) x + O2 + H20 (Plants, algae, cyanobacteria)

2H2S+ CO2 −−> (C2H0) x + 2S + H20 (Photoautotroph bacteria)

Inorganic compounds like H2, H2S203 or the organic com-pounds lactate, succinate
can be the source of reducing power in-stead of H2S.

Growth
Living organisms grow and reproduce. The growth indicates that an organism is in
active metabolism. In plants and animals one see the increase in height or size. In a
butterfly, a small larva hatching from egg grows in size, moults, pupates and
become an adult butterfly through metamorphosis. Growth in a common use refers
to increase in size but with microorganisms particularly with bacteria, this term
refers to changes in total population rather than increase in size or mass of an
individual organism. With fungi linear growth of hyphae and radial growth of
colony is observed for growth on solid media but a biomass or mycelial dry weight
on liquid media. In unicellular fungi like yeast that reproduce by fis-sion or
budding the population change is considered as growth.

The change in population in bacteria chiefly involves trans-verse binary fission in

most of the bacteria while budding is ob-served in Hyphomicrobium. In
actinomycetes, fragmentation of hyphae and sporulation results in population
change. In yeasts, budding and fission are observed that this depends upon the spe-
cies. In fungi, growth fragmentation asexual and sexual spores serve as propagates
for population increase.

The transverse binary fission, an asexual reproductive pro-cess is the most

common in the growth cycle of bacterial popula-tion. A single cell divides after
developing a transverse septum (cross wall) and continues to grow by continuous
dividing with-out cell death till it is subjected to stress.
A cell dividing by binary fission is immortal unless subjected to stress by nutrient
depletion or environmental stress. Therefore a single bacterium continuously
divides. One cell divides providing two cells and two cells divide giving four and
so on. Therefore the population increases by geometric progression.
 When a single bacterium is incubated into the liquid me-dium in flask and
incubated, bacterium divides by fission and at-tains a period of rapid growth in
which cells multiply at an expo-nential rate. If the logarithmic number of
bacterium versus time is plotted a growth curve is obtained with different phases of
growth.
Soon after transfer of an inoculum to a new medium, cells do not immediately
multiply and the population remains un-changed. The cells however increase in
size synthesizing newer protoplasm and enzyme necessary to the newer
environment. The organisms are metabolizing but require more for adjustments to
the physical environment around each cell and hence there is a lag for cell division
(lag phase).

At the end of lag phase cells divide and there is a gradual increase in the
population. When all the cells complete their lag, there is division at regular
intervals. The cells divide steadily at a constant rate in the logarithmic or
exponential phase and when log number of cells are plotted against time there is a
straight line. The population in this phase is almost uniform in chemical composi-
tion, metabolic activity and physiological characteristics.
Generation time is the time required for the population to double and this can be
determined by the number of generation that occurs at a particular time interval.
Not all bacteria have the same generation time. It varies from 15 – 20 minutes
for chia coli to many hours in others and is also dependent upon thenutrients and
physical conditions of the environment. With the growth of the bacterium, there
will be a depletion of nutrients. At high concentration of nutrients a small change
may not cause sig-nificant effect but at low concentration the growth rate decreases
significantly.

At the end of the exponential phase growth rate decreases due to exhaustion of
some nutrients or due to production of toxic products during growth. The
population remains constant due to complete cessation of division or reproduction
rate equals to death rate.

The stationary phase is followed by Decline or Death phase as bacteria divide

faster than the new cells produced. The deple-tion of nutrients, accumulation of
solubilising products like acids.

The number of viable cells decreases exponentially. G-ve Cocci divide faster but
others divide slowly but viable cells may persist for minutes or even years.
Measurement of growth
Growth refers to the magnitude of the population in bacteria. The growth can be
measured quantitatively (1) cell count (2) cell mass and (3) cell activity. Cell count
shall be made directly by microscopy or using an electronic particle counter. It can
also be made indirectly by colony count after serially diluting the sample. Cell
mass can be determined directly by weighing a known vol-ume of sample culture
broth or by measuring the cell nitrogen. It can also be determined indirectly by
finding cell activity, which can be measured by the degree of biochemical activity
to the size of population.
Petroff – Hausen counting chamber is used for direct micro-scopic count. It is a
slide accurately ruled into squares of 1/400 mm2 area over when a cover slip rests
at 1/50mm above. This gives a volume of 1/20000mm3 over one square. The liquid
can be placed in the chamber left unstained and counted using a phase contrast
microscopy. If 5 cells are present in one square there will be 5 X 20,000,000 or
108 cells/ml. This method is rapid requires simple equipment. Morphology of cells
can be simultaneously obtained but difference of viable or dead cells cannot be
made.

In electronic particle counter a bacterial suspension is passed through a tiny orifice

of 10-30 μm diameter that connects the two comparts of counter containing an
electrically conductive solu-tion. The electrical resonance between the two
compartments in-crease momentary when each bacterial cell passes in the orifice
creating an electrical signal. The signals are automatically counted. This method is
rapid but requires sophisticated electronic equip-ment.
In plate count method a known volume of bacterial suspen-sion diluted serially if
population is dense, is poured in petridishes and molten agar medium is added and
mixed thoroughly. The cells trapped immediately in the gelled medium develop
into a colony. The colonies are counted by illumination from below. The num-ber
of colonies is multiplied by the dilution factor to detect the population and
expressed as number per ml. Only those cells that grow in the medium that show
up and get counted under the con-ditions in which the plates are incubated. If
aggregate of cells are formed as in cocci in clusters, chains or pairs the resulting
counts will be lower than the individual cells. In such cases counts are referred to
as colony – forming units per ml than number per ml. The plate count is used
normally for estimation of bacterial popu-lation in milk, waste foods, soil and
many other materials.
Mernbrane filter count is used to determine the bacterial numbers in large sample
containing very small number of viable cells. Bacteria in large volume of air or
wastes shall be collected by filtering in the membrane. The membrane with filtered
bacteria is placed on plate containing a pad saturated with medium and incubated.
The organisms grow into a colony and counted. Special medium and dyes can be
used to detect certain types of organ-isms.

The bacteria in suspension absorb and scatter light passing through the cell similar
to water droplets in fogs absorbing and scattering of light. Because of this
phenomenon, a culture of more than 107 or 108 cells per ml appears turbid to the
naked eye.

The cell growth can also be measured by the nitrogen con-tent that forms the
process when is a major constituent of cell. Cells are harvested, washed free of
medium and nitrogen is analysed by standard method.
The quantitative measurement of a mass of cells is made by the dry weight
determination. Very dense suspension of cells can be washed free of extraneous
matter and weighed. In cells accu-mulating β –hydroxy butyrate cell mass may
increase without corre-sponding increase in cell growth.

The measurement of acid or any other product of metabolism shall be measured to

assess growth.

In case of yeasts, dry weight determination and nitrogen estimation can be done as
a measure of growth. In mycelial fungi, mycelial dry weights are determined by
filtering the mycelial mat in a previously weighed filter paper drying it in oven at
105oC for 24 hours and weighing it. The mycelial weight is determined by
subtracting the weight of filter paper. In agar medium, the linear growth / nodal
growth of fungi shall be measured.
Pure culture methods
In the natural environments microorganisms exist in mixed cultures. To establish the role of microbial
agent to a disease process, it is essential to demonstrate the organisms or its components in the
diseased tissues.

PURE CULTURE METHODS

In the natural environments microorganisms exist in mixed cultures. To establish

the role of microbial agent to a disease process, it is essential to demonstrate the
organisms or its components in the diseased tissues. To accomplish this, the
organism must be cultivated from the tissues. Similarly to know the kinds of
organism present in the environment it is necessary to grow them in artificial
media. Cultivation of the organism is also essential to obtain pure culture of clone
of cells derived from a single cell to perform biochemical differentiation tests
and susceptibility tests since mixed cultures give misleading results.

Artificial culture media

A medium is an environment which supplies the ingredients necessary for the

growth of the organism. Various kinds of media have been prepared in the
laboratory to isolate, grow and identify an organism. Depending on the need to
isolate and identify an

organism from a particular sample or environment, different kinds of media are

formulated.

Kinds of media Basal or supportive media

Basal medium is one that contains nutrients that allow the growth of most
nonfastidious organism without affording growth advantage to any particular
organism over others. Example is Nutrient agar, and Trypticase Soy agar.
Enrichment medium

Enrichment medium is a liquid medium which enhances the growth of certain

bacterial species, while inhibiting the growth or prolonging the lag phase of
unwanted organisms thus altering the ratio between the two in favor of the required
bacterial species.

Example is Selenite F broth for the isolation of Salmonella from stool. To get a
pure culture of the organism, any one of the solid media mentioned above is used.
In order to get discrete separate colonies, the surface of the medium must be dry.
The material is

inoculated on the surface by spreading with a sterile loop in such a way that
bacteria are ultimately deposited singly. When the bacteria are at a sufficient
distance from each other, the whole progeny of each accumulates locally during
growth to form a discrete mass or colony which is readily visible to the naked eye.
Each colony is presumed to be a pure culture, consisting exclusively of the
descendants of a single cell. It may be picked up with a sterile wire to prepare a
pure subculture in a fresh medium.

Growth and colony characteristics of Bacteria

The appearances of growths of bacteria in liquid media are generally not

distinctive. There is a uniform turbidity in the liquid and little deposit at the
bottom. Colony morphology of the isolated acteria on the solid media has much
more value. Attention

is paid to the size of the colony (diameter in mm), their outline, whether circular
and entire or indented, or wavy or rhizoid, their elevation low convex, high convex
or flat plateau-like, umbonate or nodular, their translucency, whether transparent,
translucent, or opaque, their pigmentation, colorless, white or otherwise
pigmented, and whether they produce any change in the medium (haemolysis in a
blood-containing medium).
Example: Colony characteristics of Staphylococcus aureus on Nutrient agar

After aerobic incubation at 37oC for 24 hours, colonies are 1-3 mm in diameter
and have a smooth glistening surface, an entire edge, a soft butyrous consistency
and an opaque, pigmented appearance.

Growth characteristics of yeasts

Yeasts are grown on Sabouraud Dextrose agar aerobically. Yeasts grow as typical
pasty colonies and give out yeasty odor. The colony morphology varies with
different yeasts.

Growth characteristics of filamentous fungi

The most common medium used for the isolation of fungi is Sabouraud Dextrose
agar. While observing colony morphology, one must note the colors of the surface
and the reverse of the colony, the texture of the surface (powdery, granular,
woolly, cottony, velvety or glabrous), the topography (elevation, folding, margins,
etc) and the rate of growth.
Prokaryotic cell structure
Living organisms are differentiated from nonliving matter by their (1) ability to reproduce (2) ability to
ingest or assimilate food and metabolize them for energy and growth (3) ability to excrete waste
products (4) ability to react to changes in their environment (irritability) and (5) Susceptibility to
mutation.

PROKARYOTIC CELL STRUCTURE

Living organisms are differentiated from nonliving matter by their (1) ability to
reproduce (2) ability to ingest or assimilate food and metabolize them for energy
and growth (3) ability to excrete waste products (4) ability to react to changes in
their environment (irritability) and (5) Susceptibility to mutation. The living
organisms include a variety of micro and macro organisms of differing size , shape
morphology, and behaviour. They include tiny bacteria, protozoans, worms, plants
and animals like man,

whale and elephants.

Carlous Linnaeus (1707-1778), the Swedish botanist was the first to introduce
nomenclature for plants and animals. Until 18thcentury only plant and animal
kingdoms were recognized. However some organisms are predominately plant like,
some animal like and some do not fall in both the groups. Therefore it was felt a
third kingdom was necessary. Haeckel (1866), a German zoologist suggested a
third kingdom Protista to include those organisms that are not typically plants and
animals. Bacteria, algae, fungi and protozoa are cellular organisms placed under
protista. Viruses are not cellular organisms and therefore not classified as protists.
Bacteria were lower protists while algae, fungi and protozoa were higher protists.
A satisfactory criteria to differentiate bacteria, fungi and algae could not be made
until the development of electron microscope, which depicted the internal structure
of these organisms. The absence of membrane bound internal structures in bacteria
and their presence in fungi, algae, protozoa, plants and animal cells was taken as
criterion to differentiate prokaryote and eukaryote.

Whittakar (1969) proposed five kingdoms based on three levels of cellular

organization and three principal modes of nutrition, photosynthesis, absorption and
ingestion. The prokaryotes lacking ingestive mode of nutrition are included in the
kingdom. Monera. In the kingdom protista unicellular eukaryotic microorganisms
representing all the three modes of nutrition are included. The multicellular green
plants and higher algae were placed in the kingdom plantae while multinucleate
higher fungi in the kingdom fungi and the multicellular animals in the kingdom
Animalea.

Bacteria and cyanobacteria (the blue green algae) of monera, microalgae and
protozoa of protists and yeasts molds and fungi represent the microorganisms.
Most of them are invisible to the naked eye and requires magnification. The
oratically a black dot of 4mμ in diameter on a white background can be perceived
by retina of human eye but in reality an object of above 30mμ in size only will be
visible to the eyes and objects lesser than that requires magnification.

Prokaryotes are organisms with primitive type of nucleus lacking a well-defined

membrane a less complex nuclear division than mitosis. The nuclear material is a
DNA molecule in prokaryotes compared to chromosomes of higher organisms.
Eukaryotes

are organisms with cells having true nuclei enclosed in a nuclear membrane and
are structurally more complex them prokaryotes. A varying degree of localization
of cellular functions in distinct membrane bound intracellular organelles like
nuclei, mitochondria
chloroplasts etc. The cells of living organisms are either prokaryotic or eukaryotic
in nature and there is not any intermediate condition. The size, shape, morphology
and the internal cellular organizations are different in these two groups.

The size of the microorganisms varies from unicellular tiny bacteria to large brown
algae and mushroom. Bacteria are unicellular, small 0.5-1.0mm in diameter, which
multiply by binary fission. The algae are photosynthetic simple organism
withunicellular primitive types to aggregates of similar cells and to large brown
algae with complex structure. Protozoa are unicellu-lar, most of them living freely
in soil and water while a few cause disease of man and animals.
The rigid cell wall of the bacterium confers shape. Bacteria vary in shape from
spherical (Coccus) rods (Bacillus) and heli-cally curved rods (Spirillum). Most
bacteria possess a constant shape but some exhibit polymorphism (variety of
shape).

Bacterial cells are arranged in a characteristic manner in a particular species. In

cocci the arrangement is known as diplo-cocci when cells divide in one plane and
remain attached in pairs, streptococci when divide in one plane and remain
attached to form chains; tetrococci, when they divide in two planes and form group
of four cells (tetrads), staphylococci when they divide in three planes and form
bundles, sarcinae when they divide in three planes in a regular manner and form a
cutridal arrangements.

Bacilli are not arranged in such complex form as in cocci. Most of them occur
singly or in pairs (diplobacilli), form chains (streptobacilli) form trichomes, similar
to chains but with a larger area of contact between cells and lined side by side like
match sticks (palisade arrangement) at angles to one another.

Some others form long branched multinucleate filaments called hypha as in fungi.
Hyphae ramify and collectively form mycelium. The curved bacteria are vibrioid
with less than one twist or turn of helical with one or more complete turns. Rigid
helical shape is in Spirilla and is flexible in spirochete.

Cell wall is a very rigid structure that confers shape to the cell. This prevents
expansion of cells and bursting due to uptake of water as most bacteria live in
environments of higher osmotic pressure than that exists in cells (hypotonic
environments). A cell wall is common to almost all bacteria except in mycoplasma
that lacks typical cell wall and L-forms of bacteria like Streptobacillus that are
having walls but loose them when grown in media con-taining sub lethal levels of
cell wall synthesis inhibiting antibiot-ics like penicillin. Mycoplasma lack cell wall
permanently and hence pleomorphic while L-forms of bacteria can revert back to
walled forms. The isolated cell walls without cellular constituents retain the
original contour of cells from which they are derived indicating that cell wall
confers shape. This is further strength-ened as the protoplast derived from any type
of cell cocci or bacilli show a spherical shape. Both eubacteria and archaebacteria
are grouped as Gram positive and Gram negative based on the wall thickness. As
the chemical composition of both eubacteria and archaeobacteria differ it is only
the thickness rather than the chemi-cal composition is the key factor for Gram
reaction.
Cell wall constitutes 10-40% of cell. It is essential for growth and division. Cells
without walls (protoplasts) cannot grow and divide.

The cell wall in eubacteria consists largely of an insoluble porous, cross-linked

polymer of enormous strength and rigidity viz., peptidoglycan (also called murein).

This is a bag shaped macromolecule and surrounds the cyto-plasmic membrane

and found only in prokaryotes. Although it is tough but in a dynamic state. It is a
polymer of N-acetyl glu-cosamine, N acetyl muramic acid, L-alanine, D-alanine,
D-glutamatic and a diamino acid (LL or meso diaminopimelic acid, L-lysine, L-
orthinine or L-diaminobutryic acid).
The cell wall composition of archaeobacteria is different from eubacteria. Their
walls are composed of proteins, glycoproteins or polysaccharides. But in some
genera as Methanobacterium the cell walls composed of pseudosuriein that have
some superfecial re-semblance to peptidoglycan but differs in chemical
composition.
The peptidoglycan constitutes about more than 50% of the dry weight of cells in
gram-positive eubacteria but only 10% in gram-negative bacteria. In addition to
peptidiglycan other sub-stances like polysaccharides in Streptococcus
pyrogenes teichoic acids in Staphylococcus aureus, lipids as mycolic acids
in Coryne-bacterium and Mycobacterium. The acid fast cord factor, a my-colic
acid derivative is toxic and plays a role in diseases due to Corynebacterium
diphtheriae and M. tuberculosis.

The wall of Gram negative consists of a thin peptidoglycan layer surrounded by an

outer membrane rich in lipids. The lipids in the wall constitute 11-12% of the dry
weight of the cells, The outer membrane is an impermeable barrier preventing the
escape of important enzymes from the periplasmic space between the cy-toplasmic
membrane and outer membrane. The outer membrane also prevents external
chemicals and enzymes that can destroy cells. Lysozyme, which dissolves
selectively the peptidoglycan can dam-age gram positive bacteria.

The outer membrane, a bilayered structure consisting many of phospholipids,

proteins, and polysaccharides is anchored to the peptidoglycan layer by means of
Braun’s lipoprotein. The li-popolysaccharide (LPS) layer has toxic properties and
known as endotoxin. This occurs only in outer membrane and is composed of lipid
A, core polysaccharide and O antigen. The outer mem-brane is impermeable to
large molecules like protein but allow smaller molecules like monosaccharides
peptides and amino ac-ids through channels called porins. Porins span the
membrane and are specific for different kinds of small molecules.

There are several structures external to cell wall in bacteria, which vary in structure
and composition depending upon the type of bacteria. They are flagella, pili or
fimbriae, capsules, sheath, prosthecae and stalk. Flagella are locomotory organs in
bacteria, which vary in number and arrangement. Some bacteria do not have
flagella.

Flagella are hair like helical appendages 0.01 – 0.02 nm in diameter the flagellar
arrangements vary with the organisms. It may be polar if the flagella are at one or
both the ends or lateral if they are arranged on sides. They protrude through the
cell wall. A flagellum is composed of a basal body a short hook and a helical
filament longer than the cell. The basal body is associated with cytoplasmic
membrane and cell wall.
Bacteria swim by rotating their helical flagella similar to cork screw. Bacteria with
polar flagella swim in a back and forth fash-ion. Those with lateral flagella swim in
a more complicated man-ner. Removal of flagella from a flagellate bacterium will
not result in death of bacterium and only motility will be affected Spiro-chetes, the
helical bacteria, swim even in viscous media, without any external flagella. They
have flagella like structure within the cell located just beneath the cell envelope.
They are known as periplasmic flagella (also called endoflagella, axial filament).
Spiroplasmas are also helical in shape and swim in viscous media without even
periplasmic flagella.

Some bacteria like Cytophaga exhibit a gliding motility, which is a slow sinuous
flexing motion. This occurs when the cells come in contact with solid surface.
Pili are short, hollow, non helical and filamentous append-ages. They are thinner
than flagella but more in number than fla-gella. They are found in both motile and
non motile bacteria and hence not involved in motility.
F pilus (sex pilus), a type of pilus serves as port of entry for genetic material during
bacterial mating. Some pili in pathogenic bacteria serve as attachment with host
cells in human beings fa-cilitating infection without being washed off easily by
mucous.

Capsules
A viscous substance forming a covering layer around the cell is found in some
bacteria and is known as capsule. If it is too thin it is called as microcapsule. If it is
loose and many cells are embedded in a matrix it is known as slime. The capsular
material is not water soluble in many bacteria but in some it is highly water
soluble, thus making the medium in which they grow more viscous. Capsular
material is primarily polysaccharide in most bacteria. It may be a
homopolysaccharide, made up of a single kind of sugar, synthesized outside the
cell from disaccharides. The capsule of S.mutons is a glucan (a glucose polymer)
synthesized from sucrose. Capsules composing of several kinds of sugars are
termed

heteropolysaccharides. These are synthesized from sugars within the cell,

transported and polymerized outside the cell. The capsule of Klebsiella
pneumonia is a heteropolysaccharide. The capsule of some bacteria is
polypeptides. The capsule of anthrax organism Bacillus anthracis is a polymer of
D-glutamic acid.

Sheath is a hollow tube that encloses cells in the form of chains or trichomes. This
is present in some bacteria living in fresh water and marine environment. The cells
some times move out of sheath. In a few cases the sheath is strengthened by
deposition of ferric and manganese hydroxides.

Aerobic bacteria in fresh water and marine environment possess prosthecae, which
increases the surface area of cells for nutrient absorption from the dilute aquatic
environment. They are semirigid extension of cell wall and cytoplasm membrane
and smaller than the cell. Some bacteria have single prostheca (Caulobacter) and
others have more than one (Stellar and Ancalomicrobium).

Stalks are also found in some bacteria like Gallionella or Planctomyces. They are
non-living ribbon like or tubular append-ages that are excreted by cell. These stalks
aid in attachments of cells to surface.

The structures internal to cell are cytoplasmic membrane, protoplast, intracellular

membranes, the cytoplasm, cytoplasmic inclusions and nuclear material, the DNA.

The cytoplasmic membrane is immediately beneath the cell wall and is about
7.5nm thick. It is made up of phospholipids (about20-30 percent) forming a bilayer
to which both integral pro-teins and peripheral proteins are held. The membrane
has fluidity owing due to its lipid matrix and this allows components to move
laterally.
The phospholipids of eubacteria and archaeobacteria differ in composition. The
phospholipids of eubacteria are phosphoglycerides. In this straight chain fatty acids
are linked to glycerol by ester linkage. In archaeobacteria, the lipids are
polyisoprenoid branched chain lipids. In this phytanols, (long chain branched
alcohols) are ether linked glycerols.

The cytoplasmic membrane is a barrier for penetration of most of water soluble

molecules. But the small molecules like nutrients and waste products are
transported across the membrane by specific proteins. The membrane also contains
various enzymes of respiratory metabolism and synthesis of cell wall components
and capsule. It is also the site of generation of proton motive force that drives ATP
synthesis, nutrition, transport system and flagellar motility. The damage to
membrane by physical or chemical agent lead to death of cells.

The cytoplasmic membrane and the cell material bounded by it are called
protoplast. The bacterial cell minus the cell wall is the protoplast. Protoplasts of
gram positive bacteria can be pre-pared by dissolving the cell wall by lysozyme or
by growing the bacteria in penicillin containing media. Penicillin prevents the syn-
thesis and formation of cell wall. Protoplasts thus prepared have to be suspended in
an isotonic medium, other wise the bacteria living in hypotonic environments tend
to absorb water and burst.

In Gram-negative bacteria lysozyme treatment may destroy the cell wall. The outer
membrane remains with the cytoplasmic membrane enclosing the cell content.
Such type of protoplasts with the outer membrane is called as spheroplast.

The bacteria that lack cell wall like mycoplasma are similar to protoplasts but they
are parasites of animals, plants or arthropods and hence live in osmotically
favourable or isotonic environments.

Bacteria are prokaryotes that do not contain any membrane bound organelles inside
the cells. But bacteria have specialized invagination of cytoplasmic membrane that
increase the surface area for certain functions. Mesosomes are convoluted tubules
or vesicles formed by membranous invagination in bacteria. Central mesosomes
are located near the middle of the cell and penetrates deep into the cytoplasm. It
seem to be attached to the nuclear ma-terial. Peripheral mesosomes shallowly
penerate into the cytoplasm and seen to be invalid in export of exocellular
enzymes.
The intracellular membrane is extensive in all phototrophic bacteria,
chemoautotrophs and in methane oxidizing bacteria. In phototrophic bacteria they
are the sites of photosynthesis as the increased surface area increase the light
absorbing pigments.

Thylakoids are special intracellular membranes that occur in cyanobacteria but

they are separate from cytoplasmic membrane.

The cytoplasmic membrane bound the cytoplasm. the cyto-plasm consists of a

cytoplasmic area, a chromatinic area and con-sists of Ribosome. Ribosomes are
macromolecular RNA protein bodies and are the sites of protein synthesis. The
chromatinic area is rich in DNA. The fluid proteins contain the dissolved
substances.

Ribosomes of prokaryotes have a sedimentation coef of 70 Svederberg units (70S)

and are composed of two subunits of 50S and 30S. On the other hand ribosomes of
eukaryotes have a sedimentation coefficient of 80S and are composed of 60S and
40S subunits.

Cytoplasmic inclusions are concentrated deposits of certain substances. Volutin

granules or metachromatic granules are polyphosphates deposits. It is a reserve of
phosphate. Poly-B-hy- droxy butyrate is a chloroform soluble lipid like material
and serve as carbonTheyandareenergyfoundsourceina.robic bacteria.

Polysaccharide granules viz glycogen is present as inclusion. El-emental sulfur

accumulates in certain bacteria growing in envi-ronments rich in hydrogen
sulphide.
Bacteria in aquatic habitats have gas vacuoles to provide buoyancy. Gas vacuoles
have water impermeable boundary but permeable to dissolved gases which fill the
cavity. Bacteria do not have a nucleus with a nuclear membrane. The nuclear
material is only a single circular DNA molecule. This is called as nucleoid, the
chromatin body, the nuclear equivalent or functional chromosome.
Microbiology Taxonomy
Biodiversity (Biological diversity), the variability among the galaxy of living organizations, includes
the diversity of the species between the species and of ecosystems.

TAXONOMY

Biodiversity (Biological diversity), the variability among the galaxy of living

organizations, includes the diversity of the species between the species and of
ecosystems. It is really difficult to estimate the total number of different types of
eubacteria, archaeobacteria and virus, as it is very difficult to isolate and recover
the organisms from the environment. Further the natural environment pose varying
conditions in different ecosystems rendering huge variation among the species
existing in main ecosystem. Not all environments have been investigated fully and
therefore
attempts to estimate total number of species of micro organisms become more
difficult. In complete understandings of cultural conditions required by certain
obligate parasites add to this problem. Mycoplasma are prokaryotes but have
obligate associations

with eucaryotic organisms, have remarkable diversity from some infecting insects
and some infecting plants. The soil, fresh water and marine ecosystems support a
group of diverse organisms on their ecosystem providing luxuriant microbial
diversity.

The microorganisms have species that are free living in soil and water, mostly
saprophyte in nature, a group that are parasitic on plants and a few others are
obligate pathogens of plants, animals and man. Some live in aerobic environment
and other living in anaerobic or microaerophilic conditions. Therefore there is a
wide diversity.

The advent of DNA techniques like DNA-DNA hybridization, nucleic acid finger
printing, RNA sequencing has altered the microbial diversity. The 16S r RNA
sequence and DNA fingerprinting techniques have enabled to evaluate the genetic
relatednessbetween organisms.
The smallest unit of microbial diversity is a species. Bacteriaare defined as a group
of similar strains differentiated fromother similar groups of strains by genotypic,
phenotypic and ecologicalcharacters. Bacterial strain is one with approximately
70%

or more DNA-DNA relatedness and with 5% or less in thermalstability. A bacterial

species is a genomic species based on DNADNArelatedness and this concept
differs from those of other organisms.There is an estimated total species of 40,000
bacteria,1,30,000 viruses, 1,50,000 fungi, 60,000 algae compared to
the5000,4,760,6900 and 40,000 known species which constitute4,12,5 and 67% of
known species.

The living organisms were grouped as plants, animals andprotista by

Haeckel(1866). The protista are primitive organismsincluding microbe. Based on
cell anatomy the bacteria were groupas prokaryotes. Whittaker proposed five
kingdoms plants, fungianimals, protista and Monera based on cell anatomy and
energyyielding systems. Microorganisms are in fungi, protista andmonera. Based
on the fact that bacteria are distant from plantsand animals but not that far away
from each other Woose and hiscoworkers proposed three domains
Archaea,.Bacteria and Eukaryato cover the microorganisms. The domain bacteria
include bacteria,cyanobacteria, actinonycetes etc. Archaic includesmethanogens,
extremely thermophilic organisms extremely halophilicorganisms and the Eukarya
includes molds, yeasts, algae,protozoa etc.

The microorganisms are named following the Linnaeusmethod of binomial

nomenclature. The taxonomy denotesclassification, nomenclature and
identification of organisms. Thecharacteristics or properties that are common for a
few organismsare grouped together in different groups(taxa). Bacteria were
traditionally based on morphological, biochemical and physiological
characteristics. Serological tests and Genetic tools are valuable in identification.

The general methods of classifying bacteria is by (I) intuitive method(ii) numerical

taxonomy iii. Genetic Relatedness method.
While identifying a bacteria morphological, physiological, biochemical general
and molecular characteristics of organisms are studied. It may be difficult to
classify an organism as different microbiologists may consider different
characteristics as important. This is the intuitive method.

Numerical taxonomy gives equal weightage for each character of the strain. The
percentage similarly of each strain is determined with the following formula

% S = NS / (NS+ND)

Where NS: Number of characters for each strain which are similar or dissimilar

ND = Number of characters that are different.,

%S, S = Similarly if it is high to each other placed with groups.

In Genetic relatedness classification is based on relatedness(DNA an RNA)

between organisms. The percent G+C determines the relatedness. If two bacteria
have a different not % G+C then the species are different and not related. DNA
homology
(DNA-DNA hybridization) is also determined to assesses the relatedness. The
DNA from a bacterium is isolated and the two strands are separated and a single
strand is mixed with that obtained from another. If the two bacteria are similar the
pairing of strands will occur, otherwise no pairing will occur.
Sources of water pollution - Microbiology of water
1. Sewage or municipal effluents (or) domestic effluents 2. Industrial effluents 3. Agricultural pollution

Sources of water pollution

The only source of potable fresh water nature provides is the rain water. This itself
gets polluted by the action of nature, as water falls on land and runs as stream,
gathering different types of minerals and suspended particles. In this running
water, due to anthropogenic activities, more minerals, more chemicals and organic
materials are added, that makes the water polluted. There are three main sources of
pollution of waters.

1. Sewage or municipal effluents (or) domestic effluents

2. Industrial effluents

3. Agricultural pollution

1. Sewage or Municipal effluents

The quality of water after bath, kitchen work, washing of clothes and animals etc. a
large volume of raw sewage discharged into the main stream pollute the river
waters. Among the various sources of water pollution, sewage, the domestic waste
containing decomposing organic matter is the major source and it accounts for 70
per cent of water pollution. Industrial effluents account for 15 percent of water
pollution.

2. Industrial effluents
The industrial effluents are classified under the following heads.

I. Food and drink manufacturing industries

a. Distilleries and sugar factories.

b. Food processing units.

c. Soap and oil manufacturing units.

II. Chemical Industries

a. Fertilizer and Chemicals, paints

b. Drug and pharmaceuticals

c. Insecticides and pesticides

III. Engineering Industries

a. Metallurgical industries.

b. Wire making industries.

c. Rare earths and minerals.

IV. Other industries producing organic effluents

a. Paper and rayon

b. Rubber industries

c. Textiles

d. Plywood and hardboard industries

e. Tanneries and leather industries

Factors that influence the growth of the microorganisms

Many factors that influence the growth of the microorganisms in food. Some of the factors are
intrinsic and some others are extrinsic.
Factors that influence the growth of the microorganisms
Many factors that influence the growth of the microorganisms in food. Some of the
factors are intrinsic and some others are extrinsic.

a) Intrinsic factors: The intrinsic factors include pH, moisture content, oxidation-
reduction potential, nutrient status, antimicrobial constitu-ents and biological
structures.

a) Intrinsic factors: The intrinsic factors include pH, moisture content, oxidation-
reduction potential, nutrient status, antimicrobial constitu-ents and biological
structures.

i. pH : It has been well established that most of the microorgan-isms grow

best at pH values around 7.0, while few grow below 4.0. Bacteria grow at more pH
than molds and yeasts.

ii. Moisture content: The preservation of foods by drying is a direct

consequence of removal of moisture, without which microorganisms do not grow.
The water requirements of microorganisms should be defined in terms of the water
activity (aw) in the environment. Water activity is defined by the ratio of the water
vapour pressure of food substrate to the vapour pressure of pure water at the same
tempera-ture (aw=P/po). The aw of most fresh food is above 0.99. The minimum
value of aw for the growth of the microor-ganisms in foods should be around 0.86.

iii. Oxidation reduction potential: The O/R potential of a sub-strate may be

defined generally as the ease with substrategains electrons. When an element or
compound loses elec-trons, the substrate is said to be oxidized, while a substrate
that gains electrons becomes reduced.

iv. Nutrient content: In order to grow and function normally, the

microorganisms of importance in foods require water, source of energy, source of
nitrogen, vitamins and related growth fac-tors and minerals.
v. Antimicrobial constituents: The stability of foods against attack by
microorganisms is due to the presence of certain naturally occurring substances
that have been shown to have antimicro-bial activity. Some species contain
essential oils that possess antimicrobial activity. Among these are eugenol in
cloves, alli-cin in garlic, cinnamic aldehyde and eugenol in cinanmon.

b) Extrinsic factors: These include those properties of the storageenvironment

that affect both the foods and their microorganism. The following extrinsic factors
affect the growth of microorgan-isms: Storage temperature, pH, presence and
concentration of gases in the environment.

Microbiology of milk
Milk is the white, fresh clean lateral secretion obtained from female cattle. Milk is used for the
nourishment of their younger ones.

MICROBIOLOGY OF MILK
Milk is the white, fresh clean lateral secretion obtained from female cattle.
Milk is used for the nourishment of their younger ones. It is in liquid form without
having any colostrum. The milk contains water, fat, protein and lactose. About 80-
85% of the proteins is casein protein. Due to moderate pH (6.6), good quality of
nutrients, high wa-ter contents etc. make milk an excellent nutrient for the
microbial growth.It is mainly the udder interior, teats surrounding environment and
manual The air milking process make the source of contamination.
Sources of microorganisms in milk
Milk secreted into the udder is sterile. The first few strippings of milk contain more
amount of bacteria and the population of bacteria gradually decreases. It is
observed that last strippings of milk from the udder seems to be free from bacteria.
This clearly indicates that most of the microorganisms found in the milk are from
external source. The different sources of microorganism in milk are from 1) the
udder of the cow, 2) skin of the cow, 3) utensils and equipment, 4) feeds, 5) air of
the cow shed, 6) milking persons and 7) water.
1. Udder of the cow :The milk producing animals should bekept neat and clean.
More care should be taken to keep the flanks, udder and teats clean. The interior of
the teats of the udder is warm and contains the last remains of the milk which has
more microbes which would have entered through opening of teat and multiplied.

2. Skin of the cow :Soil, faeces and dirt adhere to the skin andhairs of the cow.
Hair, dirt and dust fall in to milking utensils or into the teat cups of milking
machines. Most of the organisms from these sources are gas producers and
putrefactive types. Faeces contain enor-mous quantity of organisms and most of
them are pathogenic microor-ganisms.

3. Utensils and equipments :Milking utensils and equipmentsare the major

sources of contamination of milk. They have to be washed properly with detergent.
Further the utensils and equipments should be cleaned with hot water, air and
steam to remove all the spore forming, fluorescent and coliform microorganisms.

4. Feeds :Microorganisms are found everywhere. They arepresent in abundant

in vegetation and soil. Dry feeds have more amount of bacteria and less amount of
fungi. These organisms contaminate the milk.

5. Air of the cow shed :The air of the cow shed is greatly contaminated by dry
dirt and dust. During the mixing of feeds and during the cleaning process of the
floor, the air of the cow shed is highly contaminated and it is passed on to the milk.
6. Milking persons :Pathogenic microorganisms may enter intothe milk through
milking persons. They should wear clean clothes and properly wash their hands
before milking. Nails should be cleaned and trimmed. Discharge from sneezing,
coughing and nose blowing should not reach the atmosphere, equipment or the
milk. Some of the organ-isms may be carriers of diseases.

7. Water :Pure water should be used for cleaning purposes.Water exposed to

contamination spreads the microorganisms. Water should be free from coliform
organisms. Chlorination of water pre-vents such contamination.

Microbiological standard and grading of milk

The Indian Standard Institute (ISI) has prescribed microbiologi-cal standard for
quality of milk.
1. Coliform count in raw milk is satisfactory if coliforms are absent in 1:100
dilution.

2. Coliform count in pasteurized milk is satisfactory if coliforms are absent in

1:10 dilution.
Grading of milk
The quality of milk is judged by certain standards and it is known as grading milk.
Grading of milk is based upon regulations pertaining to production, processing and
distribution. This includes sanitation pas-teurization holding conditions and
microbiological standards. The U.S Public Health Secrine Publication ‘Milk
Ordinance and code’ shows the following chemical, bacteriological and
temperature standards for grade A milk and milk products.

Harmful microbial interactions

Harmful microbial interaction is otherwise described as negative interaction or antagonistic
interaction.

Harmful microbial interactions

Harmful microbial interaction is otherwise described as negative interaction or
antagonistic interaction. The composition of the microf-lora microfauna of any
habitat is governed by the biological balance created through interactions and
associations of all individuals present present in a community. Any inhibitory
effect of an organism created by any means to the other organisms is known as
harmful interactions or antagonistic interaction and the phenomenon of this activity
is called antagonism. Harmful interactions have three types. They are amensalism,
competition and parasitism.

Amensalism
Amensalism is the phenomenon where one microbial species is affected by the
other species, where as other species is unaffected by first one. Amensalism is
accomplished by secretion of inhibitory sub-stances such as antibiotics. Certain
organisms may be of great practical importance, since they often produce
antibiotics or other inhibitory sub-stances, which affect the normal growth of other
organisms. Antago-nistic relationships are quite common in nature. For example,

Pseudomonas aeruginosa is antagonistic towards Aspergillus terreus.

Competition
A negative association may result from competition among spe-cies for essential
nutrients. In such situations the best adapted micro-bial species will predominate or
eliminate other species which are de-pendent upon the same limited nutrient
substance.
Parasitism
Parasitism is defined as a relationship between organisms in which one organism
lives in or on another organism. The parasites feed on the cells, tissues or fluids of
another organisms, the host, which is harmed in this process. The parasite depends
on the host and lives in intimate physical and metabolic contact with the host. All
types of plants and animals are susceptible to attack by microbial parasites.

Beneficial Interactions
The beneficial interactions such as symbiosis (mutualism), proto cooperation, and
commensalism are found to operate among the soil inhabitants.

Symbiosis (Mutualism)
Mutualism is an example of symbiotic relationship in which each organism
benefits from the association. One type of mutualistic asso-ciation is that involving
the exchange of nutrients, between two species, a phenomenon called
syntrophisms. Many microorganisms synthesis vitamins and aminoacids in excess
of their nutritional requirements. Others have a requirement for one or more of
these nutrients.

Symbiosis is an obligatory relationship between two populations that benefit both

the populations. Both populations are living together for mutual benefit. The
relationship between algae and fungi that result in the formation of lichens is a
classical example of mutualistic intermicrobial relationship. Lichens are composed
of primary producer, the phycosymbiont (algae) and a consumer the mycosymbiont
(fungus).
Proto-cooperation
It is an association of mutual benefit between two populations, but not obligatory
and only complementary. Both population are capable of surviving in their natural
environment on their own, although when offered, the association offers some
mutual advantages.

For an example, the mixed culture of Proteus vulgaris (pro-duce biotin/requiring

nicotinic acid) and Bacillus polymyxa (produce nicotinic acid/ requiring biotin).
Both grow as partner bacterium syn-thesizes the missing vitamin.

Commensalism
In a commercial relationship between two microbial populations, one population is
benefited and other population remains unaffected. Commensalism is a
unidirectional relationship between two popula-tions. The unaffected population
does not benefit by the action of sec-ond population. For receiving population, the
benefit provided may be essential.

In commensalism, the unaffected population modifies the habitat in such a way

that another population benefits. For example, a popu-lation of facultative
anaerobes utilizes oxygen and creates a habitat suit-able for the growth of
anaerobes. In soil, vitamin and growth factors producing organisms benefit vitamin
and growth factors requiring organisms.
Rhizosphere
The region which is adjacent to the root system is called rhizosphere. The
microbial population on and around roots system considerably higher than that of
root-free soil or non-rhizosphere soil. This may be due to the availability of
nutrients from plant roots in the form of root nodules, secretions, lysates mucigel
and sloughed off cells. Plant roots provide shelter to soil microbes in the
rhizoplane (root surface) and endorhizosphere (inside root).

Rhizosphere effect
Bacteria predominate in rhizosphere soil and their growth is in-fluenced by
nutritional substances released from the plant tissues eg. aminoacids, vitamins and
other nutrients; the growth of the plant is in-fluenced by the products of microbial
metabolism that are released into the soil. It has been reported that aminoacid -
requiring bacteria exist in the rhizosphere in larger numbers than in the root-free
soil. It has been demonstrated that the microflora of the rhizosphere is more active
physiologically than that of non-rhizosphere soil. The rhizosphere effect improves
the physiological conditions of the plant and ultimately result in higher yield.
Greater rhizosphere effect is seen with bacteria (R:S ratio ranging from 10-20
times more) than with actinomycetes or fungi.

Phyllosphere
The Dutch Microbiologist Ruinen coined the term phyllosphere. The leaf surface
has been termed as phylloplane and the zone on leaves inhabited by the
microorganisms as phyllosphere. In forest vegetation, thick microbial epiphytic
associations exist on leaves. The dominant and useful microorganisms on the leaf
surfaces in the forest, vegetation happened to be nitrogen fixing bacteria such
as Beijerinckia and Azotobacter. Apart from these nitrogen fixing bacteria, other
generasuch as Pseudomonas, Pseudobacterium, Phytomonoas are also encountered
on the leaf surface. The quantity and quality of phyllosphere organisms vary with
the plant species and its morphological, physiological and environmental factors.
The age of plant, its leaf spread, morphology and maturity level and the
atmospheric factors greatly influence the phyllosphere microflora.

Spermosphere
The region, which is adjacent to the seed surface is termed as spermosphere.
Healthy seeds carry specific bacterial flora in respect of number and species. There
are several reports in the litera-ture on the quantity and quality of microorganisms
carried by the seeds of different plant species both externally and internally. Many
of the organisms are harmless some may be positively beneficial and very few may
be pathogenic under certain favourable conditions. It has been shown that some
organisms have beneficial effects on the germinating seed through some biological
products, such as growth hormones. It has been reported that the germinating seed
excretes some chemicals, which influence the quality and quantity of the
microorganisms on the seed. Picci defined the region of such influence around the
seed as spermosphere and the phenomenon as spermosphere effect. When the seed
is sown in soil, certain interactions between the seed-borne microflora and the soil
microorganisms take place, under the influence of chemicals exuded by the
germinating seed.

Medical Microbiology
Thousands of different kinds of microbes are present in all eco-logical niches Some are beneficial
ones, others are opportunists and some are harmful ones.

MEDICAL MICROBIOLOGY MICROBIAL DISEASES

Introduction
Thousands of different kinds of microbes are present in all eco-logical niches
Some are beneficial ones, others are opportunists and some are harmful ones.

Infection is the establishment of the organisms in the tissues re-sulting in injury or

harmful effect to the host. Infections may be endog-enous or exogenous.
Endogenous infections are contracted from the host himself from the normal flora.
Many areas of the body have nor-mal commensal flora. They have many functions.
They provide barrier to the infection by competing for nutrition with pathogens.
Some pro-duce vitamins which are useful for the host. Some produce colicins to
act against pathogens. Generally they do not cause any infection. But there are
exceptions. Streptococcus mitis is the normal flora of the mouth. It produces
infection in the previously damaged heart valves through blood stream after tooth
extraction . Streptococcus faecalis causes infective endocarditis and the source is
the urinary tract and intestine of the host. Exogenous infections are derived from
man, ani-mals and soil. Man gets the infections from patients suffering from dis-
eases. Some persons may be carriers for the pathogens and they may transmit the
diseases to others without getting affected.

Animals are important sources of infection. Such infections are known as zoonotic
diseases. Spread of these diseases is usually from animal to animal . Man may be
infected as an end host as in rabies. In some cases the infection may spread from
man to man as in pneumonic plague.
Soil has also a role in the transmission of infection. Soil is the reservoir for the
spores of Clostridium species and Bacillus anthracis.

Routes of spread of infection:

There are five main routes by which a host may become infected.
1. The respiratory route

2. The alimentary tract

3. Genital tract

4. The skin and mucous membrane

5. Placenta

Organisms causing respiratory infections are as follows. Streptococcus

pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Bordetella
pertusis are some of the bacterial pathogens. Common cold virus, influenza virus,
adno virus are some of the viruses producing respiratory infections.
The intestinal diseases like cholera, bacillary dysentery, the enteric fever and
bovine tuberculosis are contracted when the organisms are ingested. But in the case
of entero virus infections (poliomyelitis) and Hepatitis though the organisms enter
through gastro intestinal system, the effects are seen elsewhere in the body.

Organisms may be acquired from the skin as in the case of herpes virus infection or
through wounds as in tetanus. Wounds may be formed from trauma or thorn pricks
or needle stick injury. Organ-isms may also be introduced through animal bite as in
the case of rabies or by insect bites as in dengue, malaria, filariasis, and yellow
fever.
Syphilis , gonorrhea, hepatitis B and AIDS are some of the sexu-ally transmitted
diseases. Treponema pallidum, Neisseria gonorrhoeae, Hepatitis B virus and
Human Immunodeficiency Virus are the etiologic agents respectively.

Bacteria like T.pallidum, Viruses like rubella, cytomegalovirus, parasite like

Toxoplasma gondii are some of the organisms that enter through placenta and
cause disease in the newborn.

Interaction between host and microbe

Pathogenicity refers to the ability of the organism to cause dis-ease. Virulence is
the quantitative measure of this property. Virulence is not generally attributable to
a single property but depends on several parameters related to the organism, the
host and their interaction.

Microbes first enter the body, survive then multiply, elaborate many factors and
produce the disease.

Virulence Factors

1. Pili
Pili are useful for the attachement of the organisms on the epithe-lial cells.

2. Capsule
Capsules down regulate the secretion of cytokine. They inhibit leukocyte
accumulation. They also induce the suppressor T cells and inhibit
lymphoproliferation

3. Intracellular residence
The following microorganisms reside intracellularly and try to avoid host defense
mechanisms. They are M.tuberculosis, M.leprae, S.typhi, T.gondii, L.donovani,
H.capsulatum.

4. Production of enzymes

Some enzymes like proteases, DNAses, and phospholipases are produced and they
help in disruption of cell structures and to hydrolyse host tissues. In Aspergillus
species proteases help in invasion.

5.Toxins
Bacteria produce both exotoxins and endotoxins which play and important role in
the pathogenesis of disease

Exotoxins are produced by some organisms like C.diphtheriae, C.tetani,

C.botulinum. The exotoxin produced by V.cholerae acts on the intestine and is
called enterotoxin. The toxin produced by one type of Escherichia coli causes acute
gastroenteritis.

Endotoxins are lipopolysaccharide cell wall of gram negative bacteria. They induce
production of cytokines by different cells of im-mune system. Coagulation system
and complement system are acti-vated. They also affect various organs like kidney,
heart and lungs leading to organ failure.

6. Antigenic variation
Microorganisms evade the host immune responses by changing their surface
antigens. N.gonorrhoeae very often changes its outer mem-brane protein.
Antigenic drift and shift are common in influenza viruses. Trypanosoma brucei are
covered with thick protein coats which un-dergo antigenic change during infection.
Some organisms produce sur-face proteins that are similar to host proteins or coat
themselves with host proteins that they are mistaken for part of the host itself

The distinction between the commensal and the organisms asso-ciated with disease
is subtle. The definition of normal flora or pathogen is derived from the resultant
complex interaction between the organism and its host.
Routes of spread of infection
There are five main routes by which a host may become infected.

Routes of spread of infection:

There are five main routes by which a host may become infected.
1. The respiratory route

2. The alimentary tract

3. Genital tract

4. The skin and mucous membrane

5. Placenta

Organisms causing respiratory infections are as follows. Streptococcus

pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Bordetella
pertusis are some of the bacterial pathogens. Common cold virus, influenza virus,
adno virus are some of the viruses producing respiratory infections.

The intestinal diseases like cholera, bacillary dysentery, the enteric fever and
bovine tuberculosis are contracted when the organisms are ingested. But in the case
of entero virus infections (poliomyelitis) and Hepatitis though the organisms enter
through gastro intestinal system, the effects are seen elsewhere in the body.

Organisms may be acquired from the skin as in the case of herpes virus infection or
through wounds as in tetanus. Wounds may be formed from trauma or thorn pricks
or needle stick injury. Organ-isms may also be introduced through animal bite as in
the case of rabies or by insect bites as in dengue, malaria, filariasis, and yellow
fever.

Syphilis , gonorrhea, hepatitis B and AIDS are some of the sexu-ally transmitted
diseases. Treponema pallidum, Neisseria gonorrhoeae, Hepatitis B virus and
Human Immunodeficiency Virus are the etiologic agents respectively.

Bacteria like T.pallidum, Viruses like rubella, cytomegalovirus, parasite like

Toxoplasma gondii are some of the organisms that enter through placenta and
cause disease in the newborn.

Virulence Factors
Pili are useful for the attachement of the organisms on the epithe-lial cells.

Virulence Factors

1. Pili

Pili are useful for the attachement of the organisms on the epithe-lial cells.

2. Capsule
Capsules down regulate the secretion of cytokine. They inhibit leukocyte
accumulation. They also induce the suppressor T cells and inhibit
lymphoproliferation

3. Intracellular residence
The following microorganisms reside intracellularly and try to avoid host defense
mechanisms. They are M.tuberculosis, M.leprae, S.typhi, T.gondii, L.donovani,
H.capsulatum.

4. Production of enzymes

Some enzymes like proteases, DNAses, and phospholipases are produced and they
help in disruption of cell structures and to hydrolyse host tissues. In Aspergillus
species proteases help in invasion.

5.Toxins
Bacteria produce both exotoxins and endotoxins which play and important role in
the pathogenesis of disease

Exotoxins are produced by some organisms like C.diphtheriae, C.tetani,

C.botulinum. The exotoxin produced by V.cholerae acts on the intestine and is
called enterotoxin. The toxin produced by one type of Escherichia coli causes acute
gastroenteritis.

Endotoxins are lipopolysaccharide cell wall of gram negative bacteria. They induce
production of cytokines by different cells of im-mune system. Coagulation system
and complement system are acti-vated. They also affect various organs like kidney,
heart and lungs leading to organ failure.

6. Antigenic variation
Microorganisms evade the host immune responses by changing their surface
antigens. N.gonorrhoeae very often changes its outer mem-brane protein.
Antigenic drift and shift are common in influenza viruses. Trypanosoma brucei are
covered with thick protein coats which un-dergo antigenic change during infection.
Some organisms produce sur-face proteins that are similar to host proteins or coat
themselves with host proteins that they are mistaken for part of the host itself

The distinction between the commensal and the organisms asso-ciated with disease
is subtle. The definition of normal flora or pathogen is derived from the resultant
complex interaction between the organism and its host.
Respiratory tract infections
The lower respiratory tract is sterile. However the upper respiratory tract, the nose and throat are
colonized by many organisms.

RESPIRATORY TRACT INFECTIONS

Introduction
The lower respiratory tract is sterile. However the upper respiratory tract, the nose
and throat are colonized by many organisms.

Normal flora of respiratory tract

These organism are: Staphylococci, Streptococci, Pneumococci, Haemophilus and
Neisseria.

Normal defenses against infections

1. Arrangement of nose – there is no direct entry of air

2. Broncho constriction : helps the organisms to be trapped

3. Cough reflex : expels the microbes out side

4. Mucociliary blanket: traps the organisms

5. Mucosal factors: kill the organisms a. Non specific

i. Lysozyme : Cell wall of gram positive organism are lysed

ii. Influenza virus inhibitors :

iii. Resident macrophages : kill the organism

b. Specific

i. Secretory IgA antibody : gives first line of defense

Predisposing factors for respiratory tract infections

1. Ciliated epithelial cell damage due to

a. Viruses

b. Chemicals

c. Smoking

2. Fluid accumulation

3. Decreased activity of macrophages

All these factors help in the establishment of the microbes in the respiratory tract.

Alveoli
Strep.pneumoniae ; M.tuberculosis ; Mycoplasma pneumoniae

Chlamydia pneumoiae

Types of Respiratory infections (Figure 15.3)

Respiratory infections can be conveniently classified into Upper respiratory
and Lower respiratory infections.

Upper respiratory tract infections (URI)

1. Infections of the paranasal sinuses

URI may cause inflammation of the maxillary sinuses. Bacterial infection also
occurs in association with obstruction. The bacteria involved areStreptococcus
pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus
pyogenes.

2.Otitis media
This is the result of direct spread of pathogens from the throat via the Eustachian
tube. In young it may be due to Streptococcuspneumoniae, Haemophilus
influenzae, Streptococcus pyogenes. Inold people it may be due to Streptococcus
pneumoniae, Staphylo-coccus aureus.

3. Sore Throat

It occurs in the following conditions:

1. Prodromal stages of infectious diseases

2. Diphtheria

3. Vincent’s infections

4. Sore throat syndrome

Prodromal stages of infectious diseases

Many viral diseases are acquired via the respiratory tract. Signs and symptoms
appear in the mouth and throat. But the target organs may be different.
Example: In measles and chickenpox, the mucosae of the upper respiratory tract
and mouth are infected before the target organs show full clinical picture. In
measles Koplick’s spots appear in buccal mu-cosa.

Diphtheria
Diphtheria is an acute inflammatory condition of the upper respi-ratory tract
usually the throat. It is caused by Corynebacterium diphtheriae. The organisms
multiply in the throat and produce a pow-erful toxin. The toxin acts on
myocardium, adrenal glands and nerve endings.

Laboratory diagnosis

Throat swab is collected

1. Smear is stained by Gram and Albert stains

a. In positive cases Gram positive bacilli seen

b. Albert stain shows bacilli with metachromatic granules2. Material is

inoculated into Blood agar, Loeffler’s se-rum medium and Potassium tellurite
agars

3. Suspected colonies are identified by biochemical test using se-rum sugars

4. Toxigenicity test is done by agar gel precipitation test (Elek’s test) and by
guinea pig inoculation test

Prophylaxis
l Active immunization is done

l DPT (Diphtheria, Pertusis and Tetanus) immunization should be given in three

doses

l 1st dose at third month

l 2nd dose at 6-8 weeks after the first dose

l 3rd dose 4-5 months after the 2 dose

l A booster is given omitting the pertusis at school entry

Treatment
Large dose of anti toxin must be given to confirmed cases. Anti-biotics are given to
eradicate the organisms .

Vincent’s infection
Vincent’s spirochetes are Borreliae

They are present as normal flora of the mouth

In association with fusiform bacteria they can cause infection Generally infection
occurs during malnutrition

The infection is called Vincent’s angina

Sore Throat syndrome

Sour throat syndrome is caused by Streptococcus pyogenes. Based on the cell wall
polysaccharide of beta hemolytic Streptococci they are put in different groups.
Streptococcus pyogenes belongs to Group A. It causes wide range of pyogenic
infections in the respiratory tract and skin and life threatening soft tissue infections.
Post strepto-coccal infections may result in adverse immunological reactions
leading to rheumatic heart disease or acute glomerulonephritis.

Laboratory diagnosis
For the laboratory diagnosis specimens like throat swab and pus are collected and
inoculated in blood agar. The organism is identified by hemolytic property, and
serological tests.

Penicillin is the drug of choice for the treatment of streptococcal infections.

Lower respiratory tract infections

Many organisms cause lower respiratory tract infections which you will be
studying in later classes.
Infections of the Central Nervous System
Infecting agents reach the central nervous system (CNS) from the blood or by direct invasion or
by ascending through the nerves.

INFECTIONS OF THE CENTRAL NERVOUS SYSTEM

Infecting agents reach the central nervous system (CNS) from the blood or by
direct invasion or by ascending through the nerves. The infection of the CNS can
be classified as encephalitis and meningitis.

Meningitis- Etiological agents

Meningitis is the inflammation of the membranes covering the brain (meninges).
This can be caused by a wide range of micro-organisms. This can be classified as
follows:

Acute pyogenic meningitis

This condition is related to the age of the patients. Coliform ba-cilli and group B
streptococci are common cause in neonates. Haemophilus and Neisseria
meningitidis are frequently seen in children. H.influenzae and Str.pneumoniae are
commonly found in adults.N.meningitidis can cause infections in all age groups.
Other organisms involved are Str.pyogenes, S.aureus, Salmonella species, Listeria.

Pathogenesis and Epidemiology :Neonatal meningitis

In early days, it was caused by Str.pyogenes and S.aureus. But in later years
coliform bacilli are most commonly found. Coliform men-ingitis results in
congenital deformities and the source of the organisms can be genito-urinary tract,
lungs and umbilicus.

Haemophilus meningitis: This occurs mainly in young children be-tween the ages
of 3 months to 5 years. Protection before 3 months is given by maternal antibody
and after 5 months by acquired immunity. Infection spreads through blood stream.

Meningococcal meningitis (N.meningitidis) :The organism entersthe body via the

naso-pharynx, where they may produce localized in-flammatory reaction or remain
silent. By two ways they may reach the meninges. 1. They spread directly along
the spaces between the sheath and the branches of the olfactory nerve that pierce
the cribriform plate. 2. The organisms invade the blood stream, either produce
transient bacteremia or multiply in the blood. They cause lesions in the skin,
adrenal glands, joints and meninges. There are nine groups of menin-gococci.
Infections spread to one another by close contact.
Pneumococcal meningitis : Subsequent to some conditions like lo-bar pneumonia,
otitis media, or infection of the para nasal sinus, or injury to skull, Streptococcus
pneumoniae multiplies in the blood and spread to the meninges.

Meningitis due to other bacteria : Staphylococcus and Streptococ-cus

pyogenes reach the meninges via blood or directly from the exte-rior after a
trauma.

Tuberculous meningitis :This occurs in a severe form in young chil-dren and is

secondary to a tuberculous focus elsewhere in the body. The bacilli reach the
meninges via the blood.

Laboratory Diagnosis
Laboratory diagnosis is made by isolation and identification of specific organism
from blood and Cerebrospinal fluid (CSF). Antigen can be detected by counter
immunoelectrophoresis or latex agglutina-tion test. Blood cultures are useful in
50% cases. Gram stain and acid fast stains are useful in demonstrating the bacteria
in the CSF. Fluores-cence microscopy is useful for tuberculous meningitis.
Fontana’s stain-ing is useful for spirochetes like leptospira, borrelia and
treponema.

Once organisms are grown, they are identified by standard biochemi-cal tests.
Treatment will depend on the nature of the organisms and the antibiotic
susceptibility pattern
Viral meningitis
Viruses are the most frequent cause of meningitis. Most cases are due to
enteroviruses, ECHO and Coxsackie viruses. Mumps virus can cause meningitis in
children.

Pathogenesis :Viruses enter by oral or respiratory routes. They es-tablish a silent

initial focus somewhere in the naso-pharynx or in small bowel. The spread occurs
through lymph to the blood. They mainly multiply in lymphoreticular system. The
infection is detectable after 5 days. The virus is free in the plasma. They affect the
meninges and they may spread to other parts also. This time fever and neck
stiffness occur. At this time the virus appears in the throat and gut also. Serum
antibody appears about this time.

Diagnosis :Specimens of throat secretions, feces, CSF, are collectedduring acute

phase. During convalescent period feces may be obtained. Enteroviruses can be
isolated in tissue cultures. No specific therapy is available.

Sexually Transmitted Diseases

Sexually transmitted diseases are the most common communicable diseases after common cold.
They are transmitted through sexual contact. They can be classified into the following: 1. Bacterial 2.
viral 3. Parasitic 4 and fungal diseases.

SEXUALLY TRANSMITTED DISEASES

Introduction
Sexually transmitted diseases are the most common communicable diseases after
common cold. They are transmitted through sexual contact. They can be classified
into the following: 1. Bacterial 2. viral 3. Parasitic 4 and fungal diseases.

Bacterial diseases
The following are some of the sexually transmitted bacterial dis-eases. 1. Syphilis
2. Gonorrhea 3.Chancroid 4. Chlamydial diseases

The sexually transmitted viral diseases are 1. AIDS 2.Hepatitis B 3.Warts.

Trichomoniasis and genital candidiasis are the parasitic and fun-gal diseases
respectively.

Syphilis
Treponema pallidum is the organism that causes syphilis . Tre-ponemes are slender
spirochetes with fine spirals and pointed or round ends. The pathogenic
treponemes have not yet been cultivated in arti-ficial media

Pathogenesis

T.pallidum is transmitted from one partner to another through intact or damaged

mucosa. The organisms establish at the point of en-trance and multiply with in the
next 3 months and a chancre appears. This is the primary lesion and may
disappear. This stage is called pri-mary syphilis
Secondary syphilis occurs in two to 6 months after the primarysyphilis.
Because of the multiplication of the organisms secondary le-sions appear on the
skin. Spirochetes are abundant in these lesions. Usually the lesions heal
spontaneously.

In few cases tertiary syphilis appears later. This causes chronic granulomata
known as gummata in the brain, bone, skin and internal organs. Late
manifestations are degeneration of brain cells and de-struction of nerve fibres.
Tertiary lesions contain few spirochetes.

Infection during pregnancy can be transmitted to the fetus. This causes congenital
syphilis.

Laboratory diagnosis
Exudates from primary and secondary lesions are collected for examination. They
are examined by dark field microscopy for spiro-chetes and stained by silver
staining method to show the spirochetes.
Blood is collected for serological tests to demonstrate antibod-ies. Serological tests
are divided into two groups viz. nonspecific and specific tests.
In the nonspecific test antibodies to cardiolipin which develop during the infection
are demonstrated. The test is called VDRL test.

In the specific test, antibodies developed against T.pallidum are demonstrated.

Treponema pallidum immobilization test (TPI), Fluo-rescent treponemal antibody
absorption test (FTA-AB), and Treponema pallidum haemagglutination test
(TPHA) are some of the specific tests used for the diagnosis of syphilis.

Penicillin is the drug of choice for treatment. For control, all dis-covered cases
must be promptly treated and contacts also must be treated. Sex hygiene, and
prophylaxis at the time of exposure are some other control measures. Sexually
transmitted diseases can be transmit-ted simultaneously. Therefore, it is necessary
to consider the possibility of syphilis when any other sexually transmitted disease
is found.

Gonorrhea
Gonorrhea is a sexually transmitted infection of columnar and transitional
epithelium caused by Neisseria gonorrhoeae. The urethra, endocervix, anal canal,
pharynx and conjunctivae may be infected di-rectly. Systemic infection may lead
to arthritis, tenosynovitis, dermati-tis, endocarditis and meningitis.

Organism
Neisseria gonorrhoeae are gram negative diplococci, kidney shaped, the concave
side face each other, nonmotile. Grows on en-riched medium in presence of 5-10%
CO2...

Structure of male and female genital tracts (Figure 18.1)

Pathogenesis
Anterior urethra is mainly affected in men. Anterior urethra and cervix are affected
in women. In advanced infection it affects the pros-tate, seminal vesicles and
epididymis in men and uterus and fallopian tubes are affected in women. Rectal
infection and throat carriage occur in both. Gonococcal ophthalmia neonatorum is
an infection of the eye of the newborn, is acquired during the passage through
infected birth canal. The conjunctivitis progresses and if untreated results in blind-
ness. To prevent this instillation of tetracycline, erythromycin or silver nitrate
solution into the conjunctival sac of the new born is compulsory. Gonococcal
bacteremia leads to skin lesions on the hands, fore arms, feet, and tenosynovitis
and suppurative arthritis.

Laboratory diagnosis
Pus and secretions are taken from urethra, cervix, rectum, con-junctiva, throat or
synovial fluid for smear and culture. Cultures are to be done immediately after the
collection of specimens.
Treatment: Penicillin is given. If the organisms are resistant,after performing
antimicrobial susceptibility testing, appropriate drug is given.

Chancroid
Haemophilus ducreyi causes irregular ulcers in the genitalia. It produces chancroid
or soft chancre. This is a venereal disease or sexu-ally transmitted disease.
H.ducreyi is a gram negative bacilli. Chan-croid is treated with sulphonamides. If
resistant, erythromycin and cotrimoxazole are used.

Chlamydial disease

There are many serotypes in Chlamydia. Some of them cause genital infections.
Lymphogranuloma venereum is one of chlamydial sexually trans-mitted diseases.
First a vesicle develops and the lesion ulcerates in the genitals. The inguinal lymph
nodes enlarge, suppurate and release pus through multiple sinus tracts. If not
treated it will lead to other compli-cations. Sulfonamides and tetracycline are used
for the treatment.

Trichomoniasis
This is caused by Trichomonas vaginalis. Trichomonads are flagel-late protozoa
with 3-5 anterior flagella, other organelles and an undu-lating membrane.

In female the infection is limited to vulva, vagina and cervix. It usually does not
extend to uterus. The mucosal surface may be painful, inflamed, eroded and
covered with a frothy yellow or cream colored discharge. In males the prostate,
seminal vesicle and the urethra may be infected.
Trichomoniasis is treated with topical and systemic metronida-zole. The patients
sexual partner should be examined and treated si-multaneously.

Trichomoniasis is treated with topical and systemic metronida-zole. The patients

sexual partner should be examined and treated si-multaneously.

Genital candidiasis
The most common cause of genital candidiasis is due to Candida albicans.
Generally it is a commensal in the vagina. When infection oc-curs white
membranous patches are produced in the vagina and vulva. Thick or watery
vaginal discharge is seen. Gram’s stain can identify the yeast like cells. Nystatin or
miconazole or ketoconazole are used.
Viral agents

AIDS (Acquired immunodeficiency syndrome)

Human Immunodeficiency Virus (HIV) is the etiologic agent of AIDS. It belongs
to the lenti virus sub group which includes slow viruses.

The virus has central nucleoprotein core that contains single stranded RNA
genome. The enzyme reverse transcriptase is associ-ated with the viral RNA. This
RNA is transcribed into single stranded DNA and then to double stranded DNA.

The virus core is surrounded by a protein shell this is again cov-ered by a lipid
bilayer which contains envelope proteins.

Pathogenesis
Transmission is by sexual contact, through blood and blood prod-ucts- transfusion
and injection. After entry it comes in contact with T4 lymphocytes. T4 cells are
damaged and are decreased in mumber and T4:T8 ratio is reversed. Because helper
cells are affected, humoral immunity is also affected. AIDS patients are unable to
respond to new antigen. Macrophage monocyte functions are affected because of
lack of secretion of activation factors.

Within few weeks of infection, low grade gfever,malaise,head ache,

lymphadenopathy are seen. All persons pass through a period of symptomless
infection for several months or years. They show positive antibody tests and are
infectious. Lymphnodes are enlarged in some people. Then it leads to other
opportunistic infections like oral candidi-asis, salmonellosis, tuberculosis. Persons
suffer from fatigue, unexplained fever, persistent diarrhea, and weight loss. Finally
they reach the stage called AIDS.

Laboratory diagnosis :Immunological tests

Total white blood cell count: usually below 200/cmm

T4 cell count is less

Lowered cell mediated immunity is seen.

Specific tests
Viral antibody detection is performed by ELISA test and con-firmed by Western
blot test. Virus can be isolated from infected lym- phocytes.

Prevention
1. Multipartner sex should be avoided

2. Safer sex should be practiced

3. Blood should be screened before transfusion

4. Sharing of needles should be avoided

Parasitic agent

Trichomonas vaginalis is the organism that is transmitted through sexual contact.

acterial skin and wound infection
Wound can be defied as any interruption of continuity of external or internal surfaces caused by
violence

BACTERIAL SKIN AND WOUND INFECTIONS

Definition
Wound can be defied as any interruption of continuity of external or internal
surfaces caused by violence

Wounds may occur following: surgery, trauma or injections

Wound infections may occur mainly after surgical procedures

Wound sepsis is the result of cross infection from human sources and from other
outside sources.

Bacteria associated with wound infections

Many bacteria are associated with wound infection.

The normal flora may also cause infection.

The most common bacteria of the skin are: staphylococci, and various streptococci,
Sarcina spp, anaerobic Diphtheroids, gram negative rods and others

The nutrition is derived from 1. sweat, 2. aminoacids and peptides from the skin, 3.
fatty acids from the sebaceous glands of the skin.

Factors Determining the Ecology of the Skin Bacteria

Four main factors determine the ecology of skin bacteria

1. The climate: The temperature and humidity

2. The effect of free fatty acids

3. Other bacterial inhibitors

4. Maintenance of the flora by products of skin secretions

Defence Against Infection

1. Intact skin. Normal uninterrupted skin provides protection against invasion by

bacteria

2. Lysozyme in sweat: The enzyme lysozyme provides protection against gram

positive bacteria by lysing the cell wall.

3. Ig A antibodies in the sweat and secretions provide first line of defense against
infection

4. Inhibitors like unsaturated fatty acids provide protection against bacteria

5. Bacteriocins produced by the normal flora prevent the establishment of other

bacteria

Factors Responsible For Wound Infections

A. Host Factors: T

he following factors help the organisms tosurvive and produce the infections

1. Extremes of age
2. Diabetes mellitus

3. Steroid therapy

4. Obesity

5. Malnutrition

6. Immunocompromised individual

7. Presence of remote infection at the time of surgery

B. Exogenous Factors

1. Use of un sterile instruments

2. Surgeons hands / from health workers

3. Air / Hospital environments

C. Endogenous Factors

1. Wound contamination from the patient source: from the normal flora
2. Wound penetrating through structures containing normal flora

3. Surgical procedures involving mucous membranes harboring normal flora

4. Patients carrying pathogens in their nose, throat, axilla etc.

Etiological agents

Ps.aeruginosa

Staph.aureus

Proteus spp

Member of enterobacteriaceae

Anaerobic organisms

Anaerobic cocci

Bacteroides

Post Operative Infections

Gas gangrene organisms

S.aureus

Cl.tetani

Route of entry
Wounds may occur following: surgery, trauma or injections. Wound infections
may occur mainly after surgical procedures. Wound sepsis is the result of cross
infection from human sources and from other outside sources.
Mechanisms of damage
1. Organisms enter through the skin, multiply there and pro-duce the disease in
the skin. For example, impetigo, abscess and cellu-litis are caused by
Staphylococcus aureus and Streptococcus pyogenes. As soon as the organisms
enter the skin they multiple and produce various toxins that kill the cells and
produce cellulites. Further damage leads to necrosis and ulcer formation.

2. Organisms multiply in the skin and produce disease in inter-nal organs. For
example some group A streptococci multiply in the skin and produce disease
known as acute glomerulo nephritis causing damage to the kidneys. Some times
C.diphtheriae may multiply in the skin and affect the heart due to the toxin.

3. Some times organism may multiply in the skin and produce the toxin which
affect the CNS and the effects are seen. In the case of Clostridium
tetani, convulsions and paralysis occur due to the pro-duction of a powerful toxin

Laboratory diagnosis
Pus and wound swabs are cultured for the aerobic and anaero-bic organisms and
are identified using appropriate biochemical tests.
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