Reading material for the assignment of PHA204 Pharmaceutical Microbiology – I

Physical requirements for microbial growth:

Temperature Requirements for Microbial Growth
•Most microorganisms grow well at the temperatures that humans favor.
•However, certain bacteria are capable of growing at extremes of temperature that would certainly
hinder the survival of almost all eukaryotic organisms.
•Microorganisms are classified into three primary groups on the basis of their preferred range of
temperature: psychrophiles (cold-loving microbes), mesophiles (moderate-temperature
loving microbes), and thermophiles (heat-loving microbes).
Most bacteria grow only within a limited range of temperatures, and their maximum and minimum
growth temperatures are only about 30°C apart.
•They grow poorly at the high and low temperature extremes within their range.
•Each bacterial species grows at particular minimum, optimum, and maximum temperatures.
The minimum growth temperature is the lowest temperature at which the species will grow.
•The optimum growth temperature is the tempera- ture at which the species grows best.
•The maximum growth temperature is the highest temperature at which growth is possible.
Organisms that have adapted to live in the bodies of animals usually have an optimum temperature
close to that of their hosts.
•The optimum temperature for many pathogenic bacteria is about 37°C, and incubators for clinical
cultures are usually set at about this temperature.
•The mesophiles include most of the common spoilage and disease organisms.
pH Requirements for Microbial Growth
•Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5.
•Very few bacteria grow at an acidic pH below about pH 4.
•This is why a number of foods, such as sauerkraut, pickles, and many cheeses, are preserved from
spoilage by acids produced by bacterial fermentation.
•Nonetheless, some bacteria, called acidophiles, are remarkably tolerant of acidity.
•Molds and yeasts will grow over a greater pH range than bacteria will, but the optimum pH of
molds and yeasts is generally below that of bacteria, usually about pH 5 to 6.
•Alkalinity also inhibits microbial growth but is rarely used to preserve foods.
•When bacteria are cultured in the laboratory, they often produce acids that eventually interfere
with their own growth.
•To neutralize the acids and maintain the proper pH, chemical buffers are included in the growth
medium.
•The peptones and amino acids in some media act as buffers, and many media also contain
phosphate salts.
•Phosphate salts have the advantage of exhibiting their buffering effect in the pH growth range of
most bacteria.
•They are also nontoxic; in fact, they provide phosphorus, an essential nutrient.

Osmotic Pressure Requirements for Microbial Growth

•Microorganisms obtain almost all their nutrients in solution from the surrounding water.
•Thus, they require water for growth, and their composition is 80–90% water.
•High osmotic pressures have the effect of removing necessary water from a cell.
•When a microbial cell is in a solution whose concentration of solutes is higher than in the cell (the
environment is hypertonic to the cell), the cellular water passes out through the plasma
membrane to the high solute concentration.
•This osmotic loss of water causes plasmolysis, or shrinkage of the cell’s cytoplasm.
•The growth of the cell is inhibited as the plasma membrane pulls away from the cell wall.
•Thus, the addition of salts (or other solutes) to a solution, and the resulting increase in osmotic
pressure, can be used to preserve foods.
•Salted fish, honey, and sweetened condensed milk are preserved largely by this mechanism; the
high salt or sugar concentrations draw water out of any microbial cells that are present and thus
prevent their growth.
•These effects of osmotic pressure are roughly related to the number of dissolved molecules and
ions in a volume of solution.
•Some organisms, called extreme halophiles, have adapted so well to high salt concentrations that
they actually require them for growth. In this case, they may be termed obligate halophiles.
•Organisms from such saline waters as the Dead Sea often require nearly 30% salt, and the
inoculating loop (a device for handling bacteria in the laboratory) used to transfer them must
first be dipped into a saturated salt solution.
•More common are facultative halophiles, which do not require high salt concentrations but are
able to grow at salt concentrations up to 2%, a concentration that inhibits the growth of many
other organisms. A few species of facultative halophiles can tolerate even 15% salt.
•Most microorganisms, however, must be grown in a medium that is nearly all water.
•For example, the concentration of agar (a complex polysaccharide isolated from marine algae)
used to solidify microbial growth media is usually about 1.5%.
•If markedly higher concentrations are used, the increased osmotic pressure can inhibit the growth
of some bacteria.
•If the osmotic pressure is unusually low (the environment is hypotonic)—such as in distilled
water, for example—water tends to enter the cell rather than leave it.
•Some microbes that have a relatively weak cell wall may be lysed by such treatment.

Oxygen Requirements for Microbial Growth

Nutritional requirements for microbial growth (Culture Media):

Key definitions
A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture
medium.
•Some bacteria can grow well on just about any culture medium; others require special media, and
still others cannot grow on any nonliving medium yet developed.
•Microbes that are introduced into a culture medium to initiate growth are called an inoculum.
•The microbes that grow and multiply in or on a culture medium are referred to as a culture.

Necessary requirements for microbial growth in culture media

•The culture must contain the right nutrients for the specific microorganism we want to grow.
•It should also contain sufficient moisture, a properly adjusted pH, and a suitable level of oxygen,
perhaps none at all.
•The medium must initially be sterile—that is, it must initially contain no living microorganisms—
so that the culture will contain only the microbes (and their offspring) we add to the medium.
•Finally, the growing culture should be incubated at the proper temperature.
Commercially available culture media
•Most of media, are available from commercial sources, have premixed components and require
only the addition of water and then sterilization.
Chemically defined media
•A chemically defined medium is one whose exact chemical composition is known.
•Chemically defined media are usually reserved for laboratory experimental work or for the growth
of autotrophic bacteria.
Complex media
•Most heterotrophic bacteria and fungi, such as you would work with in an introductory lab
course, are routinely grown on complex media.
•It is made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins
from these and other sources.
•The exact chemical composition varies slightly from batch to batch.

•In complex media, the energy, carbon, nitrogen, and sulfur requirements of the growing
microorganisms are provided primarily by protein.
•Proteins are large, relatively insoluble molecules that only a minority of microorganisms can
utilize directly.
•Partial digestion by acids or enzymes reduces proteins to shorter chains of amino acids called
peptones.
•These small, soluble fragments can be digested by most bacteria.
•Vitamins and other organic growth factors are provided by meat extracts or yeast extracts.
•The soluble vitamins and minerals from the meats or yeasts are dissolved in the extracting water,
which is then evaporated, so these factors are concentrated. (These extracts also supplement
the organic nitrogen and carbon compounds.)
•Yeast extracts are particularly rich in the B vitamins. If a complex medium is in liquid form, it is
called nutrient broth.
•When agar is added, it is called nutrient agar. (This terminology can be confusing; just remember
that agar itself is not a nutrient.)

Anaerobic Growth Media and Methods

•Because anaerobes might be killed by exposure to oxygen, special media called reducing media
must be used.
•These media contain ingredients, such as sodium thioglycolate, that chemically combine with
dissolved oxygen and deplete the oxygen in the culture medium.
•To routinely grow and maintain pure cultures of obligate anaerobes, microbiologists use reducing
media stored in ordinary, tightly capped test tubes.
•These media are heated shortly before use to drive off absorbed oxygen.

Jar for cultivating anaerobic bacteria on Petri plates.

When water is mixed with the chemical packet containing sodium bicarbonate and sodium
borohydride, hydrogen and carbon dioxide are generated.
Reacting to the surface of a palladium catalyst in a screened reaction chamber, which may also be
incorporated into the chemical packet, the hydrogen and atmospheric oxygen in the jar combine to
form water.
The oxygen is thus removed. Also in the jar is an anaerobic indicator containing methylene blue,
which is blue when oxidized and turns colorless when the oxygen is removed.
•Laboratories that work with relatively few culture plates at a time can use systems that can
incubate the microorganisms in sealed boxes and jars in which the oxygen is chemically
removed after the culture plates have been introduced and the container sealed.
•The envelope of chemicals (the active ingredient is ascorbic acid) is simply opened to expose it
to oxygen in the container’s atmosphere.
•The atmosphere in such containers usually has less than 5% oxygen, about 18% CO2, and no
hydrogen.
•In a recently introduced system, each individual Petri plate (OxyPlate) becomes an anaerobic
chamber. The medium in the plate contains an enzyme, oxyrase, which combines oxygen with
hydrogen, removing oxygen as water is formed.
Special Culture techniques for bacteria
•Obligate intracellular bacteria, such as the rickettsias and the chlamydias, do not grow on artificial
media.
•Like viruses, they can reproduce only in a living host cell.
•These bacteria are therefore grown on cultures of living cells.
•Many clinical laboratories have special carbon dioxide incubators in which to grow aerobic
bacteria that require concentrations of carbon dioxide higher or lower than that found in the
atmosphere.
•Desired carbon dioxide levels are maintained by electronic controls.
•High carbon dioxide levels are also obtained with simple candle jars.
•Cultures are placed in a large sealed jar containing a lighted candle, which consumes oxygen.
•The candle stops burning when the air in the jar has a lowered concentration of oxygen (at about
17% O2, still adequate for the growth of aerobic bacteria). An elevated con- centration of CO2
(about 3%) is also present.
•Microbes that grow better at high CO2 concentrations are called capnophiles. The low-oxygen,
high-CO2 conditions resemble those found in the intestinal tract, respiratory tract, and other
body tissues where pathogenic bacteria grow.
•Commercially available chemical packets are used to generate carbon dioxide atmospheres in
containers.
•When only one or two Petri plates of cultures are to be incubated, clinical laboratory investigators
often use small plastic bags with self-contained chemical gas generators that are activated by
crushing the packet or moistening it with a few milliliters of water.
•These packets are sometimes specially designed to provide precise concentrations of carbon
dioxide (usually higher than can be obtained in candle jars) and oxygen for culturing organisms
such as the micro- aerophilic Campylobacter bacteria.
Biosafety Levels (BSL)
•Some microorganisms, such as the Ebola virus, are so dangerous that they can be handled only
under extraordinary systems of containment called biosafety level 4 (BSL-4).
•The lab is a sealed environment within a larger building and has an atmosphere under negative
pressure, so that aerosols containing pathogens will not escape.
•Both intake and exhaust air is filtered through high-efficiency particulate air filters (HEPA filters);
the exhaust air is filtered twice.
•All waste materials leaving the lab are rendered noninfectious.
•The personnel wear “space suits” that are connected to an air supply.
Biosafety Level 3 (BSL-3) Biosafety Level 4 (BSL-4)

Selective and Differential Media

•In clinical and public health microbiology, it is frequently necessary to detect the presence of
specific microorganisms associated with disease or poor sanitation. For this task, selective and
differential media are used.
Selective Media
•Selective media are designed to suppress the growth of unwanted bacteria and encourage the
growth of the desired microbes.
•For example, bismuth sulfite agar is one medium used to isolate the typhoid bacterium, the gram-
negative Salmonella typhi, from feces.
•Bismuth sulfite inhibits gram-positive bacteria and most gram-negative intestinal bacteria (other
than S. typhi), as well.
•Sabouraud’s dextrose agar, which has a pH of 5.6, is used to isolate fungi that outgrow most
bacteria at this pH.
Differential Media
•Differential media make it easier to distinguish colonies of the desired organism from other
colonies growing on the same plate.
•Similarly, pure cultures of microorganisms have identifiable reactions with differential media in
tubes or plates.
•Blood agar (which contains red blood cells) is a medium that microbiologists often use to identify
bacterial species that destroy red blood cells.
These species, such as Streptococcus pyogenes, the bacterium that causes strep throat, show a clear
ring around their colonies where they have lysed the surrounding blood cells.
Differential Media (Blood Agar)
Enrichment Culture
•Because bacteria present in small numbers can be missed, especially if other bacteria are present
in much larger numbers, it is sometimes necessary to use an enrichment culture.
•This is often the case for soil or fecal samples. The medium (enrichment medium) for an
enrichment culture is usually liquid and provides nutrients and environmental conditions that
favor the growth of a particular microbe but not others.
•In this sense, it is also a selective medium, but it is designed to increase very small numbers of
the desired type of organism to detectable levels.
•Suppose we want to isolate from a soil sample a microbe that can grow on phenol and is present
in much smaller numbers than other species.
•If the soil sample is placed in a liquid enrichment medium in which phenol is the only source of
carbon and energy, microbes unable to metabolize phenol will not grow.
•The culture medium is allowed to incubate for a few days, and then a small amount of it is
transferred into another flask of the same medium.
•After a series of such transfers, the surviving population will consist of bacteria capable of
metabolizing phenol.
•The bacteria are given time to grow in the medium between transfers; this is the enrichment stage.
•Any nutrients in the original inoculum are rapidly diluted out with the successive transfers.
•When the last dilution is streaked onto a solid medium of the same composition, only those
colonies of organisms capable of using phenol should grow.
•A remarkable aspect of this particular technique is that phenol is normally lethal to most bacteria.
Culture Media Summarized

Obtaining Pure cultures

•Most infectious materials, such as pus, sputum, and urine, contain several different kinds of
bacteria; so do samples of soil, water, or food.
•If these materials are plated out onto the surface of a solid medium, colonies will form that are
exact copies of the original organism.
•A visible colony theoretically arises from a single spore or vegetative cell or from a group of the
same microorganisms attached to one another in clumps or chains.
•The bacteria must be distributed widely enough so that the colonies are visibly separated from
each other.
•Most bacteriological work requires pure cultures, or clones, of bacteria.
•The isolation method most commonly used to get pure cultures is the streak plate method.
•A sterile inoculating loop is dipped into a mixed culture that contains more than one type of
microbe and is streaked in a pattern over the surface of the nutrient medium.
•As the pattern is traced, bacteria are rubbed off the loop onto the medium.
•The last cells to be rubbed off the loop are far enough apart to grow into isolated colonies.
•These colonies can be picked up with an inoculating loop and transferred to a test tube of nutrient
medium to form a pure culture containing only one type of bacterium.
•The streak plate method works well when the organism to be isolated is present in large numbers
relative to the total population. However, when the microbe to be isolated is present only in
very small numbers, its numbers must be greatly increased by selective enrichment before it
can be isolated with the streak plate method.
Obtaining Pure cultures by Plate Streaking

Preserving Bacteria
•Two common methods of preserving microbial cultures for long periods are deep-freezing and
lyophilization.
•Deep-freezing is a process in which a pure culture of microbes is placed in a suspending liquid
and quick-frozen at temperatures ranging from -50°C to -95°C.
•The culture can usually be thawed and cultured even several years later.
•During lyophilization (freeze-drying), a suspension of microbes is quickly frozen at
temperatures ranging from -54°C to -72°C, and the water is removed by a high vacuum
(sublimation).
•While under vacuum, the container is sealed by melting the glass with a high-temperature torch.
•The remaining powder-like residue that contains the surviving microbes can be stored for years.
•The organisms can be revived at any time by hydration with a suitable liquid nutrient medium.