REVIEWER

Types of Bacteriological Culture Medium

Types of Bacteriological Culture Medium
Culture media are solutions containing all of the nutrients and necessary physical
growth parameters necessary for microbial growth. All microorganisms cannot grow
in a single culture medium and, in fact, many can't grow in any known culture
medium. Organisms that cannot grow in artificial culture medium are known as
obligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema
pallidum are obligate parasites.
Bacterial culture media can be distinguished on the basis of composition,
consistency and purpose.
Classification based on consistency
1.

2.

3.

Solid medium
solid medium is media containing agar (at a concentration of 1.5-2.0%) or
some other, mostly inert solidifying agent. Solid medium has physical
structure and this allows bacteria to grow in physically informative or useful
ways (e.g. as colonies or in streaks).solid medium is useful for isolating
bacteria or for determining the characteristics of colonies.
Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have
soft custard like consistency and are useful for the cultivation of
microaerophilic bacteria or for determination of bacterial motility.
Liquid (Broth) medium
These media contains specific amounts of nutrients but dont have trace of
gelling agents such as gelatin or agar. Broth medium serves various
purposes such as propagation of large number of organisms, fermentation
studies, and various other tests. eg. Sugar fermentation tests, MR-VR broth.

Classification based on the basis of composition

1.

2.

Synthetic or chemically defined medium

A chemically defined medium is one prepared from purified ingredients and
therefore whose exact composition is known.
Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor
completely characterized nor even completely consistent from batch to batch. Often
these are partially digested proteins from various organism sources. Nutrient broth,
for example, is derived from cultures of yeasts. Synthetic medium may be simple or
complex depending up on the supplement incorporated in it. A simple non-synthetic
medium is capable of meeting the nutrient requirements of organisms requiring
relatively few growth factors where as complex non-synthetic medium support the
growth of more fastidious microorganisms.
Classification based on the basis of purpose/ functional use/ application
many special purpose media are needed to facilitate recognition, enumeration, and
isolation of certain types of bacteria. To meet these needs, numerous media are
available.
On the basis of their application or function, they are classified as follows,
1. General purpose media/ Basic media
Basal media are basically simple media that supports most non-fastidious bacteria.
Peptone water, nutrient broth and nutrient agar considered basal medium. These
media are generally used for the primary isolation of microorganisms.

2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal
medium makes them enriched media. Enriched media are used to grow nutritionally
exacting (fastidious) bacteria. Blood agar, chocolate agar, Loefflers serum slope etc
are few of the enriched media. Blood agar is preparing by adding 5-10% (by
volume) to a basal medium such as nutrient agar or other blood agar bases.
Chocolate agar is also known as heated blood agar or lysed blood agar.
3. Selective and enrichment media are designed to inhibit unwanted commensal
or contaminating bacteria and help to recover pathogen from a mixture of bacteria.
While selective media are agar based, enrichment media are liquid in consistency.
Both these media serve the same purpose. Any agar media can be made selective
by addition of certain inhibitory agents that dont affect the pathogen. Various
approaches to make a medium selective include addition of antibiotics, dyes,
chemicals, alteration of pH or a combination of these.
a. Selective medium
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms
while allowing the growth of others (i.e., they select for certain microbes).Solid
medium is employed with selective medium so that individual colonies may be
isolated.

Examples of selective media include:

Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin
and Nystatin.
Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl.
Potassium tellurite medium used to recover C.diphtheriae contains 0.04%
Potassium tellurite.

McConkeys Agar used for Enterobacteriaceae members contains Bile salt that
inhibits most gram positive bacteria.
Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide
(antiseptic agent).
Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal
violet.
Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by
incorporating Malachite green.
Wilson and Blairs Agar for recovering S.typhi is rendered selective by the addition
of dye Brilliant green.
Selective media such as TCBS Agar used for isolating V. cholerae from fecal
specimens have elevated pH (8.5-5.6), which inhibits most other bacteria.
b. Enrichment culture/ Medium
Enrichment medium is used to increase the relative concentration of certain
microorganisms in the culture prior to plating on solid selective medium. Unlike
selective media, enrichment culture is typically used as broth medium. Enrichment
media are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used
to recover pathogens from fecal specimens.
4. Differential/ indicator medium: Differential appearance:

Certain media are designed in such a way that different bacteria can be recognized
on the basis of their colony colour. Various approaches include incorporation of
dyes, metabolic substrates etc, so that those bacteria that utilize them appear as
differently coloured colonies. Such media are called differential media or indicator
media Differential media allow the growth of more than one microorganism of
interest but with morphologically distinguishable colonies.
Examples of differential media include:

Mannitol salts agar (mannitol fermentation = yellow)

Blood agar (various kinds of hemolysis i.e. , and hemolysis)
Mac Conkey agar (lactose fermenters, Pink colonies whereas non- lactose
fermenter produces pale or colorless colonies.
TCBS (Vibrio cholera produces yellow colonies due to fermentation of sucrose)
5. Transport media:
Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals. This
can be achieved by using transport media. Such media prevent drying (desiccation)
of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of
unwanted bacteria. Some of these media (Stuarts & Amies) are semi-solid in
consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair
medium and Venkatraman Ramakrishnan (VR) medium are used to transport feces
from suspected cholera patients. Sachs buffered glycerol saline is used to transport

feces from patients suspected to be suffering from bacillary dysentery. Pikes

medium is used to transport streptococci from throat specimens.
6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen
content, reduced oxidation reduction potential and extra nutrients.

Media for anaerobes may have to be supplemented with nutrients like hemin and
vitamin K. Such media may also have to be reduced by physical or chemical
means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1%
glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron
filings can render a medium reduced. Before use the medium must be boiled in
water bath to expel any dissolved oxygen and then sealed with sterile liquid
paraffin.
Robertson cooked meat that is commonly used to grow Clostridium spps medium
contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth.
Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract
and casein hydrolysate.
Methylene blue or resazurin is an oxidation-reduction potential indicator that is
incorporated in the medium. Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g.
antibiotic assay media are used for determining antibiotic potency by the
microbiological assay technique.

Other types of medium includes Media for Enumeration of Bacteria, Media for
characterization of Bacteria, Maintenance media etc.

Techniques of isolation and Enumeration of

Bacteria
Enumeration of microorganisms is especially important in dairy microbiology, food
microbiology, and water microbiology.

Direct Microscopic count/ Total cell count

direct microscopic counts are possible using special slides known as counting
chambers, consisting of a ruled slide and a cover slip. It is constructed in such a
manner that the cover slip, slide, and ruled lines delimit a known volume. The
number of bacteria in a small known volume is directly counted microscopically and
the number of bacteria in the larger original sample is determined by extrapolation.
Dead cells cannot be distinguished from living ones. Only dense suspensions can
be counted.

Bacteria can be counted easily and accurately with the petroff-Hausser counting
chamber. This is a special slide accurately ruled into squares that are 1/400 mm2 in
area; a glass cover slip rests 1/50 mm above the slide, so that the volume over a
square is 1/20,000 mm3i.e.

1/20, 000, 000 cm3. If for example, an average of five bacteria is present in each
ruled square, there is 5 x 20,000,000 or 108, bacteria per milliliter. A suspension of
unstained bacteria can be counted in the chamber, using a phase-contrast
microscope.

The formula used for the direct microscopic count is:

The number of bacteria per cc = The average numbers of bacteria per large doublelined square X The dilution factors of the large square (1,250,000) X The dilution
factor of any dilutions made prior to placing the sample in the counting chamber,
e.g., mixing the bacteria with dye
Advantage of Direct Microscopic count
1.
2.
3.

Rapid, Simple and easy method requiring minimum equipment.

Morphology of the bacteria can be observed as they counted.
Very dense suspensions can be counted if they are diluted appropriately.

Limitations of Direct Microscopic count

1.
2.

Dead cells are not distinguished from living cells.

Small cells are difficult to see under the microscope, and some cells are
probably missed.

3.
4.
5.

Precision is difficult to achieve

A phase contrast microscope is required when the sample is not stained.
The method is not usually suitable for cell suspensions of low density i.e. <
107 Cells per ml, but samples can be concentrated by centrifugation or
filtration to increase sensitivity.
Viable count/ plate count technique

Dilution:

with both the spread plate and pour plate methods, it is important that the number
of colonies developing on the plates not be too large because on crowded plates
some cells may not form colonies and some colonies may fuse, leading to
erroneous measurements. It is also essential that the number of colonies not be too
small, or the statistical significance of the calculated count will be low. The usual
practice, which is the most valid statistically, is to count colonies only on plates that
have between 30 and 300 colonies. The number of bacteria in a given sample may
be usually too great to be counted directly. To obtain the appropriate colony
number, the sample be counted must almost always diluted in such a manner
that single isolated bacteria form visible isolated colonies , the number of colonies
can be used as a measure of the number of viable (living) cells in that known
dilution. Several 10-fold dilutions of the sample are commonly used. In most cases,
serial dilutions are employed to reach the final desired dilution.
However, if the organism normally forms multiple cell arrangements, such as
chains, the colony-forming unit may consist of a chain of bacteria rather than a
single bacterium. In addition, some of the bacteria may be clumped together. The
development of one colony can occur even when the cells are in aggregates. i.e.
cocci in clusters (staphylococci), chains (streptococci), or pairs (diplococci), the
resulting counts will be lower than the number of individual cells. Each colony that
can be counted is called a colony forming unit (CFU). By extrapolation, this number
can in turn be used to calculate the number of CFUs in the original sample rather
than number of bacteria per milliliter. The assumption made in this type of counting
procedure is that each viable cell can yield one colony.
There are two ways of performing a plate count: the spread plate method and the
pour plate method.
Generally, one wants to determine the number of CFUs per milliliter (ml) of sample.
To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied
by the number of times the original ml of bacteria was diluted (the dilution factor of
the plate counted). For example, if a plate containing a 1/1,000,000 dilution of the
original ml of sample shows 150 colonies, then the number of CFUs per ml in the
original sample is found by multiplying 150 x 1,000,000 as shown in the formula
below:
The number of CFUs per ml of sample = The number of colonies (30-300 plate) X
The dilution factor of the plate counted
In the case of the example above 150 x 1,000,000 = 150,000,000 CFUs per ml
This method is used to count only live (viable) cells. A viable cell is defined as one
that is able to divide and form off springs, and the usual way to perform a viable
count is to determine the number of cells in the sample capable of forming colonies
on a suitable agar medium. For this reason, the viable count is often called the

plate count, or colony count. This method is used to enumerate bacteria in milk,
water, foods, soils; cultures etc and the number of bacteria are expressed as
colony-forming units (CFU) per ml.
Advantage of plate count method
this method is used routinely and with satisfactory results for the estimation of
bacterial populations in milk, water, foods, and many other materials.
1.

Its sensitivity (theoretically, a single cell can be detected), and it allows for
inspection and positive identification of the organism counted.
2. It is easy to perform and can be adapted to the measurement of populations
of any magnitude.
3. It is sensitive method, since small numbers of organisms can be counted. Eg.
If a specimen contains as few as one bacterium per ml, one colony should
develop up on the plating of 1 ml
Limitation of plate count technique
(1) Only living cells develop colonies that are counted;
(2) clumps or chains of cells develop into a single colony;
(3) colonies develop only from those organisms for which the cultural conditions are
suitable for growth.

Types of Techniques; Pour plate technique, Spread plate technique and

Streak plate technique
Pour plate Technique
A pour plate is a method of melted agar inoculation followed by petri dish
incubation. A known volume (usually 0.1-1.0 ml) of culture is pipette into a sterile
petri plate; melted agar medium is then added and mixed well by gently swirling the
plate on the table top. Because the sample is mixed with the molten agar medium,
a larger volume can be used than with the spread plate. However, with the pour
plate method the organism to be counted must be able to briefly withstand the
temperature of melted agar, 45oC.

The cultures are inoculated into melted agar that has been cooled to 45oC. The
liquid medium is well mixed then poured into a petri dish (or vice versa) Colonies
form within the agar matrix rather than on top as they do when streaking a plate.
Pour plates are useful for quantifying microorganisms that grow in solid medium.
Because the "pour plate" embeds colonies in agar it can supply a sufficiently
oxygen deficient environment that it can allow the growth and quantification of
microaerophiles.
Spread Plate Technique
Spread plate technique is an additional method of quantifying microorganisms
on solid medium. With the spread plate method, a volume of an appropriately
diluted culture usually no greater than 0.1 ml is spread over the surface of an agar
plate using a sterile glass spreader. The plate is than incubated until the colonies
appear, and the number of colonies counted. Instead of embedding microorganisms
into agar, as is done with the pour plate method, liquid cultures are spread on the
agar surface.

An advantage of spreading a plate over the pour plate method is that cultures are
never exposed to 45 oC (i.e. melted agar temperatures).
Note: Surface of the plate must be dry, so that the liquid that is spread soaks in.
volume greater than 0.1ml are rarely used because the excess liquid does not soak
in and may cause the colonies to coalesce as they from, making them difficult to
count.
Streak Plate Technique
For organisms that grow well on agar plate, streak plate is the method of choice for
obtaining pure culture.
The key principles of this method is that, by streaking, a dilution gradient (number
of cells decrease as they move across the agar and away from the point of
inoculation) is established across the face of the plate as bacterial cells are
deposited on the agar surface. Because of this dilution gradient, confluent growth
occurs on part of the plate where the bacterial cells are not sufficiently separated; in
other regions of the plate where few bacteria are deposited separate macroscopic
colonies develop that can easily be seen with naked eye. Each well isolated colony
is assumed to arise from a single bacterium and therefore to represent a clone of a
pure culture.

Purpose of Streak Plate Technique: The purpose of the streak plate is to obtain
isolated colonies from an inoculum by creating areas of increasing dilution on a
single plate. Isolated colonies represent a clone of cells, being derived from a
single precursor cell.

Many different streaking patterns can be used to separate individual bacterial cells
on the agar surface.
Methods of making a streak plate to obtain pure cultures.
1. Loop is sterilized, and then a loopful of inoculums is removed from tube
2. A loopful of bacterial cells is streaked across the surface of a sterile solidified
nutrient medium.
3. Following the initial streak, subsequent streaks are made at angles to it, the
loop being sterilized between streaks.
4. The plates are than incubated under favorable conditions to permit growth of
the bacteria.
5.

After incubation colonies appear along the points of the streak. It is from such
well isolated colonies that pure cultures can usually be obtained.

Maintenance and Preservation of Pure Cultures

of Bacteria
Once a microorganism has been isolated and grown in pure culture, it
becomes necessary to maintain the viability and purity of the
microorganism by keeping the pure cultures free from contamination.
Normally in laboratories, the pure cultures are transferred periodically onto
or into a fresh medium (sub culturing) to allow continuous growth and
viability of microorganisms. The transfer is always subject to aseptic
conditions to avoid contamination.
Since repeated sub culturing is time consuming, it becomes difficult to
maintain a large number of pure cultures successfully for a long time. In
addition, there is a risk of genetic changes as well as contamination.
Therefore, it is now being replaced by some modern methods that do not
need frequent sub culturing. These methods include refrigeration, paraffin
method, cryopreservation, and lyophilization (freeze drying).
Periodic Transfer to Fresh Media
Strains can be maintained by periodically preparing a fresh culture
from the previous stock culture. The culture medium, the storage
temperature, and the time interval at which the transfers are made
vary with the species and must be ascertained beforehand. The
temperature and the type of medium chosen should support a slow
rather than a rapid rate of growth so that the time interval between
transfers can be as long as possible. Many of the more common
heterotrophs remain viable for several weeks or months on a
medium like nutrient agar. The transfer method has the
disadvantage of failing to prevent changes in the characteristics of a
strain due to the development of variants and mutants.
Refrigeration
Pure cultures can be successfully stored at 0-4C either in refrigerators or
in cold-rooms. This method is applied for short duration (2-3 weeks for
bacteria and 3-4 months for fungi) because the metabolic activities of the
microorganisms are greatly slowed down but not stopped. Thus their

growth continues slowly, nutrients are utilized and waste products released
in medium. This results in, finally, the death of the microbes after
sometime.
Paraffin Method/ preservation by overlaying cultures with
mineral oil
This is a simple and most economical method of maintaining pure cultures
of bacteria and fungi. In this method, sterile liquid paraffin in poured over
the slant (slope) of culture and stored upright at room temperature. The
layer of paraffin ensures anaerobic conditions and prevents dehydration of
the medium. This condition helps microorganisms or pure culture to
remain in a dormant state and, therefore, the culture can be preserved form
months to years (varies with species). The advantage of this method is that
we can remove some of the growth under the oil with a transfer needle,
inoculate a fresh medium, and still preserve the original culture. The
simplicity of the method makes it attractive, but changes in the
characteristics of a strain can still occur.
Cryopreservation
Cryopreservation (i.e., freezing in liquid nitrogen at -196C or in the gas
phase above the liquid nitrogen at -150C) helps survival of pure cultures
for long storage times.

In this method, the microorganisms of culture are rapidly frozen in liquid

nitrogen at -196C in the presence of stabilizing agents such as glycerol or

Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to

formation of ice crystals and promote cell survival. This liquid nitrogen
method has been successful with many species that cannot be preserved by
lyophilization and most species can remain viable under these conditions
for 10 to 30 years without undergoing change in their characteristics,
however this method is expensive.
Lyophilization (Freeze-Drying)
In this method, the culture is rapidly frozen at a very low temperature (70C) and then dehydrated by vacuum. Under these conditions, the
microbial cells are dehydrated and their metabolic activities are stopped; as
a result, the microbes go into dormant state and retain viability for years.
Lyophilized or freeze-dried pure cultures and then sealed and stored in the
dark at 4C in refrigerators. Freeze-drying method is the most frequently
used technique by culture collection centers. Many species of bacteria
preserved by this method have remained viable and unchanged in their
characteristics for more than 30 years.

Advantage of Lyophilization
*Only minimal storage space is required; hundreds of lyophilized cultures
can be stored in a small area

* small vials can be sent conveniently through the mail to other

microbiology laboratories when packaged in a special sealed mailing
containers
* Lyophilized cultures can be revived by opening the vials, adding liquid
medium, and transferring the rehydrated culture to a suitable growth
medium.