MLT 202: MEDICAL MICROBIOLOGY I

PRINCIPLES OF CULTURE MEDIA

To study and identity micro-organisms, they have to be cultivated and isolated in pure
culture. Most bacteria can be cultured in artificial media if the media meet the
nutritional and physical growth requirements of the bacteria.

The media used for growing bacteria are either liquid or solid media. They must
contain water and sources of nitrogen, carbon, mineral salts and essential vitamins.
Some bacteria may require additional specific substances which may be added to the
medium.

COMPOSITION OF CULTURE MEDIA

Water: Preferably deionized or distilled water should be used to prepare culture

media.

Peptone

Peptone is the water soluble product of protein hydrolysis. Proteins in meat, milk, and
soya bean are hydrolyzed by acids or by the enzymatic actions of enzymes such as
pepsin, trypsin, and papain. All forms of peptone are heat stable. Peptone from animal
protein is the source of nitrogen in the medium, the soya bean (plant) protein is a
source of carbohydrates.

A good brand of peptone must have the following qualities:

1. Must have a neutral pH

2. Must be light in colour

3. Must be hygroscopic

4. Must contain large amount of tryptophan for indole production.

Others include meat extract, yeast extract, mineral salts and carbohydrates

CLASSIFICATION OF CULTURE MEDIA

A. Classification based on chemical form

B. Classification based on physical form

C. Classification based on function

A. Classification based on chemical form

a. Chemically defined

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b. Chemically undefined (complex medium)

Chemically defined media are those in which the concentration of each ingredient is
known. The ingredients are usually highly purified inorganic salts and simple organic
compounds such as glucose or purified amino acids. These media are valuable in that
there is little or no variation in composition from batch to batch. They are however
very expensive and not meant for routine use. They are used to determine specific
growth requirements of bacteria.

Complex media are those prepared using natural products such as meat extract or
vegetable infusion. The natural products contain essential bacterial nutrients but the
exact concentration of nutrient is unknown. Complex culture media are used routinely
because they are easy to prepare, relatively cheap and able to support the growth of
many bacteria.

B. Classification based on physical form

a. Liquid media or broth

b. Solid media

Liquid Media or Broth

In liquid media, bacteria can freely move about. Growth in liquid medium is shown
by turbidity, though some organisms show surface growth. Liquid media are used
mainly for biochemical testing, blood culture, testing for motility and as enrichment
media. The major disadvantage is that purity of the growth cannot be guaranteed.

Solid Media

When organisms are grown on solid media, they grow and multiply at the site of
inoculation and form visible colonies. Colonial appearance and any changes in the
surrounding medium help in the identification of the bacterial species.

A liquid medium is made solid by the incorporation of a solidifying agent which does
not alter the nutritional content of the medium. The most widely used solidifying
agent is the agar (formerly called agar-agar). Agar is an inert carbohydrate extract
obtained from a type of seaweed found in Japan, New Zealand, and California (USA).
It is available in powder form.

A good brand of agar must:

1. gel at a concentration of 1%

2. Melt at 98oC

3. set on cooling at 42oC

4. be easily soluble and maintain clarity in solution

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C. Classification based on function

1. Basal media

2. Enriched media

3. Selective media

4. Enrichment media

5. Differential media

6. Auxanographic media

Basal Media

Basal media are simple media that will support the growth of most microorganisms
that do not need special nutritional requirements. They contain the basic nutrients:
peptone, mineral salts and water. They are generally referred to as nutrient broths.

Nutrient broths may be in the following forms: Infusion broth and Digest broth:

Enriched Media

Enriched media are culture media that are enriched with whole or lysed blood, serum,
special extracts or nutrients to support the growth of those bacteria that cannot grow
on the basal media. Examples are blood agar, serum agar, chocolate agar:

Nutrient broth + agar = Nutrient agar

Nutrient agar + blood = Blood agar

Blood agar + heat = Chocolate agar

Chocolate agr
Selective Media

Selective media are solid media which contain substances that prevent, slow down or
inhibit the growth of micro-organisms other than those for which the media are
devised. Examples of selective media are:
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1. Tellurite medium for diphtheria organism

2. Deoxycholate citrate agar (DCA) for Salmonella and Shigella groups.

3. Salmonella Shigella Agar (SSA)

4. Mannitol Salt agar (MSA)

Antibiotics are also widely used to make a medium selective.

Enrichment Media

These are liquid media that are similar in function to the selective media. The only
difference is that selective media are solid. Example of enrichment medium is selenite
F broth which is used for the isolation of Salmonella group of bacteria.

Differential Media

These media contain substances or indicators that will differentiate one organism from
another. Examples are:

1. MacConkey agar will differentiate lactose fermenting bacteria from non-lactose

fermenting ones.

2. Blood agar will differentiate haemolytic bacteria from non-haemolytic ones.

3. Cysteine Lactose Electrolyte Deficient (CLED) Agar

CLED MACCONKAY AGAR

Auxanographic Media

These media are deficient in certain nutritional requirements. The auxanographic

media will require special growth factors in order to support the growth of some
bacteria. When the medium is inoculated with an organism, and certain growth factors
are spotted on the surface of the medium, the growth of the organism occurs only in
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or around the factors. This indicates that the organism relies on the factors for growth.
For example, Haemophilus influenza will only grow on nutrient agar in the vicinity of
X+V factors.

Transport Media

Transport media as the name indicates, are meant for the transportation of clinical
specimens containing delicate micro-organisms from the ward or clinic to the
laboratory if there is to be a delay in their delivery to the laboratory or in, processing.
They contain substances that can prevent the overgrowth of commensals and prevent
bacteria from dying as a result of change of pH or enzyme action. They are generally
semisolid. Examples are:

1. Amies transport medium for Neisseria gonorrhoeae.

2. Stuart's transport medium for delicate organisms including anaerobes.

PREPARATION OF CULTURE MEDIA

Almost all laboratories prepare their culture media from either dried or dehydrated
products or use ready-made media which are available commercially. The preparation
of media from raw materials is no longer practiced in routine laboratories.

Stages in the Preparation of Media

1. weighing and dissolving

2. pH adjustment

3. Sterilization

4. Pouring or Dispensing

1. Weighing and dissolving: Errors in weighing technique can adversely affect the
performance of the medium. The exact amount prescribed by the manufacturer should
be weighed and dissolved accordingly.

2. Adjustment of pH: the pH of most routine culture media is close to neutral, i.e.,
between 6.8 to 7.4. It is essential to adjust all media pH correctly. Usually, the pH of
commercial dehydrated media does not require adjustment if the manufacturer’s
instruction has been followed to the letter. But the pH of such media can still be tested
with a narrow range pH papers of pH meters.

3. Sterilization: Autoclave or steam the medium at the correct temperature and time
specified. Heat sensitive ingredients should be added when the medium has cooled to
about 50oC

4. Dispensing culture media: Dispensing of sterile culture media should be done in

the media 'pouring' room, using clean sterile containers and aseptic precautions. To
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pour agar into petri dishes, use the following technique:

(i) Lay out the sterile petri dishes on a flat level surface.

(ii) Mix the medium gently and flame sterilize the mouth of the bottle or flask. Pour
about 15-20 ml of medium gently into the petri dishes.

If air bubbles are formed during pouring, quickly flame the surface before the agar
begins to set. Make sure agar plates are of even and uniform depth. The surface of the
medium should be smooth.

(iii) When the media are set, stack the plate in a plate rack or seal them in a plastic
bag and store at 2-4oC.

General media control

1. Sterility test: The newly prepared media should be tested for sterility. Routine
sterility test can only detect gross contamination, and so it is of limited value.

2. Performance test: The performance test of culture media is another name for
Quality Control (QC). The objectives are to test the ability of:

 Nutrient media to support growth from small innocula,

 Differential media to ensure that organisms growing on them show
characteristic features,
 Selective and enrichment media inhibit bacteria other than those they are
designed for, and
 Biochemical test media for their ability to show the expected reactions.