Preparation of Culture Media, Inoculation

and Identification of Bacteria

Abdihakim Ismail
Ms Microbiology & MPH
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Definition
• Culture media: is an artificially prepared
medium (liquid or solid) that contains nutrients to
grow bacteria in vitro.
• Inoculum: microbes that are introduced into a
culture medium to initiate growth
• Culture: the microbes that grow and multiply in
or on a culture medium
• Colony: macroscopically visible collection of
millions of bacteria originating from a single
bacterial cell.

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 Cont’d
For a bacteria to be successful in growing, the
culture medium must provide:
• all essential nutrients, ions, and moisture
• maintain the correct pH and osmotic pressure, and
• neutralize any toxic materials produced.

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 Preparation of culture media
Materials for Culturing Bacteria

Basic materials used for culturing bacteria.

• Culture media

• Petri dishes

• Test tubes

• Inoculating loops, straight wire loops

• Bunsen burner

• Incubator
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 Common ingredients of culture media
1. H2O:
• is essential for growth,It must be free of mineral
saltswhich can inhibit bacterial growth.
• Either distilled or deionised water should be
used.
2. Peptone: contains water soluble products
obtained from breakdown of animal or plant
proteinIt
- the main source of nitrogen, some vitamins,
carbohydrates
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3. Meat extract :
- this provides amino acids, minerals, and essential growth
factors .
4. Yeast extract :
- very good growth stimulant.
- It provides amino acids, water soluble vitamins (vit. B)
5. Mineral salts :
• for metabolism
• E.g - SO4 - as a source of sulphur
- PO42- - as a source of phosphor
- NaCl - is the common ingredient
- Other trace elements include Mg, Mn, etc
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6. Carbohydrates:
- Simple sugars includes glucose, maltose, sucrose, lactose,
pentose, hexose, galactose etc.
-The complex sugar include: polysaccharides, glycerol,
mannitol etc.
Other ingredients include, blood, serum, egg yolk etc.
depending on the organisms need.
9. Solidifying agents: are agents which are used to solidify
the culture media

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 Cont’d
Agar:
• Is a mixture of two polysaccharide: agarose (70 -75%) &
agaropectin (20-25%).
• It is produced from sea weeds
Agar is a good solidifying agent due to:
- transformation temperature is very high
• Uses of agar:
1. Solidifying agent of culture media
2. Provides framework to hold moisture and nutrients
3. Not digestible for most microbes
4. Provide calcium and organic ions for the bacteria
5. Used for adding heat sensitive ingredients such as blood and serum

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 Types of culture media
Based on Physical forms into;

Solid media

Liquid media

Semi-solid media
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A. Solid media
• Are solidified by agar
• Are used mainly in Petri dishes as
plate cultures, in bottles or tubes
 The purpose of culturing on
solid medium is principally to
- isolate discrete colonies of
each organism present in the
specimen.

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B. Liquid media
• do not solidify at temperatures
above freezing C. Semi-solid media
- Growth seen as cloudiness or • Are culture media
particulates
prepared by adding small
• most commonly used as
enrichment when organisms amount of agar (0.4 -
are likely to be few. 0.5% W/V) to a liquid
E.g. blood culture broth medium.
• are used mainly as
transport media

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Types of culture media
Based on function, into;

1. Basic media

2. Enriched or enrichment media

3. Selective media

4. Indicator (differential) media

5. Transport media

6. Anaerobic media

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1. Basic Media
• These are simple media that will support the growth of
microorganisms that
- do not have special nutritional requirements.
E.g. - Nutrient agar
Purposes of basic media
- to maintain standard culture of control strains of
bacteria.
- For sub-culturing pathogens from differential and
selective media prior to performing biochemical and
serological identification tests.

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2. Enriched/ Enrichment media
• is used for a certain group of microbes
• to isolate pathogens from a mixed culture
• enrichment media selects for one microbe.
- Basic media can be enriched by addition of special
nutrients (e.g., serum, growth factors, trace elements)
- to support some fastidious bacterial growth (e.g: H.
influenzae, Neisseria sp., and some streptococcus sp.).
E.g.: - Blood agar (contain whole blood)
- Chocolate agar (contain heated blood)

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3. Selective media
• contain substances which inhibit the growth of one organism
- to allow the growth of the other
• used for culturing a specimen from a site having a normal
flora
- to prevent the growth of these unwanted contaminants.
• The medium is made selective by incorporation of certain
inhibitory substances like bile salt, crystal violet, antibiotics,

E.g. MacConkey agar

-MacConkey favors the growth of gram
negative bacteria
- inhibit the growth of Gram positives due to
the presence of bile salt.
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4. Indicator (differential) media
• dyes or other substances are added to differentiate micro-
organisms.
• Many differential media distinguish between bacteria by;
- incorporating an indicator which changes colour

• E.g. MacConkey agar

- contain neutral red as an
indicator

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5. Transport media
• mostly semisolid media contain ingredients to;
- prevent the overgrowth of commensals
-to ensure the survival of pathogens when specimens
cannot be cultured immediately after collection.
e.g.
Cary-Blair medium:
- is used for preserving and
transporting enteric pathogens.
Amies transport medium:
- is used for transportation of
gonococci.

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 The choice of culture media for isolation of bacteria
depends on;
pathogens to be isolated,
- their growth requirements
- the features by which they are recognized.
Whether the specimens sterile sites or from having
normal microbial flora.
Cost, availability, and stability of media.
Training and experience of laboratory staff

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Major procedures during Preparation of culture media

1. Weighing and dissolving of culture media

2. Sterilization

3. Addition of heat sensitive ingredients

4. pH testing of culture media

5. Dispensing of the culture media

6. Quality assurance of culture media

7. Storage of culture media

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