Practical No.

:
Date:

BACTERIOLOGICAL CULTURE MEDIA

OBJECTIVES
…………………..

MATERIALS REQUIRED:
………………………………..

THEORY
The majority of organisms to be studied in Veterinary bacteriology are either pathogens or
commensals of the animals and in order to obtain suitable growths for identifying, the media should
provide bacteria with most of important nutrition requirements for growing such as water, source of
carbon, energy, nitrogen, carbohydrates, amino acid, inorganic salts, particular growth factors like
vitamin (K) and others. These nutrients are supplied by aqueous extracts of meat and peptone.
The culture media present as:
1. Liquid media (Broth): containing of different nutrients without solidifying materials, the bacteria
colony features disappear in this medium (e.g. Nutrient broth & Brain Heart infusion broth).
2. Solid media (Agar media): containing nutrients with Agar. Agar, a solidify medium, is added to
liquid media as a result of this addition it is used for identification and isolation of pure colony
which appears well (e.g. Nutrient agar, blood agar).
3. Semi Solid media: used for special purpose. Agar is added to media in low concentration (usually
0.5% or less) to semi solidify medium (motility media like SIM media, MIL media etc.).

The routine laboratory media divided into numbers of type according to their functions.
1. Essential media (Ordinary media): (Nutrient broth, Nutrient agar) used as simple media and as
basis for supplemented enriched media.
2. Enrichment media: (Blood agar, serum agar, Chocolate agar) prepared by addition of substances
like blood, serum, glucose or egg to basal media as a requirements for cultivation of fastidious
bacteria such as Streptococci on blood agar.
3. Differential media: (MacConkey agar) Combination of enriched media with selective agents and
indicators system are used as diagnostic Laboratory media.
4. Selective media: (neomycin or gentamicin blood agar, mannitol salt agar, eosin methylene blue
agar (EMB). This media contain agent that inhibit or poison all but a few types of species from
mixed inoculums. For example high concentration of salt (in mannitol salt agar) inhibits most
bacteria but Staphylococci can grow.
5. Transport media: (e.g. Cary Blair media) they are devised to maintain the viability of pathogen
and to avoid overgrowth of the contaminants during transit from the patient to the Lab.
6. Storage media: used for the maintenance of bacterial culture for long time period eg. Yeast
extract mannitol agar medium.
PROCEDURE for the preparation of Agar Plate
The manufacturer’s instructions should always be followed but a few additional general points
include:
 Clean glassware that has been rinsed free from detergents and other chemicals should be
used.
 The glassware need not be sterile unless sterilized medium is being decanted into it.
 The appropriate amount of dehydrated medium is weighed out, placed in a flask and distilled
water added to it. Glass-distilled water must be used, because this is free from chloride and
heavy metal ions that can be inhibitory to bacteria.

We used following steps to prepare ……….. media

1. In a conical flask, ……… media is dissolved in distilled water.
2. Boil the mixture on water bath or over flame burner, gently.
3. Seal the top mouth of flask with cotton, and finally cover the cotton with loose layer of aluminum
foil.
4. Sterilize the media by autoclaving for 15 minutes at 1210C, 15psi pressure, in an autoclave.
5. Cool the sterile media to 450C and pour on sterile petri plates. Pouring at this temperature prevents
condensation forming on the lid.

Before plates are poured, every care is taken not to contaminate

1. The bench is wiped with ethanol.
2. A Bunsen burner is set up with gentle blue flame.
3. The number of plates is placed on bench with their lids.
4. The aluminum foil, cotton are removed.
5. The mouth of flask is flamed.
6. The lids of plates are lifted just enough to be poured, and is quickly half filled with media.
7. The lid replaced and left plate on bench for at least 20 min to solidify.
8. Keep plates upside down in refrigerator.

RESULT

CONCLUSION
Examples of media used in diagnostic bacteriology