Culture media 5

CULTURE MEDIA

Identification of Microorganisms
Diagnosis any infection
Biochemical test
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Media:

Media which is used for cultivation of bacteria in order to diagnose the bacteria by the culture characteristics. Colony morphology Color. Shape. Margin. Elevation.

The basic requirement of culture media are:- 1-Energy source (carbon source). 2-Nitrogen source . 3-Salts . 4-Minerals. 5-Water. 6-Adjust PH (7.2 to 7.6) .

In general we can say that culture media used for: Collection of sample . Growth of microorganism . Isolated of pure culture .

*Media are solidified by the adding of solidifying agents such as agar. Agar – agar : A complex carbohydrate obtain from a Algae.Used as solidification agent for media.Agar dissolved in aqueous solution Melts at 96 °C Solidifies at 42-44°C But will be liquid at 60 °C . Agar has no source of nutrients to the bacteria .


Table : Differentiation between agar and Gelatin
Agar
Gelatin
Agar found in 1.5% in medium.
Gelatin found in 15%.
Agar remain unmelted at all incubation temp.

Agar not decomposes by bacteria. - It is a cheep.

gelatin decomposes by bacteria. - It is expensive.

Preparation of culture media powder media

Preparation of media: 1- Dissolving powder media in distilled water and mixing of heater to boiling.

3- Cooling the media by air.

4- adding the media to petridishes or vials

6- plates are kept inverted in cold place?

5- Wait until solidify.



A- Physical state: 1-Broth media: used as transport media and for cultivation of bacteria. Contain zero% of agar. 2-Semi solid media: used for study the motility of bacteria. 3-Solid media: used to study the all culture characteristics of colony and contain 1-2% agar to solidify the media.

Classification of media

This classification according to:

B- Chemical composition. 1- Complex media : which is the exact chemical composition is unknown example, plants, animal tissue, serum, egg.

2- Synthetic: exact chemical composition is known: MacConkey agar, Nutrient agar.
3- Semi synthetic: in these media the basic media (synthetic media) added a complex substance example: blood, serum, peptone.

C- Classification according to use:

1- General media: which used for all groups of bacteria Nutrient broth, Nutrient agar, Semi solid media.
2- Differential media: The addation of certain reagent or chemical to the media may help in differentiating of certain types of bacteria from others.
Ex: MacConkey agar : To differentiate between lactose fermentation and Non lactose fermentation bacteria.

-Fermentation of bacteria will below the PH of media by producing acids, the PH indicator Neutral red will recognize the colony to :
Lactose fermentation colony pink color Ex: E.coli , klebisella spp.
Non lactose fermentation colony pale or colorless Ex: Salmonella spp. Proteus spp.



3-Selective media: in this media allow to isolation of specific group of bacteria by chemical substances that inhibit the growth of one group of bacteria. For example : Crystal violet and bile salt will prevent growth of gram positive without affecting the growth of gram negative bacteria.

a-MacConkey agar: Selective and Differential media because it s contain bile salt that inhibit all of gram positive bacteria(Selective media). And differential media because its differentiate between lactose fermentation and Non lactose fermentation bacteria, the PH indicator is Neutral red .


b-Salmonella –Shigella agar (SS agar):Selective medium inhibition the growth of all bacteria and allow to growth of Salmonella and Shigella. Selective : high bile salt inhibition G+ Sodium citrate G- (coli form)
And we can differentiate between salmonella and shigella on SS agar Neutral red (indicator).
Salmonella brown colony with black center
Shigella pale or colorless

C-Mannitol Salt agar (Selective and Differential media ).

Contains 7.5% Nacl which inhibition the growth of all bacteria except Staphylococci Spp and we can differentiate between Staph spp on this media Staph aureus Yellow color by fermentation of manitol (indicator phenol red)


4-Enrichment media:Which is used for fastidious bacteria and used by adding blood or serum as a rich substance to the nutrient agar.
A. Blood agar Prepare by adding 10% of sterile blood to sterile Nutrient agar at 45-50°C used human blood or sheep blood.Blood agar Enrichment and differential because it different between B – haemolysis Str. PyogensAnd α – haemolysis Str. Viridians

+ 10%sterile blood

Use: To culture H. influenzae & Neisseria Spp.


5-Special mediaUsed for one type of bacteriaFor example: Lowenstein – Jensen medium used for the cultivation of Mycobacterium tuberculosis This contain Malachite green which inhibition all groups of bacteria except Mycobacterium Spp. and contain glycerol inhibit M.bovis and allow to growth Mycobacterium tuberculosis

D-Classification according to O2 requirments: 1- Aerobic media All types of media, Which incubate in incubator in aerobic condition. 2- Anaerobic media These media are used to grow anaerobic organisms. Ex: Thioglycolate medium.

Fig 27.1

The Requirements for Growth in Oxygen (O2): 
Results: growth only at top = obligate aerobe growth only at bottom = obligate anaerobe , lighter growth at bottom heavy growth at top= = facultative anaerobe growth only in middle = microaerophile

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