Procedure for Preparation of Culture Media

Culture Media Biochemical Media

1. Preparation of Culture Media

Nutrient Agar (NA)

i. 37 grams of the medium is suspended in 1000 ml of distilled water and then boiled to dissolve completely.
ii. Then the medium is sterilized by autoclaving at 121 degree centigrade (15 lbs pressure) for 15 minutes.

Nutrient Broth (NB)

13 grams of the medium is dissolved in 1000 ml distilled water and autoclaved at 121°C for 15 minutes.

Mac Conkey Agar (MA)

i. 55 grams of the medium is suspended in 1000 ml of distilled water and then boiled to dissolve completely.
ii. Then the medium is sterilized by autoclaving at 121 degree centigrade (15 lbs pressure) for 15 minutes.

Blood Agar (BA)

i. 42.5 grams of the blood agar base medium is suspended in 1000 ml distilled water and sterilized by autoclaving at 121 degree centigrade (15lbs pressure) for 15 minutes.
ii. After cooling to 40-50 degree centigrade, 50 ml sterile defibrinated sheep blood is added aseptically and mixed well before pouring.

Chocolate Agar (CA)

i. The sterilized blood agar is poured in Petri plates and is allowed to solidify.
ii. After solidification it is heated at 75 degree centigrade in an oven for 30 minutes.
iii. The color changes to chocolate brown after incubation time.

Mueller Hinton Agar (MHA)

i. 38 grams of the medium is suspended in 1000 ml distilled water and the medium is warmed to dissolve.
ii. 10 ml is distributed in test tubes and sterilized by boiling in water bath for 10 minutes.

2. Preparation of Biochemical Media

Hugh and Leifson’s Medium (O/F Medium)

i. 9.4 grams of the medium is rehydrated in 1000 ml cold distilled water and then heated to boiling to dissolve completely.
ii. The medium is distributed in 100 ml amounts and sterilized in the autoclave for 15 minutes at 15 lbs pressure (121 degree centigrade).
iii. To 100 ml sterile medium aseptically added 10ml of sterile Dextrose and mixed thoroughly and dispensed in 5 ml quantities into sterile culture tubes.

Sulphide Indole Motility (SIM) Medium

i. 36 grams of the medium is suspended in 1000 ml distilled water and dissolved completely.
ii. Then it is distributed in tubes to a depth of about 3 inches and sterilized.

MR-VP Medium

i. 17 grams is dissolved in 1000 ml distilled water.
ii. 3 ml of medium is distributed in each test tube and autoclaved at 121 degree centigrade for 15 minutes.

Simmon Citrate Agar

i. 24.2 grams of the medium is dissolved in 1000 ml distilled water.
ii. 3 ml medium is distributed in test tubes and sterilized by autoclaving at 121 degree centigrade for 15 minutes. After autoclaving tubes containing medium were tilted to form slant.

Triple Sugar Iron (TSI) Agar

i. 65 grams of the medium is dissolved in 1000 ml of distilled water and sterilized by autoclaving at 15 lbs (121 degree centigrade) pressure for 15 minutes.
ii. The medium is allowed to set in sloped form with a butt about 1 inch (2.54 cm) of thickness.

Christensen Urea Agar

i. 24 grams of the medium is suspended in 950 ml distilled water and sterilized by autoclaving at 121 degree centigrade for 15 minutes.
ii. After cooling to about 45 degree centigrade, 50 ml of 40% urea is added and mixed well.
iii. Then 5 ml is dispensed in test tube and set at slant position.

Back to Culture Media Back to Biochemical Media

Some of the laboratory culture media for routine use are given below:

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Culture media are substances used in microbiology laboratories to grow and maintain microorganisms for various purposes, such as research, diagnosis, and production of biological products. These media provide the necessary nutrients, pH, and other conditions required for the growth of microorganisms under controlled laboratory conditions. Culture media can be classified based on several criteria:

1. 1. Composition: Culture media can be classified into different types based on their composition, such as:

   • Complex media: These contain a variety of nutrients, such as peptones, extracts, and carbohydrates, in an undefined composition. Examples include nutrient agar and nutrient broth.
   • Chemically defined media: These contain precise, known quantities of all components, such as specific amino acids, vitamins, and salts. They are useful for studying the nutritional requirements of microorganisms.
   • Selective media: These contain ingredients that selectively inhibit the growth of certain microorganisms while allowing the growth of others. Examples include MacConkey agar, which selects for Gram-negative bacteria, and Mannitol salt agar, which selects for staphylococci.
   • Differential media: These contain indicators that allow for the differentiation of microorganisms based on their metabolic characteristics. Examples include blood agar, which differentiates bacteria based on their hemolytic activity, and eosin methylene blue agar, which differentiates lactose-fermenting and non-lactose-fermenting bacteria.
   • Enriched media: These contain additional nutrients, such as blood or serum, to support the growth of fastidious organisms with complex nutritional requirements. Examples include blood agar and chocolate agar.

2. 2. Physical State: Culture media can be solid, liquid, or semisolid, depending on the specific requirements of the microorganisms being cultured and the purpose of the experiment.

3. 3. Purpose: Culture media can be tailored to serve specific purposes, such as isolation, enumeration, identification, or maintenance of microorganisms.

Culture media are an essential tool in microbiology laboratories, providing researchers with the means to study the growth, physiology, and behavior of microorganisms in a controlled environment. They play a crucial role in various fields, including clinical microbiology, food microbiology, environmental microbiology, and biotechnology.