Activity No.

2 Culture Media Preparation Culture media are composed of nutrients used for the growth and identification of microorganisms. They may be classified as follows : I. according to the physical state : 1.1 solid media 1.5% to 2.0% agar is added as a solidifying agent 1.2 semi-solid media with 0.5% to 1.0% agar 1.3 liquid no agar is added II. according to use : 2.1 2.2 simple media enriched media for the cultivation of non-fastidious microorganisms. e.g. nutrient agar contain additional nutrient sources for the growth of fastidious microorganisms e.g. blood agar plate, chocolate agar plate, brain heart infusion media contain nutrients that allow a particular organisms that might not be present in sufficient numbers in the specimen to be isolated and identified e.g. Selenite F for the enrichment of Salmonella typhi in a fecal specimen contain ingredients that inhibits the growth of certain bacteria while permitting the growth of others e.g. mannitol salt agar for the growth of salttolerant staphylococci allow colonies of one kind of organism to be distinguished from another. The media usually have constituents that cause observable changes in them when a particular biochemical reaction occurs. e.g. MacConkey agar allows the differentiation of lactose fermenters (red colonies) from nonlactose fermenters (colorless colonies)

2.3

enrichment media

2.4

selective media

2.5

differential media

Culture media which can not be heated are sterilized through filtration. The most popular among these at present is the membrane filter such as the cellulose nitrate filter.

Membranes with pore diameters of 0.45 u are usually used to remove bacteria and bigger contaminants, while 0.2 u filters are used to remove viruses from the filtrate. Most culture media which are heat stable are sterilized using steam under pressure i.e. autoclaving. Generally, exposure to a temperature of 121oC (250oF) for 15 to 20 minutes is sufficient to effect sterilization. This temperature is reached by applying a pressure of 15 lbs/in2 (1.06 kgs/cm2) to steam. Bigger volumes of materials require longer exposure time. Indicators for sterilization are used. These may be in the form of tapes, paper strips or bacterial spores. The following outlines the steps in the preparation of culture media which are heat stable. 1. Weigh, measure the different components e.g. nutrient agar beef extract peptone agar distilled water Adjust the pH, e.g. nutrient agar 6.8 Dissolved in a water bath. PLATED MEDIA Autoclave at 121oC for 15 to 20 mins. Cool to around 50oC* Pour into sterile petri dishes Allow to solidify TUBED MEDIA Distribute media into test tubes. Autoclave at 121oC for 15-20 minutes. Form into slant, butt and slant-slant. Test for sterility. Incubate at 37oC for 16 to 18 hours.

3 grams 5 grams 15 grams 1000 grams

2. 3.

4. 5. 6. 7.

4. 5. 6. 7.

8. Test for sterility 16 to 18 hrs.

*For blood agar plates, 5%-10% sterile, whole non-hemolyzed blood is added after cooling the medium to 50oC. For the preparation of chocolate agar plates, this blood agar is further heated to a chocolate brown color, cooled, then poured into sterile plates.

References: Alcamo, E. 1994. Fundamentals of Microbiology. 4th edition. Benjamin Cummings. Black, J. 1996. Microbiology : Principles and Applications. 3rd ed. Prentice Hall, N. Jersey. READ : Madigan, Martinko, Parker. 2000. Brock's Microbiology. Section 4.3 Sections 19.1 to 18.3