Appendix C Recommended culture media 1. FORMULAE Reference in this monograph to a culture medium identified by means of capital letters at the beginning of each word implies that the formula given in this Appendix should be employed and that the medium will have been sterilized at the stated temperature by autoclaving, unless otherwise directed. Water used for culture media should always be freshly distilled. Before use incubate samples from each batch of culture medium to check sterility and to test for growth promoting properties. 1.01 Nutrient Broth Beef extract 10 g Peptone 10 g Sodium chloride 5 g Water 1 000 ml Dissolve the ingredients by heating in the water. Adjust to pH 7.6-8.0 with 10 N NaOH and boil for 10 min. Filter. Adjust to pH 7.2-7.4 and sterilize at 115 ø for 20 min. Commercial form: Oxoid Granules CM67 or Tablets CM68. 1.02 Nutrient Agar Nutrient broth (pH 7.2-7.4) 1 1 Agar powder 15 g Dissolve the agar in the nutrient broth by autoclaving at 121 ø for 20 min. Adjust pH to 7.2. Filter through paper pulp. Distribute as required and sterilize at 121 ø for 20 min. Commercial form: Oxoid Blood Agar Base, Granules CM55 or Tablets CM56. 1.03 Blood Agar Base No. 2 Proteose peptone (Oxoid L46) 15 g Liver digest (Oxoid L27) 2.5 g Yeast extract (Oxoid L20) 5 g Sodium chloride 5 g Oxoid agar No. 3 12 g (pH 7.4 approx.) 779

780 JOURNAL OF TIlE SOCIETY OF COSMETIC CHEMISTS Add 40 g to 1 000 ml of water and soak for 15 min. Mix and sterilize at 121 ø for 15 min. Mix well before pouring. For general purpose Nutrient Agar, Blood Agar Base no. 2 is recom- mended. Commercial form: Oxoid CM271 1.04 Blood Agar Add defibrinated sterile horse blood to sterile nutrient agar that has been liquefied by heating and then cooled to `52 o. The final concentration of blood should be 5-10%. Haemolysis is best observed in layered blood agar plates. For such plates, a layer of nutrient agar is poured into the petri dish and allowed to set the blood-containing medium is then poured on top of the nutrient agar. 1.05 MacConkey Agar Peptone 20 g Sodium chloride 5 g Sodium taurocholate 5 g Water 1 000 ml Dissolve the peptone, sodium chloride and sodium taurocholate in the water by heating. Adjust to pH 8.0. Boil for 20 min. Cool and filter. Agar 20 g Lactose 10 g Neutral red, 1% aq. soln. 10 ml Add and dissolve the agar by boiling and adjust to pH 7.4. Add the lactose and indicator solution. Mix and sterilize at 11,55 ø for 20 min. The quantity of indicator required depends on the depth of colour preferred. Sodium taurocholate, sodium tauroglycocholate or other satisfactory bile salt may be used. The use of 0.1% Teepol (an anionic detergent) in place of bile salt in MacConkey agar has been recommended. Commercial form: Oxoid Granules CM7 or Tablets CM8. 1.06 MacConkey Broth (Modified) Peptone 20 g Sodium chloride 5 g Sodium taurocholate ,5 g Water 1 000 ml Bromcresol purple, 0.2% soln. ,5 ml Lactose 10 g