Penicillinase Solution

Dissolve 10 g of casein hydrolysate, 2.72 g of potassium dihydrogen orthophosphate and 5.88 g of sodium citrate in 200 mL of water, adjust the pH to 7.2 with 5m sodium hydroxide and dilute to 1000 mL with water. Dissolve 0.41 g of magnesium sulfate in 5 mL of water and add 1 mL of a 0.16% w/v solution of ammonium iron(ii) sulfate and sufficient water to produce 10 mL. Sterilise both solutions by heating in an autoclave, cool, mix, distribute in shallow layers in conical flasks and inoculate with Bacillus cereus (NCTC 9946). Incubate the flasks at 18° to 37° until growth is observed and then maintain at 35° to 37° for 16 hours, agitating constantly to ensure maximum aeration. Centrifuge and sterilise the supernatant fluid by filtration through a suitable membrane filter.

1.0 mL of Penicillinase Solution should hydrolyse benzylpenicillin to benzylpenicilloic acid at the rate of at least 500 mg per hour at 30° and pH 7, provided that the concentration of benzylpenicillin does not fall below the level necessary for enzyme saturation. The Michaelis constant for benzylpenicillin of the penicillinase in penicillinase solution is approximately 12 µg per mL.

Sterility Complies with the test for sterility, Appendix XVI A.

Store at 0° to 2° and use within 2 or 3 days. When freeze dried and kept in sealed ampoules it may be stored for several months.