After the medium is autoclaved at 121°C for 15 minutes, a filter sterilized solution of 10% solution of carbohydrate (6, 8) is aseptically added to the medium to a final concentration of 1%. The sterile medium containing the carbohydrate is aliquoted aseptically into sterile test tubes and cooled unslanted as stabs (5). Some procedures call for the addition of 10 g/liter of carbohydrate to the medium prior to sterilization. The medium is then dissolved by heating to a boil on a hot plate or by steaming for 20 minutes prior to aliquoting into test tubes. The tubed medium is then steamed for 20 minutes in place of autoclaving to prevent breaking down of the carbohydrate