Dilution bottles shall be filled with the proper amount of tap water so that after sterilization they shall contain exactly 9 cc. or 99 cc. as desired. The exact amount of water can only be determined by experiment with the particular autoclav in use. If desired, the 9 cc. dilution may be measured out from a flask of sterile water with a sterile pipette. Dilution bottles shall be sterilized in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. The sample bottle shall be shaken vigorously 25 times and 1 cc. withdrawn and added to the proper dilution bottles as required. Each dilution bottle after the addition of the 1 cc. of the sample, shall be shaken vigorously 25 times before a second dilution is made from it or before a sample is removed for plating. 5. PLATING. All sample and dilution bottles shall be shaken vigorously 25 times before samples are removed for plating. Plating shall be done immediately after the dilutions are made. One cc. of the sample or dilution shall be used for plating and shall be placed in the Petri dish, first. Ten cc. of liquefied medium at a temperature of 40° C. shall be added to the 1 cc. of water in the Petri dish. The cover of the Petri dish shall be lifted just enough for the introduction of the pipette or culture medium, and the lips of all test-tubes or flasks used for pouring the medium shall be flamed. In making litmus-lactose-agar plates, 1 cc. of sterile litmus or azolitmin solution shall be added to each 10 cc. of culture medium either in the Petri dish or before pouring into the Petri dish. The medium and sample in the Petri dish shall be thoroughly mixed and uniformly spread over the bottom of the Petri dish by tilting or rotating the dish. All plates shall be solidified as rapidly as possible after pouring and gelatin plates shall be placed immediately in the 20° C. incubator and the agar plates in the 37° C. incubator. Endo plates shall be made by placing one loopful of the material to be tested on the surface of the plate and distributing the material with a sterile loop or glass rod. 6. INCUBATION. All gelatin plates shall be incubated for 48 hours at 20 C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture.[227] All agar plates shall be incubated for 24 hours at 37° C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture. Glass covered plates shall be inverted in the incubator. Any deviation from the above described method shall be stated in making reports. 7. COUNTING.