Great care must be exercised to have the samples representative of the water to be tested and to see that no contamination occurs at the time of filling the sample bottles.
3. STORAGE AND TRANSPORTATION OF SAMPLES.
Because of the rapid and often extensive changes which may take place in the bacterial flora of bottled samples when stored even at temperatures as low as 10° C., it is urged, as of importance, that all samples be examined as promptly as possible after collection.
The time allowed for storage or transportation of a bacterial sample between the filling of the sample bottle and the beginning of the analysis should be not more than six hours for impure waters and not more than twelve hours for relatively pure waters. During the period of storage, the temperature shall be kept as near 10° C. as possible. Any deviation from the above limits shall be so stated in making reports.
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 withdrawn and added to the proper dilution bottles as required.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.
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.