Part 38

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

g. Litmus or Azolitmin Solution.

The standard litmus solution shall be a 2 per cent. aqueous solution of reagent litmus. Powder the litmus, add to the water and boil for five minutes. The solution usually needs no correction in reaction and may be at once distributed in flasks or test-tubes and sterilized as is culture media. It should give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish.

The standard azolitmin solution shall be a 1 per cent. solution of Kahlbaum’s azolitmin. Add the azolitmin powder to the water and boil for five minutes. The solution may need to be corrected in reaction by the addition of sodium hydrate solution so that it will be approximately neutral and will give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish. It may be distributed in flasks or test-tubes and sterilized as is culture media.

h. Litmus-lactose-agar.

Litmus-lactose-agar shall be prepared in the same manner as nutrient agar with the addition of 1 per cent. of lactose just before sterilization. The reaction shall be a faint pink with phenol red, or, if on titration with phenolphthalein the reaction is not already between neutral and +1, adjust to neutral. One cc. of sterilized litmus or azolitmin solution shall be added to each 10 cc. of the medium just before it is poured into the Petri dish, or the mixture may be made in the dish itself.

i. Endo’s Medium. To Make One Liter.

1. Add 5 grams of beef extract, 10 grams of peptone and 30 grams of agar dried for one-half hour at 105° C. before weighing, to 1,000 cc. of distilled water. Boil on a water bath until all the agar is dissolved and then make up the loss by evaporation.

2. Cool the mixture to 45° C. in a cold water bath, then warm to 65° C. in the same bath without stirring.

3. Make up lost weight, titrate, and if the reaction is not already between neutral and +1 adjust to neutral.

4. Filter through cloth and cotton until clear.

5. Distribute 100 cc. or larger known quantities in flasks large enough to hold the other ingredients which are to be added later.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

7. Prepare a 10 per cent. solution of basic fuchsin in 95 per cent. alcohol, allow to stand 20 hours, decant and filter the supernatant fluid. This is a stock solution.

8. When ready to make plates melt 100 cc. of agar in streaming steam or on a water bath. Dissolve 1 gram of lactose in 15 cc. of distilled water, using heat if necessary. Dissolve 0.25 gram anhydrous sodium sulphite in 10 cc. water. To the sulphite solution add 0.5 cc. of the fuchsin stock solution. Add the fuchsin-sulphite solution to the lactose solution and then add the resulting solution to the melted agar. The lactose used must be chemically pure and the sulphite solution must be made up fresh.

9. Pour plates and allow to harden thoroughly in the incubator before use.

2. COLLECTION OF SAMPLE.

Samples for bacterial analysis shall be collected in bottles which have been cleansed with great care, rinsed in clean water, and sterilized with dry heat for at least one hour and a half at 170° C., or in the autoclav at 15 lbs. (120° C.) for 15 minutes or longer after the pressure reaches 15 lbs.

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.

4. DILUTIONS.

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.