Part 36

f. CALKINS, G. N. The microscopical examination of water: Report Mass. State Board of Health, pp. 397–421, 1892.

g. JACKSON, D. D. On an improvement in the Sedgwick-Rafter method for the microscopical examination of drinking water: Tech. Quart., Vol. 9, pp. 271–4, 1896.

h. WHIPPLE, G. C. Experience with the Sedgwick-Rafter method at the Biological Laboratory of the Boston Water Works: Tech. Quart., Vol. 9, pp. 275–9, 1896.

i. ——. Microscopy of drinking water, 3d ed., John Wiley & Sons, New York, 1914. (Contains bibliography.)

BACTERIOLOGICAL EXAMINATION.

I. APPARATUS.

1. Sample Bottles.—Any size, shape or quality of bottle may be used for a bacterial sample, provided it holds a sufficient amount to carry out all the tests required and is such that it may be properly washed and sterilized and will keep the sample uncontaminated until the analysis is made. Four- or eight-ounce, ground-glass-stoppered bottles are recommended. These should be protected by being wrapped in paper, or their necks covered with tin foil, and should be placed in proper boxes for transportation.

2. Pipettes.—Pipettes may be of any convenient size or shape provided it is found by actual test that they deliver accurately the required amount in the manner in which they are used. The error of calibration shall in no case exceed 2 per cent. Protecting the pipettes with a cotton stopper is recommended.

3. Dilution Bottles.—Bottles for use in making dilutions should preferably be of tall form, of such capacity as to hold at least twice the volume of water actually used. Close-fitting ground-glass stoppers are preferable, but tight fitting cotton stoppers may be used, provided due care is taken to prevent contamination and to avoid loss of volume through wetting of the stopper before mixing has been accomplished.

4. Petri Dishes.—Petri dishes ten centimeters in diameter shall be used with glass or porous tops as preferred. The bottoms of the dishes shall be as flat as possible so that the medium shall be of uniform thickness throughout the plate.

5. Fermentation Tubes.—Any type of fermentation tube may be used provided it holds at least three times as much medium as the amount of water to be tested.

II. MATERIALS.

1. Water.—Distilled water shall be used in the preparation of all culture media and reagents.

2. Meat Extract.—Liebig’s meat extract shall be used in place of meat infusion. Other brands may be substituted for Liebig’s when comparative tests have shown that they give equivalent results.

3. Peptone.—Armour’s, Digestive Ferments Company’s, Fairchild’s, or any other peptone which gives equivalent results may be used.

4. Sugars.—All sugars used shall be of the highest purity obtainable.

5. Agar.—The agar used shall be of the best quality and shall be dried for one-half hour at 105° C. before weighing. Much of the agar on the market contains considerable amounts of sea salts. These may be removed by soaking in water and draining before use.

6. Gelatin.—The gelatin used shall be of light color, shall contain not more than a trace of arsenic, copper, sulfides, and shall be free from preservatives, and of such a melting point that a 10 per cent. standard nutrient gelatin shall have a melting point of 25° C. or over. Gelatin shall be dried for one-half hour at 105° C. before weighing.

7. Litmus.—Reagent litmus of highest purity (not litmus cubes) or azolitmin (Kahlbaum) shall be used for all media requiring a litmus indicator.

8. General Chemicals.—Special effort shall be made to have all the other ingredients used for culture media chemically pure.

III. METHODS.

1. PREPARATION OF CULTURE MEDIA.

a. Adjustment of Reaction.

aa. Phenol Red Method for adjustment to a hydrogen-ion concentration of P{H+} = 6.8–8.4._ Withdraw 5 cc. of the medium, dilute with 5 cc. of distilled water, and add 5 drops of a solution of phenol red (phenol sulphone phthalein). This solution is made by dissolving 0.04 grams of phenol red in 30 cc. of alcohol and diluting to 100 cc. with distilled water.

Titrate with a 1:10 dilution of a standard solution of NaOH (which need not be of known normality) until the phenol red shows a slight but distinct pink color. Calculate the amount of the standard NaOH solution which must be added to the medium to reach this reaction. After the addition check the reaction by adding 5 drops of phenol red to 5 cc. of the medium and 5 cc. of water.

bb. Titration with phenolphthalein. (For the convenience of those who wish to retain the use of this method for the present it is given here, but it is recommended that as soon as possible the more accurate method of determining the hydrogen-ion concentration be substituted.)

In a white porcelain dish put 5 cc. of the medium to be tested, add 45 cc. of distilled water. Boil briskly for one minute. Add 1 cc. of phenolphthalein solution (5 grams of commercial salt to one liter of 50 per cent. alcohol). Titrate immediately with a n/20 solution of sodium hydrate. A faint but distinct pink color marks the true end-point. This color may be precisely described as a combination of 25 per cent. of red (wave length approximately 658) with 75 per cent. of white as shown by the disks of the standard color top made by the Milton Bradley Educational Co., Springfield, Mass.

All reactions shall be expressed with reference to the phenolphthalein neutral point and shall be stated in percentages of normal acid or alkali solutions required to neutralize them. Alkaline media shall be recorded with a minus (-) sign before the percentage of normal acid needed for their neutralization and acid media with a plus (+) sign before the percentage of normal alkali solution needed for their neutralization.