5. Connect up the free end of the gla.s.s tube from the flask--after removing the cotton-wool plug--with the air-entry tube in the mouth of the aspirating bottle (Fig. 216).

6. Open the tap fully, and allow the water to run.

Replenish the ice from time to time if necessary.

(In emptying itself the aspirator bottle will aspirate 10 litres of air slowly through the water in the Erlenmeyer flask.)

7. When the aspiration is completed, disconnect the flask and remove it from its ice packing.

8. Liquefy three tubes of nutrient gelatine and add to them 0.5 c.c., 0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by means of a sterile graduated pipette, as in the quant.i.tative examination of water. Pour plates.

9. Pour a second similar set of gelatine plates.

10. Incubate both sets of plates at 20 C.

11. Enumerate the colonies present in the two sets of gelatine plates after three, four, or five days and average the results from the numbers so obtained; estimate the number of micro-organisms present in 1 c.c., and then in the 50 c.c. of broth in the flask.

12. The result of air examination is usually expressed as the number of bacteria present per cubic metre (i. e., kilolitre) of air; and as the number of organisms present in the 50 c.c. water only represent those contained in 10 litres of air, the resulting figure must be multiplied by 100.

Qualitative.--

1. Proceed exactly as in the quant.i.tative examination of air (_vide supra_), steps 1 to 10.

2. Pour plates of wort agar with similar quant.i.ties of the air-infected water, and incubate at 37 C.

3. Pour plates of nutrient agar with similar quant.i.ties of the water and incubate at 37 C.

4. Pour similar plates of wort gelatine and incubate at 20 C.

5. Pick off the individual colonies that appear in the several plates, subcultivate them on the various media, and identify them.

EXAMINATION OF SOIL.

The bacteriological examination of soil yields information of value to the sanitarian during the progress of the process of h.o.m.ogenisation of "made soil" (e. g., a dumping area for the refuse of town) and determines the period at which such an area may with propriety and safety be utilised for building purposes; or to the agriculturalist in informing him of the suitability of any given area for the growth of crops.

The surface of the ground, exposed as it is to the bactericidal influence of sunlight and to rapid alternations of heat and cold, rain and wind, contains but few micro-organisms. Again, owing to the density of the molecules of deep soil and lack of aeration on the one hand, and the filtering action of the upper layers of soil and bacterial antagonism on the other, bacterial life practically ceases at a depth of about 2 metres. The intermediate stratum of soil, situated from 25 to 50 cm. below the surface, invariably yields the most numerous and the most varied bacterial flora.

~Collection of Sample.~--A small copper capsule 6 cm. high by 6 cm.

diameter, with "pull-off" cap secured by a bayonet catch, previously sterilised in the hot-air oven, is the most convenient receptacle for samples of soil.

[Ill.u.s.tration: FIG. 217.--Soil scoop.]

The instrument used for the actual removal of the soil from its natural position will vary according to whether we require surface samples or soil from varying depths.

(a) For ~surface~ samples, use an iron scoop, shaped like a shoe horn, but provided with a sharp spine (Fig. 217). This is wrapped in asbestos cloth and sterilised in the hot-air oven. When removed from the oven, wrap a piece of oiled paper, silk, or gutta-percha tissue over the asbestos cloth, and secure it with string, as a further protection against contamination.

On reaching the spot whence the samples are to be taken, the coverings of the scoop are removed, and the asbestos cloth employed to brush away loose stones and debris from the selected area. The surface soil is then broken up with the point of the scoop, sc.r.a.ped up and collected in the body of the scoop, and transferred to the sterile capsule for transmission.

[Ill.u.s.tration: FIG. 218.--Fraenkel's borer.]

(b) For ~deep~ samples collected at various distances from the surface, an experimental trench may be cut to the required depth and samples collected at the required points on the face of the section. It is, however, preferable to utilise some form of borer, such as that designed by Fraenkel (Fig. 218).

_Fraenkel's Earth Borer._--This instrument consists of a stout hard-steel rod, 150 cm. long, marked in centimetres from the drill-pointed extremity. It is provided with a cross handle (adjustable at any point along the length of the rod by means of a screw nut). The terminal centimeters are thicker than the remainder of the rod, and on one side a vertical cavity about 0.5 cm. deep is cut. This is covered by a f.l.a.n.g.ed sleeve so long as the borer is driven into the soil clockwise, and is opened for the reception of the sample of soil, when the required depth is reached, by reversing the s.c.r.e.w.i.n.g motion, and again closed before withdrawal of the borer from the earth by resuming the original direction of twist. It can be sterilised in a manner similar to that adopted for the scoop, or by repeatedly filling the cavity with ether and burning it off.

~Quant.i.tative.~--Four distinct investigations are included in the complete quant.i.tative bacteriological examination of the soil:

1. The enumeration of the aerobic organisms.

2. The enumeration of the spores of aerobes.

3. The enumeration of the anaerobic organisms (including the facultative anaerobes).

4. The enumeration of the spores of anaerobes.

Further, by a combination of the results of the first and second, and of the third and fourth of these, the ratio of spores to vegetative forms is obtained.

_Apparatus Required_:

Case of sterile capsules (25 c.c. capacity).

Case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre).

Flask containing 250 c.c. sterile bouillon.

Tall cylinder containing 2 per cent. lysol solution.

Plate-levelling stand.

12 sterile plates.

Tubes of nutrient gelatine.

Tubes of wort gelatine.

Tubes of nutrient agar.

Tubes of glucose formate gelatine.

Tubes of glucose formate agar.

Water-bath regulated at 42 C.

Bunsen burner.

Grease pencil.