Examination of Soil

The bacteriological examination of soil yields information of value to the sanitarian during the progress of the process of homogenisation 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.

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 débris from the selected area. The surface soil is then broken up with the point of the scoop, scraped up and collected in the body of the scoop, and transferred to the sterile capsule for transmission.

(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 flanged 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 screwing 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.

Quantitative.—Four distinct investigations are included in the complete quantitative 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.

Sterile mortar and pestle (agate).

Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).

Sterile metal funnel with short wide bore delivery tube to just fit mouth of flask.

Solid rubber stopper to fit the flask (sterilised by boiling).

Pair of scales.

Counterpoise (Fig. 107).

Sterile metal (nickel) spoon or spatula.

Fractional steriliser (Fig. 140).

Method.—

1. Arrange four sterile capsules numbered I, II, III, and IV; pipette 9 c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of the remaining three.

2. Pipette 100 c.c. sterile bouillon into the Erlenmeyer flask.

3. Remove the cotton-wool plug from the flask and replace it by the sterile funnel.

4. Place flask and funnel on one pan of the scales, and counterpoise accurately.

5. Empty the sample of soil into the mortar and triturate thoroughly.

6. By means of the sterile spatula add 10 grammes of the earth sample to the bouillon in the flask.

The final results will be more reliable if steps 2, 3, 4, and 5 are performed under a hood—to protect from falling dust, etc.

7. Remove the funnel from the mouth of the flask; replace it by the rubber stopper and shake vigourously; then allow the solid particles to settle for about thirty minutes. One cubic centimetre of the turbid broth contains the washings from 0.1 gramme of soil.

8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil water," and add it to the contents of capsule I; mix thoroughly.

9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to capsule II, and mix.

10. In like manner add 0.1 c.c. of the contents of capsule II to capsule III, and then 0.1 c.c. of the contents of capsule III to capsule IV.

Then 1 c.c. fluid from capsule I contains soil water from .01 gm. earth.
Then 1 c.c. fluid from capsule II contains soil water from .0001 gm. earth.
Then 1 c.c. fluid from capsule III contains soil water from .000001 gm. earth.
Then 1 c.c. fluid from capsule IV contains soil water from .00000001 gm. earth.

(A) Aerobes (Vegetative Forms and Spores).—

11. Pour a set of gelatine plates from the contents of each capsule—two plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of the diluted soil water. Label and incubate.

12. Pour similar sets of wort gelatine plates from the contents of capsules II and III, label, and incubate at 20° C.

13. Pour similar sets of agar plates from the contents of capsules II and III; label and incubate at 37° C.

14. Weigh out a second sample of soil—10 grammes—dry over a water-bath until of constant weight and calculate the ratio

wet soil weight
———————
dry soil weight

15. "Count" the plates after incubation for three, four, or five days, and correcting the figures thus obtained by means of the "wet" to "dry" soil ratio estimate—

(a) The number of aerobic micro-organisms present per gramme of the soil.

(b) The number of yeasts and moulds present per gramme of the soil.

(c) The number of aerobic organisms "growing at 37° C." present per gramme of the soil.

(B) Anaerobes (Vegetative Forms and Spores).—

16. Pour similar sets of plates in glucose formate gelatine and agar and incubate in Bulloch's anaerobic apparatus.

(C) Aerobes and Anaerobes (Spores Only).—

17. Pipette 5 c.c. soil water into a sterile tube.

18. Place in the differential steriliser at 80° C. for ten minutes.

19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and 1 c.c. respectively of the soil water; label and incubate at 20° C., one set aerobically, the other anaerobically in Bulloch's apparatus.

20. "Count" the plates (delay the enumeration as long as possible) and estimate the number of spores of aerobes and anaerobes respectively present per gramme of the soil.

21. Calculate the ratio existing between spores and spores + vegetative forms under each of the two groups, aerobic and anaerobic micro-organisms.

Qualitative Examination.—The qualitative examination of soil is usually directed to the detection of one or more of the following:

Members of the Coli-typhoid group.

Streptococci.

Bacillus anthracis.

Bacillus tetani.

Bacillus œdematis maligni.

The nitrous organisms.

The nitric organisms.

1. Transfer the remainder of the soil water (88 c.c.) to a sterile Erlenmeyer flask by means of a sterile syphon.

2. Fix up the filtering apparatus as for the qualitative examination of water, and filter the soil water.

3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique similar to that described for the water sample, vide pages 434-436).

Every cubic centimetre of suspension now contains the soil water from nearly 1 gramme of earth.

The methods up to this point are identical no matter which organism or group of organisms it is desired to isolate; but from this stage onward the process is varied slightly for each particular bacterium.

I. The Coli-typhoid Group.—

II. Streptococci.—

III. Bacillus Anthracis.—

IV. Bacillus Tetani.—

The methods adopted for the isolation of these organisms are identical with those already described under water (page 437 et seq.).

V. Bacillus Œdematis Maligni.—Method precisely similar to that employed for the B. tetani.

VI. The Nitrous Organisms.—

1. Take ten tubes of Winogradsky's solution No I (vide page 198) and number them consecutively from 1 to 10.

2. Inoculate each tube with varying quantities of the material as follows:

To tube No. 1 add 1.0 c.c. of the soil water.
To tube No. 2 add 0.1 c.c. of the soil water.
To tube No. 3 add 1.0 c.c. from Capsule I.
To tube No. 4 add 0.1 c.c. from Capsule I.
To tube No. 5 add 1.0 c.c. from Capsule II.
To tube No. 6 add 0.1 c.c. from Capsule II.
To tube No. 7 add 1.0 c.c. from Capsule III.
To tube No. 8 add 0.1 c.c. from Capsule III.
To tube No. 9 add 1.0 c.c. from Capsule IV.
To tube No. 10 add 0.1 c.c. from Capsule IV.

Label and incubate at 30° C.

VII. The Nitric Organisms.—

3. Take ten tubes of Winogradsky's solution No II, number them consecutively from 1 to 10 and inoculate with quantities of soil water similar to those enumerated in section VI step 2. Label and incubate at 30° C.

4. Examine after twenty-four and forty-eight hours' incubation. From those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at 30° C.

5. Make further subcultivations from such of those tubes as show growth, and again incubate.

6. If growth occurs in these subcultures, make surface smears on plates of Winogradsky's silicate jelly (vide page 198).

7. Pick off such colonies as make their appearance and subcultivate in each of these two media.