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 oedematis 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.