Chapter XV: Appendix: 492 (13)

6. If still negative or inconclusive, kill the third guinea-pig at the end of the _sixth_ week. Make a careful post-mortem examination. Examine material from any caseous glands microscopically and inoculate freely on to Dorset's egg medium.

NOTE.--Every post-mortem examination of animals infected
with tuberculous material should include the naked eye and
microscopical examination of the popliteal, superficial and
deep inguinal, iliac, lumbar and axillary glands on each
side of the body, also the retrohepatic, bronchial and
sternal glands, the spleen, liver and lungs (Fig. 215).

(B) 1. Intimately mix all the available cream and deposit from the milk sample, and transfer to a sterile Erlenmeyer flask.

2. Treat the mixture by the antiformin method (_vide_ Appendix, page 502).

3. Inoculate each of two guinea-pigs, intraperitoneally, with half of the emulsion thus obtained.

4. Kill one of the guinea-pigs at the end of the first week and examine carefully.

5. Kill the second guinea-pig at the end of the second week and examine carefully.

6. Utilise the remainder of the deposit for microscopical examination and cultivations upon Dorset's egg medium.

NOTE.--No value whatever attaches to the result of a
microscopical examination for the presence of the B.
tuberculosis unless confirmed by the result of inoculation
experiments.

~6. Streptococcus Pyogenes Longus.~--

(A) 1. Spread serial surface plates upon nutrose agar. Also plant serial cultivations upon sloped nutrient agar (six tubes in series).

2. If the resulting growth shows colonies which resemble those of the streptococcus, make subcultivations upon agar and in bouillon, in the first instance, and study carefully.

(B) 1. Plant a large loopful of the deposit D^{2} into each of three tubes of glucose formate bouillon, and incubate anaerobically (in Buchner's tubes) for twenty-four hours at 37 deg. C.

2. If the resulting growth resembles that of the streptococcus, make subcultivations upon nutrient agar.

3. Prepare subcultivations of any suspicious colonies that appear, upon all the ordinary media, and study carefully.

If the streptococcus is successfully isolated, inoculate serum bouillon cultivations into the mouse, guinea-pig, and rabbit, to determine its pathogenicity and virulence.

~7. Staphylococcus Pyogenes Aureus.~--

1. Examine carefully the growth upon the serial blood serum cultivations prepared to isolate B. diphtheriae and the serial agar cultivations to isolate streptococci after forty-eight hours' incubation.

2. Pick off any suspicious orange coloured colonies, plant on sloped agar, and incubate at 20 deg. C. Observe pigment formation.

3. Prepare subcultivations from any suspicious growths upon all the ordinary media, study carefully and investigate their pathogenicity.

~8. Micrococcus Melitensis.~--The milk from an animal infected with M. melitensis usually contains the organisms in large numbers and but few other bacteria.

1. Spread several sets of surface plates upon nutrose agar, each from one loopful of the deposit in tube D^{1} or D^{2}.

2. Spread several sets of surface plates upon nutrose agar, each from one drop of the original milk sample.

3. Incubate aerobically at 37 deg. C. and examine daily up to the end of ten days.

4. Pick off suspicious colonies, examine them microscopically and subcultivate upon nutrose agar in tubes; upon glucose agar and in litmus milk.

5. Test the subsequent growth against the serum of an experimental animal inoculated against M. melitensis to determine its agglutinability.

6. If apparently M. melitensis, inoculate growth from a nutrose agar culture after three days incubation intracranially into the guinea-pig.

ICE CREAM.

~Collection of the Sample.~--

1. Remove the sample from the drum in the ladle or spoon with which the vendor retails the ice cream, and place it at once in a sterile copper capsule, similar to that employed for earth samples (_vide_ page 471).

2. Pack for transmission in the ice-box.

3. On arrival at the laboratory place the copper capsules containing the ice cream in the incubator at 20 deg. C. for fifteen minutes--that is, until at least some of the ice cream has become liquid.

~Qualitative and Quantitative Examination.~--Treat the fluid ice cream as milk and conduct the examination in precisely the same manner as described for milk (_vide_ page 443).

EXAMINATION OF CREAM AND BUTTER.

~Collection of the Sample.~--Collect, store, and transmit samples to the laboratory, precisely as is done in the case of ice cream.

~Quantitative.~--

_Apparatus Required_:

Sterile test-tube.
Sterilised spatula.
Water-bath regulated at 42 deg. C.
Case of sterile plates.
Case of sterile graduated pipettes, 1 c.c. (in hundredths).
Tubes of gelatine-agar (+10 reaction).
Plate-levelling stand, with its water chamber filled with water at
42 deg. C.

METHOD.--

1. Transfer a few grammes of the sample to a sterile test-tube by means of the sterilised spatula.

2. Place the tube in the water-bath at 42 deg. C. until the contents are liquid.

3. Liquefy eight tubes of gelatine-agar and place them in the water-bath at 42 deg. C, and cool down to that temperature.

4. Inoculate the gelatine-agar tubes with the following quantities of the sample by the help of a sterile pipette graduated to hundredths of a cubic centimetre--viz.,

To tube No. 1 add 1 c.c. liquefied butter.
2 add 0.5 c.c. liquefied butter.
3 add 0.3 c.c. liquefied butter.
4 add 0.2 c.c. liquefied butter.
5 add 0.1 c.c. liquefied butter.
6 add 0.05 c.c. liquefied butter.
7 add 0.03 c.c. liquefied butter.
8 add 0.02 c.c. liquefied butter.
9 add 0.01 c.c. liquefied butter.

5. Pour a plate cultivation from each of the gelatine-agar tubes and incubate at 28 deg. C.

6. "Count" the plates after three days' incubation, and from the figures thus obtained estimate the number of organisms present per cubic centimetre of the sample.

~Qualitative.~--

_Apparatus Required_:

Sterile beaker, its mouth plugged with sterile cotton-wool.

Counterpoise for beaker.

Scales and weights.

Sterilised spatula.

Water-bath regulated at 42 deg. C.

Separatory funnel, 250 c.c. capacity, its delivery tube
protected against contamination by passing it through a
cotton-wool plug into the interior of a small Erlenmeyer
flask which serves to support the funnel. This piece of
apparatus is sterilised _en masse_ in the hot-air oven.

Large centrifugal machine.

Sterile tubes (for the centrifuge) closed with solid rubber
stoppers.

Case of sterile pipettes, 10 c.c.

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

METHOD.--

1. Weigh out 100 grammes of the sample in a sterile beaker.

2. Plug the mouth of the beaker with sterile cotton-wool and immerse the beaker in a water-bath at 42 deg. C. until the contents are completely liquefied.

3. Fill the liquefied butter into the sterile separatory funnel.

4. Transfer the funnel to the incubator at 37 deg. C. and allow it to remain there for four days.

At the end of this time the contents of the funnel will have separated into two distinct strata.

(a) A superficial oily layer, practically free from bacteria.

(b) A deep watery layer, turbid and cloudy from the growth of bacteria.

5. Draw off the subnatant turbid layer into sterile centrifugal tubes, previously warned to about 42 deg. C., and centrifugalise at once.

6. Pipette off the supernatant fluid and fill the tubes with sterile 1 per cent. sodium carbonate solution previously warmed slightly; stopper the tubes and shake vigourously for a few minutes.

7. Centrifugalise again.

8. Pipette off the supernatant fluid; filling the tubes with warm sterile bouillon, shake well, and again centrifugalise, to wash the deposit.

9. Pipette off the supernatant fluid.

10. Prepare cover-slip preparations, fix and clear as for milk preparations, stain carbolic methylene-blue, Gram's method, Ziehl-Neelsen's method, and examine microscopically with a 1/12 inch oil-immersion lens.

11. Proceed with the examination of the deposit as in the case of milk deposit (see pages 450 _et seq._).

EXAMINATION OF UNSOUND MEATS.

(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)

The bacterioscopic examination of unsound food is chiefly directed to the detection of those members of the Coli-typhoid group--B. enteritidis of Gaertner and its allies--which are usually associated with epidemic outbreaks of food poisoning, and such anaerobic bacteria as initiate putrefactive changes in the food which result in the formation of poisonous ptomaines, consequently the quantitative examination pure and simple is frequently omitted.

A. Cultural Examination.

Quantitative.--

_Apparatus Required_:

Sterilised tin opener, (if necessary.)

Erlenmeyer flask (500 c.c. capacity) containing 200 c.c.
sterile bouillon and fitted with solid rubber stopper.

Counterpoise.

Scissors and forceps.

Scales and weights.

Water steriliser.

Hypodermic syringe.

Syringe with intragastric tube.

Rat forceps.

Case of sterile capsules.

Filtering apparatus as for water analysis.

Case of sterile plates.

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).

Plate-levelling stand.

Tubes of nutrient gelatine.

Tubes of nutrient agar.

Water-bath regulated at 42 deg. C.

Bulloch's apparatus.

METHOD.--

1. Place the flask containing 200 c.c. sterile broth on one pan of the scales and counterpoise accurately.

2. Mince a portion of the sample by the aid of sterile scissors and forceps, and add the minced sample to the bouillon in the flask to the extent of 20 grammes.

3. Make an extract by standing the flask in the incubator running at 42 deg. C. (or in a water-bath regulated to that temperature) for half an hour, shaking its contents from time to time. Better results are obtained if an electrical shaker is fitted inside the incubator and the flask kept in motion throughout the entire thirty minutes.

Now every centimetre contains the bacteria washed out from 0.1 gramme of the original food.

4. Inoculate tubes of liquefied gelatine as follows:

To tube No. 1 add 1.0 c.c. of the extract.
2 add 0.5 c.c. of the extract.
3 add 0.3 c.c. of the extract.
4 add 0.2 c.c. of the extract.
5 add 0.1 c.c. of the extract.

Pour plates from these tubes and incubate at 20 deg. C.

5. Prepare a precisely similar set of agar plates and incubate at 37 deg. C.

6. Pipette 5 c.c. of the extract into a sterile tube, heat in the differential steriliser at 80 deg. C. for ten minutes.

7. From the heated extract prepare duplicate sets of agar and gelatine plates and incubate anaerobically in Bulloch's apparatus at 37 deg. C. and 20 deg. C. respectively.

8. After three days' incubation examine the agar plates both aerobic and anaerobic and enumerate the colonies developed from spores (7), and from vegetative forms and spores (5), and calculate and record the numbers of each group per gramme of the original food.

9. After seven days' incubation (or earlier if compelled by the growth of liquefying colonies) enumerate the gelatine plates in the same way.

10. Subcultivate from the colonies that make their appearance and identify the various organisms.

11. Continue the investigations with reference to the detection of pathogenic organisms as described under water (page 429 _et seq._).

Qualitative.--

I. _Cultural._

The micro-organisms sought for during the examination of unsound foods comprise the following:

Members of the Coli-typhoid groups (chiefly those of the Gaertner class).

B. anthracis.

Streptococci

Anaerobic Bacteria:

B. enteritidis sporogenes.
B. botulinus.
B. cadaveris.

The methods by which these organisms if present may be identified and isolated have already been described under the corresponding section of water examination with the exception of those applicable to B. botulinus, and B. cadaveris. These can only be isolated satisfactorily from the bodies of experimentally inoculated animals.

II _Experimental._

_Tissue._--

1. Feed rats and mice on portions of the sample and observe the result.

2. If any of the animals die, make complete post-mortem examinations and endeavour to isolate the pathogenic organisms.

_Extract._--

1. Introduce various quantities of the bouillon extract into the stomachs of several rats, mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation (see page 367) and observe the result. Guinea-pigs and mice are very susceptible to infection by B. botulinus by this method; rabbits less so.

2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep pockets, and intraperitoneally with various quantities of the bouillon extract, and observe the result.

3. Filter some of the extract through a Chamberland candle and incubate the filtrate to determine the presence of soluble toxins.

4. If any of the animals succumb to either of these methods of inoculation, make careful post-mortem examinations and endeavour to isolate the pathogenic organisms.

THE EXAMINATION OF OYSTERS AND OTHER SHELLFISH.

On opening the shell of an oyster a certain amount of fluid termed "liquor" is found to be present. This varies in amount from a drop to many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the bulk of the fluid is probably the last quantum of water ingested by the bivalve before closing its shell. In order to obtain a working average of the bacteriological flora of a sample, ten oysters should be taken and the body, gastric juice and liquor should be thoroughly mixed before examination. The examination, as in dealing with other food stuffs, is directed to the search for members of the Coli-typhoid group, sewage streptococci and perhaps also B. enteritidis sporogenes.

_Apparatus Required_:

Two hard nail brushes.

Liquid soap.

Sterile water in aspirator jar with delivery nozzle
controlled by a spring clip.

Sterile oyster knives.

Sterile glass dish, with cover, sufficiently large to
accommodate ten oysters.

Sterile forceps.

Sterile scissors.

Sterile towels or large gauze pads.

Sterile graduated cylinders 1000 c.c. capacity, with either
the lid or the bottom of a sterile Petri dish inverted over
the open mouth as a cover.

Glass rods.

Corrosive sublimate solution, 1 per mille.

Bile salt broth tubes.

Litmus milk tubes.

Surface plates of nutrose agar.

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

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

Case of sterile glass capsules.

Erlenmeyer flasks, 250 c.c. capacity.

Double strength bile salt broth.

METHOD.--

1. Thoroughly clean the outside of the oyster shells by scrubbing each in turn with liquid soap and nail brush under a tap of running water. Then, holding an oyster shell in a pair of sterile forceps wash every part of the outside of the shell with a stream of sterile water running from an aspirator jar; deposit the oyster inside the sterile glass dish. Repeat the process with each of the remaining oysters.

2. Before proceeding further, cleanse the hands thoroughly with clean nail brush, soap and water, then plunge them in lysol 2 per cent. solution, and finally in sterile water.

3. Spread a sterile towel on the bench.

4. Remove one of the oysters from the sterile glass dish and place it, resting on its convex shell, on the towel. Turn a corner of the sterile towel over the upper flat shell to give a firmer grip to the left hand, which holds the shell in position.

5. With the sterile oyster knife (in the right hand) open the shell and separate the body of the oyster from the inner surface of the upper flat shell. Bend back and separate the flat shell, leaving the body of the oyster in and attached to the concave shell. Avoid spilling any of the liquor.

(Some dexterity in opening oysters should be acquired before undertaking these experiments).

6. Cut up the body of the oyster with sterile scissors into small pieces and allow the liquor freed from the body during the process to mix with the liquor previously in the shell.

7. Transfer the comminuted oyster and the liquor to the cylinder.

8. Treat each of the remaining oysters in similar fashion.

9. Mix the contents of the cylinder thoroughly by stirring with a sterile glass rod. The total volume will amount to about 100 c.c.

10. Use 0.1 c.c. of the mixed liquor to inseminate each of a series of three nutrose surface plates.

11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of litmus milk.

12. Add sterile distilled water to the contents of the cylinder up to 1000 c.c. and stir thoroughly with a sterile glass rod and allow to settle. The bacterial content of each oyster may be regarded, for all practical purposes, as comprised in 100 c.c. of fluid.

13. Arrange four glass capsules in a row and number I, II, III, IV. Pipette 9 c.c. sterile distilled water into each.

14. To capsule No. I add 1 c.c. of the diluted liquor, etc. from the cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule II to capsule III, afterwards adding 1 c.c. of fluid from capsule III to capsule IV.

15. Label tubes of bile salt broth and inoculate with the following amounts of diluted oysters:

No. 6 with 10 c.c. cylinder fluid = 0.1 oyster.
No. 5 with 1 c.c. cylinder fluid = 0.01 oyster.
No. 4 with 1 c.c. capsule I fluid = 0.001 oyster.
No. 3 with 1 c.c. capsule II fluid = 0.0001 oyster.
No. 2 with 1 c.c. capsule III fluid = 0.00001 oyster.
No. 1 with 1 c.c. capsule IV fluid = 0.000001 oyster.

16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask and add 50 c.c. double strength bile salt broth, and label 7.

17. Duplicate all the above indicated cultures.

18. Put up the tube cultures in Buchner's tubes and incubate anaerobically at 42 deg. C.

If growth occurs in tube 1 the organism finally isolated, e. g., B. coli, must have been present to the extent of one million per oyster.

19. Complete the examination for members of the Coli-typhoid group and sewage streptococci, as directed under Water Examination, page 429 (steps 11-21).

20. Inoculate a series of 6 tubes of litmus milk with quantities of the material similar to those indicated in step 15; heat to 80 deg. C. for ten minutes, and incubate under anaerobic conditions at 37 deg. C. Examine for the presence of B. enteritidis sporogenes as directed under Water Examination, page 438 (steps 7-10).

EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.

Quantitative.--

_Collection of the Sample._--As only small quantities of material are needed, the samples should be collected in a manner similar to that described under water for quantitative examination and transmitted in the ice apparatus used in packing those samples.

_Apparatus Required._--As for water (_vide_ page 420).

METHOD.--

1. Arrange four sterile capsules in a row and number them I, II, III, IV.

2. Pipette 9 c.c. sterile bouillon into capsule No. I.

3. Pipette 9.9 c.c. sterile bouillon into capsules II, III, and IV.

4. Add 1 c.c. of the sewage to capsule No. I by means of a sterile pipette, and mix thoroughly.

5. Take a fresh sterile pipette and transfer 0.1 c.c. of the mixture from No. I to No. II and mix thoroughly.

6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1 c.c. from No. III to No. IV.

Now 1 c.c. of dilution No. I contains 0.1 c.c. of the original sewage.
1 c.c. of dilution No. II contains 0.001 c.c. of the original sewage.
1 c.c. of dilution No. III contains 0.00001 c.c. of the original sewage.
1 c.c. of dilution No. IV contains 0.0000001 c.c. of the original sewage.

7. Pour a set of gelatine plates from the contents of each capsule, three plates in a set, and containing respectively 0.2, 0.3, and 0.5 c.c. of the dilution. Label carefully; incubate at 20 deg. C. for three, four, or five days.

8. Enumerate the organisms present in those sets of plates which have not liquefied, probably those from dilution III or IV, and calculate therefrom the number present per cubic centimetre of the original sample of sewage.

Qualitative.--The qualitative examination of sewage is concerned with the identification and enumeration of the same bacteria dealt with under the corresponding section of water examination; it is consequently conducted on precisely similar lines to those already indicated (_vide_ pages 426 to 441).

EXAMINATION OF AIR.

Quantitative.--

_Apparatus Required_:

Aspirator bottle, 10 litres capacity, fitted with a delivery
tube, and having its mouth closed by a perforated rubber
stopper, through which passes a short length of glass
tubing.

Erlenmeyer flask, 250 c.c. capacity (having a wide mouth
properly plugged with wool), containing 50 c.c. sterile
water.

Rubber stopper to fit the mouth of the flask, perforated
with two holes, and fitted as follows:

Take a 9 cm. length of glass tubing and bend up 3 cm. at one
end at right angles to the main length of tubing. Pass the
long arm of the angle through one of the perforations in the
stopper; plug the open end of the short arm with
cotton-wool.

Take a glass funnel 5 or 6 cm. in diameter with a stem 12
cm. in length and bend the stem close up to the apex of the
funnel, in a gentle curve through a quarter of a circle;
pass the long stem through the other perforation in the
rubber stopper.

A battery jar or a small water-bath to hold the Erlenmeyer
flask when packed round with ice.

Supply of broken ice.

Rubber tubing.

Screw clamps and spring clips, for tubing.

Water steriliser.

Retort stand and clamps.

Apparatus for plating (as for enumeration of water
organisms, _vide_ page 420).

METHOD.--

1. Fill 10 litres of water into the aspirating bottle and attach a piece of rubber tubing with a screw clamp to the delivery tube. Open the taps fully and regulate the screw clamp, by actual experiment, so that the tube delivers 1 c.c. of water every second. The screw clamp is not touched again during the experiment.

At this rate the aspirator bottle will empty itself in just under three hours. Shut off the tap and make up the contents of the aspirator bottle to 10 litres again.

2. Sterilise the fitted rubber cork, with its funnel and tubing, by boiling in the water steriliser for ten minutes.

3. Remove the cotton-wool plug from the flask, and replace it by the rubber stopper with its fittings. Make sure that the end of the stem of the funnel is immersed in the bouillon.

4. Place the flask in a glass or metal vessel and pack it round with pounded ice. Arrange the flask with its ice casing just above the neck of the aspirator bottle.

5. Connect up the free end of the glass 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 quantitative examination of water. Pour plates.

9. Pour a second similar set of gelatine plates.

10. Incubate both sets of plates at 20 deg. 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 quantitative examination of air (_vide supra_), steps 1 to 10.

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

3. Pour plates of nutrient agar with similar quantities of the water and incubate at 37 deg. C.

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

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

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 Oedematis 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 deg. 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 deg. 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 deg. 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.

TESTING FILTERS.

Porcelain filter candles are examined with reference to their power of holding back _all_ the micro-organisms suspended in the fluids which are filtered through them, and permitting only the passage of germ-free filtrates. In order to determine the freedom of the filter from flaws and cracks which would permit the passage of bacteria no matter how perfect the general structure of the candle might be, the candle must first be attached by means of a long piece of pressure tubing, to a powerful pump, such as a foot bicycle pump, fitted with a manometer. The candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump. Any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it.

The examination for permeability is conducted as follows:

_Apparatus Required_:

Filtering apparatus: The actual filter candle that is used
must be the one it is intended to test and must be
previously carefully sterilised; the arrangement of the
apparatus will naturally vary with each different form of
filter, one or other of those already described (_vide_
pages 42-48).

Plate-levelling stand.

Case of sterile plates.

Case of sterile pipettes, 10 c.c. (in tenths).

Case of sterile pipettes, 1 c.c. (in tenths).

Tubes of nutrient gelatine.

Flask containing sterile normal saline solution.

Sterile measuring flask, 1000 c.c. capacity.

METHOD.--

1. Prepare surface cultivations, on nutrient agar in a culture bottle, of the Bacillus mycoides, and incubate at 20 deg. C., for forty-eight hours.

2. Pipette 5 c.c. sterile normal saline into the culture bottle and emulsify the entire surface growth in it.

3. Pipette the emulsion into the sterile measuring flask and dilute up to 1000 c.c. by the addition of sterile water.

4. Pour the emulsion into the filter reservoir and start the filtration.

5. When the filtration is completed, pour six agar plates each containing 1 c.c. of the filtrate.

6. Incubate at 37 deg. C. until, if necessary, the completion of seven days.

7. If the filtrate is not sterile, subcultivate the organism passed and determine its identity with the test bacterium before rejecting the filter--since the filtrate may have been accidentally contaminated.

8. If the filtrate is sterile, resterilise the candle and repeat the test now substituting a cultivation of B. prodigiosus--a bacillus of smaller size.

9. If the second test is satisfactory, test the candle against a cultivation of a very small coccus, e. g., Micrococcus melitensis, in a similar manner; in this instance continuing the incubation of cultivations from the filtrate for fourteen days.

TESTING OF DISINFECTANTS.

Methods have already been detailed (page 310) for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides. But it frequently happens that the bacteriologist has to determine the relative efficiency of "disinfectants" from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view. In pursuing this line of investigation, it is convenient to compare the efficiency, under laboratory conditions, of the proposed disinfectant with that of some standard germicide, such as pure phenol. In so doing, and in order that the work of different observers may be compared, conditions as nearly uniform as possible should be aimed at. The method described is one that has been in use by the writer for many years past, modified recently by the adoption of some of the recommendations of the Lancet Commission on the Standardisation of Disinfectants--particularly of the calculation for determining the phenol coefficient.

This method has many points in common with that modification of the "drop" method known as the Rideal-Walker test.

~General Considerations.~--

These may be grouped under three headings: Test Germ, Germicide, and Environment.

1. _Test Germ._--~B. coli.~

As disinfectants are tested for sanitary purposes, it is obvious that a member of the coli-typhoid group should be selected as the test germ. B. coli is selected on account of its relative nonpathogenicity, the ease with which it can be isolated and identified by different observers in various parts of the world, the stability of its fundamental characters, and evenness of its resistance when utilised for these tests; finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group, a margin of safety is introduced into the test which certainly enhances its value.

B. coli should be recently isolated from a normal stool, and plated at least twice to ensure the purity of the strain; and a stock agar culture prepared which should be used throughout any particular test. For any particular experiment prepare a smear culture on agar and incubate at 37 deg. C. for 24 hours anaerobically. Then emulsify the whole of the surface growth in 10 c.c. of sterile water. Transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion. Transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads. Pipette off the supernatant emulsion for use in the test.

_2. Germicide._--

_a. Disinfectant to be tested._--

The first essential point is to test the unknown disinfectant, which may be referred to as germicide-x, on the lines set out on page 311 to determine its inhibition coefficient.

This constant having been fixed, prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods, commencing with a solution somewhat stronger than that representing the inhibition coefficient. The solutions must be prepared in fairly large bulk, not less than 5 c.c. of the disinfectant being utilised for the preparation of any given percentage solution.

_b. Standard Control._--~Phenol.~

The standard germicide used for comparison should be one which is not subject to variation in its chemical composition, and the one which has obtained almost universal use is Phenol.

The following table shows the effect of different percentages of carbolic acid upon B. coli for varying contact times, compiled from an experiment conducted under the standard conditions referred to under Environment. The results closely correspond to those recorded by the Lancet Commission on Disinfectants, 1909.

---------------------+----------------------------------- | Contact time in minutes. Percentage of phenol +------+---+---+---+---+---+---+---- | 2-1/2| 5 |10 | 15| 20| 25| 30| 35 ---------------------+------+---+---+---+---+---+---+---- 1.20 | - | - | - | - | - | - | - | - 1.10 | - | - | - | - | - | - | - | - 1.0 | + | - | - | - | - | - | - | - 0.9 | + | - | - | - | - | - | - | - 0.85 | + | + | - | - | - | - | - | - 0.80 | + | + | + | - | - | - | - | - 0.75 | + | + | + | + | + | - | - | - 0.7 | + | + | + | + | + | + | - | - 0.65 | + | + | + | + | + | + | + | - ---------------------+------+---+---+---+---+---+---+----

- = No growth, i. e., bacteria killed. + = Growth, i. e., bacteria still living.

From this it will be seen that the following percentage solutions will need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.

Prepare solutions of varying percentages by weighing out the quantity of carbolic acid required for each and dissolving in 100 c.c. of pure distilled water in an accurately standardised measuring flask. The solutions must be prepared freshly as required each day.

~Environment.~--

_a. General._--

Close the windows and doors of the laboratory in which the investigation is carried out, to avoid draughts. Flush over the work bench and adjacent floor with 1:1000 solution of corrosive sublimate. Caution the assistant, if one is employed, to avoid unnecessary movement or speech.

_b. Contact Temperature_, ~15-18 deg. C.~--

This is the temperature at which contact between the germicide and the test germ takes place, and is of importance, since some germicides (_e. g._, Phenol) appear to be more powerful at high temperatures. 18 deg. C.--practically the ordinary room temperature--is a temperature at which the multiplication of B. coli is a comparatively slow process, but variation of a degree above this temperature or of two or three degrees below is of no moment. If the room temperature is below 15 deg. C. when the experiments are in progress, arrange a water-bath regulated at 18 deg. C. for the reception of the tubes containing the mixture of germ and germicide; if above 19 deg. C. immerse the tubes in cold water, to which small pieces of ice are added from time to time to prevent the temperature rising above 18 deg. C.

_c. Relative Proportional Bulk of Test Germ and Germicide_, ~50:1.~--

Five cubic centimetres is a convenient amount of germicidal solution to employ, and to this 0.1 c.c. of the emulsion of test germ should be added.

_d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the Time Periods_, ~0.1 c.c.~--

This is sufficient to afford a fair sample of the germ content of the mixture, and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium.

_e. Subculture Medium._ ~Bile Salt Broth.~--

A _fluid_ medium is essential in order to obtain immediate dilution of the germicide carried over; at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation, and if possible one which affords characteristic cultural appearances.

Bile Salt Broth (page 180) combines these desiderata; it permits only the growth of intestinal bacteria, whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living B. coli.

The amount of medium present in each test-tube is a matter of importance, since the medium not only provides pabulum for the test germ, but also acts as a diluent to the germicide, to reduce its strength below its inhibition coefficient. For routine work each subculture tube contains 10 c.c. of medium, but it is obvious that if germicide-x possesses an inhibition coefficient of 0.1 per cent. the addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality. Hence the preliminary tests may in some instances indicate the necessity for the presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture tubes.

_f. Incubation Temperature_, ~37 deg. C.~--

_g. Observation Period of the Subcultivations_, ~Seven Days.~--

In order to determine whether or no the test germs have been destroyed, observations must always be continued--when growth appears to be absent--up to the end of seven days before recording "no growth."

_h. Identification of the Organisms Developing in the Subcultivations after Contact in the Germ + Germicide Solution._--

This is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments. The sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place.

METHOD.--

_Apparatus Required_:

Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).

Test-tube rack (Fig. 219).

Sterile graduated pipettes in case, 1 c.c. (in tenths).

Sterile graduated pipettes in case, 5 c.c. (in c.c.).

Circular rubber washers, 2.5 cm. diameter with central hole,
sterilised by boiling immediately before use, then
transferred to sterilised glass double dish.

Electric signal clock or stop watch.

Sterile forceps.

Sterilised glass beads.

Shaking machine.

Grease pencil.

_Material Required_:

Percentage solutions of germicide-x (_vide_ page 481).

Percentage solutions of pure phenol (_vide_ page 482).

Aqueous emulsion of B. coli (_vide_ page 481).

Tubes of bile salt broth.

~Preliminary Tests.~--

_a. Inhibition Coefficient._--

Determine the lowest percentage of germicide-x which inhibits growth of B. coli in the bile salt broth, and the highest percentage which fails to inhibit (page 311). On the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip. Assuming the inhibition coefficient to be 1:1000, it will be quite safe to employ the ordinary culture tubes containing 10 c.c. medium in the subsequent experiments.

_b. Trial Trip._--

Determine the lethal effect of a series of five solutions of germicide-x (say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25 and 30 minutes in the following manner:

1. Arrange five test-tubes marked A to E in the lower tier of the test-tube rack.

2. Into tube A pipette 5 c.c. germicide-x 1:100 solution.

Into tube B pipette 5 c.c. germicide-x 1:200 solution.

Into tube C pipette 5 c.c. germicide-x 1:300 solution.

Into tube D pipette 5 c.c. germicide-x 1:500 solution.

Into tube E pipette 5 c.c. germicide-x 1:600 solution.

3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.

4. Place a square wire basket of about 50 tubes capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.

5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole.

6. Put down the forceps on the bench with the sterile points projecting over the edge. Without taking the tube from the rack remove the cotton-wool plug from tube A, and lower the pipette, with the rubber washer affixed, on to the open mouth of the tube; with the help of the forceps to steady the washer, push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube (see fig. 219).

7. Adjust in the same way a pipette and a washer in the mouth of each of the other tubes, B, C, D and E.

8. Set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.

9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by.

10. As soon as the bell rings lift the pipette from tube A with the left hand and from the charged pipette held in the right hand deliver 0.1 c.c. of B. coli emulsion into the 1:100 solution. Then replace the pipette and washer.

11. Raise the tube with the left hand and shake it to mix germ and germicide, whilst returning the delivery pipette in the right hand.

12. Repeat the process with tubes B, C, D and E; then drop the infected delivery pipette in the lysol jar. The inoculation of the five tubes can be carried out very expeditiously, but a period of 10 seconds must be allowed for each tube.

13. When the bell rings at 2-1/2 minutes blow through the pipette in tube A (this agitates the germ + germicide mixture and ensures the collection of a fair sample); allow the mixture to enter the pipette, and as the column of fluid extends well above the terminal graduation, the right forefinger adjusted over the butt-end of the pipette before it is lifted will retain more than 0.1 c.c. of the mixture within the bore when the point of the pipette is clear of the fluid in the tube. Touch the point of the pipette on the inner wall of the tube, and allow any excess of fluid to escape, only retaining 0.1 c.c. in the pipette.

14. At the same time, with the left hand remove Bile Salt Tube No. 1 from the upper tier of the rack, take out the cotton-wool plug with the hand already holding the pipette (the relative positions of pipette, plug and culture tubes being practically the same as those of platinum loop, plug and culture tube shown in Fig. 68, page 74).

15. Insert the point of the pipette into the subculture tube, and blow out the mixture into the medium--replug the tube and drop it into the wire basket. Replace the washer-pipette in tube A.

As soon as the point of the pipette has entered the mouth of tube A it may be released, since it has already been so adjusted that it just clears the bottom of the test-tube, and the elastic washer will prevent any damage to the tube.

Steps 13, 14 and 15 occupy on an average 10 seconds.

16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and E.

17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30 minutes.

18. Place all the inoculated tubes in the incubator at 37 deg. C.

19. Examine the tubes at intervals of 24 hours, and record the results in tabular form as shown in Table page 491 (the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of B. coli first appeared).

20. If a consideration of the tabulated results indicates strengths of Germicide-x lethal at 2-1/2 and 30 minutes the final test can be arranged, but if this result has not been attained, sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information.

~Final Test.~--

c. _Determination of Phenol Coefficient._--

_X-Disinfectant._--This comprises two distinct tests, one of the Germicide-x, the other of the standard phenol.

1. Arrange five test-tubes clearly marked in the lower tier of the rack.