Chapter XIV: Appendix: 492 (12)

26. Calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days--the presence of liquefying bacteria may render the calculation necessary at an earlier date, hence the importance of daily observations.

_Method of Counting._--The most accurate method of counting the colonies on each of the plates is by means of either Jeffery's or Pakes' counting disc. Each of these discs consists of a piece of paper, upon which is printed a dead black disc, subdivided by concentric circles and radii, printed in white. In Jeffery's counter (Fig. 207), each subdivision has an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii divide the circle into sixteen equal sectors, and counting is facilitated by concentric circles equidistant from the centre.

(a) In the final counting of each plate, place the plate over the counting disc, and centre it, if possible, making its periphery coincide with one or other of the concentric circles.

(b) Remove the cover of the plate, and by means of a hand lens count the colonies appearing in each of the sectors in turn. Make a note of the number present in each.

(c) If the colonies present are fewer than 500, the entire plate should be counted. If, however, they exceed this number, enumerate one-half, or one-quarter of the plate, or count a sector here and there, and from these figures estimate the number of colonies present on the entire plate. In practice it will be found that Pakes' disc is more suitable for the former class of plate; Jeffery's disc for the latter. It should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas--since where the medium is thick all the bacteria will develop, where the layer is a thin one, only a few bacteria will find sufficient pabulum for the production of visible colonies.

It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. Again the counts of the plates in a measure control each other; for example, the second and third plates of each gelatine series should together contain as many colonies as the first, and the second should contain about half as many more than the third and so on.

2. Qualitative Examination.--

_Collection of Sample._--The water sample required for the routine examination, which it will be convenient to consider first, amounts to about 110 c.c. It is collected in the manner previously described (_vide_ page 416); similar bottles are used, and if four are filled the combined contents, amounting to about 240 c.c., will provide ample material for both the qualitative and quantitative examinations. Unless the examination is to be commenced at once, the ice-box must be employed, otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution, and so increase the difficulties of the investigation.

In the routine examination of water supplies it is customary to limit the qualitative examination to a search for

A. B. coli and its near allies.

B. Streptococci,

organisms which are frequently spoken of as microbes of indication, as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary canal, and so to constitute a danger signal.

C. Some observers still attach importance to the presence of B. enteritidis sporogenes, but as the search for this bacterium, (relatively scarce in water) necessitates the collection of a fairly large quantity of water it is not usually included in the routine examination.

In the case of water samples examined during the progress of an epidemic, of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group; and on occasion the question of the presence or absence of Vibrio cholerae or (more rarely) such bacteria as B. anthracis or B. tetani, may need investigation.

When pathogenic or excremental bacteria are present in water, their numbers are relatively few, owing to the dilution they have undergone, and it is usual in commencing the examination, to adopt one or other of the following methods:

A. _Enrichment_, in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited, whilst the growth of the parasitic bacteria is encouraged.

This is attained by so arranging the environment, (i. e., Media, incubation temperature, and atmosphere) as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes.

B. _Concentration_, whereby all the bacteria present in the sample of water, pathogenic or otherwise, are concentrated in a small bulk of fluid.

This is usually effected by filtration of the water sample through a porcelain filter candle, and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quantity of sterile bouillon.

A. ~Enrichment Method.~

(Dealing with the demonstration of bacteria of intestinal origin.)

_Apparatus Required_ (_Preliminary Stage_):

Incubator running at 42 deg. C.
Case of sterile pipettes, 1 c.c. graduated in tenths.
Case of sterile pipettes, 10 c.c. graduated in c.c.
Case of sterile pipettes, graduated to deliver 25 c.c.
Tubes of bile salt broth (_vide_ page 180).
Flask of double strength bile salt broth (_vide_ page 199).
Tubes of litmus silk.
Sterile flasks, 250 c.c. capacity.
Buchner's tubes.
Tabloids pyrogallic acid.
Tabloids sodium hydrate.
Bunsen burner.
Grease pencil.

(_Later stage_):

Incubator running at 37 deg. C.
Surface plates of nutrose agar (see page 232).
Aluminium spreader.
Tubes of various media, including carbohydrate media.
Agglutinating sera, etc.

METHOD.--

1. Number a set of bile salt broth, tubes 1-5, and a duplicate set 1a-5a.

2. Number one flask 7 and another 8.

3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.

To Tubes No. 2 and 2a add 1 c.c. water sample.

To Tubes No. 3 and 3a add 2 c.c. water sample.

To Tubes No. 4 and 4a add 5 c.c. water sample.

To Tubes No. 5 and 5a add 10 c.c. water sample.

4. Put up all the tubes in Buchner's tubes and incubate anaerobically at 42 deg. C.

NOTE.--The bile salt medium is particularly suitable for the
cultivation of bacteria of intestinal origin, and at the
same time inhibits the growth of bacteria derived from other
sources.

The anaerobic conditions likewise favor the multiplication of intestinal bacteria, and also their fermentative activity. The temperature 42 deg. C. destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria.

5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and 50 c.c. double strength bile salt broth into flask 7.

6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample into flask 7.

7. Incubate the two flasks aerobically at 42 deg. C.

8. After twenty-four hours incubation note in each culture:

a. The presence or absence of visible growth.

b. The reaction of the medium as indicated by the colour change, if any, the litmus has undergone.

c. The presence or absence of gas formation, as indicated by a froth on the surface of the medium, and the collection of gas in the inner "gas" tube.

9. Replace those tubes which show no signs of growth in the incubator. Examine after another period of twenty-four hours (total forty-eight hours incubation) with reference to the same points.

10. Remove culture tubes which show visible growth from the Buchner's tubes, whether acid production and gas formation are present or not.

11. Examine all tubes which show growth by hanging-drop preparations. Note such as show the presence of chains of cocci.

12. Prepare surface plate cultivations upon nutrose agar from each tube that shows growth either macroscopically or microscopically, and incubate for twenty-four hours aerobically at 37 deg. C.

13. Examine the growth on the plates either with the naked eye or with the help of a small hand lens. Practice will facilitate the recognition of colonies of the coli group, the typhoid group and the paratyphoid group; also those due to the growth of streptococci. The investigation from this stage proceeds along two divergent lines of enquiry--the first being concerned with the identity of the bacilli--typhoid bacilli, the second with that of the cocci.

A. _B. Coli and its allies._

14. Pick off coliform or typhiform colonies; make streak or smear subcultivations upon nutrient agar; incubate aerobically for twenty-four hours at 37 deg. C.

15. Examine the growth in each tube carefully both macroscopically and microscopically. If the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. When the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it.

16. Utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below, using the ordinary loop to make the subcultures upon solid media, but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette.

Gelatine streak.
Agar streak.
Potato.
Nutrient broth.
Litmus milk.
Dextrose peptone solution.
Laevulose peptone solution.
Galactose peptone solution.
Maltose peptone solution.
Lactose peptone solution.
Saccharose peptone solution.
Raffinose peptone solution.
Dulcite peptone solution.
Mannite peptone solution.
Glycerin peptone solution.
Inulin peptone solution.
Dextrin peptone solution.

17. Differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of:

I. The Escherich Group.

B. coli communis.
B. coli communior.
B. lactis aerogenes.
B. cloacae.

II. The Gaertner Group.

Bacillus enteritidis (of Gaertner).
B. paratyphosus A.
B. paratyphosus B.
Bacillus cholerae suum.

III. The Eberth Group.

B. typhosus.
B. dysenteriae (Shiga).
B. dysenteriae (Flexner).
B. faecalis alcaligines.

18. Confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals.

If a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity (down to 0.1 c.c.) of the sample that contains at least one of the microbes of indication. For instance, if growth occurs in all the tubes from 4 to 10, and that growth is subsequently proved to be due to the multiplication of B. coli, then it follows that at least one colon bacillus is present in every 10 c.c. of the water sample, but not in every 5 c.c. If, on the other hand, the presence of the B. coli can only be proved in flask No. 7, then the average number of colon bacilli present in the sample is at least one in every 50 c.c. (i. e., twenty per litre), but not one in every 25 c.c. and so on.

The general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema--whilst the full differential details are set out in tabular form.

ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID GROUPS.

Nutrose agar.
|
-----------------------------------
| |
Red colonies. Blue colonies.
Escherich group. Gaertner and Eberth groups.
|| |
====================---------------
||
Lactose peptone solution.
||
====================---------------
|| |
Gas. No gas.
|| |
B. coli communis and its allies. |
|| Gaertner and Eberth groups.
Acid and gas in glucose peptone solution. |
Acid and coagulation in milk. |
General turbidity and indol in bouillon. Glucose peptone solution.
|
==================================|
|| |
|| |
Gas. No gas.
|| |
Gaertner group. Eberth group.
|| |
=================== ----------------
|| || | |
|| || | |
Litmus milk. Peptone solution. Litmus milk. Peptone solution.
|| || | |
Acid at first. General turbidity. Acid. General turbidity.
Alkaline later. No indol. No coagulation. No indol.
No coagulation. Serum reaction. Serum reaction.

_B. Streptococci._

19. Pick off streptococcus colonies and subcultivate upon nutrient agar exactly as directed in steps 14, 15 and 16.

20. Differentiate the streptococci isolated into members of the saprophytic group of short-chained cocci, or members of the parasitic (pathogenic) group of long-chained cocci, by means of their cultural characters, and record their numerical frequency in the manner indicated for the members of the coli-typhoid group.

DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP

Transcriber's note: Table split to fit 80 spaces.

+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | | M | D | L | G | M | L | S | R | D | |A = acid reaction | o | e | a | a | a | a | a | a | e | |G = gas formation | t | x | e | l | l | c | c | f | t | | | i | t | v | a | t | t | c | f | r | | | l | r | u | c | o | o | h | i | i | | | i | o | l | t | s | s | a | n | n | | | t | s | o | o | e | e | r | o | | | | y | e | s | s | | | o | s | | | | | | e | e | | | s | e | | | | | | | | | | e | | | | | +-----+-----+-----+-----+-----+-----+-----+-----+ | | | A G | A G | A G | A G | A G | A G | A G | A G | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ |_The Escherich Group._ | | | | | | | | | | | B. coli communis | + | + + | + + | + + | + + | + + | O | + + | + + | | B. coli communior | + | + + | + + | + + | + + | + + | + + | + + | + + | | B. lactis aerogenes | - | + + | + + | + + | + + | + + | O | O | + + | | B. acidi lactici | - | + + | + + | + + | + + | + + | O | O | O | | B. pneumoniae | - | + + | + + | + + | + + | + + | + + | + + | + + | | B cloaceae(A) | + | + + | + + | + + | + + | + + | + + | + + | + + | | | | | | | | | | | | |_The Gaertner Group._ | | | | | | | | | | | B. enteritidis | + | + + | + + | + + | + + | O | O | O | O | | B. paratyphosus A | + | + + | + + | + + | + + | O | O | O | O | | B. paratyphosus B | + | + + | + + | + + | + + | O | O | O | O | | B. cholerae suum | + | + + | + + | + + | + + | O | O | | O | | B. suipestifer | + | + + | + + | + + | + + | O | O | | O | | | | | | | | | | | | |_The Eberth Group._ | | | | | | | | | | | B. typhosus | + | + | + | + | + | O | O | O | + | | B. dysenteriae (Shiga) | - | + | + | + | O | O | O | O | O | | B. dysenteriae (Flexner)| - | + | + | + | + | O | O | +/- | O | | B. faecalis alkaligines | + | O | O | O | O | O | O | O | O | | | | | | | | | | | | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | Table Notes: |(B)| (C) | +-------------------------+---+-----------------------------------------------+

+-------------------------+-----+-----+-----+-----+-----+-----+---+-----------+ | | I | S | G | D | M | S | I |Litmus Milk| |A=acid reaction | n | a | l | u | a | o | n | | |G=gas formation | u | l | y | l | n | r | d | | | | l | i | c | c | n | b | o +-----+-----+ | | i | c | e | i | i | i | l |Early|Late | | | n | i | r | t | t | t | | | | | | | n | i | e | e | e | | | | | | | | n | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |-----+-----+-----+-----+-----+-----+ | | | | | A G | A G | A G | A G | A G | A G | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ |_The Escherich Group_ | | | | | | | | | | | B. coli communis | O | O | + + | + + | + + | + + | + | + | + C | | B. coli communior | O | O | + + | + + | + + | + + | + | + | + C | | B. lactis aerogenes | O | O | O | O | + + | + + | - | + | + C | | B. acidi lactici | O | O | O | + + | + + | + + | + | + | + C | | B. pneumoniae | O | O | + + | + + | + + | + + | - | + | + C | | B cloaceae[A] | O | O | + + | O | + + | - + | + | + | + C | | | | | | | | | | | | |_The Gaertner Group._ | | | | | | | | | | | B. enteritidis | O | O | O | + + | + + | + + | - | +/- | - | | B. paratyphosus A | O | +/- | O | + + | + + | + + | - | + | O | | B. paratyphosus B | O | O | O | + + | + + | + + | - | + | - | | B. cholerae suum | O | O | O | O | O | + + |+/-| + | - | | B. suipestifer | O | O | O | + + | + + | + + | - | + | - | | | | | | | | | | | | |_The Eberth Group._ | | | | | | | | | | | B. typhosus | O | O | O | O | + | + | - | + | + | | B. dysenteriae (Shiga) | O | O | O | O | O | O | - | + | - | | B. dysenteriae (Flexner)| O | O | O | O | + | O |+/-| + | - | | B. faecalis alkaligines | O | O | O | O | O | O | - | - | - | | | | | | | | | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ | Table Notes: | |(D)| (E) | +-------------------------+-----------------------------------+---+-----------+

Table Notes:

(A) * Liquefies gelatine.

(B) + = motile. - = non-motile.

(C) + = acid or gas production. +/- = slight acid production. O = no change.

(D) + = indol production. +/- = slight indol production. - = no indol formed.

(E) + = acid production. - = alkali production. O = no change in reaction. C = clot.

21. Determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated.

On the facing insert page is reproduced a blank from the author's Laboratory Water Analysis Book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined.

B. ~Concentration Method.~

The remaining organisms referred to on page 426 are more conveniently sought for by the concentration method.

_Collection of the Sample._--The quantity of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue glass Winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. The bottle may be packed in a wooden box or in an ordinary wicker case. The method of collecting the sample is identical with that described under the heading of Quantitative Examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory.

_Apparatus required_:

Sterile Chamberland or Doulton "white" porcelain open mouth
filter candle, fitted with rubber washer.

Rubber cork to fit mouth of the filter candle, perforated
with one hole.

Kitasato serum flask, 2500 c.c. capacity.

Geryk air pump or water force pump.

Wulff's bottle, fitted as wash-bottle, and containing
sulphuric acid (to act as a safety valve between filter and
pump).

Pressure tubing, clamps, pinch-cock.

Retort stand, with ring and clamp.

Rubber cork for the neck of Winchester quart, perforated
with two holes and fitted with one 6 cm. length of straight
glass tubing, and one V-shaped piece of glass tubing, one
arm 32 cm. in length, the other 52 cm., the shorter arm
being plugged with cotton-wool. The rubber stopper must be
sterilised by boiling and the glass tubing by hot air,
before use.

Flask containing 250 c.c. sterile broth.

Test-tube brush to fit the lumen of the candle, enclosed in
a sterile test-tube (and previously sterilised by dry heat
or by boiling).

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

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

Case of sterile pipettes, 1 c.c. in hundredths.

Tubes of various nutrient media (according to requirements).

Twelve Buchner's tubes with rubber stoppers.

Pyrogallic acid tablets.

Caustic soda tablets.

]

METHOD.--

1. Fit up the filtering apparatus as in the accompanying diagram (Fig. 209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle.

2. Filter the entire 2000 c.c. of water through the filter candle.

3. When the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing.

4. Reverse the position of the glass tubes in the Wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid.

5. Slowly open the metal clamps and allow air to gradually pass through the acid, and enter filter flask, and so restore the pressure.

6. Unship the apparatus, remove the cork from the mouth of the candle.

7. Pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (Fig. 210) emulsify the slimy residue which lines the candle, with the broth.

Practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (Some bacteria will of course be left behind on the walls of the filter and in its pores.)

Up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria.

The Coli-Typhoid Group.--

1. Number nine tubes of bile salt broth (_vide_ page 180), consecutively from 1 to 9.

2. To No 1 add 1 c.c. } of the original water sample
2 add 2 c.c. } before the nitration is commenced.
3 add 5 c.c. }

3. To the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes, as follows:

No. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample). No. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample). No. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample). No. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample). No. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample). No. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample).

4. Put up each tube anaerobically in a Buchner's tube and incubate at 42 deg. C.

5. The subsequent steps are identical with those described under the Enrichment method (see page 428 to 431; Steps 8 to 18).

_Alternative Methods._--

A few of the older methods for the isolation of the members
of the coli-typhoid groups are referred to but they are
distinctly inferior to those already described.

(A) The Carbolic Method:

1. Take ten tubes of carbolised bouillon (_vide_ page 202)
and number them consecutively from 1 to 10.

2. Inoculate each tube with a different amount of the water
sample or suspension, as in the previous method.

3. Incubate aerobically at 37 deg. C.

4. Examine the culture tubes after twenty-four hours'
incubation.

5. From those tubes which shows signs of growth, pour plates
in the usual manner, using carbolised gelatine (_vide_ page
202) in place of the ordinary gelatine, and incubate at 20 deg.
C. for three, four, or five days as may be necessary.

6. Subcultivate from any colonies that make their
appearance, and determine their identity on the lines laid
down in the previous method.

(B) Parietti's Method:

1. Take nine tubes of Parietti's bouillon (_vide_ page
202)--i. e., three each of those containing 0.1 c.c., 0.2
c.c., and 0.5 c.c. of Parietti's solution respectively.
Mark plainly on the outside of each tube the quantity of
Parietti's solution it contains.

2. To each tube add a different amount of the original
water, or of the suspension, and incubate at 37 deg. C.

3. Examine the culture tubes after twenty-four and
forty-eight hours' incubation, and plate in nutrient
carbolised or potato gelatine from such as have grown.

4. Pick off suspicious colonies, if any such appear on the
plates, subcultivate them upon the various media, and
identify them.

(C) Elsner's Method: This method simply consists in
substituting Elsner's potato gelatine (_vide_ page 204) for
ordinary nutrient gelatine in any of the previously
mentioned methods.

(D) Cambier's Candle Method:

Treat a large volume of the water sample by the
concentration method (_vide_ page 434).

1. Remove the rubber stopper from the mouth of the filter
candle, introduce 10 c.c. sterile bouillon into its
interior, and emulsify the bacterial sediment; replug the
mouth of the candle with a wad of sterile cotton-wool.

2. Remove the filter candle from the filter flask and insert
it into the mouth of a flask or a glass cylinder containing
sterile bouillon sufficient to reach nearly up to the rubber
washer on the candle.

3. Incubate for twenty-four to thirty-six hours at 37 deg. C.

4. From the now turbid bouillon in the glass cylinder pour
gelatine plates and incubate at 20 deg. C.

5. Subcultivate and identify any suspicious colonies that
appear.

(The method depends upon the assumption that members of the
typhoid and coli groups find their way through the porcelain
filter from the interior to the surrounding bouillon at a
quicker rate than the associated bacteria.)

B. ~Enteritidis Sporogenes.~--

1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80 deg. C. for twenty minutes.

3. Number ten tubes of litmus milk consecutively from 1 to 10.

4. Remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid.

5. To each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group (_vide_ page 437).

6. Incubate each tube anaerobically at 37 deg. C. Anaerobic conditions can be obtained by putting the cultures up in Buchner's tubes or in Bulloch's apparatus. If, however, whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis; whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid.

7. Examine after twenty-four hours' incubation. Note (if B. enteritidis sporogenes is present)--

(a) Acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation.

(b) Presence of clotting, and the separation of clear whey.

(c) Presence of gas, as indicated by fissures and bubbles in the coagulum, and possibly masses of coagulum driven up the tube almost to the plug.

8. Replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points.

9. Remove those tubes which give evidence of growth from the Buchner's tubes and carefully pipette off the whey; examine the whey microscopically.

10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the whey and observe the result.

~Vibrio Cholerae.~--

1. Number ten tubes of peptone water consecutively from 1 to 10.

2. To each of the tubes of peptone water add a measured quantity of the suspension, corresponding to those amounts employed in isolating the members of the coli group (_vide_ page 437).

3. Incubate aerobically at 37 deg. C. for twenty-four hours. Examine the tubes carefully for visible growth, especially delicate pellicle formation, which if present should be examined microscopically for vibrios, both by stained preparations or by fresh specimens with dark ground illumination.

4. Inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation--from the pellicle itself--and incubate at 37 deg. C. for twenty-four hours.

5. Test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated H_{2}SO_{4}.

5. Prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation.

6. Pick off from the plates any colonies resembling those of the Vibrio cholerae and subcultivate upon all the ordinary laboratory media.

7. Test the vibrio isolated against the serum of an animal immunised to the Vibrio cholerae for agglutination.

~B. Anthracis.~--

1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80 deg. C. for twenty minutes.

3. Inoculate a _young_ white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. Observe during life, and, if the animal succumbs, make a complete post-mortem examination.

4. Melt three tubes of nutrient agar in boiling water and cool to 42 deg. C.

5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates therefrom.

6. Incubate at 37 deg. C. for twenty-four or forty-eight hours.

7. Pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media.

8. Inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe during life, and if the animal succumbs, make a complete post-mortem examination.

~B. Tetani.~--

1. Proceed as detailed above in steps 1 and 2 for the isolation of the B. anthracis.

2. Add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in Buchner's tubes at 37 deg. C.

3. From such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. Observe carefully during life, and, if death occurs, make a complete post-mortem examination.

4. From the same tubes pour agar plates and incubate anaerobically in Bulloch's apparatus, at 37 deg. C.

5. Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths.

EXAMINATION OF MILK.

"One-cow" or "whole" milk, if taken from the apparently healthy animal (that is, an animal without any obvious lesion of the udder or teats) with ordinary precautions as to cleanliness, avoidance of dust, etc., contains but few organisms. In dealing with one-cow milk, from a suspected, or an obviously diseased animal, a complete analysis should include the examination (both qualitative and quantitative) of samples of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible, from each quarter of the udder. "Mixed" milk, on the other hand, by the time it leaves the retailer's hands, usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such.

It is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed 5000 micro-organisms per cubic centimetre. Such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation (heating the milk to 65 deg. C. for twenty minutes or to 77 deg. C. for one minute) or the simpler plan of adding preservatives to the milk. Information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples, and in this connection the following tests will be found useful:

1. _Raw Milk_ (Saul).

To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol (ortho-methyl-amino-phenol sulphate), recently prepared and mix. Next add 0.2 c.c. of a 3 per cent. peroxide of hydrogen solution. The appearance of a brick red color within 30 seconds indicates raw milk. Milk heated to 74 deg. C. for thirty minutes undergoes no alteration in color; if heated to 75 deg. C. for ten minutes only, the brick red color appears after standing for about two minutes.

2. _Boric Acid._

Evaporate to dryness, 50 c.c. of the milk which has been rendered slightly alkaline to litmus, then incinerate.

Dissolve in distilled water, add slight excess of dilute hydrochloric acid and again evaporate to dryness.

Dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution. Dry the turmeric paper. _Rose_ or _cherry-red_ color = borax or boric acid.

3. _Formaldehyde_ (Hehner).

To 10 c.c. milk in a test tube add 5 c.c. concentrated _commercial_ sulphuric acid slowly, so that the two fluids do not mix. Hold the tube vertically and agitate very gently. _Violet zone_ at the junction of the two liquids = formaldehyde.

4. _Hydrogen Peroxide._

To 10 c.c. milk (diluted with equal quantities of water) in a test tube add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2 c.c. acetic acid. _Blue coloration_ of the mixture = hydrogen peroxide.

5. _Salicylic Acid._

Precipitate the caseinogen by the addition of acetic acid and filter. To the filtrate add a few drops of 1 per cent. aqueous solution of ferric chloride. _Purple coloration_ = salicylic acid.

6. _Sodium Carbonate or Bicarbonate._

To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3 c.c. of a 1 per cent. alcoholic solution of rosolic acid. _Brownish_ color = pure milk; _rose_ color = preserved milk.

Quantitative.--

_Collection of Sample._--

The apparatus used for the collection of a retail mixed milk sample consists of a cylindrical copper case, 16 cm. high and 9 cm. in diameter, provided with a "pull-off" lid, containing a milk dipper, also made of copper; and inside this, again, a wide-mouthed, stoppered glass bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter), having a tablet for notes, sand-blasted on the side. The copper cylinder and its contents, secured from shaking by packing with cotton-wool, are sterilised in the hot-air oven (Fig. 26).

When collecting a sample,

1. Remove the cap from the cylinder.

2. Draw out the cotton-wool.

3. Lift out the bottle and dipper together.

4. Receive the milk in the sterile dipper, and pour it directly into the sterile bottle.

5. Enter the particulars necessary for the identification of the specimen, on the tablet, with a lead pencil, or pen and ink.

6. Pack the apparatus in the ice-box for transmission to the laboratory in precisely the same manner as an ordinary water sample.

"Whole" milk may with advantage be collected in the sterile bottle directly since the mouth is sufficiently wide for the milker to direct the stream of milk into it.

~Condensed milk~ must be diluted with sterile distilled water in accordance with the directions printed upon the label, then treated as ordinary milk.

_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.
Case of sterile plates.
Tubes nutrient gelatine or gelatine agar.
Tubes of wort gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42 deg. C.
Bunsen burner.
Grease pencil.

METHOD.--

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

2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon into each of the three remaining capsules.

3. Remove 1 c.c. milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule I; mix thoroughly by repeatedly filling and emptying the pipette.

4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the contents of capsule II, and mix as before.

5. 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. of dilution I contains 0.1 c.c. milk sample.
1 c.c. of dilution II contains 0.001 c.c. milk sample.
1 c.c. of dilution III contains 0.00001 c.c. milk sample.
1 c.c. of dilution IV contains 0.0000001 c.c. milk sample.

6. Melt the gelatine and the agar tubes in boiling water; then transfer to the water-bath and cool them down to 42 deg. C.

7. Number the gelatine tubes consecutively 1 to 12.

8. Inoculate the tubes with varying quantities of the material as follows:

To tube No. 1 add 1.0 c.c. of the milk sample.
2 add 0.1 c.c. of the milk sample.
{ 3 add 1.0 c.c. from capsule I.
{ 4 add 0.1 c.c. from capsule I.
{ 5 add 1.0 c.c. from capsule II.
{ 6 add 0.1 c.c. from capsule II.
{ 7 add 0.5 c.c. from capsule III.
{ 8 add 0.3 c.c. from capsule III.
{ 9 add 0.2 c.c. from capsule III.
{ 10 add 0.5 c.c. from capsule IV.
{ 11 add 0.3 c.c. from capsule IV.
{ 12 add 0.2 c.c. from capsule IV.

9. Pour plates from the gelatine tubes; label, and incubate at 20 deg. C.

10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the milk sample and a similar quantity of the diluted milk from capsules I, II, and III and IV respectively.

11. Pour plates from the wort gelatine; label, and incubate at 20 deg. C.

12. Inoculate the liquefied agar tubes as follows:

To tube No. 1 add 0.1 c.c. of the milk sample.
2 add 0.1 c.c. from capsule I.
3 add 0.1 c.c. from capsule II.
4 add 0.1 c.c. from capsule III.
5 add 1.0 c.c. from capsule IV. }
6 add 0.1 c.c. from capsule IV. }

13. Pour plates from the agar tubes; label, and incubate at 37 deg. C.

14. After twenty-four hours' incubation "inspect," and after forty-eight hours' incubation, "count" the agar plates and estimate the number of "organisms growing at 37 deg. C." present per cubic centimetre of the sample of milk.

15. After three, four, or five days' incubation, "count" the gelatine plates and estimate therefrom the number of "organisms growing at 20 deg. C." present per cubic centimetre of the sample of milk.

16. After a similar interval "count" the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk.

NOTE.--Many observers prefer to employ gelatine agar (see
page 193) for the quantitative examination. In this case
gelatine-agar plates should be poured from tubes containing
the quantities of material indicated in step 8, incubated at
28 deg. C. to 30 deg. C. and after five days the "total number
of organisms developing at 28 deg. C." recorded.

~Qualitative.~--The qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency.

Members of the Coli-typhoid group.
Vibrio cholerae.
Streptococcus pyogenes longus.
Micrococcus melitensis.
Staphylococcus pyogenes aureus.
Bacillus enteritidis sporogenes.
Bacillus diphtheriae.
Bacillus tuberculosis.

Some of these occur as accidental contaminations, either from the water supply to the cow farm, or from the farm employees, whilst others are derived directly from the cow.

In milk, as in water examinations, two methods are available, viz.: Enrichment and Concentration--the former is used for the demonstration of bacteria of intestinal origin, the latter for the isolation of the micro-organisms of diphtheria and tubercle. The first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compass; but in the case of milk, thorough centrifugalisation is substituted for filtration.

_Apparatus Required_:

A large centrifugal machine. This machine, to be of real
service in the bacteriological examination of milk, must
conform to the following requirements:

1. The centrifugal machine must be of such size, and should
carry tubes or bottles of such capacity, as to enable from
200 to 500 c.c. of milk to be manipulated at one time.

2. The rate of centrifugalisation should be from 2500 to
3000 revolutions per minute.

3. The portion of the machine destined to carry the tubes
should be a metal disc, of sufficient weight to ensure good
"flank" movement, continuing over a considerable period of
time. In other words, the machine should run down very
gradually and slowly after the motive power is removed, thus
obviating any disturbance of the relative positions of
particulate matter in the solution that is being
centrifugalised.

4. The machine should preferably be driven by electricity,
or by power, but in the case of hand-driven machines--

(a) The gearing should be so arranged that the requisite
speed is obtained by not more than forty or fifty
revolutions of the crank handle per minute, so that it may
be maintained for periods of twenty or thirty minutes
without undue exertion.

(b) The handle employed should be provided with a special
fastening (e. g., a clutch similar to that employed for
the free wheel of a bicycle), or should be readily
detachable so that, on ceasing to turn, the handle should
not, by its weight and air resistance, act as a brake and
stop the machine too suddenly.

One of the few satisfactory machines of this class is shown
in figure 212.

Sterile centrifugal tubes, of some 60-70 c.c. capacity,
tapering to a point at the closed end, plugged with
cotton-wool.

Small centrifugal machine to run two tubes of 10 c.c.
capacity at 2500 to 3000 revolutions per minute preferably
driven by electricity, of the type figured on page 327 (Fig.
162).

Sterile centrifugal tubes of 10 c.c. capacity with the
distal extremity contracted to a narrow tube and graduated
in hundredths of a cubic centimetre (Fig. 213).

Sterilised cork borer.

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

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

Sterile teat pipettes.

Flask of sterile normal saline solution.

METHOD.--

1. Fill 50 c.c. of the milk sample into each of four tubes, and replace the cotton-wool plugs by solid rubber stoppers (sterilised by boiling), and fit the tubes in the centrifugal machine.

NOTE.--One or two cubic centimetres of paraffinum liquidum
introduced into the buckets of the centrifuge before the
glass tubes are inserted will obviate any risk of breakage
to the latter.

2. Centrifugalise the milk sample for thirty minutes at a speed of 2500 revolutions per minute.

3. Remove the motive power and allow the machine to slow down gradually.

4. Remove the tubes of milk from the centrifuge. Each tube will now show (Fig. 214):

(a) A superficial layer of cream (varying in thickness with different samples) condensed into a semi-solid mass, which can be shown to contain some organisms and a few leucocytes.

(b) A central layer of separated milk, thin, watery, and opalescent, and containing extremely few bacteria.

(c) A sediment or deposit consisting of the great majority of the contained bacteria and leucocytes, together with adventitious matter, such as dirt, hair, epithelial cells, faecal debris, etc.

5. Withdraw the rubber stopper and remove a central plug of cream from each tube by means of a sterile cork borer; place these masses of cream in two sterile capsules. Label C^{1} and C^{2}.

6. Remove all but the last one or two c.c. of separated milk from each tube, by means of sterile pipettes.

7. Mix the deposits thoroughly with the residual milk, pipette the mixture from each pair of tubes into one sterile 10 c.c. tube (graduated) by means of sterile teat pipettes, then fill to the 10 c.c. mark with sterile normal saline solution and mix together. Label D^{1} and D^{2}.

8. Place the two tubes of mixed deposit in the centrifuge, adjust by the addition or subtraction of saline solution so that they counterpoise exactly, and centrifugalise for ten minutes.

NOTE.--Each tube now contains the deposit from 100 c.c. of
the milk sample and the amount can be read off in hundredths
of a centimetre. The multiplication of this figure by 100
will give the amount of "Apparent Filth," in "parts per
million"--the usual method of recording this quality of
milk.

9. Pipette off all the supernatant fluid and invert the tube to drain on to a pad of sterilised cotton-wool, contained in a beaker. (This wool is subsequently cremated.)

10. Examine both cream (C^{1}) and deposit (D^{1}) microscopically--

(a) In hanging-drop preparations.

(b) In film preparations stained carbolic methylene-blue, by Gram's method, by Neisser's method, and by Ziehl-Neelsen's method.

Note the presence or absence of altered and unaltered vegetable fibres; pus cells, blood discs; cocci in groups or chains, diphtheroid bacilli, Gram negative bacilli or cocci, spores and acid fast bacteria.

11. Adapt the final stages of the investigation to the special requirements of each individual sample, thus:

~1. Members of the Coli-typhoid Group.~--

1. Emulsify the deposit from the second centrifugal tube (D^{2}) with 10 c.c. sterile bouillon and inoculate three tubes of bile salt broth as follows:

To Tube No. 1 add 2.5 c.c. milk deposit emulsion
(=25 c.c. original milk.)
To Tube No. 2 add 1.0 c.c. milk deposit emulsion
(=10 c.c. original milk.)
To Tube No. 3 add 0.5 c.c. milk deposit emulsion
(= 5 c.c. original milk.)

2. Inoculate tube of bile salt broth No. 4 with 1 c.c. of the original milk.

3. Inoculate further tubes of bile salt broth with previously prepared dilutions (see page 445) as follows:

To tube No. 5 add 1.0 c.c. from capsule I.
To tube No. 6 add 0.1 c.c. from capsule I.
To tube No. 7 add 1.0 c.c. from capsule II.
To tube No. 8 add 0.1 c.c. from capsule II.
To tube No. 9 add 1.0 c.c. from capsule III.
To tube No. 10 add 0.1 c.c. from capsule III.
To tube No. 11 add 1.0 c.c. from capsule IV.
To tube No. 12 add 0.1 c.c. from capsule IV.

and incubate anaerobically (in Buchner's tubes) at 42 deg. C. for a maximum period of forty-eight hours.

4. If growth occurs complete the investigation as detailed under the corresponding section of water examination (see pages 428 to 431).

NOTE.--The B. coli communis, derived from the alvine
discharges of the cow, is almost universally present in
large or small numbers, in retail milk. Its detection,
therefore, unless in enormous numbers, (when it indicates
want of cleanliness), is of little value.

~2. Vibrio Cholerae.~--Inoculate tubes of peptone water by using the same amounts as in the search for members of the Coli-typhoid groups (_vide ante_ 1-3); incubate aerobically at 37 deg. C. and complete the examination as detailed under the corresponding section of water examination (see page 439).

~3. B. Enteritidis Sporogenes.~--Inoculate tubes of litmus milk with similar amounts to those used in the previous searches, omitting tube No. 1 (_vide ante_ 1-3) place in the differential steriliser at 80 deg. C. for ten minutes and then incubate anaerobically at 37 deg. C. for a maximum period of forty-eight hours. Complete the investigation as detailed under the corresponding section of water examination (see page 438).

~4. B. Diphtheriae.~--

(A) 1. Plant three sets of serial cultivations, twelve tubes in each set, from (a) cream C^{2}, (b) deposit D^{1} upon oblique inspissated blood-serum, and incubate at 37 deg. C.

2. Pick off any suspicious colonies which may have made their appearance twelve hours after incubation, examine microscopically and subcultivate upon blood-serum and place in the incubator; return the original tubes to the incubator.

3. Repeat this after eighteen hours' incubation.

4. From the resulting growths make cover-slip preparations and stain carbolic methylene-blue, Neisser's method, Gram's method. Subcultivate such as appear to be composed of diphtheria bacilli in glucose peptone solution. Note those in which acid production takes place.

5. Inoculate guinea-pigs subcutaneously with one or two cubic centimetres forty-eight-hour-old glucose bouillon cultivation derived from the first subcultivation of each glucose fermenter, and observe the result.

6. If death, apparently from diphtheritic toxaemia, ensues, inoculate two more guinea pigs with a similar quantity of the lethal culture. Reserve one animal as a control and into the other inject 1000 units of antidiphtheritic serum. If the control dies and the treated animal survives, the proof of the identity of the organism isolated with the Klebs-Loeffler bacillus becomes absolute.

7. Inoculate guinea-pigs subcutaneously with filtered glucose bouillon cultivations (toxins?) and observe the result.

(B) 1. Emulsify the remainder of the deposit with 5 c.c. sterile bouillon and inoculate two guinea-pigs, thus: guinea-pig a, subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with 2 c.c. emulsion; and observe the result.

2. If either or both of the inoculated animals succumb, make complete post-mortem examination and endeavour to isolate the pathogenic organisms from the local lesion. Confirm their identity as in A5 and 6 (_vide supra_).

~5. Bacillus Tuberculosis.~--

(A) 1. Inoculate each of three guinea-pigs (previously tested with tuberculin, to prove their freedom from spontaneous tuberculosis) subcutaneously at the inner aspect of the bend of the left knee, with 1 c.c. of the deposit emulsion remaining in one or other tube (D^{1} or D^{2}).

2. Introduce a small quantity of the cream into a subcutaneous pocket prepared at the inner aspect of the bend of the right knee of each of these three animals. Place a sealed dressing on the wound.

3. Observe carefully, and weigh accurately each day.

4. Kill one guinea-pig at the end of the second week and make a complete post-mortem examination.

5. If the result of the examination is negative or inconclusive, kill a second guinea-pig at the end of the third week and examine carefully.