15. Examine the growth in each tube carefully both macroscop_The elements of bacteriological technique

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 |

+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+

| B. coli communis | + | + + | + + | + + | + + | + + | O | + + | + + |

| B. coli communior | + | + + | + + | + + | + + | + + | + + | + + | + + |

| B. lactis aerogenes | - | + + | + + | + + | + + | + + | O | O | + + |

| B. acidi lactici | - | + + | + + | + + | + + | + + | O | O | O |

| B. pneumoniae | - | + + | + + | + + | + + | + + | + + | + + | + + |

| B cloaceae(A) | + | + + | + + | + + | + + | + + | + + | + + | + + |

| | | | | | | | | | |

| 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 |

| | | | | | | | | | |

| 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 | | | |

+-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+

| 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 |

| | | | | | | | | | |

| 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 | + + | + + | + + | - | + | - |

| | | | | | | | | | |

| 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.

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.

[Illustration: Sample form]]

[Illustration: FIG. 209.--Water filtering apparatus. That portion of the

figure to the left of the vertical line is drawn to a larger scale than

that on the right, in order to show details of Sprengel's pump.]

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.

[Illustration: FIG. 210. Sterile test-tube brush.]

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.

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.

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.

[Illustration: FIG. 211.--Milk-collecting bottle and dipper in case.]

Quantitative.--

_Collection of Sample._--

15. Examine the growth in each tube carefully both macroscopically and