The Elements of Bacteriological Technique Part 116

[Ill.u.s.tration: FIG. 212.--Electrically driven centrifugal machine, with flexible (broken) spindle encircled by the field magnets of the motor.]

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 gla.s.s tubes are inserted will obviate any risk of breakage to the latter.

[Ill.u.s.tration: FIG. 213.--Milk sedimenting tubes.]

[Ill.u.s.tration: FIG. 214.--Milk in centrifuge tube.]

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 ma.s.s, 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 advent.i.tious 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 ma.s.ses 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 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 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 C.

for ten minutes and then incubate anaerobically at 37 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 insp.i.s.sated blood-serum, and incubate at 37 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.