13. Pour plates from the agar tubes; label, and incubate at 37° 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° 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° 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° C. to 30° C. and after five days the "total number of organisms developing at 28° 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.
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, fæcal débris, 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 C1 and C2.
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 D1 and D2.
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 (C1) and deposit (D1) 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 (D2) with 10 c.c. sterile bouillon and inoculate three tubes of bile salt broth as follows:
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:
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 Choleræ.—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. Diphtheriæ.—
(A) 1. Plant three sets of serial cultivations, twelve tubes in each set, from (a) cream C2, (b) deposit D1 upon oblique inspissated 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.
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 toxæmia, 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-Lœffler 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 (D1 or D2).
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.
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 D2 into each of three tubes of glucose formate bouillon, and incubate anaerobically (in Buchner's tubes) for twenty-four hours at 37° 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. diphtheriæ 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° 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 D1 or D2.
2. Spread several sets of surface plates upon nutrose agar, each from one drop of the original milk sample.
3. Incubate aerobically at 37° 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.
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° 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).
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:
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° C. until the contents are liquid.
3. Liquefy eight tubes of gelatine-agar and place them in the water-bath at 42° 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.,
5. Pour a plate cultivation from each of the gelatine-agar tubes and incubate at 28° 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° 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° 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° 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° 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.).
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.
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° 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° 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:
Pour plates from these tubes and incubate at 20° C.
5. Prepare a precisely similar set of agar plates and incubate at 37° C.
6. Pipette 5 c.c. of the extract into a sterile tube, heat in the differential steriliser at 80° 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° C. and 20° 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:
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.
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:
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° 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° C. for ten minutes, and incubate under anaerobic conditions at 37° C. Examine for the presence of B. enteritidis sporogenes as directed under Water Examination, page 438 (steps 7-10).
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° 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).
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.