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Read Ebook: The Elements of Bacteriological Technique A Laboratory Guide for Medical Dental and Technical Students. Second Edition Rewritten and Enlarged. by Eyre J W H John William Henry
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Ebook has 1212 lines and 48649 words, and 25 pages
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 .
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 .
Twelve Buchner's tubes with rubber stoppers.
Pyrogallic acid tablets.
Caustic soda tablets.
METHOD.--
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.
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.--
No. 4 0.05 c.c. . No. 5 0.125 c.c. . No. 6 0.25 c.c. . No. 7 0.5 c.c. . No. 8 1.0 c.c. . No. 9 2.5 c.c. .
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.
The Carbolic Method:
Parietti's Method:
Elsner's Method: This method simply consists in substituting Elsner's potato gelatine for ordinary nutrient gelatine in any of the previously mentioned methods.
Cambier's Candle Method:
Treat a large volume of the water sample by the concentration method .
B. ~Enteritidis Sporogenes.~--
Acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation.
Presence of clotting, and the separation of clear whey.
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.
~Vibrio Cholerae.~--
~B. Anthracis.~--
~B. Tetani.~--
EXAMINATION OF MILK.
"One-cow" or "whole" milk, if taken from the apparently healthy animal 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 of samples of fore-milk, mid-milk, 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 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:
To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol , 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? C. for thirty minutes undergoes no alteration in color; if heated to 75? C. for ten minutes only, the brick red color appears after standing for about two minutes.
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.
Quantitative.--
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 , 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 .
When collecting a 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.
Case of sterile capsules . Case of sterile graduated pipettes, 10 c.c. . Case of sterile graduated pipettes, 1 c.c. . 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? C. Bunsen burner. Grease pencil.
METHOD.--
NOTE.--Many observers prefer to employ gelatine agar 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.
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.
A large centrifugal machine. This machine, to be of real service in the bacteriological examination of milk, must conform to the following requirements:
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.
The handle employed should be provided with a special fastening , 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.
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