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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
the bottle in the vertical position .
The apparatus being arranged is lowered to the required depth, a sharp jerk is then given to the suspending cord, which detaches the rubber tube and so opens the two glass tubes. Water enters through the longer tube and the air is expelled through the shorter tube. The bubbles of air can be seen or heard rising through the water, until the bottle is nearly full, a small volume of compressed air remaining in the neck of the bottle.
As the apparatus is raised, the air thus imprisoned expands, and prevents the entry of more water from nearer the surface.
On reaching the laboratory, the method of examination consists in adding measured quantities of the water sample to several tubes of nutrient media previously liquefied by heat, pouring plate cultivations from each of these tubes, incubating at a suitable temperature, and finally counting the colonies which make their appearance on the plates.
Plate-levelling stand. Case of sterile plates. Case of sterile pipettes, 1 c.c. . Case of sterile pipettes, 10 c.c. . Case of sterile capsules, 25 c.c. capacity. Tubes of nutrient gelatine. Tubes of nutrient agar. Tubes of wort gelatine. One 250 c.c. flask of sterile distilled water. Tall cylinder containing 2 per cent. lysol solution. Bunsen burner. Grease pencil. Water-bath regulated at 42? C.
METHOD.--
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.
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.
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.
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, 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 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:
This is attained by so arranging the environment, as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes.
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.
Incubator running at 42? 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 . Flask of double strength bile salt broth . Tubes of litmus silk. Sterile flasks, 250 c.c. capacity. Buchner's tubes. Tabloids pyrogallic acid. Tabloids sodium hydrate. Bunsen burner. Grease pencil.
Incubator running at 37? C. Surface plates of nutrose agar . Aluminium spreader. Tubes of various media, including carbohydrate media. Agglutinating sera, etc.
METHOD.--
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.
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? C. destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria.
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.
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.
B. coli communis. B. coli communior. B. lactis aerogenes. B. cloacae.
Bacillus enteritidis . B. paratyphosus A. B. paratyphosus B. Bacillus cholerae suum.
B. typhosus. B. dysenteriae . B. dysenteriae . B. faecalis alcaligines.
If a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity 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. , 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.
DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP
Transcriber's note: Table split to fit 80 spaces.
Table Notes:
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.
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 .
Pressure tubing, clamps, pinch-cock.
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