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The foregoing cultures are called primary cultures. They are incubated for 48 hours at 37 deg. C.
The primary cultures which, after incubation for 48 hours at 37 deg. C., fail to develop "gas" are discarded
because this negative result conclusively proves the absence from such cultures of glucose fermenting coli-like microbes,
of any kind. On the other hand, the occurrence of gas in the primary cultures constitutes the prima facie or presumptive
test for B. coli, and indicates the necessity for making from the liquid primary cultures secondary cultures on solid
media. Surface gelatine plate cultures are, I think, the best for this purpose. Usually, it is advisable to inoculate one
to three loopfuls of the primary culture into 10 c.c. of sterile water, and then to spread one to three loopfuls of this
inoculated water over the surface of a gelatine plate by means of a sterile glass rod previously bent to an angle of
about 45 deg. at one end. The gelatine plates are incubated for two days at 20 deg. C., and the coli-like colonies (filmy
in preference) sub-cultured into gelatine and "shake" cultures made. If "gas" is developed in the gelatine " shake
culture without any liquefaction of the gelatine within the first few days, the micro-organism is classed as a coli-like
microbe. Further tests are then applied, and if these yield negative results the symbol 0 is applied to the microbe in
question. The further tests are—
Neutral red broth cultures (for greenish yellow fluorescence, symbol "fl").
Lactose peptone cultures (for acid and gas formation, symbol "ag").
Broth cultures (for indol formation, symbol "in").
Litmus milk cultures (for acidity and clot, symbol "ae ").
If a microbe fulfils all these tests, it is termed a "flaginac" B. coli. Failure to yield a positive result as regards
any of the tests is indicated by the omission of the corresponding letters. For example, a "flagac" B. coli would
mean a B. coli typical except as regards indol production. A "weakness" of attribute is indicated by placing the
corresponding letters in brackets. For example, a flag(in)(ac) B. coli would mean B. coli typical except that the
attributes of indol production and acid clotting of milk were feebly displayed.
The original gelatine "shake" culture is kept preferably for one month for observation as regards liquefaction.
If slow liquefaction occurs, the letters S.L. are appended. [L.Y. = a liquefying chromogonic (yellow) microbe].
Reverting to the stage of the process at which the primary cultures representing the first "washings" of the
mixture of cress and water have been made: The next step is to pour off the remaining water in the flask, to add fresh
clean water, shake the flask, then pour off the water, again to add fresh water and again to pour off the water. Repeat
this procedure several times; then add to the flask sterile water, shake the flask and pour off the water. This should be
repeated several times. Finally add 900 c.c. of sterile water to the flask, Bhake it repeatedly, and proceed on exactly the
same lines as before to examine the liquid (called the final "washings"). During the process of washing, any simple
device, such as holding a piece of sterile wire gauze over the mouth of the flask will prevent loss of small floating pieces
of cress.
As the final "washings" liquid is examined in exactly the same way as the first washings, it is unnecessary to
repeat what has been already described.
Mention has only been made so far of the B. coli tests, but of course the first and final "washings" of the cress
may be examined by the streptococcus, B. enteritidis sporogenes, or other tests.
(B)—The results, as regards subsequent isolation of B. coli or coli-like microbes, of the addition of cress in known
quantities directly to liquid culture media both before and also after prolonged washing.
Several methods have been tried, but the following will be found satisfactory:—
Unwashed cress.
(a).—Weigh out 1 gramme of cress, cut it up into small pieces with a sterile pair of scissors, and drop the
pieces with a sterile pair of forceps into a wide tube (8 in. by 1J in.) containing at least 30 c.c. of bile-salt glucose
peptone medium. Incubate for two days at 37 deg. C.; and if there is aeid and gas production make secondary
plate cultures and proceed on the lines explained in the preceding paragraphs. It is desirable to make several
cultures.
(b).—As above, but weigh out 10 grammes instead of 1 gramme of cress, use 100 instead of 30 c.c. of bilesalt
glucose peptone medium and a wider tube (8 in. by 1£ in.). It is desirable to make several cultures.
Washed cress.
(c).—Weigh out 1 gramme of cress and affix a thin strip of sterile sheet lead for sinking purposes.
(d).—Weigh out 10 grammes of cress and fasten the pieces together with a thin strip of sterile sheet lead.
In either case wash first in clean water and finally in sterile water, then cut up the cress with sterile scissors
and add the pieces to culture tubes with a sterile pair of forceps, as previously explained. Incubate at 37 deg. C., and
proceed as under (a), (6).
(C).—Comparison as regards the B coli test between the leaf and the stalk of cress.
With a sterile pair of scissors and a pair of forceps separate the leaves from the stalk.
(а).—Introduce 1 gramme by weight of the leaves into a wide-mouthed tube containing 30 c.c. of bile-salt
glucose peptone medium.
(б).—Ditto with the stalk.
(c).—As in (a), but weigh out 10 grammes.
(d).—As in (b), do.
Some experiments were also carried out in which 1 leaf, 10 (leaves, and 100 leaves were severally introduced into
wide-mouthed tubes (e, f, g), containing each 30 c.c. of bile-salt glucose peptone medium.
Cultures (a, b, c, d, e, f, g) are incubated at 37 deg. C. If no gas develops the results are negative ; if gas formation
occurs, secondary plate cultures are made in the ordinary way.