In order to avoid fictitious accuracy and yet to express the numerical
results by a method consistent with the precision of the work, the
numbers of colonies of bacteria per cubic centimeter shall be recorded
as follows:[212]
Number of bacteria per cc.
From 1 to 50 shall be recorded as found
" 51 " 100 " " " to the nearest 5
" 101 " 250 " " " " " " 10
" 251 " 500 " " " " " " 25
" 501 " 1,000 " " " " " " 50
" 1,001 " 10,000 " " " " " " 100
" 10,001 " 50,000 " " " " " " 500
" 50,001 " 100,000 " " " " " " 1,000
" 100,001 " 500,000 " " " " " " 10,000
" 500,001 " 1,000,000 " " " " " " 50,000
" 1,000,001 " 10,000,000 " " " " " " 100,000
This applies to the gelatin count at 20° C. and to the agar count at 37°
C.
SUMMARY OF STEPS INVOLVED IN MAKING PRESUMPTIVE, PARTIALLY CONFIRMED AND
COMPLETED TESTS FOR B. COLI.
────────────────────────────────────────────────────────────┬─────────
Steps in procedure. │ Further
│procedure
│required.
────────────────────────────────────────────────────────────┼─────────
I. Inoculate lactose broth fermentation tubes; incubate 24 │
hours at 37° C.; observe gas-formation in each tube. │
1. Gas-formation, 10 per cent. or more; constitutes │
positive presumptive test. │
(a) For other than smallest portion of any sample │
showing gas at this time, and for all portions, │
including smallest, of sewage and raw water this│
test is sufficient. │None
(b) For smallest gas-forming portion, except in │
examinations of sewage and raw water. │III
2. Gas-formation less than 10 per cent. in 24 hours; │
inconclusive. │II
II. Incubate an additional 24 hours, making a total of 48 │
hours’ incubation; observe gas-formation. │
1. Gas-formation, any amount; constitutes doubtful │
test, which must always be carried further. │III
2. No gas-formation in 48 hours; constitutes final │
negative test. │None
III. Make plate from smallest gas-forming portion of sample │
under examination; incubate 18 to 24 hours; observe │
colonies. │
1. One or more colonies typical in appearance. │
(a) If only “partially confirmed” test is required│None
(b) If completed test is required, select two │
typical colonies for identification. │V
2. No typical colonies. │IV
IV. Replace plate in incubator for an additional 18 to 24 │
hours; then, whether colonies appear typical or not, │
select at least two of those which most nearly resemble B.│
coli. │V
V. Transfer each colony fished to: │
1. Lactose broth fermentation tube; incubate not more │
than 48 hours at 37° C. Observe gas-formation. │None
2. Agar slant; incubate 48 hours at 37° C. │
(a) If gas formed in lactose broth tube inoculated│
with corresponding culture │VI
(b) If no gas formed in corresponding lactose │
broth tube, test is completed and negative. │None
VI. Make stained cover-slip or slide preparation, and │
examine microscopically. │
1. If preparation shows non-spore-forming bacilli in │
apparently pure culture, demonstration of B. coli is │
completed. │None
2. If preparation fails to show non-spore-forming │
bacilli or shows them mixed with spore-bearing forms │
or bacteria of other morphology. │VII
VII. Replate, to obtain assuredly pure culture, select │
several colonies of bacilli and repeat steps V and VI. │
────────────────────────────────────────────────────────────┴─────────
In order that tests for B. coli may have quantitative significance, the
following general principles and rules should be observed:
Ordinarily not less than three portions of each sample should be tested,
the portions being even decimal multiples or fractions of a cubic
centimeter; for example, 10 cc., 1 cc., 0.1 cc., .01 cc., etc. It is
essential that the dilutions should be such that the largest amount
gives a positive test (unless the water is such as to give negative
tests in 10 cc.), and the smallest dilution, a negative result. To
insure this result, it is often necessary to plant four or five
dilutions, especially in the examination of a sample of entirely unknown
quality. The quantitative value of a series of tests is lost, unless all
or at least a large proportion of the smallest dilutions tested have
given negative results.
In reporting a single test, it is preferable merely to record results as
observed, indicating the amounts tested and the result in each, rather
than to attempt expression of the result in numbers of B. coli per cc.
In summarizing the results of a series of tests, however, it is
desirable, for the sake of simplicity, to express the results in terms
of the numbers of B. coli per cc., or per 100 cc. To convert results of
fermentation tests to this form, the result of each test is recorded as
indicating a number of B. coli per cc. equal to the reciprocal of the
smallest decimal or multiple fraction of a cubic centimeter giving a
positive result. For example, the result: 10 cc. +; 1 cc. +; 0.1 cc. -;
would be recorded as indicating one B. coli per cc. An exception should
be made in the case where a negative result is obtained in an amount
larger than the smallest portion giving a positive result; for example,
in a result such as: 10 cc. +; 1 cc. -; 0.1 cc. +. In such case, the
result should be recorded as indicating a number of B. coli per cc.
equal to the reciprocal of the dilution next larger than the smallest
one giving a positive test, this being a more probable result.
Where tests are made in amounts larger than 1 cc., giving average
results less than one B. coli per cc., it is more convenient to express
results in terms of the numbers of B. coli per 100 cc.
The following table illustrates the method of recording and averaging
results of B. coli tests:
Result of Tests in Amounts Designated. Indicated Number of B.
coli.
10 cc. 1 cc. 0.1 cc. .01 cc. per cc. per 100 cc.
+ − − − 0.1 10.
+ + − − 1.0 100.
+ + + − 10.0 1,000.
+ + + + 100.0 10,000.
+ + − + 10.0 1,000.
————— —————
Totals (for estimating averages) 121.1 12,110.
Average of 5 tests 24.0 2,422.
The above method of expressing results is not mathematically altogether
correct. The average number of B. coli per cc., as thus estimated, is
not precisely the most probable number calculated by application of the
theory of probability.[220] To apply this theory to a correct
mathematical solution of any considerable series of results involves,
however, mathematical calculations so complex as to be impracticable of
application in general practice. The simpler method given is therefore
considered preferable, since it is easily applied and the results so
expressed are readily comprehensible.
In order that results as reported may be checked and carefully valuated,
it is necessary that the report should show not only the average number
of B. coli per cc., but also the number of samples examined; and, for
each dilution, the total number of tests made, and the number (or per
cent.) positive.