---------------
dry soil weight
15. "Count" the plates after incubation for three, four, or five days,
and correcting the figures thus obtained by means of the "wet" to "dry"
soil ratio estimate--
(a) The number of aerobic micro-organisms present per gramme of the
soil.
(b) The number of yeasts and moulds present per gramme of the soil.
(c) The number of aerobic organisms "growing at 37 deg. C." present per
gramme of the soil.
(B) _Anaerobes (Vegetative Forms and Spores)._--
16. Pour similar sets of plates in glucose formate gelatine and agar and
incubate in Bulloch's anaerobic apparatus.
(C) _Aerobes and Anaerobes (Spores Only)._--
17. Pipette 5 c.c. soil water into a sterile tube.
18. Place in the differential steriliser at 80 deg. C. for ten minutes.
19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and
1 c.c. respectively of the soil water; label and incubate at 20 deg. C.,
one set aerobically, the other anaerobically in Bulloch's apparatus.
20. "Count" the plates (delay the enumeration as long as possible) and
estimate the number of spores of aerobes and anaerobes respectively
present per gramme of the soil.
21. Calculate the ratio existing between spores and spores + vegetative
forms under each of the two groups, aerobic and anaerobic
micro-organisms.
~Qualitative Examination.~--The qualitative examination of soil is usually
directed to the detection of one or more of the following:
Members of the Coli-typhoid group.
Streptococci.
Bacillus anthracis.
Bacillus tetani.
Bacillus oedematis maligni.
The nitrous organisms.
The nitric organisms.
1. Transfer the remainder of the soil water (88 c.c.) to a sterile
Erlenmeyer flask by means of a sterile syphon.
2. Fix up the filtering apparatus as for the qualitative examination of
water, and filter the soil water.
3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique
similar to that described for the water sample, _vide_ pages 434-436).
Every cubic centimetre of suspension now contains the soil water from
nearly 1 gramme of earth.
The methods up to this point are identical no matter which organism or
group of organisms it is desired to isolate; but from this stage onward
the process is varied slightly for each particular bacterium.
~I. The Coli-typhoid Group.~--
~II. Streptococci.~--
~III. Bacillus Anthracis.~--
~IV. Bacillus Tetani.~--
The methods adopted for the isolation of these organisms are identical
with those already described under water (page 437 _et seq._).
~V. Bacillus Oedematis Maligni.~--Method precisely similar to that
employed for the B. tetani.
~VI. The Nitrous Organisms.~--
1. Take ten tubes of Winogradsky's solution No I (_vide_ page 198) and
number them consecutively from 1 to 10.
2. Inoculate each tube with varying quantities of the material as
follows:
To tube No. 1 add 1.0 c.c. of the soil water.
To tube No. 2 add 0.1 c.c. of the soil water.
To tube No. 3 add 1.0 c.c. from Capsule I.
To tube No. 4 add 0.1 c.c. from Capsule I.
To tube No. 5 add 1.0 c.c. from Capsule II.
To tube No. 6 add 0.1 c.c. from Capsule II.
To tube No. 7 add 1.0 c.c. from Capsule III.
To tube No. 8 add 0.1 c.c. from Capsule III.
To tube No. 9 add 1.0 c.c. from Capsule IV.
To tube No. 10 add 0.1 c.c. from Capsule IV.
Label and incubate at 30 deg. C.
~VII. The Nitric Organisms.~--
3. Take ten tubes of Winogradsky's solution No II, number them
consecutively from 1 to 10 and inoculate with quantities of soil water
similar to those enumerated in section VI step 2. Label and incubate at
30 deg. C.
4. Examine after twenty-four and forty-eight hours' incubation. From
those tubes that show signs of growth make subcultivations in fresh
tubes of the same medium and incubate at 30 deg. C.
5. Make further subcultivations from such of those tubes as show growth,
and again incubate.
6. If growth occurs in these subcultures, make surface smears on plates
of Winogradsky's silicate jelly (_vide_ page 198).
7. Pick off such colonies as make their appearance and subcultivate in
each of these two media.
TESTING FILTERS.
Porcelain filter candles are examined with reference to their power of
holding back _all_ the micro-organisms suspended in the fluids which are
filtered through them, and permitting only the passage of germ-free
filtrates. In order to determine the freedom of the filter from flaws
and cracks which would permit the passage of bacteria no matter how
perfect the general structure of the candle might be, the candle must
first be attached by means of a long piece of pressure tubing, to a
powerful pump, such as a foot bicycle pump, fitted with a manometer. The
candle is then immersed in a jar of water and held completely submerged
whilst the internal pressure is gradually raised to two atmospheres by
the action of the pump. Any crack or flaw will at once become obvious by
reason of the stream of air bubbles issuing from it.
The examination for permeability is conducted as follows:
_Apparatus Required_:
Filtering apparatus: The actual filter candle that is used
must be the one it is intended to test and must be
previously carefully sterilised; the arrangement of the
apparatus will naturally vary with each different form of
filter, one or other of those already described (_vide_
pages 42-48).
Plate-levelling stand.
Case of sterile plates.
Case of sterile pipettes, 10 c.c. (in tenths).
Case of sterile pipettes, 1 c.c. (in tenths).
Tubes of nutrient gelatine.
Flask containing sterile normal saline solution.
Sterile measuring flask, 1000 c.c. capacity.
METHOD.--
1. Prepare surface cultivations, on nutrient agar in a culture bottle,
of the Bacillus mycoides, and incubate at 20 deg. C., for forty-eight
hours.
2. Pipette 5 c.c. sterile normal saline into the culture bottle and
emulsify the entire surface growth in it.
3. Pipette the emulsion into the sterile measuring flask and dilute up
to 1000 c.c. by the addition of sterile water.
4. Pour the emulsion into the filter reservoir and start the filtration.
5. When the filtration is completed, pour six agar plates each
containing 1 c.c. of the filtrate.
6. Incubate at 37 deg. C. until, if necessary, the completion of seven
days.
7. If the filtrate is not sterile, subcultivate the organism passed and
determine its identity with the test bacterium before rejecting the
filter--since the filtrate may have been accidentally contaminated.
8. If the filtrate is sterile, resterilise the candle and repeat the
test now substituting a cultivation of B. prodigiosus--a bacillus of
smaller size.
9. If the second test is satisfactory, test the candle against a
cultivation of a very small coccus, e. g., Micrococcus melitensis, in
a similar manner; in this instance continuing the incubation of
cultivations from the filtrate for fourteen days.
TESTING OF DISINFECTANTS.
Methods have already been detailed (page 310) for the purpose of
studying the vital resistance offered by micro-organisms to the lethal
effect of germicides. But it frequently happens that the bacteriologist
has to determine the relative efficiency of "disinfectants" from the
standpoints of the sanitarian and commercial man rather than from the
research worker's point of view. In pursuing this line of investigation,
it is convenient to compare the efficiency, under laboratory conditions,
of the proposed disinfectant with that of some standard germicide, such
as pure phenol. In so doing, and in order that the work of different
observers may be compared, conditions as nearly uniform as possible
should be aimed at. The method described is one that has been in use by
the writer for many years past, modified recently by the adoption of
some of the recommendations of the Lancet Commission on the
Standardisation of Disinfectants--particularly of the calculation for
determining the phenol coefficient.
This method has many points in common with that modification of the
"drop" method known as the Rideal-Walker test.
~General Considerations.~--
These may be grouped under three headings: Test Germ, Germicide, and
Environment.
1. _Test Germ._--~B. coli.~
As disinfectants are tested for sanitary purposes, it is obvious that a
member of the coli-typhoid group should be selected as the test germ. B.
coli is selected on account of its relative nonpathogenicity, the ease
with which it can be isolated and identified by different observers in
various parts of the world, the stability of its fundamental characters,
and evenness of its resistance when utilised for these tests; finally
since the colon bacillus is an organism which is slightly more
resistant to the lethal action of germicides than the more pathogenic
members of this group, a margin of safety is introduced into the test
which certainly enhances its value.
B. coli should be recently isolated from a normal stool, and plated at
least twice to ensure the purity of the strain; and a stock agar culture
prepared which should be used throughout any particular test. For any
particular experiment prepare a smear culture on agar and incubate at
37 deg. C. for 24 hours anaerobically. Then emulsify the whole of the
surface growth in 10 c.c. of sterile water. Transfer the emulsion to a
sterile test-tube with some sterile glass beads and shake thoroughly to
ensure homogenous emulsion. Transfer to a centrifuge tube and
centrifugalise the emulsion to throw down any masses of bacteria which
may have escaped the disintegrating action of the beads. Pipette off the
supernatant emulsion for use in the test.
_2. Germicide._--
_a. Disinfectant to be tested._--
The first essential point is to test the unknown disinfectant, which may
be referred to as germicide-x, on the lines set out on page 311 to
determine its inhibition coefficient.
This constant having been fixed, prepare various solutions of
germicide-x with sterilised distilled water by accurate volumetric
methods, commencing with a solution somewhat stronger than that
representing the inhibition coefficient. The solutions must be prepared
in fairly large bulk, not less than 5 c.c. of the disinfectant being
utilised for the preparation of any given percentage solution.
_b. Standard Control._--~Phenol.~
The standard germicide used for comparison should be one which is not
subject to variation in its chemical composition, and the one which has
obtained almost universal use is Phenol.
The following table shows the effect of different percentages of
carbolic acid upon B. coli for varying contact times, compiled from an
experiment conducted under the standard conditions referred to under
Environment. The results closely correspond to those recorded by the
Lancet Commission on Disinfectants, 1909.
---------------------+-----------------------------------
| Contact time in minutes.
Percentage of phenol +------+---+---+---+---+---+---+----
| 2-1/2| 5 |10 | 15| 20| 25| 30| 35
---------------------+------+---+---+---+---+---+---+----
1.20 | - | - | - | - | - | - | - | -
1.10 | - | - | - | - | - | - | - | -
1.0 | + | - | - | - | - | - | - | -
0.9 | + | - | - | - | - | - | - | -
0.85 | + | + | - | - | - | - | - | -
0.80 | + | + | + | - | - | - | - | -
0.75 | + | + | + | + | + | - | - | -
0.7 | + | + | + | + | + | + | - | -
0.65 | + | + | + | + | + | + | + | -
---------------------+------+---+---+---+---+---+---+----
- = No growth, i. e., bacteria killed.
+ = Growth, i. e., bacteria still living.
From this it will be seen that the following percentage solutions will
need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per
cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.
Prepare solutions of varying percentages by weighing out the quantity of
carbolic acid required for each and dissolving in 100 c.c. of pure
distilled water in an accurately standardised measuring flask. The
solutions must be prepared freshly as required each day.
~Environment.~--
_a. General._--
Close the windows and doors of the laboratory in which the investigation
is carried out, to avoid draughts. Flush over the work bench and
adjacent floor with 1:1000 solution of corrosive sublimate. Caution the
assistant, if one is employed, to avoid unnecessary movement or speech.
_b. Contact Temperature_, ~15-18 deg. C.~--
This is the temperature at which contact between the germicide and the
test germ takes place, and is of importance, since some germicides (_e.
g._, Phenol) appear to be more powerful at high temperatures. 18 deg.
C.--practically the ordinary room temperature--is a temperature at which
the multiplication of B. coli is a comparatively slow process, but
variation of a degree above this temperature or of two or three degrees
below is of no moment. If the room temperature is below 15 deg. C. when
the experiments are in progress, arrange a water-bath regulated at 18
deg. C. for the reception of the tubes containing the mixture of germ and
germicide; if above 19 deg. C. immerse the tubes in cold water, to which
small pieces of ice are added from time to time to prevent the
temperature rising above 18 deg. C.
_c. Relative Proportional Bulk of Test Germ and Germicide_, ~50:1.~--
Five cubic centimetres is a convenient amount of germicidal solution to
employ, and to this 0.1 c.c. of the emulsion of test germ should be
added.
_d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the
Time Periods_, ~0.1 c.c.~--
This is sufficient to afford a fair sample of the germ content of the
mixture, and at the same time is insufficient to exert any inhibitory
action when transferred to the subculture medium.
_e. Subculture Medium._ ~Bile Salt Broth.~--
A _fluid_ medium is essential in order to obtain immediate dilution of
the germicide carried over; at the same time it is advantageous to
employ a selective medium which favours the growth of the test germ to
the exclusion of organisms likely to contaminate the preparation, and
if possible one which affords characteristic cultural appearances.
Bile Salt Broth (page 180) combines these desiderata; it permits only
the growth of intestinal bacteria, whilst the formation of an acid
reaction and the production of gas in subcultures prepared from the
germ-germicide mixture is fairly complete evidence of the presence of
living B. coli.
The amount of medium present in each test-tube is a matter of
importance, since the medium not only provides pabulum for the test
germ, but also acts as a diluent to the germicide, to reduce its
strength below its inhibition coefficient. For routine work each
subculture tube contains 10 c.c. of medium, but it is obvious that if
germicide-x possesses an inhibition coefficient of 0.1 per cent. the
addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium
would effectually prevent the subsequent growth of the test germ after a
contact period insufficient to destroy its vitality. Hence the
preliminary tests may in some instances indicate the necessity for the
presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture
tubes.
_f. Incubation Temperature_, ~37 deg. C.~--
_g. Observation Period of the Subcultivations_, ~Seven Days.~--
In order to determine whether or no the test germs have been destroyed,
observations must always be continued--when growth appears to be
absent--up to the end of seven days before recording "no growth."
_h. Identification of the Organisms Developing in the Subcultivations
after Contact in the Germ + Germicide Solution._--
This is based on the naked eye characters of the growth in the bile salt
broth, supplemented where necessary by plating methods, further
subcultivations upon carbohydrate media and agglutination experiments.
The sign (+) is used to indicate that growth of the test organism
occurred in the subcultivations, and the sign (-) to indicate that the
test germs have been destroyed and no subsequent growth has taken place.
METHOD.--
_Apparatus Required_:
Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).
Test-tube rack (Fig. 219).
Sterile graduated pipettes in case, 1 c.c. (in tenths).
Sterile graduated pipettes in case, 5 c.c. (in c.c.).
Circular rubber washers, 2.5 cm. diameter with central hole,
sterilised by boiling immediately before use, then
transferred to sterilised glass double dish.
Electric signal clock or stop watch.
Sterile forceps.
Sterilised glass beads.
Shaking machine.
Grease pencil.
_Material Required_:
Percentage solutions of germicide-x (_vide_ page 481).
Percentage solutions of pure phenol (_vide_ page 482).
Aqueous emulsion of B. coli (_vide_ page 481).
Tubes of bile salt broth.
~Preliminary Tests.~--
_a. Inhibition Coefficient._--
Determine the lowest percentage of germicide-x which inhibits growth of
B. coli in the bile salt broth, and the highest percentage which fails
to inhibit (page 311). On the result of this experiment determine the
bulk of medium required in the subculture tubes and the percentage
solutions to be employed in the trial trip. Assuming the inhibition
coefficient to be 1:1000, it will be quite safe to employ the ordinary
culture tubes containing 10 c.c. medium in the subsequent experiments.
_b. Trial Trip._--
Determine the lethal effect of a series of five solutions of germicide-x
(say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25
and 30 minutes in the following manner:
1. Arrange five test-tubes marked A to E in the lower tier of the
test-tube rack.
2. Into tube A pipette 5 c.c. germicide-x 1:100 solution.
Into tube B pipette 5 c.c. germicide-x 1:200 solution.
Into tube C pipette 5 c.c. germicide-x 1:300 solution.
Into tube D pipette 5 c.c. germicide-x 1:500 solution.
Into tube E pipette 5 c.c. germicide-x 1:600 solution.
3. Arrange 20 tubes of bile salt broth in the upper tier of the
test-tube rack in two rows, those in the front row numbered
consecutively from left to right 1-10, those in the back row 11-20.
4. Place a square wire basket of about 50 tubes capacity close to the
left of the test-tube rack, for the reception of the inoculated tubes.
5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber
washer with forceps and push the point of the pipette into the central
hole.
6. Put down the forceps on the bench with the sterile points projecting
over the edge. Without taking the tube from the rack remove the
cotton-wool plug from tube A, and lower the pipette, with the rubber
washer affixed, on to the open mouth of the tube; with the help of the
forceps to steady the washer, push the pipette on through the hole until
the point of the pipette has reached to within a few millimetres of the
bottom of the tube (see fig. 219).
7. Adjust in the same way a pipette and a washer in the mouth of each of
the other tubes, B, C, D and E.
8. Set the electric signal clock to ring for the commencement of the
experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.
9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in
tenths of a cubic centimetre and stand by.
10. As soon as the bell rings lift the pipette from tube A with the left
hand and from the charged pipette held in the right hand deliver 0.1
c.c. of B. coli emulsion into the 1:100 solution. Then replace the
pipette and washer.
[Illustration: FIG. 219.--Test-tube rack.]
11. Raise the tube with the left hand and shake it to mix germ and
germicide, whilst returning the delivery pipette in the right hand.
12. Repeat the process with tubes B, C, D and E; then drop the infected
delivery pipette in the lysol jar. The inoculation of the five tubes can
be carried out very expeditiously, but a period of 10 seconds must be
allowed for each tube.
13. When the bell rings at 2-1/2 minutes blow through the pipette in
tube A (this agitates the germ + germicide mixture and ensures the
collection of a fair sample); allow the mixture to enter the pipette,
and as the column of fluid extends well above the terminal graduation,
the right forefinger adjusted over the butt-end of the pipette before it
is lifted will retain more than 0.1 c.c. of the mixture within the bore
when the point of the pipette is clear of the fluid in the tube. Touch
the point of the pipette on the inner wall of the tube, and allow any
excess of fluid to escape, only retaining 0.1 c.c. in the pipette.
14. At the same time, with the left hand remove Bile Salt Tube No. 1
from the upper tier of the rack, take out the cotton-wool plug with the
hand already holding the pipette (the relative positions of pipette,
plug and culture tubes being practically the same as those of platinum
loop, plug and culture tube shown in Fig. 68, page 74).
15. Insert the point of the pipette into the subculture tube, and blow
out the mixture into the medium--replug the tube and drop it into the
wire basket. Replace the washer-pipette in tube A.
As soon as the point of the pipette has entered the mouth of tube A it
may be released, since it has already been so adjusted that it just
clears the bottom of the test-tube, and the elastic washer will prevent
any damage to the tube.
Steps 13, 14 and 15 occupy on an average 10 seconds.
16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and
E.
17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30
minutes.
18. Place all the inoculated tubes in the incubator at 37 deg. C.
19. Examine the tubes at intervals of 24 hours, and record the results
in tabular form as shown in Table page 491 (the figures in the squares
indicate the number of hours at which the changes in the medium due to
the growth of B. coli first appeared).
20. If a consideration of the tabulated results indicates strengths of
Germicide-x lethal at 2-1/2 and 30 minutes the final test can be
arranged, but if this result has not been attained, sufficient evidence
will probably be available to enable a second trial test to be planned
which will give the required information.
~Final Test.~--
c. _Determination of Phenol Coefficient._--
_X-Disinfectant._--This comprises two distinct tests, one of the
Germicide-x, the other of the standard phenol.
1. Arrange five test-tubes clearly marked in the lower tier of the rack.
2. Pipette into each 5 c.c. respectively of the five percentage
solutions of x-disinfectant which the trial run has already shown will
include those affording lethal values at 2-1/2 and 30 minutes.
3. Arrange 20 tubes of bile salt broth in the upper tier of the
test-tube rack in two rows, those in the front row numbered
consecutively from left to right 1-10, those in the back row 11-20.
4. Arrange further 20 tubes of bile salt broth numbered 21-40 in two
rows in a second smaller rack which can be stood on the upper tier of
the rack as soon as the first 20 tubes have been inoculated.
5. Place a square wire basket of about 50 tube capacity close to the
left of the test-tube rack, for the reception of the inoculated tubes.
6. Adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, A,
B, C, D and E, by means of a washer, as previously described.
7. Set the electric signal clock to ring for the commencement of the
experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35
minutes.
8. Complete precisely as indicated in Trial Runs, steps 9-19.
_Control Phenol._--
Immediately the subculture tube from the 30-minute contact period have
been inoculated, carry out a precisely similar experiment, in which
five percentage strengths of Phenol, (e. g., 1.1, 1.0, 0.9, 0.75, 0.7)
are arranged in the lower tier of the test-tube rack in place of the
five strengths of Germicide-x.
Calculate the phenol coefficient by the following method:
(a) Divide the figure representing the percentage strength of the
weakest lethal dilution of the carbolic acid control at the 2-1/2-minute
contact period by the figure representing the percentage strength of the
weakest lethal dilution of the x-disinfectant at the same period. The
quotient = phenol coefficient at 2-1/2 minutes.
(b) Similarly obtain the phenol coefficient at 30 minutes contact
period.
(c) Record the mean of the two coefficients obtained in (a) and (b) as
the _mean phenol coefficient_, or simply as the ~Phenol Coefficient~.
The details of the Final Test of an actual determination are set out in
the accompanying table.
TABLE 27