Bacillus Hoffmanni.
Bacillus xerosis.
Bacillus septus.
VIII. _Coli-typhoid group._
B. typhi abdominalis (B. typhosus).
B. coli communis.
B. enteritidis (Gaertner).
to contrast with
B. aquatilis sulcatus.
IX. _Escherich group._
B. coli communis (Escherich).
B. coli communior.
B. lactis aerogenes.
B. cloacae.
X. _Gaertner group._
Bacillus enteritidis (Gaertner).
B. paratyphosus A.
B. paratyphosus B.
Bacillus cholerae suum (Hog Cholera).
B. psittacosis.
XI. _Eberth group._
B. typhosus (Eberth).
B. dysenteriae (Shiga).
B. dysenteriae (Flexner).
B. faecalis alcaligines.
XII. _Spirillum group._
Vibrio cholerae.
Vibrio metschnikovi.
to contrast with
Vibrio proteus (Finkler and Prior).
Spirillum rubrum.
Spirillum rugula.
XIII. _Anthrax group._
Bacillus anthracis.
to contrast with
Bacillus subtilis.
Bacillus mycoides.
Bacillus mesentericus fuscus.
XIV. _Acid fast group._
Bacillus tuberculosis (human).
" " (bovine).
" " (avian).
" " (fish).
to contrast with
Bacillus phlei (Timothy grass bacillus).
Butter bacillus of Rabinowitch.
XV. _Plague group._
Bacillus pestis.
B. septicaemiae haemorrhagicae.
B. suipestifer.
XVI. _Influenzae group._
B. influenzae.
Bacillus aegypticus (Koch-Weeks).
Bacillus pertussis.
XVII. _Miscellaneous._
Bacillus leprae.
Bacillus mallei.
Micrococcus melitensis.
XVIII. _Streptothrix group._
Streptothrix actinomycotica.
Streptothrix madurae.
to contrast with
Cladothrix nivea.
XIX. _Tetanus group._
Bacillus tetani.
Bacillus oedematis maligni.
Bacillus chauvei (symptomatic anthrax).
XX. _Enteritidis sporogenes group._
Bacillus enteritidis sporogenes.
B. botulinus.
B. butyricus.
B. cadaveris.
FOOTNOTES:
[15] See note on Vivisection License, page 334.
XXI. BACTERIOLOGICAL ANALYSES.
Each bacteriological or bacterioscopical analysis of air, earth, sewage,
various food-stuffs, etc., includes, as a general rule, two distinct
investigations yielding results of very unequal value:
1. Quantitative.
2. Qualitative.
The first is purely quantitative and as such is of minor importance as
it aims simply at enumerating (approximately) the total number of
bacteria present in any given unit of volume irrespective of the nature
and character of individual organisms.
The second and more important is both qualitative and quantitative in
character since it seeks to accurately identify such pathogenic bacteria
as may be present while, incidentally, the methods advocated are
calculated to indicate, with a fair degree of accuracy, the numerical
frequency of such bacteria, in the sample under examination.
The general principles underlying the bacteriological analyses of water,
sewage, air and dust, soil, milk, ice cream, meat, and other tinned
stuffs, as exemplified by the methods used by the author, are indicated
in the following pages, together with the methods of testing filters and
chemical germicides; and the technique there set out will be found to be
capable of expansion and adaptation to any circumstance or set of
circumstances which may confront the student.
~Controls.~--The necessity for the existence of adequate controls in all
experimental work cannot be too urgently insisted upon. Every batch of
plates that is poured should include at least one of the presumably
"sterile" medium; plate or tube cultures should be made from the various
diluting fluids; every tube of carbohydrate medium that is inoculated
should go into the incubator in company with a similar but uninoculated
tube, and so on.
BACTERIOLOGICAL EXAMINATION OF WATER.
The bacteria present in the water may comprise not only varieties which
have their normal habitat in the water and will consequently develop at
20 deg. C., but also if the water has been contaminated with excremental
matter, varieties which have been derived from, or are pathogenic for,
the animal body, and which will only develop well at a temperature of
37 deg. C. In order to demonstrate the presence of each of these classes
it will be necessary to incubate the various cultivations at each of these
temperatures.
Further, the sample of water may contain moulds, yeasts, or torulae, and
the development of these will be best secured by plating in wort
gelatine and incubating at 20 deg. C.
~1. Quantitative.~--
_Collection of the Sample._--The most suitable vessels for the reception
of the water sample are small glass bottles, 60 c.c. capacity, with
narrow necks and overhanging glass stoppers (to prevent contamination of
the bottle necks by falling dust). These must be carefully sterilised in
the hot-air steriliser (_vide_ page 31).
(a) If the sample is obtained from a ~tap~ or ~pipe~, turn on the water
and allow it to run for a few minutes. Remove the stopper from the
bottle and retain it in the hand whilst the water is allowed to run into
the bottle and three parts fill it. Replace the stopper and tie it down,
but _do not seal it_.
(b) If the sample is obtained from a ~stream~, ~tank~, or ~reservoir~,
fasten a piece of stout wire around the neck of the bottle, remove the
stopper, and retain it in the hand. Then, using the wire as a handle,
plunge the bottle into the water, mouth downward, until it is well
beneath the surface; then reverse it, allow it to fill, and withdraw it
from the water. Pour out a few cubic centimetres of water from the
bottle, replace the stopper, and tie it down.
[Illustration: FIG. 203.--Esmarch's collecting bottle for water
samples.]
(c) If the sample is obtained from a ~lake~, ~river~ or the ~sea~; or when
it is desired to compare samples taken at varying depths, the apparatus
designed by v. Esmarch (Fig. 203) is employed. In this the sterilised
bottle is enclosed in a weighted metal cage which can be lowered, by
means of a graduated line, until the required depth is reached. At this
point the bottle is opened by a thin wire cord attached to the stopper;
when the bottle is full (as judged by the air bubbles ceasing to rise)
the pull on the cord is released and the tension of the spiral spring
above the stopper again forces it into the neck of the bottle. When the
apparatus is taken out of the water, the small bottles are filled from
it, and packed in the ice-box mentioned below.
An inexpensive substitute for Esmarch's bottle can be made in the
laboratory thus:
Select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity
(about 20 cm. high and 8 cm. in diameter).
Remove the glass stopper and insert a rubber cork with two perforations
in its place.
Through one perforation pass a piece of glass tubing about 5 cm. long
and through the other a piece 22 cm. long, reaching to near the bottom
of the bottle, each tube projecting about 2.5 cm. above the rubber
stopper. Plug the open ends of the tubes with cotton wool. Secure the
stopper in place with thin copper wire.
[Illustration: FIG. 204.--Thresh's deep water sampling bottle.]
Sterilise the fitted bottle in the autoclave. Remove the cotton wool
plugs and connect the projecting tubes by a piece of loosely fitting
stout rubber pressure tubing about 5 cm. long, previously sterilised by
boiling.
Take a piece of stout rubber cord about 33 cm. long, and of 10 mm.
diameter (such as is used for door springs) thread a steel split ring
upon it and secure the free ends tightly to the neck of the bottle by
cord or catgut.
Attach the cord used for lowering the bottle into the water to the split
ring on the rubber suspender. The best material for this purpose is
cotton insulated electric wire knotted at every metre.
Connect the split ring also with the short piece of rubber tubing
uniting the two glass tubes by a piece of catgut (or thin copper wire)
of such length that when the bottle is suspended there is no pull upon
the rubber tube, but which, however, will be easily jerked off when a
sharp pull is given to the suspending cord.
Now wind heavy lead tubing about 1 cm. diameter around the upper part of
the bottle, starting at the neck just above the shoulder. This ensures
the sinking of the bottle in the vertical position (Fig. 204).
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.
[Illustration: FIG. 205.--Ice-box for transmission of water samples,
etc.]
_Transport of Sample._--If the examination of the sample cannot be
commenced immediately, steps must be taken to prevent the multiplication
of the bacteria contained in the water during the interval occupied in
transit from the place of collection to the laboratory. To this end an
ice-box such as that shown (in Fig. 205) is essential. It consists of a
double-walled metal cylinder into which slides a cylindrical chamber of
sufficient capacity to accommodate four of the 60 c.c. bottles; this in
turn is covered by a metal disc--the three portions being bolted
together by thumb screws through the overhanging flanges. When in use,
place the bottles, rolled in cotton-wool, in the central chamber, pack
the space between the walls with pounded ice, securely close the metal
box by screwing down the fly nuts, and place it in a felt-lined wooden
case. (It has been shown that whilst bacteria will survive exposure to
the temperature of melting ice, practically none will multiply at this
temperature.)
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.
_Apparatus Required_:
Plate-levelling stand.
Case of sterile plates.
Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).
Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).
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 deg. C.
METHOD.--
1. Arrange the plate-levelling platform with its water compartment
filled with water, at 45 deg. C.
2. Number the agar tubes, consecutively, 1 to 6; the gelatine tubes,
consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs
and see that they are not adherent to the lips of the tubes.
3. Place the agar tubes in boiling water until the medium is melted,
then transfer them to the water-bath regulated at 42 deg. C. Liquefy the
nutrient gelatine and wort gelatine tubes by immersing them in the same
water-bath.
4. Remove the bottle containing the water sample from the ice-box,
distribute the bacterial contents evenly throughout the water by
shaking, cut the string securing the stopper, and loosen the stopper,
but do not take it out.
[Illustration: FIG. 206.--Withdrawing water from water sample bottle.]
5. Remove one of the 1 c.c. pipettes from the case, holding it by the
plain portion of the tube. Pass the graduated portion twice through the
Bunsen flame. Tilt the bottle containing the water sample on the bench
holding the neck between the middle and ring fingers of the left hand;
grasp the head of the stopper between the forefinger and thumb, and
remove it from the bottle.
6. Pass the pipette into the mouth of the bottle, holding its point well
below the surface of the water (Fig. 206). Suck up rather more than 1
c.c. into the pipette and allow the pipette to empty; this moistens the
interior of the pipette and renders accurate measurement possible. Now
draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the
bottle, replace the stopper, and stand the bottle upright.
7. Take the first melted agar tube in the left hand, remove the
cotton-wool plug, and add to its contents 0.5 c.c. of the water sample
from the pipette; replug the tube and replace it in the water-bath. In a
similar manner add 0.3 c.c. water to the contents of the second tube,
and 0.2 c.c. to the contents of the third.
8. In a similar manner add 1 c.c. of the sample to the contents of the
fourth tube.
9. Similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of
the fifth and sixth tubes.
10. Drop the pipette into the cylinder containing lysol solution.
11. Mix the water sample with the medium in each tube in the manner
described under plate cultivations; pour a plate from each tube. Label
each plate with (a) the distinctive number of the sample, (b) the
quantity of water sample it contains, and (c) the date.
12. Pour the contents of a tube of liquefied agar--not inoculated--into
a Petri dish to act as a control to demonstrate the sterility of the
batch of agar employed.
13. Allow the plates to set, and incubate at 37 deg. C.
14. Empty the water chamber of the levelling apparatus and refill it
with ice-water.
15. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile
distilled water into a sterile glass capsule.
16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in
the capsule. This will give a dilution of 1 in 100.
17. Plant the six tubes of nutrient gelatine in the following manner: To
the first tube add 0.5 c.c. of the water sample direct from the bottle;
to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of
each tube. To the fourth tube add 0.5 c.c. of the diluted water sample
from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.;
and pour a plate from each.
18. Label each plate with the quantity of the water sample it
contains--that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c.,
and 0.002 c.c.
19. Pour a control (uninoculated) gelatine plate.
20. Allow the plates to set, and incubate at 20 deg. C.
21. To the first tube of liquefied wort gelatine add 0.5 c.c. water
sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c.
22. Label the plates, allow them to set, and incubate at 20 deg. C.
23. Count and record the number of colonies that have developed upon the
agar at 37 deg. C. after forty-eight hours' incubation.
24. Note the number of colonies present on each of the gelatine and wort
gelatine plates after forty-eight hours' incubation.
25. Replace the gelatine and wort plates in the incubator; observe again
at three days, four days, and five days.
26. Calculate and record the number of organisms present per cubic
centimetre of the original water from the average of the six gelatine
plates at the latest date possible up to seven days--the presence of
liquefying bacteria may render the calculation necessary at an earlier
date, hence the importance of daily observations.
_Method of Counting._--The most accurate method of counting the colonies
on each of the plates is by means of either Jeffery's or Pakes' counting
disc. Each of these discs consists of a piece of paper, upon which is
printed a dead black disc, subdivided by concentric circles and radii,
printed in white. In Jeffery's counter (Fig. 207), each subdivision has
an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii
divide the circle into sixteen equal sectors, and counting is
facilitated by concentric circles equidistant from the centre.
[Illustration: FIG. 207.--Jeffery's disc, reduced.]
[Illustration: FIG. 208.--Pakes' disc, reduced.]
(a) 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.
(b) 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.
(c) 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.
2. Qualitative Examination.--
_Collection of Sample._--The water sample required for the routine
examination, which it will be convenient to consider first, amounts to
about 110 c.c. It is collected in the manner previously described
(_vide_ page 416); similar bottles are used, and if four are filled the
combined contents, amounting to about 240 c.c., will provide ample
material for both the qualitative and quantitative examinations. Unless
the examination is to be commenced at once, the ice-box must be
employed, otherwise water bacteria and other saprophytes will probably
multiply at the expense of the microbes indicative of pollution, and so
increase the difficulties of the investigation.
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,
(relatively scarce in water) 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 (more rarely)
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:
A. _Enrichment_, in which the harmless non-pathogenic bacteria may be
destroyed or their growth inhibited, whilst the growth of the parasitic
bacteria is encouraged.
This is attained by so arranging the environment, (i. e., Media,
incubation temperature, and atmosphere) as to favor the growth of the
pathogenic organisms at the expense of the harmless saprophytes.
B. _Concentration_, whereby all the bacteria present in the sample of
water, pathogenic or otherwise, are concentrated in a small bulk of
fluid.
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.~
(Dealing with the demonstration of bacteria of intestinal origin.)
_Apparatus Required_ (_Preliminary Stage_):
Incubator running at 42 deg. 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 (_vide_ page 180).
Flask of double strength bile salt broth (_vide_ page 199).
Tubes of litmus silk.
Sterile flasks, 250 c.c. capacity.
Buchner's tubes.
Tabloids pyrogallic acid.
Tabloids sodium hydrate.
Bunsen burner.
Grease pencil.
(_Later stage_):
Incubator running at 37 deg. C.
Surface plates of nutrose agar (see page 232).
Aluminium spreader.
Tubes of various media, including carbohydrate media.
Agglutinating sera, etc.
METHOD.--
1. Number a set of bile salt broth, tubes 1-5, and a duplicate set
1a-5a.
2. Number one flask 7 and another 8.
3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.
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.
4. Put up all the tubes in Buchner's tubes and incubate anaerobically at
42 deg. C.
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 deg. C.
destroys ordinary water bacteria and inhibits the growth of many
ordinary mesophilic bacteria.
5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and
50 c.c. double strength bile salt broth into flask 7.
6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample
into flask 7.
7. Incubate the two flasks aerobically at 42 deg. C.
8. After twenty-four hours incubation note in each culture:
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.
9. Replace those tubes which show no signs of growth in the incubator.
Examine after another period of twenty-four hours (total forty-eight
hours incubation) with reference to the same points.
10. Remove culture tubes which show visible growth from the Buchner's
tubes, whether acid production and gas formation are present or not.
11. Examine all tubes which show growth by hanging-drop preparations.
Note such as show the presence of chains of cocci.
12. Prepare surface plate cultivations upon nutrose agar from each tube
that shows growth either macroscopically or microscopically, and
incubate for twenty-four hours aerobically at 37 deg. C.
13. Examine the growth on the plates either with the naked eye or with
the help of a small hand lens. Practice will facilitate the recognition
of colonies of the coli group, the typhoid group and the paratyphoid
group; also those due to the growth of streptococci. The investigation
from this stage proceeds along two divergent lines of enquiry--the first
being concerned with the identity of the bacilli--typhoid bacilli, the
second with that of the cocci.
A. _B. Coli and its allies._
14. Pick off coliform or typhiform colonies; make streak or smear
subcultivations upon nutrient agar; incubate aerobically for twenty-four
hours at 37 deg. C.