per cent. sodium carbonate solution previously warmed slightly; stopper
the tubes and shake vigourously for a few minutes.
7. Centrifugalise again.
8. Pipette off the supernatant fluid; filling the tubes with warm
sterile bouillon, shake well, and again centrifugalise, to wash the
deposit.
9. Pipette off the supernatant fluid.
10. Prepare cover-slip preparations, fix and clear as for milk
preparations, stain carbolic methylene-blue, Gram's method,
Ziehl-Neelsen's method, and examine microscopically with a 1/12 inch
oil-immersion lens.
11. Proceed with the examination of the deposit as in the case of milk
deposit (see pages 450 _et seq._).
EXAMINATION OF UNSOUND MEATS.
(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)
The bacterioscopic examination of unsound food is chiefly directed to
the detection of those members of the Coli-typhoid group--B. enteritidis
of Gaertner and its allies--which are usually associated with epidemic
outbreaks of food poisoning, and such anaerobic bacteria as initiate
putrefactive changes in the food which result in the formation of
poisonous ptomaines, consequently the quantitative examination pure and
simple is frequently omitted.
A. Cultural Examination.
Quantitative.--
_Apparatus Required_:
Sterilised tin opener, (if necessary.)
Erlenmeyer flask (500 c.c. capacity) containing 200 c.c.
sterile bouillon and fitted with solid rubber stopper.
Counterpoise.
Scissors and forceps.
Scales and weights.
Water steriliser.
Hypodermic syringe.
Syringe with intragastric tube.
Rat forceps.
Case of sterile capsules.
Filtering apparatus as for water analysis.
Case of sterile plates.
Case of sterile graduated pipettes, 10 c.c. (in tenths of a
cubic centimetre).
Case of sterile graduated pipettes, 1 c.c. (in tenths of a
cubic centimetre).
Plate-levelling stand.
Tubes of nutrient gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42 deg. C.
Bulloch's apparatus.
METHOD.--
1. Place the flask containing 200 c.c. sterile broth on one pan of the
scales and counterpoise accurately.
2. Mince a portion of the sample by the aid of sterile scissors and
forceps, and add the minced sample to the bouillon in the flask to the
extent of 20 grammes.
3. Make an extract by standing the flask in the incubator running at 42
deg. C. (or in a water-bath regulated to that temperature) for half an
hour, shaking its contents from time to time. Better results are obtained
if an electrical shaker is fitted inside the incubator and the flask kept
in motion throughout the entire thirty minutes.
Now every centimetre contains the bacteria washed out from 0.1 gramme of
the original food.
4. Inoculate tubes of liquefied gelatine as follows:
To tube No. 1 add 1.0 c.c. of the extract.
2 add 0.5 c.c. of the extract.
3 add 0.3 c.c. of the extract.
4 add 0.2 c.c. of the extract.
5 add 0.1 c.c. of the extract.
Pour plates from these tubes and incubate at 20 deg. C.
5. Prepare a precisely similar set of agar plates and incubate at 37
deg. C.
6. Pipette 5 c.c. of the extract into a sterile tube, heat in the
differential steriliser at 80 deg. C. for ten minutes.
7. From the heated extract prepare duplicate sets of agar and gelatine
plates and incubate anaerobically in Bulloch's apparatus at 37 deg. C. and
20 deg. C. respectively.
8. After three days' incubation examine the agar plates both aerobic and
anaerobic and enumerate the colonies developed from spores (7), and from
vegetative forms and spores (5), and calculate and record the numbers of
each group per gramme of the original food.
9. After seven days' incubation (or earlier if compelled by the growth
of liquefying colonies) enumerate the gelatine plates in the same way.
10. Subcultivate from the colonies that make their appearance and
identify the various organisms.
11. Continue the investigations with reference to the detection of
pathogenic organisms as described under water (page 429 _et seq._).
Qualitative.--
I. _Cultural._
The micro-organisms sought for during the examination of unsound foods
comprise the following:
Members of the Coli-typhoid groups (chiefly those of the Gaertner
class).
B. anthracis.
Streptococci
Anaerobic Bacteria:
B. enteritidis sporogenes.
B. botulinus.
B. cadaveris.
The methods by which these organisms if present may be identified and
isolated have already been described under the corresponding section of
water examination with the exception of those applicable to B.
botulinus, and B. cadaveris. These can only be isolated satisfactorily
from the bodies of experimentally inoculated animals.
II _Experimental._
_Tissue._--
1. Feed rats and mice on portions of the sample and observe the result.
2. If any of the animals die, make complete post-mortem examinations and
endeavour to isolate the pathogenic organisms.
_Extract._--
1. Introduce various quantities of the bouillon extract into the
stomachs of several rats, mice and guinea-pigs repeatedly over a period
of two or three days by the intragastric method of inoculation (see page
367) and observe the result. Guinea-pigs and mice are very susceptible
to infection by B. botulinus by this method; rabbits less so.
2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep
pockets, and intraperitoneally with various quantities of the bouillon
extract, and observe the result.
3. Filter some of the extract through a Chamberland candle and incubate
the filtrate to determine the presence of soluble toxins.
4. If any of the animals succumb to either of these methods of
inoculation, make careful post-mortem examinations and endeavour to
isolate the pathogenic organisms.
THE EXAMINATION OF OYSTERS AND OTHER SHELLFISH.
On opening the shell of an oyster a certain amount of fluid termed
"liquor" is found to be present. This varies in amount from a drop to
many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the
bulk of the fluid is probably the last quantum of water ingested by the
bivalve before closing its shell. In order to obtain a working average
of the bacteriological flora of a sample, ten oysters should be taken
and the body, gastric juice and liquor should be thoroughly mixed before
examination. The examination, as in dealing with other food stuffs, is
directed to the search for members of the Coli-typhoid group, sewage
streptococci and perhaps also B. enteritidis sporogenes.
_Apparatus Required_:
Two hard nail brushes.
Liquid soap.
Sterile water in aspirator jar with delivery nozzle
controlled by a spring clip.
Sterile oyster knives.
Sterile glass dish, with cover, sufficiently large to
accommodate ten oysters.
Sterile forceps.
Sterile scissors.
Sterile towels or large gauze pads.
Sterile graduated cylinders 1000 c.c. capacity, with either
the lid or the bottom of a sterile Petri dish inverted over
the open mouth as a cover.
Glass rods.
Corrosive sublimate solution, 1 per mille.
Bile salt broth tubes.
Litmus milk tubes.
Surface plates of nutrose agar.
Case of sterile pipettes, 1 c.c. (in tenths of a c.c.)
Case of sterile pipettes, 10 c.c. (in tenths of a c.c.)
Case of sterile glass capsules.
Erlenmeyer flasks, 250 c.c. capacity.
Double strength bile salt broth.
METHOD.--
1. Thoroughly clean the outside of the oyster shells by scrubbing each
in turn with liquid soap and nail brush under a tap of running water.
Then, holding an oyster shell in a pair of sterile forceps wash every
part of the outside of the shell with a stream of sterile water running
from an aspirator jar; deposit the oyster inside the sterile glass dish.
Repeat the process with each of the remaining oysters.
2. Before proceeding further, cleanse the hands thoroughly with clean
nail brush, soap and water, then plunge them in lysol 2 per cent.
solution, and finally in sterile water.
3. Spread a sterile towel on the bench.
4. Remove one of the oysters from the sterile glass dish and place it,
resting on its convex shell, on the towel. Turn a corner of the sterile
towel over the upper flat shell to give a firmer grip to the left hand,
which holds the shell in position.
5. With the sterile oyster knife (in the right hand) open the shell and
separate the body of the oyster from the inner surface of the upper flat
shell. Bend back and separate the flat shell, leaving the body of the
oyster in and attached to the concave shell. Avoid spilling any of the
liquor.
(Some dexterity in opening oysters should be acquired before undertaking
these experiments).
6. Cut up the body of the oyster with sterile scissors into small pieces
and allow the liquor freed from the body during the process to mix with
the liquor previously in the shell.
7. Transfer the comminuted oyster and the liquor to the cylinder.
8. Treat each of the remaining oysters in similar fashion.
9. Mix the contents of the cylinder thoroughly by stirring with a
sterile glass rod. The total volume will amount to about 100 c.c.
10. Use 0.1 c.c. of the mixed liquor to inseminate each of a series of
three nutrose surface plates.
11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of
litmus milk.
12. Add sterile distilled water to the contents of the cylinder up to
1000 c.c. and stir thoroughly with a sterile glass rod and allow to
settle. The bacterial content of each oyster may be regarded, for all
practical purposes, as comprised in 100 c.c. of fluid.
13. Arrange four glass capsules in a row and number I, II, III, IV.
Pipette 9 c.c. sterile distilled water into each.
14. To capsule No. I add 1 c.c. of the diluted liquor, etc. from the
cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in
capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule
II to capsule III, afterwards adding 1 c.c. of fluid from capsule III to
capsule IV.
15. Label tubes of bile salt broth and inoculate with the following
amounts of diluted oysters:
No. 6 with 10 c.c. cylinder fluid = 0.1 oyster.
No. 5 with 1 c.c. cylinder fluid = 0.01 oyster.
No. 4 with 1 c.c. capsule I fluid = 0.001 oyster.
No. 3 with 1 c.c. capsule II fluid = 0.0001 oyster.
No. 2 with 1 c.c. capsule III fluid = 0.00001 oyster.
No. 1 with 1 c.c. capsule IV fluid = 0.000001 oyster.
16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask
and add 50 c.c. double strength bile salt broth, and label 7.
17. Duplicate all the above indicated cultures.
18. Put up the tube cultures in Buchner's tubes and incubate
anaerobically at 42 deg. C.
If growth occurs in tube 1 the organism finally isolated, e. g., B.
coli, must have been present to the extent of one million per oyster.
19. Complete the examination for members of the Coli-typhoid group and
sewage streptococci, as directed under Water Examination, page 429
(steps 11-21).
20. Inoculate a series of 6 tubes of litmus milk with quantities of the
material similar to those indicated in step 15; heat to 80 deg. C. for ten
minutes, and incubate under anaerobic conditions at 37 deg. C. Examine for
the presence of B. enteritidis sporogenes as directed under Water
Examination, page 438 (steps 7-10).
EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.
Quantitative.--
_Collection of the Sample._--As only small quantities of material are
needed, the samples should be collected in a manner similar to that
described under water for quantitative examination and transmitted in
the ice apparatus used in packing those samples.
_Apparatus Required._--As for water (_vide_ page 420).
METHOD.--
1. Arrange four sterile capsules in a row and number them I, II, III,
IV.
2. Pipette 9 c.c. sterile bouillon into capsule No. I.
3. Pipette 9.9 c.c. sterile bouillon into capsules II, III, and IV.
4. Add 1 c.c. of the sewage to capsule No. I by means of a sterile
pipette, and mix thoroughly.
5. Take a fresh sterile pipette and transfer 0.1 c.c. of the mixture
from No. I to No. II and mix thoroughly.
6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1
c.c. from No. III to No. IV.
Now 1 c.c. of dilution No. I contains 0.1 c.c. of the original sewage.
1 c.c. of dilution No. II contains 0.001 c.c. of the original sewage.
1 c.c. of dilution No. III contains 0.00001 c.c. of the original sewage.
1 c.c. of dilution No. IV contains 0.0000001 c.c. of the original sewage.
7. Pour a set of gelatine plates from the contents of each capsule,
three plates in a set, and containing respectively 0.2, 0.3, and 0.5
c.c. of the dilution. Label carefully; incubate at 20 deg. C. for three,
four, or five days.
8. Enumerate the organisms present in those sets of plates which have
not liquefied, probably those from dilution III or IV, and calculate
therefrom the number present per cubic centimetre of the original sample
of sewage.
Qualitative.--The qualitative examination of sewage is concerned with
the identification and enumeration of the same bacteria dealt with under
the corresponding section of water examination; it is consequently
conducted on precisely similar lines to those already indicated (_vide_
pages 426 to 441).
EXAMINATION OF AIR.
Quantitative.--
_Apparatus Required_:
Aspirator bottle, 10 litres capacity, fitted with a delivery
tube, and having its mouth closed by a perforated rubber
stopper, through which passes a short length of glass
tubing.
Erlenmeyer flask, 250 c.c. capacity (having a wide mouth
properly plugged with wool), containing 50 c.c. sterile
water.
Rubber stopper to fit the mouth of the flask, perforated
with two holes, and fitted as follows:
Take a 9 cm. length of glass tubing and bend up 3 cm. at one
end at right angles to the main length of tubing. Pass the
long arm of the angle through one of the perforations in the
stopper; plug the open end of the short arm with
cotton-wool.
Take a glass funnel 5 or 6 cm. in diameter with a stem 12
cm. in length and bend the stem close up to the apex of the
funnel, in a gentle curve through a quarter of a circle;
pass the long stem through the other perforation in the
rubber stopper.
A battery jar or a small water-bath to hold the Erlenmeyer
flask when packed round with ice.
Supply of broken ice.
Rubber tubing.
Screw clamps and spring clips, for tubing.
Water steriliser.
Retort stand and clamps.
Apparatus for plating (as for enumeration of water
organisms, _vide_ page 420).
METHOD.--
1. Fill 10 litres of water into the aspirating bottle and attach a piece
of rubber tubing with a screw clamp to the delivery tube. Open the taps
fully and regulate the screw clamp, by actual experiment, so that the
tube delivers 1 c.c. of water every second. The screw clamp is not
touched again during the experiment.
At this rate the aspirator bottle will empty itself in just under three
hours. Shut off the tap and make up the contents of the aspirator bottle
to 10 litres again.
2. Sterilise the fitted rubber cork, with its funnel and tubing, by
boiling in the water steriliser for ten minutes.
3. Remove the cotton-wool plug from the flask, and replace it by the
rubber stopper with its fittings. Make sure that the end of the stem of
the funnel is immersed in the bouillon.
4. Place the flask in a glass or metal vessel and pack it round with
pounded ice. Arrange the flask with its ice casing just above the neck
of the aspirator bottle.
[Illustration: FIG. 216.--Arrangement of apparatus for air analysis.]
5. Connect up the free end of the glass tube from the flask--after
removing the cotton-wool plug--with the air-entry tube in the mouth of
the aspirating bottle (Fig. 216).
6. Open the tap fully, and allow the water to run.
Replenish the ice from time to time if necessary.
(In emptying itself the aspirator bottle will aspirate 10 litres of air
slowly through the water in the Erlenmeyer flask.)
7. When the aspiration is completed, disconnect the flask and remove it
from its ice packing.
8. Liquefy three tubes of nutrient gelatine and add to them 0.5 c.c.,
0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by
means of a sterile graduated pipette, as in the quantitative examination
of water. Pour plates.
9. Pour a second similar set of gelatine plates.
10. Incubate both sets of plates at 20 deg. C.
11. Enumerate the colonies present in the two sets of gelatine plates
after three, four, or five days and average the results from the numbers
so obtained; estimate the number of micro-organisms present in 1 c.c.,
and then in the 50 c.c. of broth in the flask.
12. The result of air examination is usually expressed as the number of
bacteria present per cubic metre (i. e., kilolitre) of air; and as the
number of organisms present in the 50 c.c. water only represent those
contained in 10 litres of air, the resulting figure must be multiplied
by 100.
Qualitative.--
1. Proceed exactly as in the quantitative examination of air (_vide
supra_), steps 1 to 10.
2. Pour plates of wort agar with similar quantities of the air-infected
water, and incubate at 37 deg. C.
3. Pour plates of nutrient agar with similar quantities of the water and
incubate at 37 deg. C.
4. Pour similar plates of wort gelatine and incubate at 20 deg. C.
5. Pick off the individual colonies that appear in the several plates,
subcultivate them on the various media, and identify them.
EXAMINATION OF SOIL.
The bacteriological examination of soil yields information of value to
the sanitarian during the progress of the process of homogenisation of
"made soil" (e. g., a dumping area for the refuse of town) and
determines the period at which such an area may with propriety and
safety be utilised for building purposes; or to the agriculturalist in
informing him of the suitability of any given area for the growth of
crops.
The surface of the ground, exposed as it is to the bactericidal
influence of sunlight and to rapid alternations of heat and cold, rain
and wind, contains but few micro-organisms. Again, owing to the density
of the molecules of deep soil and lack of aeration on the one hand, and
the filtering action of the upper layers of soil and bacterial
antagonism on the other, bacterial life practically ceases at a depth of
about 2 metres. The intermediate stratum of soil, situated from 25 to 50
cm. below the surface, invariably yields the most numerous and the most
varied bacterial flora.
~Collection of Sample.~--A small copper capsule 6 cm. high by 6 cm.
diameter, with "pull-off" cap secured by a bayonet catch, previously
sterilised in the hot-air oven, is the most convenient receptacle for
samples of soil.
[Illustration: FIG. 217.--Soil scoop.]
The instrument used for the actual removal of the soil from its natural
position will vary according to whether we require surface samples or
soil from varying depths.
(a) For ~surface~ samples, use an iron scoop, shaped like a shoe horn,
but provided with a sharp spine (Fig. 217). This is wrapped in asbestos
cloth and sterilised in the hot-air oven. When removed from the oven,
wrap a piece of oiled paper, silk, or gutta-percha tissue over the
asbestos cloth, and secure it with string, as a further protection
against contamination.
On reaching the spot whence the samples are to be taken, the coverings
of the scoop are removed, and the asbestos cloth employed to brush away
loose stones and debris from the selected area. The surface soil is then
broken up with the point of the scoop, scraped up and collected in the
body of the scoop, and transferred to the sterile capsule for
transmission.
[Illustration: FIG. 218.--Fraenkel's borer.]
(b) For ~deep~ samples collected at various distances from the surface,
an experimental trench may be cut to the required depth and samples
collected at the required points on the face of the section. It is,
however, preferable to utilise some form of borer, such as that designed
by Fraenkel (Fig. 218).
_Fraenkel's Earth Borer._--This instrument consists of a stout
hard-steel rod, 150 cm. long, marked in centimetres from the
drill-pointed extremity. It is provided with a cross handle (adjustable
at any point along the length of the rod by means of a screw nut). The
terminal centimeters are thicker than the remainder of the rod, and on
one side a vertical cavity about 0.5 cm. deep is cut. This is covered by
a flanged sleeve so long as the borer is driven into the soil clockwise,
and is opened for the reception of the sample of soil, when the required
depth is reached, by reversing the screwing motion, and again closed
before withdrawal of the borer from the earth by resuming the original
direction of twist. It can be sterilised in a manner similar to that
adopted for the scoop, or by repeatedly filling the cavity with ether
and burning it off.
~Quantitative.~--Four distinct investigations are included in the complete
quantitative bacteriological examination of the soil:
1. The enumeration of the aerobic organisms.
2. The enumeration of the spores of aerobes.
3. The enumeration of the anaerobic organisms (including the facultative
anaerobes).
4. The enumeration of the spores of anaerobes.
Further, by a combination of the results of the first and second, and of
the third and fourth of these, the ratio of spores to vegetative forms
is obtained.
_Apparatus Required_:
Case of sterile capsules (25 c.c. capacity).
Case of sterile graduated pipettes, 10 c.c. (in tenths of a
cubic centimetre).
Case of sterile graduated pipettes, 1 c.c. (in tenths of a
cubic centimetre).
Flask containing 250 c.c. sterile bouillon.
Tall cylinder containing 2 per cent. lysol solution.
Plate-levelling stand.
12 sterile plates.
Tubes of nutrient gelatine.
Tubes of wort gelatine.
Tubes of nutrient agar.
Tubes of glucose formate gelatine.
Tubes of glucose formate agar.
Water-bath regulated at 42 deg. C.
Bunsen burner.
Grease pencil.
Sterile mortar and pestle (agate).
Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).
Sterile metal funnel with short wide bore delivery tube to
just fit mouth of flask.
Solid rubber stopper to fit the flask (sterilised by
boiling).
Pair of scales.
Counterpoise (Fig. 107).
Sterile metal (nickel) spoon or spatula.
Fractional steriliser (Fig. 140).
METHOD.--
1. Arrange four sterile capsules numbered I, II, III, and IV; pipette 9
c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of
the remaining three.
2. Pipette 100 c.c. sterile bouillon into the Erlenmeyer flask.
3. Remove the cotton-wool plug from the flask and replace it by the
sterile funnel.
4. Place flask and funnel on one pan of the scales, and counterpoise
accurately.
5. Empty the sample of soil into the mortar and triturate thoroughly.
6. By means of the sterile spatula add 10 grammes of the earth sample to
the bouillon in the flask.
The final results will be more reliable if steps 2, 3, 4, and 5 are
performed under a hood--to protect from falling dust, etc.
7. Remove the funnel from the mouth of the flask; replace it by the
rubber stopper and shake vigourously; then allow the solid particles to
settle for about thirty minutes. One cubic centimetre of the turbid
broth contains the washings from 0.1 gramme of soil.
8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil
water," and add it to the contents of capsule I; mix thoroughly.
9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to
capsule II, and mix.
10. In like manner add 0.1 c.c. of the contents of capsule II to capsule
III, and then 0.1 c.c. of the contents of capsule III to capsule IV.
Then 1 c.c. fluid from capsule I contains soil water
from .01 gm. earth.
Then 1 c.c. fluid from capsule II contains soil water
from .0001 gm. earth.
Then 1 c.c. fluid from capsule III contains soil water
from .000001 gm. earth.
Then 1 c.c. fluid from capsule IV contains soil water
from .00000001 gm. earth.
(A) _Aerobes (Vegetative Forms and Spores)._--
11. Pour a set of gelatine plates from the contents of each capsule--two
plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of
the diluted soil water. Label and incubate.
12. Pour similar sets of wort gelatine plates from the contents of
capsules II and III, label, and incubate at 20 deg. C.
13. Pour similar sets of agar plates from the contents of capsules II
and III; label and incubate at 37 deg. C.
14. Weigh out a second sample of soil--10 grammes--dry over a water-bath
until of constant weight and calculate the ratio