 |
Transfer to the second trough containing the diluted stain for ten minutes.
Transfer to the third trough containing distilled water, and holding the trough over a sink, run in a stream of distilled water until washing is complete. Remove slides from the rack and dry.
Leishman's stain is the best for routine work for all bacteria other than B. tuberculosis. Films containing tubercle bacilli must of course be stained by the Ziehl Neelsen method.
13. Examine specific serum slide microscopically with 1/12 inch oil immersion. Find the edge of the blood film—along this the bulk of the leucocytes will be collected. Starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria. The sum of the contents of the first 50 consecutive polymorphonuclears that are encountered is marked down. (The average number of bacilli ingested per leucocyte = the "phagocytic index.")
14. In precisely similar manner enumerate the bacteria present in the first 50 cells of the control preparation. This number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum. This fraction, expressed as a percentage of unity = the opsonic index.
IMMUNE BODY.
Immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. The presence of the immune body in the serum can be demonstrated in vitro by the reaction elaborated by Bordet and Gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of haemolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate haemolysin—two of the three essentials in the haemolytic system, vide page 326).
Apparatus Required:
Sterile pipettes 1 c.c., (graduated in tenths).
16 x 2 cm. test-tubes.
9 x 1 cm. test-tubes.
Test-tube racks for each size of test-tube.
Reagents Required:
Normal saline solution.
Erythrocyte solution (human red cells, page 329) = E.
Haemolytic serum (for human cells) = H.S.
Complement (fresh guinea-pig serum) = C.
Specific serum from inoculated animal, inactivated = S.S.
Control pooled serum from normal animals of same species, Inactivated = P.S.
Antigen (cultivation upon solid medium of the organism (e. g., B. typhosus) which has already served as antigen in the inoculation of the experimental animal) = A.
To prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution.
Shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion, and sterilise by heating to 60 deg. C. in a water-bath for one hour.
METHOD.—
1. Take five small test-tubes, and number them 1 to 5 with a grease pencil.
2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.
3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.
4. Into tube No. 4 pipette 0.2 c.c. of control serum.
5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen.
6. Place the whole set of tubes in the incubator at 37 deg. C. for a period of one hour.
7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal haemolytic doses of the corresponding haemolysin into each tube.
8. Mix thoroughly and return the tubes to the incubator at 37 deg. C. for further period of one hour.
9. At the expiration of that time transfer the tubes to the ice chest, and allow them to stand for three hours.
10. Examine the tubes.
Tubes 3, 4 and 5 should show complete haemolysis; tube 2 should give no evidence whatever of haemolysis.
These tubes form the controls to the first tube, which contains the serum to be tested.
In tube No. 1 the absence of haemolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations; since it shows that the complement has been bound by the immune body to the bacterial antigen, and none has been left free to enter into the haemolytic system; on the other hand the presence of haemolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. In other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and haemolytic serum, and was ready to combine with those reagents to complete the haemolytic system.
The method may be shown diagramatically as under using the symbols already indicated
Test-tubes.
1 2 3 4 5
0.1 c.c. C. ........ 0.1 c.c. C. 0.1 c.c. C. 0.1 c.c. C.
0.2 c.c. S.S. 0.2 c.c. S.S. ......... 0.2 c.c. P.S. ........
A. A. A. A. ........ ————————————————————————————————————— Incubate at 37 deg. C. for one hour. —————————————————————————————————————
1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.
H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} Incubate at 37 deg. C. for one hour. (?) No haemolysis.
Haemolysis.
NOTE.—It is sometimes more convenient to sensitise the erythrocytes just before they are needed. This is done forty-five minutes after the experiment has been started (page 394, step 6), that is to say, before the completion of the first period of incubation, thus:
1. Measure out into a sterile test-tube (or flask) five c.c. of erythrocyte solution.
2. Measure out twenty minimal haemolytic doses of haemolysin, add to the erythrocyte solution on the test-tube.
3. Allow the erythrocyte and haemolysin to remain in contact for fifteen minutes at room temperature. The red cells are then sensitised and ready for use.
4. When the tubes are removed from the incubator at the end of the first hour (i. e., step 7) add 1 c.c. sensitised red cells to each tube by means of a graduated pipette.
5. Mix thoroughly, return the tubes to the incubator at 37 deg. C. and complete the experiment as previously described (steps 8 onward).
XIX. POST-MORTEM EXAMINATIONS OF EXPERIMENTAL ANIMALS.
The post-mortem examination should be carried out as soon as possible after the death of the animal, for it must be remembered that even in cold weather the tissues are rapidly invaded by numerous bacteria derived from the alimentary tract or the cavities of the body, and from external sources.
The following outlines refer to a complete and exhaustive necropsy, and in routine work the examination will rarely need to be carried out in its entirety.
NOTE.—Throughout the autopsy the searing irons must be freely employed, and it must be recollected that one instrument is only to be employed to seize or cut one structure. This done, it must be regarded as contaminated and a fresh instrument taken for the next step.
Apparatus Required:
Water steriliser.
{ Scalpels. Surgical instruments: { Scissors. { Forceps. { Bone forceps.
Spear-headed platinum spatula (Fig. 199).
Searing irons (Fig. 198).
Tubes of media—bouillon and sloped agar.
Surface plates in petri dishes (of agar or one of its derivatives).
Platinum loop.
Aluminium "spreader."
Grease pencil.
Sterile capillary pipettes (Fig. 13, a).
Sterile glass capsules, large and small.
Cover-slips or slides.
Bottles of fixing fluid (vide page 114) for pieces of tissue intended for sectioning.
1. Place the various instruments, forceps, scissors, scalpels, etc., needed for the autopsy inside the steriliser and sterilise by boiling for ten minutes; then open the steriliser, raise the tray from the interior and rest it crosswise on the edges.
2. Heat the searing irons to redness in a separate gas stove.
3. Drench the fur (or feathers) with lysol solution, 2 per cent. This serves the twofold purpose of preventing the hairs from flying about and entering the body cavities during the autopsy, and of rendering innocuous any vermin that may be present on the animal.
4. Examine the cadaver carefully. Recollect that laboratory animals are not always hardy; death may be due to exposure to heat or cold, to starvation or over- or improper feeding or to the attack of rats—and not to the bacterial infection.
5. Fasten the body of the animal, ventral surface upward (unless there is some special reason for having the dorsum exposed), out on a board by means of copper nails driven through the extremities.
6. With sterile forceps and scalpel incise the skin in the middle line from the top of the sternum to the pubes. Make other incisions at right angles to the first out to the axillae and groins, and reflect the skin in two lateral flaps. (Place the now infected instruments on the board by the side of the body or support them on a porcelain knife rest.)
Seat of Inoculation.—
7. Inspect the seat of inoculation. If any local lesion is visible, sear its exposed surface and with the platinum loop, remove material from the deeper parts to make tube and surface plate cultivations and cover-slip preparations.
Collect specimens of pus or other exudation in capillary pipettes for subsequent examination.
8. Inspect the neighbouring lymphatic glands and endeavour to trace the path of the virus.
9. Sear the whole of the exposed surface of the thorax with the searing irons.
Pleural Cavity.—
10. Divide the ribs on either side of the sternum and remove a rectangular portion of the anterior chest wall with sterile scissors and a fresh pair of forceps, exposing the heart. Place the infected instruments by the side of the first set.
11. Observe the condition of the anterior mediastinal glands, the thymus and the lungs. Collect a quantity of pleuritic effusion, if such is present, in a pipette for further examination later.
12. Raise the pericardial sac in a fresh pair of forceps and burn through this structure with a searing iron.
Collect a sample of pericardial fluid in a pipette for microscopical and cultural examination.
13. Grasp the apex of the heart in the forceps and sear the surface of the right ventricle.
14. Plunge the open point of a capillary pipette through the seared area into the ventricle and fill with blood.
Make cultivations and cover-slip preparations of the heart blood.
15. Collect a further sample of blood or serum for subsequent investigation as to the presence of antibodies.
Peritoneal Cavity.—
16. Sear a broad track in the middle line of the abdominal wall; open the peritoneal cavity by an incision in the centre of the seared line. Observe the condition of the omentum, the mesentery, the viscera and the peritoneal surface of the intestines.
17. Collect a specimen of the peritoneal fluid (or pus, if present) in a capillary pipette. Make cultivations, tube and surface plate, and cover-slip preparations from this situation.
18. Collect a specimen of the urine from the distended bladder in a large pipette (in the manner indicated for heart blood), for further examination, by cultivations, microscopical preparations, and chemical analysis.
19. Collect a specimen of bile from the gall bladder in similar manner.
20. Excise the spleen and place it in a sterile capsule. Later, sear the surface of this organ; plunge the spear-headed spatula through the centre of the seared area, twist it round between the finger and thumb, and remove it from the organ. Sufficient material will be brought away in the eye in its head to make cultivations. A repetition of the process will afford material for cover-slip preparations.
21. Seize one end of the spleen with sterile forceps. Sear a narrow band of tissue, right around the organ and divide the spleen in this situation with a pair of scissors. Holding the piece of spleen in the forceps, dab the cut surface on to a surface plate in a number of different spots.
22. In like manner examine the other organs—liver, lungs, kidneys, lymphatic glands (mesenteric, hepatic, lumbar, etc), etc. Prepare cultivations and cover-slip preparations.
23. Dissect out a long bone from one upper and one lower limb and one of the largest ribs. Prepare cultures from the bone marrow in each case. Set aside these bones for the subsequent preparation of marrow films.
24. Film preparations of bone marrow are best made by the Price-Jones method. Seize the bone in a pair of pliers and squeeze out some of the marrow; receive it in a platinum loop, and transfer to a watch glass of dissociating fluid and emulsify. The dissociating fluid is a neutral 10 per cent. solution of glycerine prepared as follows:—
Measure out 10 c.c. Price's best glycerine and 90 c.c. sterile ammonia-free distilled water. Mix. Titrate against n/10 sodic hydrate solution using phenolphthalein as the indicator. The initial reaction is usually + 0.1 to + 0.5; add the calculated amount of n/10 sodic hydrate solution to neutralise.
25. Place a loopful of fresh desiccating fluid on a 3 x 1 glass slide; add a similar loopful of the marrow emulsion, and spread very gently over the surface of the slip.
26. Allow film to dry in the air (protected from dust) without heating.
27. Stain with Jenner's polychrome stain (page 97) for two and a half minutes.
28. Wash with ammonia-free distilled water, dry thoroughly and mount in xylol balsam.
Cranial and Spinal Cavities.—
29. In some instances it may be necessary (e. g., experimental inoculation of rabies) to examine the cranial cavity or to remove the spinal cord. Return the viscera to the abdominal cavity; draw the flaps of skin together and secure with Michel's steel clips. Draw the copper nails securing the limbs to the board, reverse the animal and again nail the limbs down—the body now being dorsum uppermost.
30. Make a longitudinal incision in the mesial line from snout to root of tail, and four transverse incisions—one joining the roots of the two ears, one across the body at the level of the spinis of the scapulae, another at the level of the costal margin and the last across the upper level of the pelvis. Reflect these flaps of skin.
31. With forceps and scalpel dissect out the muscles lying in the furrow on either side of the spinal processes.
32. Cut through the bases of the transverse processes with bone forceps. Cut away the vault of the skull, cut through the roots of the nerves and remove the brain and spinal cord, place in a large glass dish for examination. Prepare cultivations from the cerebro-spinal fluid. The removal of the brain and cord is a tedious process and during the dissection it is difficult to avoid injury to these structures.
The operation is, however, carried out very expeditiously and neatly with the aid of the surgical engine (vide page 361). A small circular saw is fitted to the hand piece. The bones of the skull are cut through and the whole of the vault removed, exposing the entire vertex of the brain. Similarly all the spinous processes can be removed in one string by running the saw down first one side of the spinal column and then the other. In this way ample space for the removal of the nervous tissues is obtained with a minimum of labour.
33. Having completed the preparation of cultures remove small portions of various organs at leisure and place each in separate bottles of fixing fluid for future sectioning. Affix to each bottle a label bearing all necessary details as to its contents.
34. If necessary, remove portions of the organs for preservation and display as museum specimens (vide page 404).
35. Gather up all the infected instruments, return them to the steriliser, and disinfect by boiling for ten minutes.
36. Sprinkle dry sawdust into the exposed body cavities to absorb blood and fluid. Cover the body with blotting or filter paper, moistened with 2 per cent. lysol solution. Place in a galvanised iron pail, provided with a lid, ready for transport to the crematorium.
37. Cremate the cadaver together with the board upon which it is fixed.
38. Stain the cover-slip preparations by suitable methods and examine microscopically.
39. Incubate the cultivations and examine carefully from day to day.
40. Make full notes of the condition of the various body cavities and of the viscera immediately the autopsy is completed; and add the result of the microscopical and cultural investigation when available.
As part of the card index system in use in the author's laboratory already referred to (vide page 335) there is a special yellow card for P-M notes. On the face of the card are printed headings for various data—some of which are sometimes unintentionally omitted—and on the reverse is a schematic figure which can be utilised for indicating the position of the chief lesions in the cadaver of any of the laboratory animals.
AUTOPSY CARD Laboratory No.
Date
Animal No. in Series [Symbols: male female] Weight ———————————————————————————————————— Died (or killed) o'clock m. Autopsy made o'clock m. ———————————————————————————————————— Notes on Post Mortem Examinations.
General.
A. Seat of Inoculation.
B. Thoracic Cavity.
C. Abdominal Cavity.
D. Cranial Cavity.
- - Bacteriological Histological Organs Preserved. Examination. Examination. A. B. C. D.
41. Finally, the results of the action of the organism or organisms isolated may be correlated with the symptoms observed during life and the observations summarised under the following headings:
Tissue changes:
1. Local—i. e., produced in the neighbourhood of the bacteria.
Position: (a) At primary lesion.
(b) At secondary foci.
Character: (a) Vascular changes and tissue } Acute reactions. } or (b) Degeneration and necrosis. } chronic.
2. General (i. e., produced at a distance from the bacteria, by absorption of toxins):
(a) In special tissues—e. g., nerve cells and fibres, secreting cells, vessel walls, etc.
(b) General effects of malnutrition, etc.
Symptoms:
(a) Associated with known tissue changes.
(b) Without known tissue changes.
Permanent Preparations—Museum Specimens.—
I. Tissues.—The naked-eye appearances of morbid tissues may be preserved by the following method:
1. Remove the tissue or organ from the cadaver as soon after death as possible, using great care to avoid distortion or injury.
2. Place it in a wide-mouthed stoppered jar, large enough to hold it conveniently, resting on a pad of cotton-wool, and arrange it in the position it is intended to occupy (but if it is intended to show a section of the tissue or organ, do not incise it yet).
3. Cover with the Kaiserling fixing solution, and stopper the jar; allow the tissues to remain in this solution for from forty-eight hours to seven days (according to size) to fix. Make any necessary sections.
Kaiserling modified solution is prepared as follows:
Weigh out
Potassium acetate 30 grammes. Potassium nitrate 15 grammes.
and dissolve in
Distilled water 1000 c.c.
then add
Formalin 150 c.c.
Filter.
This fixing solution can be used repeatedly so long as it remains clear. Even when it has become turbid, if simple filtration is sufficient to render it clear, the filtrate may be used again.
4. Transfer the tissue to a bath of methylated spirit (95 per cent.) for thirty minutes to one hour.
5. Remove to a fresh bath of spirit and watch carefully. When the natural colours show in their original tints, average time three to six hours, remove the tissues from the spirit bath, dry off the spirit from the cut surfaces by mopping with a soft cloth, then transfer to the mounting solution.
Jore's mounting solution (modified) consists of
Glycerine 500 c.c. Distilled water 750 c.c. Formalin 2 c.c.
Equally good but much cheaper is Frost's mounting solution:
Potassium acetate 160 grammes. Sodium fluoride 80 grammes. Chloral hydrate 80 grammes. Cane sugar (Tate's cubes) 3,500 grammes. Saturated thymol water 8,000 c.c.
6. After twenty-four hours in this solution, or as soon as the tissue sinks, transfer to a museum jar, fill with fresh mounting solution, and seal.
6a. Or transfer to museum jar and fill with liquefied gelatine, to which has been added 1 per cent. formalin. Cover the jar and allow the gelatine to set. When solid, seal the cover of the jar in place.
7. To seal the museum preparation first warm the glass plate which forms the cover. This is most conveniently done by placing the cleaned and polished cover-plate upon a piece of asbestos millboard over a bunsen flame turned low.
8. Smear an even layer of hot cement over the flange of the jar. The cement is prepared as follows:
Weigh out and mix in an iron ladle
Gutta percha (pure) 4 parts. Asphaltum 5 parts.
and melt together over a bunsen flame, stirring with an iron rod until solution is complete.
9. Invert the glass plate over the jar and press down firmly into the cement. Place a piece of asbestos board on the top and on that rest a suitable weight until the cement is cold and has thoroughly set.
10. Trim off any projecting pieces of cement with an old knife, burr over the joint between jar and cover-plate with a hot smooth piece of metal (e. g., the searing iron).
11. Paint a narrow band of Japan black to finish off, round the joint, overlapping on to the cover-plate.
II. Tube Cultivations of Bacteria.—When showing typical appearances these may be preserved, if not permanently, at least for many years, as museum specimens, by the following method:
1. Take a large glass jar 25 cm. high by 18 cm. diameter, with a firm base and a broad flange, carefully ground, around the mouth. The jar must be fitted with a disc of plate glass ground on one side, to serve as a lid.
2. Smear a thick layer of resin ointment (B.P.) on the flange around the mouth of the jar.
3. Cover the bottom of the jar with a layer of cotton-wool and saturate it with formalin.
4. Remove the cotton-wool plug from the culture tubes and place them, mouth upward, inside the jar. (If water of condensation is present in any of the culture tubes, it should be removed by means of a capillary pipette before placing the tubes in the formalin chamber.)
5. Adjust the glass disc, ground side downward, over the mouth of the jar and secure it by pressing it firmly down into the ointment, with a rotary movement.
6. Remove the tubes from the formalin chamber after the lapse of a week, and dry the exterior of each.
7. Seal the open mouth of each tube in the blowpipe flame and label.
If the cultivations are intended for museum purposes when they are first planted, it is more convenient to employ Bulloch's tubes. These are slightly longer than the ordinary tubes, and are provided with a constriction some 2 cm. below the mouth (Fig. 202)—a feature which renders sealing in the blowpipe flame an easy matter.
XX. THE STUDY OF THE PATHOGENIC BACTERIA.
The student, who has conscientiously worked out the methods, etc., previously dealt with, is in a position to make accurate observations and to write precise descriptions of the results of such observations. He is, therefore, now entrusted with pure cultivations of the various pathogenic bacteria, in order that he may study the life-history of each and record the results of his own observations—to be subsequently corrected or amplified by the demonstrator. In this way he is rendered independent of text-book descriptions, the statements in which he is otherwise too liable to take for granted, without personally attempting to verify their accuracy.
During the course of this work attention must also be directed, as occasion arises, to such other bacteria, pathogenic or saprophytic, as are allied to the particular organisms under observation, or so resemble them as to become possible sources of error, by working them through on parallel lines—in other words the various bacteria should be studied in "groups." In the following pages the grouping in use in the author's elementary classes for medical and dental students and for candidates for the Public Health service is adopted, since a fairly long experience has completely vindicated the value and utility of this arrangement, and by its means a fund of information is obtained with regard to the resemblances and differences, morphological and cultural, of a large number of bacteria. The fact that some bacteria appear in more than one of these groups, so far from being a disadvantage, is a positive gain to the student, since with repetition alone will the necessary familiarity with the cultural characters of important bacteria be acquired. The study of the various groups will of course vary in detail with individual demonstrators, and with the student's requirements—the general line it should take is indicated briefly in connection with the first group only (pages 410-411). This section should be carefully worked through before the student proceeds to the study of bacterioscopical analysis.
It is customary to commence the study of the pathogenic bacteria with the Organisms of Suppuration. This is a large group, for all the pathogenic bacteria possess the power, under certain conditions, of initiating purely pyogenic processes in place of or in addition to their specific lesions, (e. g., Bacillus tuberculosis, Streptococcus lanceolatus, Bacillus typhosus, etc.). There are, however, a certain few organisms which commonly express their pathogenicity in the formation of pus. These are usually grouped together under the title of "pyogenic bacteria," as distinct from those which only occasionally exercise a pyogenic role.
The organisms included in this group are:
1. Staphylococcus pyogenes albus. 2. Staphylococcus pyogenes aureus. 3. Staphylococcus pyogenes citreus. 4. Streptococcus pyogenes longus. 5. Micrococcus tetragenus. 6. Bacillus pyocyaneus. 7. Bacillus pneumoniae.
and in certain special tissues
8. Micrococcus gonorrhoeae. 9. Micrococcus intracellularis meningitidis (Meningococcus). 10. Micrococcus catarrhalis. 11. Bacillus aegypticus (Koch-Weeks Bacillus).
The group may with advantage be subdivided as indicated in the following pages:
I. Pyogenic cocci.
Staphylococcus pyogenes albus. Staphylococcus pyogenes aureus. Staphylococcus pyogenes citreus. to contrast with Micrococcus candicans. Micrococcus agilis.
1. Prepare subcultivations from each:
Bouillon, } Agar streak, } Blood serum, } Litmus milk. } and incubate at 37 deg. C. Agar streak, } Gelatine stab, } Potato. } and incubate at 20 deg. C.
Compare the naked-eye appearances of the cultures from day to day. Note M. agilis refuses to grow at 37 deg. C.
2. Make hanging-drop preparations from the bouillon and agar cultivations after twenty-four hours' incubation. Examine microscopically and compare. Note the locomotive activity of M. agilis and the Brownian movement of the remaining micrococci.
3. Prepare cover-slip films from the agar cultures, after twenty-four hours' incubation. Stain for flagella by the modified Pitfield's method. Note M. agilis is the only micrococcus showing flagella.
4. Make microscopical preparations of each from all the various media after twenty-four and forty-eight hours and three days' incubation. Stain carbolic methylene-blue, carbolic fuchsin, and Gram's method. Examine the films microscopically and compare. Note in the Gram preparation, the Gram negative character of certain individual cocci in each film prepared from the three days' growth—such cocci are dead.
5. Stain section of kidney tissue provided (showing abscess formation by Staphylococcus aureus) by Gram's method, and counterstain with cosin.
6. Stain film preparation of pus from an abscess (containing Staphylococcus pyogenes aureus) with carbolic methylene-blue and also by Gram's method, counterstained with cosin.
7. Inoculate[15] a white mouse subcutaneously with three loopfuls of a forty-eight-hour agar cultivation of the Staphylococcus aureus, emulsified with 0.2 c.c. sterile broth.
Observe carefully during life, and when death occurs make a careful post-mortem examination.
II. Pyogenic cocci.
Micrococcus gonorrhoeae. Micrococcus intracellularis meningitidis (meningococcus). Micrococcus catarrhalis. Micrococcus tetragenus. Micrococcus paratetragenus.
III. Pyogenic cocci.
Streptococcus pyogenes longus. Streptococcus of bovine mastitis. Streptococcus lanceolatus (Diplococcus pneumoniae or pneumococcus). to contrast with Streptococcus brevis. Streptococcus lebensis.
IV. Pyogenic bacilli.
Bacillus pneumoniae (Friedlaender). Bacillus rhinoscleromatis. Bacillus lactis aerogenes.
V. Pyogenic bacilli.
Bacillus pyocyaneus. to contrast with Bacillus fluorescens liquefaciens. Bacillus fluorescens non-liquefaciens.
VI. Pneumonia group.
Streptococcus lanceolatus (pneumococcus). Bacillus pneumoniae (Friedlaender). Streptococcus pyogenes longus.
VII. Diphtheroid group.
Bacillus diphtheriae (Klebs-Loeffler). 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.
(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.
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.
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.
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.
(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.
15. Examine the growth in each tube carefully both macroscopically and microscopically. If the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. When the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it.
16. Utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below, using the ordinary loop to make the subcultures upon solid media, but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette.
Gelatine streak. Agar streak. Potato. Nutrient broth. Litmus milk. Dextrose peptone solution. Laevulose peptone solution. Galactose peptone solution. Maltose peptone solution. Lactose peptone solution. Saccharose peptone solution. Raffinose peptone solution. Dulcite peptone solution. Mannite peptone solution. Glycerin peptone solution. Inulin peptone solution. Dextrin peptone solution.
17. Differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of:
I. The Escherich Group.
B. coli communis. B. coli communior. B. lactis aerogenes. B. cloacae.
II. The Gaertner Group.
Bacillus enteritidis (of Gaertner). B. paratyphosus A. B. paratyphosus B. Bacillus cholerae suum.
III. The Eberth Group.
B. typhosus. B. dysenteriae (Shiga). B. dysenteriae (Flexner). B. faecalis alcaligines.
18. Confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals.
If a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity (down to 0.1 c.c.) of the sample that contains at least one of the microbes of indication. For instance, if growth occurs in all the tubes from 4 to 10, and that growth is subsequently proved to be due to the multiplication of B. coli, then it follows that at least one colon bacillus is present in every 10 c.c. of the water sample, but not in every 5 c.c. If, on the other hand, the presence of the B. coli can only be proved in flask No. 7, then the average number of colon bacilli present in the sample is at least one in every 50 c.c. (i. e., twenty per litre), but not one in every 25 c.c. and so on.
The general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema—whilst the full differential details are set out in tabular form.
ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID GROUPS.
Nutrose agar. - Red colonies. Blue colonies. Escherich group. Gaertner and Eberth groups. ==================== - Lactose peptone solution. ==================== - Gas. No gas. B. coli communis and its allies. Gaertner and Eberth groups. Acid and gas in glucose peptone solution. Acid and coagulation in milk. General turbidity and indol in bouillon. Glucose peptone solution. ================================== Gas. No gas. Gaertner group. Eberth group. =================== Litmus milk. Peptone solution. Litmus milk. Peptone solution. Acid at first. General turbidity. Acid. General turbidity. Alkaline later. No indol. No coagulation. No indol. No coagulation. Serum reaction. Serum reaction.
B. Streptococci.
19. Pick off streptococcus colonies and subcultivate upon nutrient agar exactly as directed in steps 14, 15 and 16.
20. Differentiate the streptococci isolated into members of the saprophytic group of short-chained cocci, or members of the parasitic (pathogenic) group of long-chained cocci, by means of their cultural characters, and record their numerical frequency in the manner indicated for the members of the coli-typhoid group.
DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP
Transcriber's note: Table split to fit 80 spaces.
+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ M D L G M L S R D A = acid reaction o e a a a a a a e G = gas formation t x e l l c c f t i t v a t t c f r l r u c o o h i i i o l t s s a n n t s o o e e r o y e s s o s e e s e e + -+ -+ -+ -+ -+ -+ -+ -+ A G A G A G A G A G A G A G A G + -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ The Escherich Group. B. coli communis + + + + + + + + + + + O + + + + B. coli communior + + + + + + + + + + + + + + + + + B. lactis aerogenes - + + + + + + + + + + O O + + B. acidi lactici - + + + + + + + + + + O O O B. pneumoniae - + + + + + + + + + + + + + + + + B cloaceae(A) + + + + + + + + + + + + + + + + + The Gaertner Group. B. enteritidis + + + + + + + + + O O O O B. paratyphosus A + + + + + + + + + O O O O B. paratyphosus B + + + + + + + + + O O O O B. cholerae suum + + + + + + + + + O O O B. suipestifer + + + + + + + + + O O O The Eberth Group. B. typhosus + + + + + O O O + B. dysenteriae (Shiga) - + + + O O O O O B. dysenteriae (Flexner) - + + + + O O +/- O B. faecalis alkaligines + O O O O O O O O + -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ Table Notes: (B) (C) + -+ -+ -+
- - - - - - - - - I S G D M S I Litmus Milk A=acid reaction n a l u a o n G=gas formation u l y l n r d l i c c n b o - -+ i c e i i i l Early Late n i r t t t n i e e e n -+ - - - - -+ A G A G A G A G A G A G + - - - - - - - - - - The Escherich Group B. coli communis O O + + + + + + + + + + + C B. coli communior O O + + + + + + + + + + + C B. lactis aerogenes O O O O + + + + - + + C B. acidi lactici O O O + + + + + + + + + C B. pneumoniae O O + + + + + + + + - + + C B cloaceae[A] O O + + O + + - + + + + C The Gaertner Group. B. enteritidis O O O + + + + + + - +/- - B. paratyphosus A O +/- O + + + + + + - + O B. paratyphosus B O O O + + + + + + - + - B. cholerae suum O O O O O + + +/- + - B. suipestifer O O O + + + + + + - + - The Eberth Group. B. typhosus O O O O + + - + + B. dysenteriae (Shiga) O O O O O O - + - B. dysenteriae (Flexner) O O O O + O +/- + - B. faecalis alkaligines O O O O O O - - - - - - - - - - - - -+ Table Notes: (D) (E) + - - - -
Table Notes:
(A) * Liquefies gelatine.
(B) + = motile. - = non-motile.
(C) + = acid or gas production. +/- = slight acid production. O = no change.
(D) + = indol production. +/- = slight indol production. - = no indol formed.
(E) + = acid production. - = alkali production. O = no change in reaction. C = clot.
21. Determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated.
On the facing insert page is reproduced a blank from the author's Laboratory Water Analysis Book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined.
B. Concentration Method.
The remaining organisms referred to on page 426 are more conveniently sought for by the concentration method.
Collection of the Sample.—The quantity of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue glass Winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. The bottle may be packed in a wooden box or in an ordinary wicker case. The method of collecting the sample is identical with that described under the heading of Quantitative Examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory.
Apparatus required:
Sterile Chamberland or Doulton "white" porcelain open mouth filter candle, fitted with rubber washer.
Rubber cork to fit mouth of the filter candle, perforated with one hole.
Kitasato serum flask, 2500 c.c. capacity.
Geryk air pump or water force pump.
Wulff's bottle, fitted as wash-bottle, and containing sulphuric acid (to act as a safety valve between filter and pump).
Pressure tubing, clamps, pinch-cock.
Retort stand, with ring and clamp.
Rubber cork for the neck of Winchester quart, perforated with two holes and fitted with one 6 cm. length of straight glass tubing, and one V-shaped piece of glass tubing, one arm 32 cm. in length, the other 52 cm., the shorter arm being plugged with cotton-wool. The rubber stopper must be sterilised by boiling and the glass tubing by hot air, before use.
Flask containing 250 c.c. sterile broth.
Test-tube brush to fit the lumen of the candle, enclosed in a sterile test-tube (and previously sterilised by dry heat or by boiling).
Case of sterile pipettes, 10 c.c. in tenths.
Case of sterile pipettes, 1 c.c. in tenths.
Case of sterile pipettes, 1 c.c. in hundredths.
Tubes of various nutrient media (according to requirements).
Twelve Buchner's tubes with rubber stoppers.
Pyrogallic acid tablets.
Caustic soda tablets.
]
METHOD.—
1. Fit up the filtering apparatus as in the accompanying diagram (Fig. 209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle.
2. Filter the entire 2000 c.c. of water through the filter candle.
3. When the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing.
4. Reverse the position of the glass tubes in the Wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid.
5. Slowly open the metal clamps and allow air to gradually pass through the acid, and enter filter flask, and so restore the pressure.
6. Unship the apparatus, remove the cork from the mouth of the candle.
7. Pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (Fig. 210) emulsify the slimy residue which lines the candle, with the broth.
Practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (Some bacteria will of course be left behind on the walls of the filter and in its pores.)
Up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria.
The Coli-Typhoid Group.—
1. Number nine tubes of bile salt broth (vide page 180), consecutively from 1 to 9.
2. To No 1 add 1 c.c. } of the original water sample 2 add 2 c.c. } before the nitration is commenced. 3 add 5 c.c. }
3. To the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes, as follows:
No. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample). No. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample). No. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample). No. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample). No. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample). No. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample).
4. Put up each tube anaerobically in a Buchner's tube and incubate at 42 deg. C.
5. The subsequent steps are identical with those described under the Enrichment method (see page 428 to 431; Steps 8 to 18).
Alternative Methods.—
A few of the older methods for the isolation of the members of the coli-typhoid groups are referred to but they are distinctly inferior to those already described.
(A) The Carbolic Method:
1. Take ten tubes of carbolised bouillon (vide page 202) and number them consecutively from 1 to 10.
2. Inoculate each tube with a different amount of the water sample or suspension, as in the previous method.
3. Incubate aerobically at 37 deg. C.
4. Examine the culture tubes after twenty-four hours' incubation.
5. From those tubes which shows signs of growth, pour plates in the usual manner, using carbolised gelatine (vide page 202) in place of the ordinary gelatine, and incubate at 20 deg. C. for three, four, or five days as may be necessary.
6. Subcultivate from any colonies that make their appearance, and determine their identity on the lines laid down in the previous method.
(B) Parietti's Method:
1. Take nine tubes of Parietti's bouillon (vide page 202)—i. e., three each of those containing 0.1 c.c., 0.2 c.c., and 0.5 c.c. of Parietti's solution respectively. Mark plainly on the outside of each tube the quantity of Parietti's solution it contains.
2. To each tube add a different amount of the original water, or of the suspension, and incubate at 37 deg. C.
3. Examine the culture tubes after twenty-four and forty-eight hours' incubation, and plate in nutrient carbolised or potato gelatine from such as have grown.
4. Pick off suspicious colonies, if any such appear on the plates, subcultivate them upon the various media, and identify them.
(C) Elsner's Method: This method simply consists in substituting Elsner's potato gelatine (vide page 204) for ordinary nutrient gelatine in any of the previously mentioned methods.
(D) Cambier's Candle Method:
Treat a large volume of the water sample by the concentration method (vide page 434).
1. Remove the rubber stopper from the mouth of the filter candle, introduce 10 c.c. sterile bouillon into its interior, and emulsify the bacterial sediment; replug the mouth of the candle with a wad of sterile cotton-wool.
2. Remove the filter candle from the filter flask and insert it into the mouth of a flask or a glass cylinder containing sterile bouillon sufficient to reach nearly up to the rubber washer on the candle.
3. Incubate for twenty-four to thirty-six hours at 37 deg. C.
4. From the now turbid bouillon in the glass cylinder pour gelatine plates and incubate at 20 deg. C.
5. Subcultivate and identify any suspicious colonies that appear.
(The method depends upon the assumption that members of the typhoid and coli groups find their way through the porcelain filter from the interior to the surrounding bouillon at a quicker rate than the associated bacteria.)
B. Enteritidis Sporogenes.—
1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.
2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (vide page 257), and expose to a temperature of 80 deg. C. for twenty minutes.
3. Number ten tubes of litmus milk consecutively from 1 to 10.
4. Remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid.
5. To each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group (vide page 437).
6. Incubate each tube anaerobically at 37 deg. C. Anaerobic conditions can be obtained by putting the cultures up in Buchner's tubes or in Bulloch's apparatus. If, however, whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis; whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid.
7. Examine after twenty-four hours' incubation. Note (if B. enteritidis sporogenes is present)—
(a) Acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation.
(b) Presence of clotting, and the separation of clear whey.
(c) Presence of gas, as indicated by fissures and bubbles in the coagulum, and possibly masses of coagulum driven up the tube almost to the plug.
8. Replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points.
9. Remove those tubes which give evidence of growth from the Buchner's tubes and carefully pipette off the whey; examine the whey microscopically.
10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the whey and observe the result.
Vibrio Cholerae.—
1. Number ten tubes of peptone water consecutively from 1 to 10.
2. To each of the tubes of peptone water add a measured quantity of the suspension, corresponding to those amounts employed in isolating the members of the coli group (vide page 437).
3. Incubate aerobically at 37 deg. C. for twenty-four hours. Examine the tubes carefully for visible growth, especially delicate pellicle formation, which if present should be examined microscopically for vibrios, both by stained preparations or by fresh specimens with dark ground illumination.
4. Inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation—from the pellicle itself—and incubate at 37 deg. C. for twenty-four hours.
5. Test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated H{2}SO{4}.
5. Prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation.
6. Pick off from the plates any colonies resembling those of the Vibrio cholerae and subcultivate upon all the ordinary laboratory media.
7. Test the vibrio isolated against the serum of an animal immunised to the Vibrio cholerae for agglutination.
B. Anthracis.—
1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.
2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (vide page 257), and expose to a temperature of 80 deg. C. for twenty minutes.
3. Inoculate a young white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. Observe during life, and, if the animal succumbs, make a complete post-mortem examination.
4. Melt three tubes of nutrient agar in boiling water and cool to 42 deg. C.
5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates therefrom.
6. Incubate at 37 deg. C. for twenty-four or forty-eight hours.
7. Pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media.
8. Inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe during life, and if the animal succumbs, make a complete post-mortem examination.
B. Tetani.—
1. Proceed as detailed above in steps 1 and 2 for the isolation of the B. anthracis.
2. Add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in Buchner's tubes at 37 deg. C.
3. From such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. Observe carefully during life, and, if death occurs, make a complete post-mortem examination.
4. From the same tubes pour agar plates and incubate anaerobically in Bulloch's apparatus, at 37 deg. C.
5. Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths.
EXAMINATION OF MILK.
"One-cow" or "whole" milk, if taken from the apparently healthy animal (that is, an animal without any obvious lesion of the udder or teats) with ordinary precautions as to cleanliness, avoidance of dust, etc., contains but few organisms. In dealing with one-cow milk, from a suspected, or an obviously diseased animal, a complete analysis should include the examination (both qualitative and quantitative) of samples of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible, from each quarter of the udder. "Mixed" milk, on the other hand, by the time it leaves the retailer's hands, usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such.
It is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed 5000 micro-organisms per cubic centimetre. Such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation (heating the milk to 65 deg. C. for twenty minutes or to 77 deg. C. for one minute) or the simpler plan of adding preservatives to the milk. Information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples, and in this connection the following tests will be found useful:
1. Raw Milk (Saul).
To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol (ortho-methyl-amino-phenol sulphate), recently prepared and mix. Next add 0.2 c.c. of a 3 per cent. peroxide of hydrogen solution. The appearance of a brick red color within 30 seconds indicates raw milk. Milk heated to 74 deg. C. for thirty minutes undergoes no alteration in color; if heated to 75 deg. C. for ten minutes only, the brick red color appears after standing for about two minutes.
2. Boric Acid.
Evaporate to dryness, 50 c.c. of the milk which has been rendered slightly alkaline to litmus, then incinerate.
Dissolve in distilled water, add slight excess of dilute hydrochloric acid and again evaporate to dryness.
Dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution. Dry the turmeric paper. Rose or cherry-red color = borax or boric acid.
3. Formaldehyde (Hehner).
To 10 c.c. milk in a test tube add 5 c.c. concentrated commercial sulphuric acid slowly, so that the two fluids do not mix. Hold the tube vertically and agitate very gently. Violet zone at the junction of the two liquids = formaldehyde.
4. Hydrogen Peroxide.
To 10 c.c. milk (diluted with equal quantities of water) in a test tube add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2 c.c. acetic acid. Blue coloration of the mixture = hydrogen peroxide.
5. Salicylic Acid.
Precipitate the caseinogen by the addition of acetic acid and filter. To the filtrate add a few drops of 1 per cent. aqueous solution of ferric chloride. Purple coloration = salicylic acid.
6. Sodium Carbonate or Bicarbonate.
To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3 c.c. of a 1 per cent. alcoholic solution of rosolic acid. Brownish color = pure milk; rose color = preserved milk.
Quantitative.—
Collection of Sample.—
The apparatus used for the collection of a retail mixed milk sample consists of a cylindrical copper case, 16 cm. high and 9 cm. in diameter, provided with a "pull-off" lid, containing a milk dipper, also made of copper; and inside this, again, a wide-mouthed, stoppered glass bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter), having a tablet for notes, sand-blasted on the side. The copper cylinder and its contents, secured from shaking by packing with cotton-wool, are sterilised in the hot-air oven (Fig. 26).
When collecting a sample,
1. Remove the cap from the cylinder.
2. Draw out the cotton-wool.
3. Lift out the bottle and dipper together.
4. Receive the milk in the sterile dipper, and pour it directly into the sterile bottle.
5. Enter the particulars necessary for the identification of the specimen, on the tablet, with a lead pencil, or pen and ink.
6. Pack the apparatus in the ice-box for transmission to the laboratory in precisely the same manner as an ordinary water sample.
"Whole" milk may with advantage be collected in the sterile bottle directly since the mouth is sufficiently wide for the milker to direct the stream of milk into it.
Condensed milk must be diluted with sterile distilled water in accordance with the directions printed upon the label, then treated as ordinary milk.
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. Case of sterile plates. Tubes nutrient gelatine or gelatine agar. Tubes of wort gelatine. Tubes of nutrient agar. Water-bath regulated at 42 deg. C. Bunsen burner. Grease pencil.
METHOD.—
1. Arrange four sterile capsules in a row; number them I, II, III, and IV.
2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon into each of the three remaining capsules.
3. Remove 1 c.c. milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule I; mix thoroughly by repeatedly filling and emptying the pipette.
4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the contents of capsule II, and mix as before.
5. 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. of dilution I contains 0.1 c.c. milk sample. 1 c.c. of dilution II contains 0.001 c.c. milk sample. 1 c.c. of dilution III contains 0.00001 c.c. milk sample. 1 c.c. of dilution IV contains 0.0000001 c.c. milk sample.
6. Melt the gelatine and the agar tubes in boiling water; then transfer to the water-bath and cool them down to 42 deg. C.
7. Number the gelatine tubes consecutively 1 to 12.
8. Inoculate the tubes with varying quantities of the material as follows:
To tube No. 1 add 1.0 c.c. of the milk sample. 2 add 0.1 c.c. of the milk sample. { 3 add 1.0 c.c. from capsule I. { 4 add 0.1 c.c. from capsule I. { 5 add 1.0 c.c. from capsule II. { 6 add 0.1 c.c. from capsule II. { 7 add 0.5 c.c. from capsule III. { 8 add 0.3 c.c. from capsule III. { 9 add 0.2 c.c. from capsule III. { 10 add 0.5 c.c. from capsule IV. { 11 add 0.3 c.c. from capsule IV. { 12 add 0.2 c.c. from capsule IV.
9. Pour plates from the gelatine tubes; label, and incubate at 20 deg. C.
10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the milk sample and a similar quantity of the diluted milk from capsules I, II, and III and IV respectively.
11. Pour plates from the wort gelatine; label, and incubate at 20 deg. C.
12. Inoculate the liquefied agar tubes as follows:
To tube No. 1 add 0.1 c.c. of the milk sample. 2 add 0.1 c.c. from capsule I. 3 add 0.1 c.c. from capsule II. 4 add 0.1 c.c. from capsule III. 5 add 1.0 c.c. from capsule IV. } 6 add 0.1 c.c. from capsule IV. }
13. Pour plates from the agar tubes; label, and incubate at 37 deg. C.
14. After twenty-four hours' incubation "inspect," and after forty-eight hours' incubation, "count" the agar plates and estimate the number of "organisms growing at 37 deg. C." present per cubic centimetre of the sample of milk.
15. After three, four, or five days' incubation, "count" the gelatine plates and estimate therefrom the number of "organisms growing at 20 deg. C." present per cubic centimetre of the sample of milk.
16. After a similar interval "count" the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk.
NOTE.—Many observers prefer to employ gelatine agar (see page 193) for the quantitative examination. In this case gelatine-agar plates should be poured from tubes containing the quantities of material indicated in step 8, incubated at 28 deg. C. to 30 deg. C. and after five days the "total number of organisms developing at 28 deg. C." recorded.
Qualitative.—The qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency.
Members of the Coli-typhoid group. Vibrio cholerae. Streptococcus pyogenes longus. Micrococcus melitensis. Staphylococcus pyogenes aureus. Bacillus enteritidis sporogenes. Bacillus diphtheriae. Bacillus tuberculosis.
Some of these occur as accidental contaminations, either from the water supply to the cow farm, or from the farm employees, whilst others are derived directly from the cow.
In milk, as in water examinations, two methods are available, viz.: Enrichment and Concentration—the former is used for the demonstration of bacteria of intestinal origin, the latter for the isolation of the micro-organisms of diphtheria and tubercle. The first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compass; but in the case of milk, thorough centrifugalisation is substituted for filtration.
Apparatus Required:
A large centrifugal machine. This machine, to be of real service in the bacteriological examination of milk, must conform to the following requirements:
1. The centrifugal machine must be of such size, and should carry tubes or bottles of such capacity, as to enable from 200 to 500 c.c. of milk to be manipulated at one time.
2. The rate of centrifugalisation should be from 2500 to 3000 revolutions per minute.
3. The portion of the machine destined to carry the tubes should be a metal disc, of sufficient weight to ensure good "flank" movement, continuing over a considerable period of time. In other words, the machine should run down very gradually and slowly after the motive power is removed, thus obviating any disturbance of the relative positions of particulate matter in the solution that is being centrifugalised.
4. The machine should preferably be driven by electricity, or by power, but in the case of hand-driven machines—
(a) The gearing should be so arranged that the requisite speed is obtained by not more than forty or fifty revolutions of the crank handle per minute, so that it may be maintained for periods of twenty or thirty minutes without undue exertion.
(b) The handle employed should be provided with a special fastening (e. g., a clutch similar to that employed for the free wheel of a bicycle), or should be readily detachable so that, on ceasing to turn, the handle should not, by its weight and air resistance, act as a brake and stop the machine too suddenly.
One of the few satisfactory machines of this class is shown in figure 212.
Sterile centrifugal tubes, of some 60-70 c.c. capacity, tapering to a point at the closed end, plugged with cotton-wool.
Small centrifugal machine to run two tubes of 10 c.c. capacity at 2500 to 3000 revolutions per minute preferably driven by electricity, of the type figured on page 327 (Fig. 162).
Sterile centrifugal tubes of 10 c.c. capacity with the distal extremity contracted to a narrow tube and graduated in hundredths of a cubic centimetre (Fig. 213). |
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