XVIII The Study of Experimental Infections During Life
The possession of pathogenetic properties by an organism under study is indicated by the “infection” of the experimental animal—a term which is employed to summarise the condition resulting from the successful invasion of the tissues of the experimental animal by the micro-organisms inoculated and by their multiplication therein. Infection is considered to have taken place:
1. When the death of the animal is produced as a direct consequence of the inoculation.
2. When without necessarily producing death the inoculation causes local or general changes of a pathological character.
3. When either with or without death, or local or general changes occurring, certain substances make their appearance in the body fluids, which can be shown (in vitro or in vivo) to exert some profound and specific effect when brought into contact with subcultivations of the organism originally inoculated.
The important factors in the production of infection are:
A. Seed. Virulence of organism.
Dose of organism.
B. Soil. Resistance offered by the cells of the experimental animal.
The first two factors, although variable, are to a certain extent under the control of the experimenter. Thus by suitable means the virulence of an organism can be exalted or attenuated, whilst the size of the dose may be increased or diminished. The third factor also varies, not only amongst different species of animals, but also amongst different individuals of the same species. The essential causes of this variation are not so obvious, so that beyond selecting the animals intended for similar experiments with regard to such points as age, size or sex, but little can be done to standardise cell resistance.
Immediately an animal has been inoculated a period of clinical observation must be entered upon, which should only terminate with the death of the animal. The general observations should at first and if the infection is an acute one, be made daily—later, and if the animal appears to be unaffected or if the infection is chronic, both general and special observations should be carried out at weekly intervals. If the animal appears to be still unaffected, it should be killed with chloroform vapour at the end of two or three months and a complete post-mortem carried out.
A. The general observations should take cognisance of:
1. General appearance. The experimental animal should be inspected daily, not only with a view to detecting symptoms due to the experimental infection, but also to prevent any intercurrent infection, naturally acquired, from escaping notice (vide page 337).
2. The weight of the inoculated animal should be observed and recorded each day during the course of an experimental infection at precisely the same hour, preferably just before the morning feed.
3. The temperature should similarly be recorded daily, if not more frequently, during the whole period the animal is under observation, and carefully charted—individual variations will at once become apparent. It should be borne in mind that the temperature regarded as normal for man (37.5° C.) is not the normal average temperature of any of the lower animals save the rat and mouse. The accompanying table of normal averages for the animals usually employed in bacteriological research may be of use in preventing the erroneous assumption that pyrexia is present in an animal, which merely shows its own normal temperature.
| NORMAL AVERAGES. | |||
| Rectal Temp. °C. | Pulse. | Respirations. | |
| Animal. | Rate per minute. | ||
| Frog | 8.9-17.2 | 80 | 12 |
| Mouse | 37.4 | 120 | ... |
| Rat | 37.5 | ... | 210 |
| Guinea pig | 38.6 | 150 | 80 |
| Rabbit | 38.7 | 135 | 55 |
| Cat | 38.7 | 130 | 24 |
| Dog | 38.6 | 95 | 15 |
| Goat | 40.0 | 75 | 16 |
| Ox | 38.8 | 45 | .. |
| Horse | 37.9 | 38 | 11 |
| Monkey (Rhesus) | 38.4 | 100 | 19 |
| Pigeon | 40.9 | 136 | 30 |
| Fowl | 41.6 | 140 | 12 |
B. Special observations comprise some or all of the following, according to the method of inoculation and the character of the virus.
1. The site of inoculation should be minutely examined at least at weekly intervals, and the neighbouring lymphatic glands palpated.
2. Any local reaction at the site of inoculation and any other readily accessible lesion should be carefully investigated. Any suppurative process which may occur, whether in the subcutaneous tissues or in joints, should be explored and the pus carefully examined both microscopically and culturally.
Fluid secretions and excretions, such as pus or serous exudates when accessible are collected direct from the body in sterile capillary pipettes (vide Fig. 13a,) in the following manner:
1. Open the case containing the pipettes, grasp one by the plugged end, remove it from the case, and replace the lid of the latter.
2. Attach a rubber teat (vide page 10) to the plugged end of the pipette and use the teat as the handle of the pipette.
3. Pass the entire length of the pipette twice or thrice through the flame of the Bunsen burner.
4. Snap off the sealed end of the pipette with a pair of sterile forceps.
5. Compress the india-rubber teat, thrust the point of the pipette into the secretion; now relax the pressure on the teat and allow the pipette to fill.
6. Remove the point of the pipette from the secretion, allow the fluid to run a short distance up the capillary stem and seal the point of the pipette in the flame. (If using a pipette with a constriction below the plugged mouthpiece (Fig 13b), this portion of the pipette may also be sealed in the flame.)
When ready to examine the morbid material snap off the sealed end of the pipette with sterile forceps and eject the contents of the pipette into a sterile capsule. The material can now be utilized for cover-slip preparations, cultivations and inoculation experiment.
3. The peripheral blood should be examined from time to time for from this tissue is often obtained the fullest information as to the course and progress of an infection.
a. The histological examination of the blood should be directed chiefly to observations on the number and kind of white cells; and since but few bacteriologists are at the same time expert comparative hæmatologists, some notes on the normal characters of the blood of the commoner laboratory animals, contrasted with those of man, are inserted for reference. These have been very kindly compiled for me by my friend and one time colleague Dr. Cecil Price Jones.
Comparative Hæmocytology of Laboratory Animals
| Totals | Percentages | |||||||
| Animal | Red cells | White cells | Hb, per cent. | Lymphocytes, per cent. | Large monos, per cent. | Polymorph, per cent. | Eosinoph, per cent. | Mast cells, per cent. |
| Frog | 490,000 | 8,000 | 58 | 40 | 10.0 | 22.0 | 15 | 13 |
| Mouse | 8,700,000 | 8,000 | 78 | 60 | 21.5 | 17.0 | 1.4 | 0.1 |
| Rat | 9,000,000 | 9,000 | 85 | 54 | 7.0 | 37.5 | 1.3 | 0.2 |
| Guinea-pig | 5,700,000 | 10,000 | 99 | 55 | 9.0 | 32.8 | 3.0 | 0.2 |
| Rabbit | 6,000,000 | 7,000 | 70 | 50 | 2.0 | 46.0 | 0.6 | 1.4 |
| Rhesus | 4,500,000 | 13,000 | 77 | 43 | 5.0 | 50.0 | 1.3 | 0.7 |
| Goat | 14,600,000 | 15,000 | 58 | 35 | 6.3 | 56.7 | 1.25 | 0.75 |
| Fowl | 3,500,000 | 30,000 | 100 | 49 | 3.0 | 42.0 | 1.0 | 5.0 |
| Pigeon | 3,500,000 | 20,000 | 101 | 43 | 9.0 | 43.0 | 3.0 | 2.0 |
| Man(adult) Normal limits. | 5,000,000 (4.5-5) millions. | 7,500 (7-9) thousands. | 100 (95-101) | 25 (20-30) | 5.5 (4-8) | 65 (55-68) | 4.0 (3-5) | 0.5 (0.5-2) |
The above table represents in each case the average of a large number of counts.
Remarks.
Frog.—The red cells are large oval nucleated (20-25µ by 12-15µ) discs, the nucleus relatively small and irregularly elongated or oval, about 10µ in length. Many primitive and developing forms are usually observed—also free nuclei and many cells in various stages of degeneration. Hæmoglobin estimation is difficult owing to turbidity of the blood after dilution with water. The polymorphonuclear leucocytes are large cells, about 20µ; no definite granules can be observed. The eosinophile cells contain large deeply staining coccal-shaped granules.
Mouse.—The granules of the polymorphonuclear leucocytes are usually not stained, or only very faintly so. The nucleus of the eosinophile cell is ring-shaped or much divided, and the granules are coccal and stain oxyphile. The remarkable character of the blood is the high percentage of large mononuclear cells.
Rat.—The fine rod-shaped granules of the polymorphonuclear leucocytes are usually very faintly stained. The granules of eosinophile cells are well stained and coccal-shaped, the nucleus is often ring shaped. The basophile granular cells are few—but the granules are large, and stain deeply basophile.
Guinea-pig.—Polychromasia and punctate basophilia of red cells are very commonly observed—nucleated red cells are also frequent. The large mononuclear cells often contain vacuoles—“Kurlow cells”—possibly of a parasitic nature.
Rabbit.—It is not uncommon to find nucleated red cells in films from quite healthy animals. The granules of the polymorphonuclear leucocytes stain oxyphile. The coarse granules of the eosinophile cells appear to stain less deeply oxyphile, probably owing to the basophile staining of the cytoplasm.
Rhesus monkey.—The blood cells resemble those met with in human blood. The minute neutrophile granules of the polymorphonuclear leucocytes are often very scanty, and sometimes apparently absent. The eosinophile cells are not so densely packed with coarse oxpyhile granules as in the human eosinophile, and the nuclei of these cells are usually much divided, or polymorphous.
Goat.—The red cells are small, nonnucleated discs, only about 4.5µ diameter, not much more than half that of the human red cell. The polymorphonuclear leucocytes have only a few very minute coccal-shaped oxyphile granules, the nucleus is polymorphous. The eosinophile cells are large cells up to 20µ, the cytoplasm is basophile and contains coarse coccal-shaped oxyphile granules, and the nucleus is often much divided.
Fowl.—The red cells are oval nucleated discs about 12µ by 6µ, the nucleus being relatively small (about 4µ long), irregularly elongated or oval; round, more deeply stained cells with round or diffuse nuclei, also free nuclei and degenerated forms of red cells are often present. The granules of the cells corresponding to the polymorphonuclear leucocytes are rod-shaped, often beaded or with clubbed ends. The nucleus is not polymorphous, but usually divided into two, though it may be single. The cells probably corresponding to eosinophile leucocytes have fine coccal-shaped granules, faintly staining eosinophile or neutrophile. The basophile granules of the “mast” cells are coccal-shaped, of various size—often quite powdery.
Pigeon.—Red cells resemble those of the fowl, and similar varieties of appearance may be noted. The granules of those cells which correspond to polymorphonuclear leucocytes are rod-shaped, but smaller and finer than in the fowl, and do not show clubbed appearances. The nucleus is not polymorphous, and only occasionally divided. The coccal-shaped granules of the eosinophile cells are stained more deeply oxyphile than those of the corresponding cells of the fowl.
The preparation of dried films for this histological examination of the blood is carried out as follows:
1. Small samples of blood for the preparation of blood films are most conveniently obtained from the veins of the ear in most of the ordinary laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in the pigeon and fowl the axillary vein should be punctured; in the rat and mouse either a vein in the ear or preferably by wounding the tip of the tail; in the frog, the web of the foot should be selected.
2. Puncture the selected vein with a sharp needle. A flat Hagedorn needle (size No. 8) with a cutting edge is the most useful for this purpose. If the vein cannot be distended by proximal compression, vigourous friction with a piece of dry lint may have the desired effect—or a test-tube full of water at about 40°C. may be placed close to the vein. Failing these methods, a drop or two of xylol may be dropped on the skin just over the vein, left on for a few seconds and then wiped off with a piece of dry lint.
3. One of the short ends of a 3 by 1 glass slip is brought into contact with the exuding drop of blood, so that it picks up a small drop.
4. The slide is then lowered transversely on to the surface of a second 3 by 1 slip, which rests on the bench near to one end at an angle of about 45°, and retained in this position for a few seconds, while the drop of blood spreads along the whole of the line of contact (see also Fig. 69).
5. Draw the first slide firmly and evenly along the entire length of the lower slide, leaving a thin regular film which will probably show the blood cells only one layer thick.
6. Allow the film to dry in the air.
7. Stain with one of the polychrome blood stains (see page 97).
8. Examine microscopically.
b. The bacteriological examination of the blood is directed solely to the demonstration of the presence in the circulating blood of the organisms previously injected into the animal. For this purpose several cubic centimetres of blood should be taken in an all-glass syringe from an accessible vein corresponding to one of those suggested as the site of intravenous inoculation—and under similar aseptic precautions.
1. Sterilise an all-glass syringe of suitable size, and when cool draw into the syringe some sterile sodium citrate solution and moisten the whole of the interior of the barrel; then eject all the citrate solution if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are required retain about half a cubic centimetre of the fluid in the syringe. This prevents coagulation of the blood.
The sodium citrate solution is prepared by dissolving:
| Sodium citrate | 10 gramme. |
| Sodium chloride | 0.75 grammes. |
| In distilled water | 100 c.c. |
Sterilise by Boiling
2. Prepare the animal as for intravenous inoculation (see page 363) and introduce the syringe needle into the lumen of the selected vein.
3. Slowly withdraw the piston of the syringe. When sufficient blood has been collected direct the assistant to release the proximal compression of the vein; and withdraw the needle.
4. Remove the needle from the nozzle of the syringe and deliver the citrated blood into a small Ehlenmeyer flask containing about 250 c.c. of nutrient broth.
5. Label, incubate and examine daily until growth occurs or until the expiration of ten days.
c. The serological examination of the blood is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals, and within certain limits to an estimation of their amounts.
The chief of these bodies are:
Antitoxin.
Agglutinin.
Precipitin.
Opsonin.
Immune body or Bacteriolysin.
None of these substances are capable of isolation in a state of purity apart from the blood serum, consequently special methods have been elaborated to permit of their recognition. In every instance the behaviour of serum from the experimental animal, which may be termed “specific” serum, is studied in comparison with that of serum from an uninoculated animal of the same species, and which is termed “normal” serum. In view of minor differences in constitution exhibited by the serum of various individuals of the same series, it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal, under the term “pooled serum.” The method of collecting blood (e. g., from the rabbit) for serological tests is as follows:
Collection of Serum.
Apparatus required:
Razor.
Liquid soap.
Cotton-wool.
Lysol 2 per cent. solution, in drop bottle.
Ether in drop bottle.
Flat Hagedorn needles.
Blood pipettes (Fig. 16, page 12).
Centrifugal machine.
Centrifuge tubes.
Glass cutting knife.
Bunsen flame.
Writing diamond or grease pencil.
Method.
1. Shave the dorsal surface of the ear over the course of the posterior auricular vein (see Fig. 192).
2. Sterilise the skin by washing with lysol.
The lysol should be applied with sterile cotton-wool and the ear vigourously rubbed, not only to remove superficial scales of epithelium, but also to render the ear hyperæmic and the vein prominent.
3. Remove the lysol with ether dropped from a drop bottle, and allow the ether to evaporate.
4. Puncture the vein with a sterile Hagedorn needle.
5. Take a small blood-collecting pipette (Fig. 161) and hold it at an angle to the ear, one end touching the issuing drop of blood, the other depressed.
The blood will now enter the pipette at first by capillarity; afterward gravity will also come into play and the pipette can be two-thirds filled without difficulty.
6. Hold the tube by the end containing the blood, the clean end pointing obliquely upward—warm this end at the bunsen flame to expel some of the contained air; then seal the clean point in the flame.
7. Place the pipette down on a cool surface (e. g., a glass slide). The rapid cooling of the air in the clean end of the pipette creates a negative pressure, and the blood is sucked back into the pipette, leaving the soiled end free from blood. Seal this end in the bunsen flame.
8. Mark the distinctive title of the specimen (e. g., animal’s number) upon the pipette with a writing diamond or grease pencil.
9. When the sealed ends are cold and the blood has clotted, place the pipette on the centrifuge, clean end downward; counterpoise and centrifugalise thoroughly. On removing the pipette from the centrifuge, the red cells will be collected in a firm mass at one end, and above them will appear the clear serum.
10. By marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes.
If larger quantities of blood are required, the animal, after puncturing the vein, should be inverted, an assistant holding it up by the legs. Blood to the volume of several cubic centimetres will now drop from the punctured vein, and should be caught in a tapering centrifuge tube, the tube transferred to the incubator at 37° C. for two hours, then placed in the centrifugal machine, counterpoised and centrifugalised thoroughly. The three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin, opsonin and bacteriolysin; and the methods of testing for these bodies will now be considered.
Agglutinin
Agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. This substance possesses the power of collecting together in clumps and masses, or agglutinating watery suspensions of that particular microbe.
Dilution of the Specific Serum:
Apparatus required:
Sterile graduated capillary pipettes to contain 10 c. mm. (Fig. 17).
Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).
Small sterile test-tubes 5 × 0.5 cm.
Normal saline solution in flask or test-tube.
Pipette of specific serum.
Glass cutting knife, or three-square file.
Glass capsule, nearly full of dry silver sand, or roll of plasticine.
Grease pencil.
Method.—
1. Take three sterile test-tubes and number them 1, 2 and 3.
2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block.
3. Make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube.
4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube No. 1; and label s.s., (specific serum), 10 per cent.
5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh pipette, and mix it with the contents of tube No. 2; and label s.s., 1 per cent.
6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh pipette, and mix it with the contents of tube No. 3; and label s.s., 0.1 per cent.
When the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by Wright’s method in a capillary teat pipette.
Dilution of Serum by Means of a Teat Pipette.
Materials required:
Blood pipette containing sample of specific serum after centrifugalisation.
Capsule of diluting fluid—normal saline solution.
Supply of Pasteur pipettes (Fig. 13a).
India-rubber teats.
Small test-tubes.
A block of plasticine to act as a test-tube stand.
Grease pencil.
Method:
1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block.
2. Take a Pasteur pipette, nick the capillary stem just above the sealed end with a glass cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem—cut “square”, in fact. Prepare several, say a dozen, in this manner.
3. Fit a rubber teat to the barrel of each of the pipettes.
4. Make a mark with the grease pencil on the stem of one of the pipettes about 2 or 3 cm. from the open extremity.
5. Compress the teat between the finger and thumb (Fig. 193) to such an extent as to drive out the greater part of the contained air.
6. Maintaining the pressure on the teat pass the stem of the pipette into the capsule holding the saline solution, until the open end of the pipette is below the level of the fluid.
7. Now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark. As soon as this point is reached, check the movement of the column of fluid by maintaining the pressure on the teat, neither relaxing nor increasing it.
8. Withdraw the point of the pipette clear of the fluid, and again relax the pressure on the teat very slightly. The column of saline solution rises higher in the stem, and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one.
9. Again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark, and follow this with a second air index.
10. In like manner take up seven more equal volumes of saline solution and their following air bubbles. There are now nine equal volumes of normal saline in the pipette.
11. Now pass the point of the pipette into the blood tube and dip the open end below the surface of the serum. Proceeding as before, aspirate a volume of serum into the capillary stem up to the level of the pencil mark.
12. Eject the contents of the pipette into the small tube marked 10 per cent. by compressing the rubber teat between thumb and finger.
13. Mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube.
14. Now take a clean pipette and proceed precisely as before, 4 to 10.
15. Having aspirated nine equal volumes of saline into this second pipette, now take up one similar volume of the fluid in the “10 per cent. tube.”
16. Eject the contents of this pipette into the second tube marked 1 per cent. and mix thoroughly as before.
17. In similar fashion make the 0.1 per cent. solution and transfer to the third tube.
18. Further dilutions in multiples of ten can be prepared in the same way, and by varying the number of volumes of diluting fluid or serum any required dilution can be made (see Appendix, Dilution Tables).
Note.—The saline diluting fluid must always be taken into the pipette first, otherwise if the serum contains a very large amount of agglutinin the traces of this serum added to the saline solution may be sufficient to entirely vitiate the subsequent observations—whilst if more than one sample of serum is diluted from the same saline solution serious errors may be introduced into the experiments.
The Microscopical Reaction:
Apparatus Required:
Five hanging-drop slides (or preferably two slide), with two cells mounted side by side on each (Fig. 62, a), and one slide with one cell only.
Vaseline.
Cover-slips.
Platinum loop.
Grease pencil.
Eighteen to twenty-four-hour-old bouillon cultivation of the organism to be tested (e. g., Bacillus typhi abdominalis)
Pipette end with the remainder of the specific serum labelled s.s.
Tubes containing the three solutions of the specific serum, 10, 1, and 0.1 per cent. respectively.
Pipette end with pooled normal serum labelled p.s.
Method.—
1. Make five hanging-drop preparations, thus:
(a) One loopful of bouillon cultivation + one loopful pooled serum; label “Control.”
(b) One loopful culture + one loopful undiluted specific serum; label 50 per cent.
Mount these two cover-slips on a double-celled slide.
(c) One loopful bouillon culture + one loopful 10 per cent. serum; label 5 per cent.
Mount this on single-cell slide.
(d) One loopful bouillon culture + one loopful 1 per cent. serum; label 0.5 per cent.
(e) One loopful bouillon culture + one loopful 0.1 per cent. serum; label 0.05 per cent.
Mount these two cover-slips on a double-celled slide.
2. Note the time: Examine the control to determine that the bacilli are motile and uniformly scattered over the field—not collected into masses.
3. Next examine the 50 per cent. serum preparation.
If agglutinin is present and the test is giving a positive reaction, the bacilli will be collected in large clumps.
If the test is giving a negative reaction, the bacilli may be collected in large clumps owing to the viscosity of the concentrated serum.
4. Observe the 5 per cent. preparation microscopically.
If the bacilli are aggregated into clumps, positive reaction.
If the bacilli are not aggregated into clumps, observe until thirty minutes from the time of preparation before recording a negative reaction.
5. Examine the 0.5 and 0.05 per cent. preparations.
These may or may not show agglutination when the result of the examination of the 5 per cent. preparation is positive, according to the potency of the specific serum; and by the examination of a series of dilutions a quantitative comparison of the valency of specific sera from different sources, or of serum from the same animal at different periods during the course of active immunisation may be obtained.
Note.—The graduated pipettes supplied with Thoma’s hæmatocytometer (intended for the collection of the specimen of blood required for the enumeration of leucocytes), giving a dilution of 1 in 10—i. e., 10 per cent.—may be substituted for the graduated capillary pipettes referred to above, if the vessel in which the serum has been separated is of sufficiently large diameter to permit of their use.
The Macroscopical Reaction:
Sterile graduated capillary pipettes to contain 90 c. mm.
Eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested.
Three test-tubes containing the 10, 1, and 0.1 per cent. solutions of specific serum (about 90 c. mm. remaining in each).
Tube containing 50 per cent. solution of pooled serum.
Sedimentation pipettes (vide page 17) or teat pipettes.
Method.
1. Pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quantity into the tube containing the pooled serum.
2. Fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. Seal off the lower ends of the sedimentation tubes in the Bunsen flame.
3. Label each tube with the dilution of serum that it contains—viz., 5, 0.5, and 0.05 per cent.
4. Place the pipettes in a vertical position, in a beaker, in the incubator at 37°C., for one or two hours.
5. Observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum.
Opsonin
Opsonin is the term applied by Wright to a substance, present in the serum of an inoculated animal, which is able to act upon or sensitise bacteria of the species originally injected, so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes. In the method for demonstrating opsonin about to be described, a comparison is made between the opsonic “power” of the pooled serum and the specific serum.
Apparatus:
Small centrifuge and tubes for same (made from the barrels of broken capillary pipettes by sealing the conical ends in the bunsen flame).
Capillary Pasteur pipettes.
India-rubber teats.
Grease pencil.
Bunsen burner with peep flame.
Electrical signal clock (see page 39) stop watch, or watch.
Rectangular glass box or tray to hold pipettes.
Incubator regulated at 37°C.
3 × 1 slides.
Piece of light rubber tubing.
Rectangular block of plasticine.
Flask of normal saline solution.
Flask of sodium citrate (1.5 per cent.) in normal saline solution.
Materials required, and their preparation:
Small tube of “washed cells” (red blood discs and leucocytes); human cells are used in estimating the opsonising power of the serum of experimental animals.
Small tube of emulsion of bacteria of the species responsible for the infection of the experimental animal.
Blood pipette containing specific serum.
Blood pipette containing “pooled” serum.
Washed Cells.—
1. Take a small centrifuge tube and half fill it with sodium citrate solution. Mark with the grease pencil the upper limit of the fluid.
2. Cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether. Wind the rubber tubing tightly round the second phalanx; puncture with a sterile Hagedorn needle through the cleansed area of skin.
3. Take up a sufficiency of the issuing blood (more or less according to the number of tests to be performed) with a teat pipette, transfer it to the tube of citrate solution and mix thoroughly. Make a second mark on the tube at the upper level of the mixed citrate solution and blood.
4. Place the tube in the centrifuge, counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks.
The column of fluid in the tube now shows clear supernatant fluid (citrate solution and blood plasma) separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes.
5. Remove the supernatant column of citrate solution by means of a teat pipette, fill normal saline solution into the tube up to the upper pencil mark, and distribute the blood cells throughout the saline by means of the teat pipette. Centrifugalise as before.
6. Again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more.
7. Remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes.
8. Next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands—just as is done with a tube of liquefied medium prior to pouring a plate.
9. Set the tube upright in the plasticine block near to one end.
Bacterial Emulsion.—
1. Take an 18- to 24-hour culture of the required bacterium (e. g., Diplococcus pneumoniæ) grown upon sloped blood agar at 37° C. Pour over the surface of the medium some 5 c.c. of normal saline solution.
2. With a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution.
3. Allow the tube to stand for a few minutes so that the large masses of growth may settle down; transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly.
4. Examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses. If unsatisfactory prepare another emulsion, this time scraping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quantities of normal saline. If satisfactory insert the tube in the plasticine block next to that containing the washed cells.
Specific Serum.—
Pooled Serum.—
These sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block.
The plasticine block now presents the appearances shown in Fig. 194.
Method for Determining the Opsonic Index.—
1. Take a capillary pipette fitted with a teat, cut the distal end square and make a pencil mark about 2 cm. from the end.
2. Aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see Fig. 195).
3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. These two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem.
4. Seal the point of the pipette in the peep flame of the bunsen burner and remove teat.
5. Mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray.
6. Take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. Treat precisely as in 3 and 4, label it “control” or “N.S.” (normal serum) and place in the box by the side of the specific serum preparation.
7. Place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch).
8. At the expiration of the incubation time remove the pipettes from the incubator.
9. Cut off the sealed end of the specific serum preparation. Mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (vide page 376) on each in such a way that only one-half of the surface of each slide is covered with blood—the free edge of the blood film approximating to the longitudinal axis of the slide.
Allow films to dry and label the slides with writing diamond.
10. Treat the contents of the control pipette in similar fashion.
11. Select the better film from each pair for fixing and staining.
12. Fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (Fig. 196). Place the rack in the first trough which contains the alcoholic solution of Leishman’s stain for two minutes to fix.
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 hæmolysis on the subsequent addition of “sensitised” red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate hæmolysin—two of the three essentials in the hæmolytic system, vide page 326).
Apparatus Required:
Sterile pipettes 1 c.c., (graduated in tenths).
16 × 2 cm. test-tubes.
9 × 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.
Hæmolytic 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° 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° C. for a period of one hour.
7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal hæmolytic doses of the corresponding hæmolysin into each tube.
8. Mix thoroughly and return the tubes to the incubator at 37° 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 hæmolysis; tube 2 should give no evidence whatever of hæmolysis.
These tubes form the controls to the first tube, which contains the serum to be tested.
In tube No. 1 the absence of hæmolysis 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 hæmolytic system; on the other hand the presence of hæmolysis 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 hæmolytic serum, and was ready to combine with those reagents to complete the hæmolytic system.
The method may be shown diagramatically as under using the symbols already indicated
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 hæmolytic doses of hæmolysin, add to the erythrocyte solution on the test-tube.
3. Allow the erythrocyte and hæmolysin 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°C. and complete the experiment as previously described (steps 8 onward).