The Fundamentals of Bacteriology

by Charles Bradfield Morrey

The chemical nature of opsonins is not certainly determined, but they appear to be a distinct class of antibodies and to possess two groups, a combining or haptophore and a preparing or opsonic group and hence are similar to antibodies of Ehrlich’s second order—agglutinins and precipitins. Wright also showed that opsonins are just as specific as agglutinins are—that is, a micrococcus opsonin prepares micrococci only for phagocytosis and not streptococci or any other bacteria.

Wright showed that opsonins for many bacteria are present in normal serum and that in the serum of an animal which has been immunized against such bacteria the opsonins are increased in amount. Also that in a person infected with certain bacteria the opsonins are either increased or diminished, depending on whether the progress of the infection is favorable or unfavorable. The opsonic power of a serum normal or otherwise is determined by mixing an emulsion of fresh leukocytes in normal saline solution with a suspension of the bacteria and with the serum to be tested. The leukocytes must first be washed in several changes of normal salt solution to free them from any adherent plasma or serum. The mixture is incubated for about fifteen minutes and then slides are made, stained with a good differential blood stain, Wright’s or other, and the average number of bacteria taken up by at least fifty phagocytes taken in order in a field is determined by counting under the microscope. The number so obtained Wright calls the phagocytic index of the serum tested. The phagocytic index of a given serum divided by the phagocytic index of a normal serum gives the opsonic index of the serum tested. Assuming the normal opsonic index to be 1, Wright asserts that in healthy individuals the range should be not more than from 0.8 to 1.2, and that an index below 0.8 may show a great susceptibility for the organism tested, infection with the given organism if derived from the individual, or improper dosage in case attempts have been made to immunize by using killed cultures, vaccines, of the organism.

On the occasion of the author’s visit to Wright’s clinic (1911) he stated that he used the determination of the opsonic index chiefly as a guide to the dosage in the use of vaccines.

Most workers outside the Wright school have failed to recognize any essential value of determinations of the opsonic index in the use of vaccines. Some of the reasons for this are as follows: The limit of error in phagocytic counts may be as great as 50 per cent. in different series of fifty, hence several hundred must be counted, which adds greatly to the tediousness and time involved; the variation in apparently healthy individuals is frequently great, hence the “normal” is too uncertain; finally the opsonic index and the clinical course of the disease do not by any means run parallel. Undoubtedly the method has decided value in the hands of an individual who makes opsonic determinations his chief work, as Wright’s assistants do, but it can scarcely be maintained at the present time that such determinations are necessary in vaccine therapy. Nevertheless that opsonins actually exist and that they play an essential part in phagocytosis, and hence in immunity, is now generally recognized.

BACTERIAL VACCINES.

Whether determinations of opsonic index are useful or not is largely a matter of individual opinion, but there is scarcely room to doubt that Wright has conferred a lasting benefit by his revival of the use of dead cultures of bacteria, bacterial vaccines, both for protective inoculation and for treatment. It is perhaps better to use the older terms “vaccination” and “vaccine” (though the cow, vacca, is not concerned) than to use Wright’s term “opsonic method” in this connection, bearing in mind that the idea of a vaccine is that it contains the causative organism of the infection as indicated on [p. 253].

As early as 1880 Touissant proposed the use of dead cultures of bacteria to produce immunity. But because injections of such cultures were so frequently followed by abscess formation, doubtless due to the high temperatures used to kill the bacteria, the method was abandoned. Further, Pasteur and the French school persistently denied the possibility of success with such a procedure, and some of them even maintain this attitude at the present time. The successes of Wright and the English school which are being repeated so generally wherever properly attempted, leave no doubt in the unprejudiced of the very great value of the method and have unquestionably opened a most promising field both for preventive inoculation and for treatment in many infectious diseases. That the practice is no more universally applicable than are immune serums and that it has been and is still being grossly overexploited is undoubted.

The use of a vaccine is based on two fundamental principles. The first of these is that the cell introduced must not be in a condition to cause serious injury to the animal by its multiplication and consequent elaboration of injurious substances. The second is that, on the other hand, it must contain antigens in such condition that they will act as stimuli to the body cells to produce the necessary antibodies, whether these be opsonins, bactericidal substances, or anti-endotoxins. In the introduction of living organisms there is always more or less risk of the organism not being sufficiently attenuated and hence of the possibility of its producing too severe an infection. In using killed cultures, great care must be exercised in destroying the organisms, so that the antigens are not at the same time rendered inactive. Hence in the preparation of bacterial vaccines by Wright’s method the temperature and the length of time used to kill the bacteria are most important factors. This method is in general to grow the organisms on an agar medium, rub off the culture and emulsify in sterile normal salt solution (0.85 per cent. NaCl). The number of bacteria per cc. is determined by staining a slide made from a small volume of the emulsion mixed with an equal volume of human blood drawn from the finger and counting the relative number of bacteria and of red blood corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., a simple calculation gives the number of bacteria. The emulsion of bacteria is then diluted so that a certain number of millions shall be contained in each cc., “standardized” as it is called, then heated to the proper temperature for the necessary time and it is ready for use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added unless the vaccine is to be used up at once. The amounts of culture, salt solution, etc., vary with the purpose for which the vaccine is to be used, from one or two agar slant cultures and a few cc. of solution, when a single animal is to be treated, to bulk agar cultures and liters of solution as in preparing antityphoid vaccine on a large scale.

Agar surface cultures are used so that there will be as little admixture of foreign protein as possible (see [Anaphylaxis], p. 289 et seq.). Normal saline solution is isotonic with the body cells and hence is employed as the vehicle.

Lipovaccines.—The suspension of bacteria in neutral oil was first used by Le Moignac and Pinoy who gave the name “lipovaccines” (λιπος = fat) to them. It was claimed that the reaction following injection of these vaccines was less severe than with saline vaccines in many instances; also, that the bacteria were much more slowly absorbed. For these two reasons it was hoped that much larger numbers of bacteria could be injected at one dose and one injection would suffice instead of three or more as ordinarily used. The technique of preparation, standardization and killing of the organisms has not as yet been sufficiently well established to warrant the general substitution of lipovaccines for ordinary saline suspensions.