The Fundamentals of Bacteriology

by Charles Bradfield Morrey

The agglutinins in the serum may be removed from it by treating it with a suspension of the cells for which agglutinins are present. If the “chief” cell is used all the agglutinins will be absorbed. If related cells are used, only the agglutinins for this particular kind are removed. These “absorption tests” furnish another means of determining specificity of serum, or rather of determining the “chief agglutinin” present.

Just as an unidentified disease in an animal may be determined by testing its serum as above described against known kinds of bacteria, so unknown bacteria isolated from an animal, from water, etc., may be identified by testing them against the blood sera of different animals, each of which has been properly inoculated with a different kind of known bacteria. If the unknown organism is agglutinated by the blood of one of the animals in high dilution, and not by the others, evidently the bacterium is the same as that with which the animal has been inoculated, or immunized, as is usually stated. This method of identifying cultures of bacteria is of wide application, but is used practically only in those cases where other methods of identification are not readily applied, and especially where other methods are not sufficient as in the “intestinal group” of organisms in human practice.

The diagnosis of disease in an animal by testing its serum is also a valuable and much used procedure. This is the method of the “Widal” or “Gruber-Widal” test for typhoid fever in man and is used in veterinary practice in testing for glanders, contagious abortion, etc. In some cases a dilution of the serum of from 20 to 50 times is sufficient for diagnosis (Malta fever), in most cases, however, 50 times is the lowest limit. Evidently the greater the dilution, that is, the higher the “titer,” the more specific is the reaction.

PRECIPITINS.

Since agglutinins act on bacteria, probably through the presence of substances within the bacterial cell, it is reasonable to expect that if these substances be dissolved out of the cell, there would be some reaction between their (colloidal) solution and the same serum. As a matter of fact Kraus (1897) showed that broth cultures freed from bacteria by porcelain filters do show a precipitate when mixed with the serum of an animal immunized against the particular bacterium and that the reaction is specific under proper conditions of dilution. It was not long after Kraus’s work until the experiments were tried of “immunizing” an animal not against a bacterium or its filtered culture, but against (colloidal) solutions of proteins, such as white of egg, casein of milk, proteins of meat and of blood serum, vegetable proteins, etc. It was ascertained that in all these cases the animal’s serum contains a substance which causes a precipitate with solutions of the protein used for immunization. The number of such precipitating serums that have been made experimentally is very large and it appears that protein from any source when properly introduced into the blood or tissues of an animal will cause the formation of a precipitating substance for its solutions. This substance is known, technically as a “precipitin.” The protein used as antigen to stimulate its formation, or some part of the protein molecule (haptophore group), which acts as stimulus to the cell is spoken of as a “precipitinogen,” both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific precipitation and agglutination are strictly analogous phenomena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on substances within cells which cells are in suspension in a fluid and cause the cells to settle out. Ehrlich’s theory of the formation of precipitins is similar to that of agglutinins, and need not be repeated. Substitute the corresponding words in the theory of formation of agglutinins as above given and the theory applies.

The precipitin reaction has not found much practical use in bacteriology largely because the “agglutination test” takes its place as simpler of performance and just as accurate. The reaction is, however, generally applicable to filtrates of bacterial cultures and could be used if needed. The so-called “mallease” reaction in glanders is an instance.

Precipitins find their greatest usefulness in legal medicine and in food adulteration work. As was noted above, if animals, rabbits for example, are immunized with the blood of another animal (human beings) precipitins are developed which are specific for the injected blood with proper dilution. This forms an extremely valuable means of determining the kind of blood present in a given spot shown by chemical and spectroscopic tests to be blood and has been adopted as a legal test in countries where such rules of procedure are applied. Similarly the test has been used to identify the different kinds of meat in sausage, and different kinds of milk in a mixture. An extract of the sausage is made and tested against the serum of an animal previously treated with extract of horse meat, or hog meat, or beef, etc., the specific precipitate occurring with the specific serum. Such reactions have been obtained where the protein to be tested was diluted 100,000 times and more. Biological relationships and differences have been detected by the reaction. Human immune serum shows no reaction with the blood of any animals except to a slight extent with that of various monkeys, most with the higher, very slight with the lower Old World and scarcely any with New World monkeys.

It is a fact of theoretical interest mainly that if agglutinins and precipitins themselves be injected into an animal they will act as antigens and cause the formation of antiagglutinins or antiprecipitins, which are therefore receptors of the first order since they simply combine with these immune bodies to neutralize their action, have only a combining or haptophore group. Also if agglutinins or precipitins be heated to the proper temperature they may retain their combining power but cause no agglutination or precipitation, i.e., they are converted into agglutinoid or precipitinoid respectively after the analogy of toxin and toxoid.

Precipitins like agglutinins possess at least two groups—a combining or haptophore group and a precipitating (sometimes called zymophore) group. Hence they are somewhat more complex than antitoxins or antienzymes which have a combining group only. For this reason Ehrlich classes agglutinins and precipitins as receptors of the second order.