Tuberculin

Tuberculin was obtained by Koch in 1891. This first preparation is called the old Koch's T. (OT). Koch's prescription for preparing OT is as follows: 4-6 week cultures of tubercle bacilli in 5% glycerin broth are sterilized in flowing steam for 1 hour and then evaporated to ¹/₁₀ of the volume at a lower temperature. After evaporation, the fluid is freed from microbial bodies by filtration. The Paris Pasteur Institute uses for preparing OT calf broth with 1% peptone, 0.5% NaCl and 5% glycerin; pH before sterilization=7.2. Instead of sterilization at 100°, some institutes use filtration through Berkefeld candles. Concentration to 1/10 volume is performed on a water bath at a temperature of about 90°. After filtration through a paper filter, sterilization is performed again (at 60°, 100° or even 110°) and then 0.5% phenol is added or phenol is added without repeated sterilization. For use in humans, a mixed T. is also prepared from typus humanus and typus bovinus; mixing is done after concentration (1/10 hum. and 7/10 bov. and others). Besides OT, Koch also prepared protein-free T. (instead of glycerin broth, the tubercle bacilli are grown on a synthetic medium of the following composition: monopotassium phosphate 0.5, magnesium sulfate 0.04, magnesium citrate 0.25, asparagine 0.5, glycerin 2.0, soda 0.25, distilled water 100 cm³), residual T. (RT; well-dried tubercle bacilli are ground for a long time in an agate mortar, the mass is weighed in physiological solution and centrifuged; the upper layer-TO-consists of glycerin-soluble components of the tubercle bacilli, the precipitate represents RT) and new T.-bacterial suspension-BE (ground tubercle bacilli are suspended in physiological solution and allowed to settle; to the supernatant 50% glycerin is added). Landman's tuberculin is a mixture of extracts of tubercle bacilli in physiological solution or glycerin water, obtained at temperatures from 40° to 100°; 1 cm³ of T. kills a guinea pig. Beranek's tuberculin (Vega-neck) is a mixture of filtrate of tubercle bacilli culture on a protein-poor medium and extract of tubercle bacilli in 1% orthophosphoric acid. Rosenbach prepared T., using the principle of mixed culture: 6-8 week cultures of tubercle bacilli are seeded with a piece of the fungus Trichophyton holosericum album. At a temperature of 20-22° after 10-12 days, the tubercle bacilli cultures are covered with a white coating of overgrown fungus. The culture is separated from the medium, suspended in a solution of glycerin with carbolic acid and filtered. Tuberculin Denys (B.F.) is the filtrate of broth culture of tubercle bacilli; high temperatures during preparation are not allowed. Endotin Gavrilovich is prepared from tubercle bacilli typus hum.; the bacilli are previously treated with alcohol, xylene, ether and chloroform; then decantation, centrifugation and treatment with hot alkali solution follow. Weleminsky prepared T. (tuberculinomucin), based on a strain of tubercle bacilli which, after 8 years of cultivation, began to produce mucin in culture. Tuberculinomucin is a mucin-containing broth, freed from bacterial bodies by filtration through paper. Antiphthisin of Klebs--a substance obtained from the liquid part of tuberculosis bacilli culture by precipitation with a solution of sodium-bismuth-iodide in acetic acid and absolute alcohol. Iron tuberculin (Ditthorn and Schultz) is obtained from OT, from defatted tubercle bacilli and others by precipitation with ferric chloride and treatment of the precipitate with dilute soda solution. Iodotuberculin (Cantani) is prepared from tubercle bacilli culture treated with iodine. Spengler for use in humans prepares tuberculins (according to Koch's principle) from bacilli typus bovinus. Negre and Boquet for therapeutic purposes recommend methyl extract of tubercle bacilli, prepared as follows: tubercle bacilli are killed by heating at 120° for half an hour, dried and extracted with acetone (0.01 tubercle bacilli per 1 cm³) for 24-36 hours; followed by filtration, drying of bacilli and extraction with 99% methyl alcohol (1 cm³ per 0.01 tubercle bacilli) for 10-12 days at a temperature of 37-38° with occasional shaking. The filtrate is the methyl antigen used for therapeutic purposes. Partial antigens of Deyke-Muha are prepared as follows. Tubercle bacilli are treated with 0.5-1% solution of lactic acid for one month, resulting in a soluble part (MTbL) and an insoluble precipitate (MTbR). Subsequent treatment of the precipitate with alcohol and ether yields three fractions: A-proteins, F-fatty acids and lipoids, N-neutral fats and waxy substances. MTbL-"pure" tuberculin; A, F and N-antigens; they are devoid of toxic properties.-The list of T. shows that under this name are combined preparations not similar to each other. The prototype of all T., OT, is a water-glycerin extract of tubercle bacilli, BE-a suspension of mechanically ground tubercle bacilli, B.F. (tuberculin Denys)-filtrate of broth culture of tubercle bacilli, which may contain tubercle ultravirus, etc. Common to all these preparations is their biological action. T. is not a primary toxin; for healthy guinea pigs and healthy humans it is not poisonous. For tuberculous animals and humans, T. is a strong poison; a tuberculous guinea pig dies from 0.3-0.5 cm³ within 24 hours with symptoms of hyperemia and hemorrhages at the site of injection and around the tuberculous foci. Causing death in large doses, T. in small doses causes a specific reaction, the presence of which indicates the presence of tuberculous tissue in the body. The tuberculin reaction consists of general (increase in temperature and general symptoms), local (papule or inflammatory changes at the site of T. injection) and focal reactions (intensification of inflammatory changes in existing tuberculous foci in the body).-As for the chemistry of tuberculin, Long believes that the active substance of T. should be attributed to proteins or high-molecular products of its breakdown. Zinsser disagrees with this; he managed to obtain from T. a substance that has the property of giving a skin reaction, but giving no protein reactions ("residual" substance). According to Billing, the active substance of T. cannot be attributed to proteins. On the other hand, Muller, Laidlaw and Dudley isolated from T. a polysaccharide (identical to that obtained from tubercle bacilli) that gives a precipitation reaction with immune serum in very high dilutions; the skin reaction is caused by albumin that does not give a precipitation reaction. At present, the question of the nature of the active principle of tuberculin can be considered solved, since F.B. Seibert obtained from T. a protein preparation (P.P.D.-Purified protein derivative), not containing polysaccharide and having the ability to give skin reactions. To obtain P.P.D., tuberculin is prepared on Dorset medium; then by ultrafiltration and treatment with trichloroacetic acid, T. is freed from ballast proteins and polysaccharides. In its final form, P.P.D. is a yellowish-brown light powder, which before use is dissolved in physiological solution of NaCl with the addition of alkali. P.P.D. is a stable preparation and is therefore a standard preparation. It is released in tablets containing ten doses of P.P.D. Doses: 0.00002 mg for the first reaction and 0.005 mg for the second, if the first is negative. There is no theory that would explain the action of T. Koch's opinion about the primary toxic nature of T. does not correspond to the facts. The theories of Wassermann and Brook (the existence of antituberculin in the tuberculous organism), M. Nicola-Wolf-Eisner (lytic theory) and Friedberger (anaphylactic theory) admit the presence of a specific antibody in the reacting organism; all these theories are contradicted by the fact that passive transfer of tuberculin sensitivity has not yet been achieved by anyone. Zelter reduces the mechanism of action of T. to irritation; T. manifests its inherent action unchanged and leaves the body unchanged. Finally, there are views that deny T. its specific action. Such an opinion can hardly be considered correct; in any case, there are large quantitative differences between the action of tuberculin on the tuberculous organism and non-specific substances. G.E. Platov showed that the tuberculin reaction is a complex phenomenon that changes under the influence of various substances. From an immunobiological point of view, tuberculin is not a complete antigen; its introduction into the body is not accompanied by the development of immunity; the proteins of the tubercle bacillus contained in it appear to be deeply denatured. Control of T., besides research for sterility and harmlessness (see Control of bacterial preparations), also includes testing for strength (toxicity and allergizing ability). Donitz's method is as follows. Tuberculous guinea pigs, dying from 0.3-0.5 cm³ of standard T.

(preliminary test), ascending doses of standard T. are injected; another series of such guinea pigs receives the same doses of the test T. (0.05-0.1-0.15-0.2 and 0.3 cm³). The test T. is considered satisfactory if its minimum lethal dose is equal to that of the standard. The determination of T.'s ability to cause a skin reaction in allergic animals is carried out by the Calmette-Potter method. A tuberculous guinea pig, tested for sensitivity to T. by intradermal injection of standard T., receives intradermal injections of the standard and test T. (0.1 cm³ of different dilutions from 1:10 to 1:2,500) into a widely shaved area of the abdomen. The nature of the reaction (redness, swelling, infiltration) is noted. The guinea pigs should have white or pink skin on the abdomen. T. intended for human use, in addition to the above method, is tested on tuberculous patients (at least 10) using the Pirke reaction (see). Dilutions of 1%, 3%, 10%, 30% and 100%. For diagnostic purposes, T. is used by various methods. The Pirke skin reaction represents the most convenient test, which is still widely used. The Mantu intradermal test consists of injecting ¹/₁₀₀ cm³ of TAK [dilution of T. 1:5,000 (original prescription)] into the skin. The papule reaches maximum development after 48 hours; its size is up to 15-20 mm. The Moro patch test consists in rubbing into the skin an ointment of the following composition: TAK-1.0, Lanolini anhydrici-1.0. A piece the size of a pea is rubbed in. In case of a strong reaction, redness and papules appear. Rarely used. The ophthalmoreaction (Cadlmet-Wolf-Eisner) consists of introducing 1 drop of a 1% solution of TAK into the conjunctival sac. The result is redness and swelling of the conjunctiva. Not used in humans. It is used in cattle. In humans, one can also do without the subcutaneous test (first injection-0.0001 cm³ of TAK; maximum-0.005 cm³, there are many other variants). Therapeutic use of T. see Tuberculosis of the lungs, therapy.