Bacteriophagy

BACTERIOPHAGY, the process of dissolution of bacteria by an agent of unknown nature. In 1915, the phenomenon of B. was discovered by Twort in cultures of white staphylococcus from smallpox vaccine. The name "bacteriophage" and a detailed description of its properties was given by d'Hérelle, who obtained it in 1918 from the feces of a patient recovering from dysentery. Under the influence of the bacteriophage, after only 30 minutes, weakly stained specimens are found among normally staining bacteria; after 45 minutes, the number of the latter increases, and after 2 hours only a few microbes stain normally, while the entire mass of them is transformed into granules, amorphous clumps, and finally into spherical forms; after 4 hours-complete dissolution. When sown on solid media, cultures containing bacteriophages show colonies with uneven, as if eaten-away, edges or with a lightened center; among the solid growth, sterile areas are noted. d'Hérelle considers these areas as negative colonies of bacteriophages. If one touches such an area with a loop and transfers it to fresh nutrient media, the change in bacteria in them is again observed. Such "transfers" can be continued indefinitely, with lysis becoming more and more active. The entire process is called bacteriophagy, or the Twort-d'Hérelle phenomenon. Bacteriophage is very widespread in nature. It is found in soil, in old laboratory cultures, in river, well, and sea water, in wastewater, in the feces of sick and healthy people, and mainly in recovering patients. - Method of obtaining. 1-2 g of formed fecal masses are ground in 2-2^th/₈ ^cm of physiological solution and transferred to 100 cubic cm of meat broth. Liquid fecal masses are diluted 2-3 times with physiological solution before sowing. At the same time, a sowing is made on Petri dishes. After 18-20 hours in an incubator at 37°, the culture is filtered through a Chamberland or Berkefeld candle; the filtrate is examined for the presence of bacteriophages. Using a modified d'Hérelle method, 0.1 cubic cm of filtrate is added to a broth simultaneously seeded with the corresponding microbe. After 18-20 hours, the results are examined. Depending on the activity of the bacteriophages, either complete dissolution may occur-the broth is transparent, or partial dissolution-the broth is slightly cloudy. In the latter case, a sowing is made on dishes and the presence of bacteriophages is determined by the presence of sterile areas or abnormal colonies. Doerr, Otto, Bail use this method without resorting to preliminary sowing in broth. The dishes are seeded with the corresponding culture. After 5-10 minutes, a drop of filtrate is applied to the center of the seeded area; by tilting the dish, the drop is allowed to flow. After 18-20 hours, the results are examined. The presence of sterile areas at the site of the drop among solid bacterial growth provides a basis for a positive answer. Upon detecting the presence of bacteriophages, their activity is determined by titration. Titration by the Appelman method. In a series of test tubes, the filtrate is diluted from 1:10 to 1:10⁹ (one billionth); 0.1 cubic cm of each dilution is added to freshly seeded broth. The highest dilution in which the broth remains transparent is taken as the titer of the bacteriophages. Otto titrates bacteriophages on dishes, applying drops from different dilutions. The presence of at least one abnormal colony or sterile area determines the titer of the bacteriophages. A transparent broth in which bacterial dissolution has occurred may become cloudy after 1-2 days due to the appearance of so-called "secondary" cultures, resistant ("lyso-resistant") to bacteriophages. The biological properties of these cultures differ sharply from the maternal strain. They lose the ability to agglutinate in specific serum, become more virulent, produce ugly polymorphic giant forms, lose the ability to gas formation, etc. The action of bacteriophages is associated with a number of conditions: acidic reaction of the nutrient medium, absence of dissociated salts, significant viscosity of the medium, some chemical agents (tetralin, quinine and its salts, etc.) reduce the activity of bacteriophages or completely destroy them. In sealed ampoules, the activity of bacteriophage is preserved for years; when exposed to air, it is destroyed in 6 weeks. When exposed to sunlight or diffused light, the bacteriophage dies within 2-3 days. Resistance to heating depends on the individuality of the bacteriophage type; according to Doerr and Otto, inactivation of bacteriophages occurs within 58-75°. An inactivated bacteriophage can have its activity restored by successive passages with the corresponding microbe culture. According to Hauduroy, bacteriophage is destroyed only at 102° in a moist medium and at 135° in a dried state. The optimal time for bacteriophage formation is from 3 to 18 hours, the temperature optimum is 37°. At temperatures below 8° and above 46°, the formation of bacteriophages ceases. The formation of bacteriophage and its activity depend on the quantitative ratios with bacteria. The bacteriophage develops best at pH=7.6-7.8. The addition of gelatin, agar, gum, egg white and other colloids inhibits the action of bacteriophage; it is energetically adsorbed by electronegative colloids (silicate scale, kaolin) and poorly by electropositive ones. Bacteriophage is capillary active: when added to liquid agar, after the latter solidifies, it diffuses to its surface, passes through thin colloidal bags and membranes that allow proteins to pass through. Based on the ability of bacteriophage to pass through filters of different porosity, its size is determined to be equal to a particle of collargol (20-35 mμ). From the filtrate, bacteriophage can be precipitated by centrifugation, ammonium sulfate and alcohol. Bacteriophage causes the formation in the animal body of so-called antibodies against itself and against the corresponding type of bacteria (bacteriolysins, agglutinins, antitoxins). Anti-bacteriophage serum arrests bacteriophagy but does not destroy the bacteriophage. - B. undoubtedly plays some role in immunity, infection and the development of epidemics. However, this role is not sufficiently clarified. Therapeutic value of bacteriophage. d'Hérelle describes 7 cases of severe dysentery and 4 cases of bubonic plague, cured by the introduction of bacteriophages; da Costa Kruz reports on the use of bacteriophage per os in 10,000 cases of dysentery-also with good results. Hauduroy successfully treated typhoid fever with bacteriophages, Gratia and others-staphylococcal diseases. Other authors (Otto, Winkler, Meissner), when treating the same diseases with bacteriophages, did not see good results. With repeated injections of bacteriophages into the animal body, the serum of the latter easily acquires anti-bacteriophage properties, lyso-resistant forms of bacteria arise, which, in the opinion of some authors, makes the use of bacteriophages unsafe for the infected organism.

There are a number of opinions regarding the nature of bacteriophage, united around three theories: 1) the theory of the ultramicroscopic virus of d'Hérelle, 2) the theory of autolysis of Bordet and Ciuca, and 3) the enzyme theory of Otto and Munter. Based on the infinite transferability of bacteriophages, the increase in "virulence" during passages, adaptability to harmful agents, wide distribution in nature and a number of other properties inherent in a living organism, d'Hérelle considers bacteriophage a microbial parasite. This view is supported by Prausnitz, Eliava, Ashe-show, Reichert. Bordet and Ciuca consider B. as a result of a hereditary disease of bacteria, arising on the basis of a violation of the balance of assimilation and exchange. Otto and Munter believe that bacteriophage originates from the bacteria themselves, breaking down into colloidal particles with enzymatic ability. The authors see confirmation of their theory in the experiments of Ehrenberg, who managed to multiply protein enzymes by filtering them through bacterial filters and develop specificity in them. Gamaleya believes that the phenomenon of B. is associated with the action of the division hormone of bacteria-"clastin." In certain cases, clastin is produced in excess-bacteria split and pass into filterable forms; when sown in the filtrate, clastin is transferred to fresh portions, the phenomenon of splitting occurs again and so on.