OBTAINING AND USING BACTERIOPHAGES

1. Bacteriophages are very widespread in nature, they are found where the bacteria sensitive to them ("accompany bacteria") - in water, soil, milk, in bowel movements of intestinal diseases, in pus and sputum of patients with other infections. Especially often the phage is released during the recovery period.

Most often used to isolate bacteriophages by sewage by filtering the latter through bacterial filters. The presence of a bacteriophage is judged on the basis of clarification of the corresponding broth culture of bacteria or the formation of "sterile spots" on dense media.

2. To obtain a phage, the phage-containing material (for example, stool of patients with dysentery) is inoculated into the liquid medium (broth) and kept in a thermostat at 37 ° C for 18-20 hours to accumulate the phage. The broth culture is then filtered through bacterial filters to separate the phage from the bacterial cells. The filtrate is added to the fresh culture of the dysentery bacillus, and after its clarification (evidence of the presence and multiplication of the phage and lysis of the bacterial cells), filtration is again performed and added to the fresh culture of the dysentery rod, i.e. pass multiple passages through a culture of phage-sensitive bacteria, resulting in an increase in the amount (titer) of the released phage. After the accumulation of a sufficient amount of phage, the broth culture is again filtered and a phage preparation is obtained. Thus, phage preparations are filtrates of broth cultures of bacteria lysed by them (clear liquid of light yellow color). Phages are also released in the form of tablets with an acid-resistant coating, in the form of ointments, aerosols and candles (prepared from filtrates).

3. On preparations of a bacteriophage, the phage titer or the number of phage particles per unit volume or 1 mg (or 1 tablet) of the drug is necessarily indicated.

The number of phage particles can be determined by inoculating on a sensitive bacterial culture growing with a continuous lawn on a dense nutrient medium. As a result of the multiplication of phage causing bacterial lysis, "sterile spots" ("plaques") are formed - negative colonies of phage (they have a characteristic for this phage form). The number of colonies is equal to the number of phage particles. one phage infects one bacterial cell, and a sterile spot is formed as a result of its reproduction.

4. Phage titre - the largest phage dilution, which can still cause lysis of bacteria sensitive to it.

Titration - establishment of a titer - can be carried out on liquid and dense nutrient media. For this, the bacteriophage preparation is diluted from 10^-1 to 10^-9 and more. To each dilution, the same amount of daily broth culture of the corresponding bacteria is added. The titer is determined after standing in the thermostat to clarify the broth in the last test tube. For example, if the first six tubes remained clear, and in subsequent dilutions and in a control tube there is a culture growth, then the phage titer is 10^-6. When titrated on dense media, phage dilutions are applied to a bacterial culture sown with a continuous lawn. The titer is determined by the last cup, where there are still "sterile spots". In medical practice, most often used are bacteriophages with a titer of 10^-7 and 10^-8.

5. The use of phage is based on its strict specificity. They are used for:

a) diagnosis of infectious diseases - with the help of a known (diagnostic) phage, it is possible to determine the species or type belonging of the isolated culture. The definition of phage - phage typing - is of great importance for identifying variants (types) within the species, it allows to establish the source and ways of infection spreading. At the present time, phage typing has been developed. Salmonella, r. Vibrio, Staphylococcus aureus. With the help of a known bacterial test culture, it is possible to determine the unknown phage in the material under study, which indicates the presence of appropriate pathogens in it. The presence of pathogens in the test material can be established by increasing the phage titer when such a phage is introduced into the material under study.

b) treatment and prevention of diseases. The therapeutic effect of the drugs is greater the earlier they are started from the onset of the disease. A greater effectiveness of prophylactic phage use has been established. With the preventive purpose, they are prescribed to persons who have come into contact with a patient or bacterium carrier. For the production of therapeutic and prophylactic drugs, we use proven production strains of phages and, accordingly, typical cultures of microorganisms. Bacterial culture in the liquid nutrient medium, which is in the stage of logarithmic growth, is infected with the mother liquor of the phage. The phage-lysed culture is filtered through bacterial filters and the preparation is prepared.

In liquid and dense nutrient media, bacteriophages very quickly dissolve the corresponding bacteria, but in the human body, the bacterial lysis does not fully occur (the action is inhibited in the presence of blood, pus, lymph, intestinal contents), but the bacteria become more malleable to the protective factors of the body.

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