From: "RESULTS OF BACTERIOPHAGE TREATMENT OF SUPPURATIVE BACTERIAL INFECTIONS: GENERAL EVALUATION OF THE RESULTS"S. Ślopek, I. Durlakowa, B. Weber-Dąbrowska, A. Kucharewicz-Krukowska, M. Dąbrowski, R. Bisikiewicz, Arch. Immunol. Ther. Exp., 1983, 31, 267
In this scientific work the method description for preparing Bacteria and Bacteriophages used for Phage Therapy is not sufficiently clear.
I have schematized the method below:
Origin of bacteriophages:
259 virulent bacteriophages including:
-116 for Staphylococcus ( human and animal origin)
-42 for Klebsiella
-39 for Escherichia
-30 for Shigella
-20 for Pseudomonas
-11 for Proteus
-1 for Salmonella enteridis
Phages really used in the course of therapy
-52 for Staphylococcus ( human and animal origin)
-17 for Klebsiella
-20 for Escherichia
-1 for Shigella
-17 for Pseudomonas
-4 for Proteus
-1 for Salmonella enteridis
lysates of virulent bacteriophages which caused a total lysis of the sensitive bacterial strains isolated from the patients are used.
Isolation of bacterial strains from patients and bacterial sensitivity to selected bacteriophages
Bacteria from 18 h broth culture
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Bacteria inoculated in new broth and incubate for 4 h at 37°C(shaker)
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2-3 ml of suspension are plated on agar plates:
Staphylococcus :Agar plates with Wahl medium
Gram negative bacteria: Agar plates with Phosphate buffer
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excess of suspension is removed and agar plates are used after 30 min
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One drop of the selected bacteriophage
( diluited 1:10) for each plate
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Incubation at 37°C
Staphylococcus : 4h
Pseudomonas :4-5 h
Escherichia,Klebsiella ,Shigella:2-3 h
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Transfer into a refrigerator and reading results the following day
Growth of Staphylococcus bacteriophages
Two 0,5L flasks:
a) flask (300ml broth + 5ml 30%glucose+2,5 ml of 18 h Staphylococcus broth culture)
b)Control: (300ml broth + 5ml 30%glucose+2,5 ml of 18 h Staphylococcus broth culture)
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Incubation at 37°C,1h
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5ml of Phage Lysate or from agar spot are added only in the flask a
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The flasks are reincubate at 37°C ( shaker) for 3 h
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After the lysis the flasks are stored overnight in a refrigerator
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The day after the lysate is tested for the phage presence and if it is turbid is centrifuged 1h at 3,500 rpm. After it is titrated and Thymol crystal is added
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the lysate, after 3 days in refrigerator , is collected and poured into ampouls (10ml) and tested fo sterility.
Growth of Bacteriophages for Gram negative baciili( Proteus,Klebsiella ,Escherichia,Shigella , Salmonella and Pseudomonas)
Pseudomonas phages
Two flasks
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a)10ml peptone water+ 1-2 ml of 4 h Pseudomonas broth culture
b)Control: 10ml peptone water+ 1-2 ml of 4 h Pseudomonas broth culture
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Incubation at 37°C, 2h
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5ml of Phage Lysate or from agar spot are added only in the flask a
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The flasks are reincubate at 37°C ( shaker) for 3 h
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After the lysis the flasks are stored overnight in a refrigerator
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The day after the lysate is tested for the phage presence and 2ml of Chloroform is added and shaken 2 min. and left fo 2 h at room temperature and after is stored overnight in a refrigerator.
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The day after the lysate is collected and poured into ampouls (10ml) and tested fo sterility.
Remaining Phages
Two flasks
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a)10ml Broth+ 1-2 ml of 4 h Strain broth culture
b)Control: 10ml peptone water+ 1-2 ml of 4 h Strain broth culture
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Incubation at 37°C, 2h
↓
5ml of Phage Lysate or from agar spot are added only in the flask a
↓
The flasks are reincubate at 37°C ( shaker) for 3 h
↓
After the Lysis the flasks are stored overnight in a refrigerator
↓
The day after the lysate is tested for the phage presence and 2ml of Chloroform is added and shaken 2 min. and left fo 2 h at room temperature and after is stored overnight in a refrigerator.
↓
The day after the lysate is collected and poured into ampouls (10ml) and tested fo sterility.
Therapeutic use of Bacteriophages
Orally:
3 times a day,one 10 ml ampoule,30min. before meal after neutralization of the gastric juice (gelatum, baking soda or a glass of Vichy water)
Directly on a wound (not washed with any antiseptics):
3 times a day per 24 h
