Streptomycin solution preparation

Assay Technique. Sample dilutions and standard dilutions may be prepared exactly as for penicillin (Section IV,1). By the addition of 0.6 ml. of standard streptomycin solution to the first two tubes in the series followed by dilution through tube 12, concentrations are obtained which range from 10.0 pg. in tube 1 to 0.0045 pg, in 12. 

Standard Solution. Prepare the working standard from stock as for streptomycin (Section IV,2). 

If the preparation is soluble in water dissolve 1 to 2 g, accurately weighed, in 100 ml of water. If the preparation is not soluble in water, suspend a weighed amount in acetone and then add about four volumes of water. From these solutions prepare final dilutions for use in the assay of 1 in 100,000 and 1 in 400,000 of hexachlorophane, or of 1 in 10,000 and 1 in 40,000 of dichlorophen with phosphated buffer at pH 8. The assay medium is a peptone Lemco agar at pH 8, and the test organism a suspension of spores of Bacillus pumilus (B. subtilis) as used in the assay of streptomycin. 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

Before DMEM is used for culture, add 400pL each of glutamine and penicillin-streptomycin solntion to 40 mL of DMEM. Eresh glntamine and penidUin-streptomycin shonld be added to the DMEM working solntion after 2wk. The working solution is good for 4 wk after preparation when kept at 4°C. 

Standard Solution. Streptomycin levels in biologic materials (as reported in pg.) run numerically higher than known unit levels for penicillin. It is, therefore, suggested that a standard streptomycin working solution be freshly prepared prior to the test, with a concentration of 20 pg. per milliliter. 

Pipette a volume of sample containing 2 5 g (2,500,000 units) of streptomycin sulphate into a 100-ml graduated fiask and dilute to volume with water. Dilute a 5-ml aliquot of this solution to 500 ml with water in a graduated flask, then transfer a 5-ml aliquot of this final dilution to a test-tube and add exactly 1 ml of 2N sodium hydroxide. Mix and place the tube in a water-bath. Exactly three minutes after the addition of the alkali, remove the tube from the bath and cool under a cold-water tap for exactly three minutes. At the end of this time add, by pipette, 4 ml of a 1 per cent solution of ferric ammonium sulphate in 0 75 N sulphuric acid, shake and allow to stand. After exactly ten minutes, measure the extinction at 550 mju, using 2-cm cells with water in the comparison cell. Read the concentration of streptomycin in the final 5-ml aliquot prepared from the sample from a standard curve. 

The standard curve is prepared as follows Transfer suitable volumes, covering the range 0 to 2 0 mg of a standard solution of streptomycin sulphate containing 0 5 mg per ml, to a series of test-tubes and dilute each to 5 ml with water. Treat the contents of each tube exactly as described above from add exactly 1 ml of 2N sodium hydroxide. . . and ending with .. . water in the comparison cell. Prepare a curve by plotting streptomycin sulphate content against extinction. 

Nomi, Nimi and Miyazaki (1966) reported that, upon incubation of supernatants from suspensions of S. griseus in glucose and sodium chloride, streptomycin production was observed. When the pH of the suspension fluid reached 6.8 to 7.0, the suspension was centrifuged and the supernatant removed. When the supernatant preparations were adjusted to a pH of about 9 2tnd shaken for 24 hrs. the antibiotic potency, as determined by bioassay, increased considerably. This increase in potency was inhibited by the addition of phosphate or EDTA to the supernatant solution. The system was heat labile above 50 C. 

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