Culture media sterility testing

Liquid Culture Medium Dissolve 15 g of peptonized milk, 5 g of water-soluble yeast extract, 10 g of anhydrous glucose, and 2 g of anhydrous potassium dihydrogen phosphate in about 600 mL of water. Add 100 mL of filtered tomato juice (filtered through Whatman No. 1 filter paper, or equivalent), and adjust to pH 6.5 by the dropwise addition of 1.0 N sodium hydroxide. Add, with mixing, 10 mL of the Polysorbate 80 Solution. Dilute with water to a final volume of 1000 mL. Add 10-mL portions of this Liquid Culture Medium to test tubes, cover to prevent contamination, and sterilize by heating in an autoclave at 121° for 15 min. Cool the tubes rapidly to keep color formation to a minimum, and store at 10° in the dark. 

For each test compound concentration, vehicle, negative or positive controls, make 8.25 mL of serum culture medium containing 70% rat serum, 30% sterile Tyrode s salt buffer with less than 1 mg/500 mL phenol red (free acid indicator) and 35 p,g/mL streptomycin sulfates. 

Sterilization After a suitable culture medium is selected for the cultivation of a specific microorganism, it is poured into a culture vessel. If you use test tubes or flasks as your culture vessel, the ends of test tubes or flasks should be covered with a suitable closure to allow for the exchange of gases with the atmosphere, yet to keep foreign organisms out of the media. Various types of closures are used in the modern laboratory including cotton plugs, plastic foam, screw caps, metal caps, and aluminum foil. 

Figure 7.5 Apparatus for testing the microbial retention characteristics of membrane filters. The whole apparatus is sterilized, and initially the flask contains 140 mL of double-strength culture medium. The culture to be tested (100 mL) is passed through the filter with clamp 1 open and clamp 2 closed. The sides of the filter apparatus are washed with two 20 mL portions of sterile broth. Clamp 2 is then opened, the vacuum released, and clamp 1 closed. The filter apparatus is replaced by a sterile rubber stopper and the flask incubated. Absence of turbidity in the flask indicates that the filter has retained the test organism. From Brock [4]. Courtesy of Thomas D. Brock...

After sterilizing, and adjusting to pH 7.0, 100 ml of the medium was placed in each of several test tubes, 500 ml capacity, and sterilized. Streptomyc carzinostaticus var.neocarzinostaticus strain F-41 was inoculated therein, and fermented with agitation for 24 h at 27°C and then used as the stock culture. Next, an aqueous production culture medium was prepared containing (%) glucose 3.0, peptone 0.5, meat extract 0.5, sodium chloride 0.5, calcium carbonate 0.2. 

After sterilization, the medium was adjusted to pH 7.0. 100 ml of the production medium was placed in each of 70 test tubes, 500 ml capacity and sterilized. 5 % by volume of the above-mentioned stock culture was added to the production culture medium in each test tube and fermented with agitation at 27°C. The pH be came 6.6 after an incubation period of 36 h, and 6.8-7.2 after 48 h. After that, the pH showed no further change. When the amount of... 

Apart from standardization of potency, which also serves as an identity test, the material must be checked for sterility and for the absence of viable mycobacteria. Because of their slow growth the latter may not be detected by conventional sterility tests and it is usual to perform check tests by guinea-pig inoculation, or by prolonged culture on Lowen-stein-Jensen medium. The product is also checked for absence of reactogenicity in unsensitized guinea-pigs and if required by the regulatory authority, for abnormal toxicity. 

A test for sterility is laid down in the European Pharmacopeia for all parenteral products, and two techniques for testing are described, either membrane filtration of the product with subsequent incubation of the filter in suitable culture media (which is the preferred technique), or direct inoculation of the product into the culture medium, followed by incubation at the appropriate temperature for the specified time. Suitable media are described in the European Pharmacopeia although, it is also recognized that other media may be used. In every case, however, it is necessary to demonstrate that the medium is capable of supporting the growth of microorganisms both in the presence and absence of the material to be tested. 

The membrane filtration technique is technically more elaborate and requires that the radiopharmaceutical under test, after aseptic dilution, is passed through a membrane filter with a pore size of 0.45 m, which has been moistened with a sterile nutrient diluent. After filtration, the membrane is either transferred to a suitable culture medium or aseptically cut into two equal parts and one half placed in each of two suitable media. Incubation at the appropriate temperature is required for at least 7 days. 

The eggs should be broken and the contents homogenised aseptically. One volume of homogenate should be added to five volumes of sterile culture medium to dilute the enzyme lysozyme, which dissolves bacterial cell walls. The preparation is then tested for Salmonella,... 

Liquid products and water soluble solids may be tested by membrane filtration. Solids would first be dissolved in a suitable diluent, such as 1 /4 strength Ringers solution. After the liquid has been passed through the sterile filter (pore size < 0.45 pm), the filter is cut up aseptically, and pieces are placed into an appropriate culture medium for incubation. Liquid products may also be tested by adding a concentrated preparation of culture medium to a container with the product in situ. The concentration of medium added is such that the combination of medium and product gives single strength medium. The whole container is then incubated at the appropriate temperature. 

Sterile graduated pipettes of 10, 5, 2 and 1 ml, sterile capped 7.5 x 1.3 cm tubes/ small screw-capped bottles, Pasteur pipettes, an overnight broth culture of the test and control organisms (same as for disc diffusion tests), the required antibiotic in powder form, the required solvent for the antibiotic, sterile distilled water - 500 ml and suitable nutrient broth medium. A suitable rack to hold 22 tubes in two rows, i.e., 11 tubes in each row. 

Test Compounds. Nickel subsulfide (crystalline oNi3S2, particle size < 5 ym) was provided by Dr. Edward Kostiner, University of Connecticut, and its purity and crystal structure were verified by emission spectroscopy and X-ray diffractometry as previously described (L5 - Amorphous nickel monosulfide (NiS) was precipitated by addition of aimnonium sulfide to a solution of NiCl2 that was prepared from carbonyl-derived Ni dust and ultrapure HCl. The amorphous NiS was devoid of crystal structure, based upon X-ray diffractometry. The aNi3 2 and NiS powders were sterilized by washing in acetone immediately prior to suspension in tissue culture medium. 

The cytotoxicity assay is usually performed by determining the viability of suitable cell lines in the presence of polymers. For this test, 3-5 mm discs of polymer film are cut and sterilised under standard conditions (at 121 C and 6.8 kg (15 lb) pressure for 15 min). The cell growth in the presence of the polymer films is measured under a controlled atmosphere (CO2 incubator, 37"C) using an appropriate culture medium, supplemented by 10% fetal bovine serum and penicillin-streptomycin antibiotic solution. Confluent monolayers are propagated by trypsinisation (0.25% trypsin and 0.02% EDTA, ethylene diamine tetraacetic acid) and re-plated at 2 x 10 cells/mL in a sterile polystyrene cell culture plate, then incubated for 24,48 and 72 h. The morphology of the cells is analysed by light naicroscopy (Leica) after... 

The endoagar culture medium is not poured into test tubes. Instead it is sterilized in stages (3 times 20 minutes with intervals of 2 hours between each stage) in an Erlenmeyer flask and is then poured directly from this flask into sterile Petri dishes. The pouring out operation must be carried... 

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