• Zone sizes should be interpreted by referring to a table of critical diameters. Zone diameters for each test organism should fall within the limits given in Table 14. • Only pure cultures of rapidly growing bacteria give reliable readings. • The three standard control strains 1 are: - Staphylococcus aureus (ATCC 25923; NCTC 6571); - Escherichia coli (ATCC 25922; NCTC 10418); - Pseudomonas aeruginosa (ATCC 27853; NCTC 10622). 1 These strains can be obtained from: (ATCC) American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA; or (NCTC) National Collection of Type Cultures, Public Health Laboratory Service, London NW9, England. • Tests should be carried out with the three standard strains: - when a new batch of discs is put into use; - when a new batch of medium is put into use; - once a week, in parallel with the routine antibiograms. • Use the quality control chart shown in Fig. 11 for recording and evaluating performance tests.
Maintenance and use of stock cultures Selection and origin Select the strains so that the maximum number of morphological, metabolic, and serological characteristics can be tested with the minimum number of cultures; a suggested list is given in Table 2. These strains can be obtained from a combination of the following sources: - properly documented isolates from clinical specimens; - official culture collections; - commercial producers; - external quality assessment surveys; - reference laboratories. Preservation Long-term preservation Long-term preservation methods permit intervals of months or even years between subcultures. The best methods are lyophilization (freeze-drying), or storage at - 70 °C or below, in an electric freezer or in liquid nitrogen. Alternative methods are described below. 1. Glycerol at -20°C. • Grow a pure culture on an appropriate solid medium. • When the culture is fully developed, scrape it off with a loop. • Suspend small clumps of the culture in sterile neutral glycerol. • Distribute in quantities of 1-2 ml in screw-capped tubes or vials. • Store at -20°C. • Avoid repeated freezing and thawing. • Transfer after 12-18 months. 2. Mineral oil at room temperature. 1 • Prepare tubes of heart infusion agar with a short slant. For fastidious organisms, add fresh native or heated blood. Sterilize mineral oil (liquid petrolatum) in hot air (170°C for 1 hour). Grow a pure culture on the agar slant. • When good growth is seen, add sterile mineral oil to about 1 cm above the tip of the slant. • Subculture when needed by scraping growth from under the oil. • Store at room temperature.
• Transfer after 6-12 months. 1 MORTON, H. E. & PULASKI, E. J. The preservation of bacterial cultures. Journal of bacteriology, 38: 163 -183 (1938). 3. Stab cultures at room temperature (use for non-fastidious organisms only, such as staphylococci and Enterobacteriaceae). • Prepare tubes with a deep butt of carbohydrate-free agar. Tryptic soy agar (soybean casein digest agar) is recommended. • Stab the organism into the agar. • Incubate overnight at 35 °C.
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- Growth medium, Agar plate, • Store