Samples are typically diluted 10-fold prior to counting, and the diluting fluid should be of such a pH and osmolarity that there is no possibility of viability loss before the sample reaches the Petri dish. It is therefore necessary to confirm this as part of the validation program. Water is unsuitable as a diluent, not only because its pH can be outside the range of 6 to 8 — normally considered acceptable, but also because some sensitive organisms can suffer osmotic shock and die rapidly. Phosphate-buffered saline (PBS), saline-peptone or fluid soyabean casein digest medium (tryptone soya broth), are among the commonly recommended pharmacopoeial diluents. Wetting agents may be added to any of them if necessary, as may chemical inactivators, to neutralize antimicrobial activity. Of these, PBS will not support any significant microbial growth, whereas diluents containing peptone or protein hydrolysates will. Thus it is important to avoid substantial time delays between preparation of the diluted specimen and the final filtration or plating; the USP puts a limit of one hour on this interval.
Soyabean casein digest agar (tryptone soya agar; TSA) is the recommended and most frequently used plating medium for bacteria. It will also support the growth of yeasts and molds so the possibility exists of conducting both counts on the same plate simply by incubating at 30-35°C for 48 hours for bacteria and then at 20-25° for five days thereafter. Again this strategy would have to be validated. The more common approach is to use Sabouraud-dextrose agar (SDA) with (EP), or without (USP), added antibacterial antibiotics for the yeast and mold count. The USP also suggests potato-dextrose agar for this purpose, and its lower pH (3.5) compared with SDA (5.6) makes it more effective for the exclusion of bacteria. Two of the antibiotics the EP recommends for addition to SDA, benzylpenicillin and tetracycline, are thermosensitive, and must be added as sterile solutions after autoclaving; the alternative — chloramphenicol — may be added before. The value of adding antibiotics is questionable anyway, simply because most samples do not contain sufficient bacteria for the presence of bacterial colonies on the plate to pose a problem when enumerating the yeast and mold colonies. The EP defines the total aerobic count as the sum of the bacterial and fungal counts, but a correction may be made for any organisms growing on both media. The use of antibacterial antibiotics in the fungal medium renders this possibility very unlikely, but it could be the best justification for the practice of using just one medium and incubating at two different temperatures. Other categories of organisms that may be quantified in the bioburden include coliforms (usually plated on MacConkey's agar) and anaerobes (on reinforced clostridial medium, or one of several alternatives).
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