The most commonly used placebo for media fills is TSB used to simulate aqueous injections. It is a reasonably good all-round, general-purpose microbiological medium, which can support growth of aerobic bacteria when incubated at temperatures in the range 20-35°C. Equally, it is a reasonably good medium for supporting the growth of yeasts and fungi, when incubated at 20-25°C. It is the recommended test for sterility in all of the major pharmacopoeias.
However, many microorganisms will not readily grow in TSB and some will not grow at all. It is a good recovery medium for Gram-positive and human commensal-type bacteria, but not the best recovery medium for Gram-negative bacteria. The latter grow better with lower nutrient concentrations, and at lower incubation temperatures, than those recommended in the pharmacopoeias for the test for sterility.
TSB is not the best recovery media for yeasts and fungi. A mycology specialist would not use TSB as the first choice for surveying an environment for yeasts and fungi. It the not best recovery medium for anaerobic and microaerophilic microorganisms such as the common skin commensal, Propionibacterium acnes.
Why is TSB used so widely if it displays so many limitations? The answer is, quite simply, that it is a compromise medium, commercially available, uncomplicated and robust. It is supported by the reflected authority of the pharmacopoeias. Most importantly, it has become the industry standard.
In the interests of academic science, it could be desirable to use a better medium for media fills, or more types of media for each media fill, but in practical terms there is little benefit. The media fill is not an exhaustive search for every microorganism that could be contaminating an aseptic process — it is a "snapshot" in time with a recognised and limited "focal range."
A wider variety of placebos is used for solid dosage forms. Generally, the placebo is filled into the unit containers and then TSB is added, either on- or off-line. It is possible to add the TSB before the placebo, but it is not general practice. The placebo is dissolved in the TSB and incubated.
The chosen placebo should have similar flow characteristics to the product or products that it has been chosen to represent. If it does not have these similar characteristics, it might be effectively impossible to simulate the intended process.
It must be sterilizable. Gamma irradiation is the method of choice for sterilizing solids provided they have a low moisture content; it is unlikely to induce chemical or physical changes through radiation. Irradiation is reliable and penetrative through bulk quantities.
The placebo must be soluble in TSB, and must not inhibit the growth of microorganisms. The practical application of this principle is complicated by the fact that the amount of space available for TSB in each container is restricted by the amount of placebo already added. This in turn must be sufficient to stimulate the process.
Polyethylene glycol, mannitol and lactose are the most widely used placebos for solid dosage forms.
Performing media fills on ophthalmic ointments is a nightmare. Placebos are based on TSB made viscous by the addition of a substance such as carboxymethyl cellulose at about 65 g/l, although this concentration may differ according to the process settings applicable to the range of ointments being simulated.
The nightmarish aspects of ointment media fills are three-fold.
First, there is cleaning up behind them. Actual ointments generally present a sticky mess, which is difficult but obviously not impossible to clean from production equipment. But, add a microbiological growth medium to that sticky mess, and cleaning becomes highly critical, especially if the creation of foci for microbiological growth in the equipment and in the facility is to be avoided — as it must. Good clean-room practices are difficult to maintain in ointment media fills.
Second, ointment tubes are rarely transparent, therefore inspection of thousands of placebo-filled tubes for growth after incubation is difficult. The tubes are opened and squeezed out, although some users of plastic tubes special-order transparent tubes purchased solely for media fills.
Third, microorganisms grow as colonies in carboxymethyl cellulose-thickened TSB rather than displaying a general opacity. Carboxymethyl cellulose-thickened TSB is not a clear transparent medium in which colonies can be easily discerned. This is addressed by inclusion of a metabolic indicator, such as 2,3-tri-phenyltetrazolium chloride in the medium, at or around 0.0025%. Tetrazolium chloride is a metabolic indicator that changes to a red or purple color when microorganisms respire.
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