Depending on the nature of the product in which microorganisms are to be enumerated or isolated, there are a number of "traditional" long-standing techniques. Many of these are firmly established in the seminal work of the pioneers in the field during the 1800s. The methods are still valid in the traditional sense today and used in countless laboratories throughout the world, providing a relatively simple means of assessing the number and type of microorganisms in pharmaceutical products, or in the environment in which they are manufactured.
There are, however, a number of drawbacks, some of which are shared by rapid methods.
• For the purposes of enumeration, it is assumed that one visible colony is derived from one bacterial cell. This may lead to underestimation by a factor of x10 to x100, depending on the nature of the organism and the environment from which it is isolated.
• The approach is very much "one size fits all." It is assumed that the media selected will recover most types of organisms under the given incubation temperatures. Allowance is sometimes made for sublethally damaged cells by including a resuscitation step, but this may preclude enumeration.
• Different enumeration methods can give vastly different counts, which can be impacted by the technician's skill as well as media and incubation variables.
• They are time-consuming. From isolation to identification may take 7 to 10 days (or longer) for more esoteric isolates.
• They may lack sensitivity due, for example, to the high dilutions needed to remove the inherent inhibitory effects of the product.
• The product itself may make it difficult to discriminate between bacterial or fungal growth and dispersed product.
• Considerable infrastructure is required to support the methods, e.g., autoclaves, media steamers, laminar flow or biosafety cabinets, separate laboratory areas etc., an infrastructure making cost comparisons between traditional and rapid methods difficult.
Notwithstanding these issues, the techniques have stood the test of time and many billions of pharmaceutical doses have been released to the market using such techniques to confirm the absence of objectionable microorganisms.
Traditional techniques most commonly used are:
• Pour plates — the product or a dilution of the product is carefully mixed with molten agar media at approximately 45°C or below, poured into Petri dishes, allowed to solidify, and incubated at appropriate times and temperatures. An additional drawback of this method is the effect of agar medium at approximately 45°C on sublethally damaged microorganisms.
• Spread plating — normally used for difficult-to-homogenize materials such as fats or where fungi are to be enumerated. Samples or a dilution thereof are simply uniformly spread over the surface of a solidified agar medium and then incubated. While this overcomes the impact of warm agar on sublethally damaged cells it has the drawback of little or no dilution of any product inhibitory impact, such that bacteria or fungi may be present but, because they do not proliferate, cannot be counted. Spreading over the surface uniformly is a difficult technique, leading to underestimation of the counts due to confluent growth.
• Drop counts (Miles Misra) — here a low dilution of highly soluble or aqueous formulations is applied to the surface of a predried plate using a calibrated dropper, the plate is incubated and the number of colonies per drop counted. The principal disadvantage is its low sensitivity, which limits it to product containing more than approximately 500 colonies per ml, and the small area covered by the absorbed drop that results in counts of no more than 30 to 50 per drop, over which count confluent growth and under estimation is the outcome.
• Most probable number (MPN) — basically the dilution of a sample in a range of 1 in 10 to 1 in 1000 added to triplicate 9-ml volumes of tryptone soya broth. Using statistical tables and the number of tubes at each dilution step showing growth after incubation, the number of bacteria can be estimated. The main drawbacks are the sensitivity (published tables provide, in theory, the ability to enumerate down to 3 cfU; however, as this is only at 95% confidence then the true result could be as high as 17); and that the whole technique is based on the assumption that microorganisms are normally distributed in a sample, although this is not proven on a case-by-case basis.
• Direct or microscopic counts — as the title implies, this is the examination of the product or dilutions thereof in a counting chamber under a microscope. The drawback here is that the technique requires considerable microscopy skills to establish good clear solutions.
• Membrane filtration — the product where possible or dilutions thereof are filtered through a bacterially retentive membrane, which is then washed and aseptically transferred either to broth for presence or absence, or agar for enumeration following incubation. Drawbacks are the expense of the apparatus, the need for a laminar flow unit (although this can be overcome by the use of closed, disposable membrane filtration units), the effect the product may have on the membrane rendering it porous or causing degradation, and its limitations when trying to filter fatty or very viscous solutions. Care must also be taken when a vacuum is applied to assist the filtration, that the membrane is not excessively dried, leading to death of microorganisms due to desiccation. Current practice is to use a membrane of porosity of 0.45 micron, though there are bacteria known to be capable of passing such filters; either because they are habitually small, or the substrate in which they are growing exerts pressure on their ability to grow to normal (i.e., greater than 0.45 micron) size. This may lead to underestimation of the count.
One further limitation common to all the above methods is their ability to be validated when compared with modern standards applied to other analytical techniques. This can often lead to unfair comparisons drawn by the regulatory bodies when assessing the substitution of a traditional method with a rapid one.
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