Having prepared the sample in a suitable form for a TVC, the choice of counting method is the next consideration. The methods described in the pharmacopoeias are listed in Table 5.3, which also identifies their relative merits. Other methods e.g., surface drop (Miles and Misra method) and semiautomated techniques (e.g., spiral plating) may be used, if validated, and are considered elsewhere. 9,10
The EP and USP differ in the guidance they offer on choice of method. The EP directs that membrane filtration or a plating method (pour plate or surface spread) should be used, and that the most probable number (MPN) method (called the multiple tube method in the USP) should only be selected if there is no satisfactory alternative. By contrast the USP directs that pour plates should be used for all sufficiently soluble or translucent specimens, and the MPN method used otherwise. Surface spread techniques are not mentioned at all in the USP. Membrane filtration, despite its widespread use in the industry — and its acceptability to the FDA — is not a technique specifically recommended in the USP section entitled Total aerobic microbial count, although it is mentioned in the preparatory testing section as a technique that may be used to deal with inhibitory substances.
The principal criterion for selecting a method should be its suitability for the specimen in question. Considerations such as speed, ease of operation and cost are secondary. Suitability, in this context, means how well the method will deal with
Table 5.3 Relative Merits of Different TVC Methods
Detects lower concentrations than all other methods
Removes inhibitory (antimicrobial) components
1 Less suitable for viscous samples, emulsions or high particulate loads 1 Filter integrity may be compromised by solubilizers Relatively expensive consumables
Detects lower concentrations than surface spread or MPN
Possibility of thermosensitive organisms being killed Possibility of strict aerobes producing small colonies that are overlooked
Unsuitable for samples with high particulate loads and emulsions
Surface spread plate
Colonies of aerobes and facultative anaerobes are relatively large and easy to count
Best for emulsions, insoluble solids and fungi
Requires surface drying of agar to soak up sample Difficult to obtain uniform spread of colonies: some may be confluent
Relatively inaccurate and imprecise 1 Recommended by EP as method of last resort problems like elimination of antimicrobial activity in the specimen and the accuracy and precision of the result. It is well established that the methods available do not all give the same numerical result and that the precision of each varies, but it is not possible to quote relative values for accuracy and reproducibility simply because these will depend upon the organism used for testing and the skill and experience of the operator. Because the various methods have the potential to exhibit different detection limits and different degrees of accuracy and reproducibility, once a method is established for a product it cannot be substituted at will by another, unless validation data show equivalence.
Although not specified routinely in the current USP, membrane filtration was described in a proposed new chapter <61> Microbial Enumeration Tests, as the most accurate method for TVCs.12 Membrane filtration is stated to be the preferred technique for sterility testing because it is the most effective means by which intrinsic antimicrobial activity may be removed; the same logic applies in TVC determinations. The sample is passed through a filter, and soluble antimicrobial agents should be physically separated from organisms retained on the membrane. It is possible for the antimicrobial chemical to be adsorbed onto the surface of the organisms, although the EP recommendation of passing three x 100 ml volumes of rinsing fluid through the membrane is intended to address this problem. All TVC determinations are best performed in a laminar flow hood. This is particularly important in membrane filtration, because the use of vacuum pumps means that surrounding air may be drawn through the membrane before or after the liquid sample, and the recorded bioburden may be artificially high due to the airborne microorganisms. Filtration will detect lower concentrations of microorganisms than plating or MPN methods, and since the usual recommendation is that TVCs should be based upon plates containing not less than 30 colonies and sample volumes for filtration are typically 100 ml, the lower detection limit is approximately 0.3 CFU/ml. It is, of course, possible to detect lower concentrations by use of larger sample volumes providing that the filter remains unblocked. Slow filtration rates or membrane blocking may render filtration an unsuitable method for samples that are viscous or contain a high concentration of solid materials, and samples containing nonaqueous solvents or surfactants (solubilizers) may alter the membrane structure or porosity.
If the sample is known not to possess antimicrobial activity, a pour plate or surface spread plate is likely to be preferred to membrane filtration, because plating methods are easier to conduct, and usually quicker and less expensive since there is no expenditure on filter manifolds, membranes and rinsing fluids. The choice between pour plates and surface spread plates is often a matter of personal preference and are equally suitable for many types of samples. Pour plates can accommodate larger sample volumes (usually 1 ml, but up to 5 ml provided dilution of the medium is shown not to influence recovery) so they will detect lower cell concentrations. Thirty colonies derived from a 5 ml sample correspond to a lower limit of 6 CFU/ml. This contrasts with the situation for surface spread plates where the maximum volume of liquid that can be absorbed is 0.5 ml (corresponding to 60 CFU/ml) although smaller volumes of 0.1 to 0.25 ml (detection limits of 300 to 120 CFU/ml) are more commonly used. Other disadvantages of the surface spread method are that the agar surface needs to be dried in order for the inoculum liquid to soak into the agar and so provide discrete colonies. Control of surface drying (also referred to as overdrying) is important. If the agar is dried excessively the microbial recovery might be low, but failure to ensure adequate drying may result in bacterial multiplication in the liquid on the agar surface, and the plate becoming uncountable due to confluent growth. Confluence may also result from nonuniform spreading of the liquid over the surface. Against this, the spread plate eliminates the possibility inherent in the pour plate method that an artificially low result may arise if the bioburden contains either thermosensitive organisms that are damaged by the hot agar or a high proportion of strict aerobes (some fungi, Bacillus and Pseudomonas spp., for example) that produce colonies which, at the bottom of the agar, are so small due to inadequate oxygen diffusion that they are overlooked. The surface spread method might also be better when the sample is an emulsion or it contains suspended solids making it difficult to visualize small colonies within the agar. One aspect of the pour plate technique and the associated risk of killing thermosensitive cells is the problem arising from the use of microwave ovens to remelt solidified agar. This practice can result in the liquid at the very centre of the bottle becoming appreciably hotter than that nearest to the glass walls of the vessel. A prudent precaution to eliminate this problem is to cool the agar for a sufficient period in a 45°C bath to ensure a uniform temperature throughout.
The MPN method has little to commend it other than as a technique of last resort. It is relatively imprecise and has poor sensitivity. The table in the EP from which results are calculated indicates that as few as 3 CFU/ml may be detected, but as Green and Randell4 pointed out, this means the real result could be as high as 17. The corresponding table in the USP is curtailed at the lower detectable limit of 23 CFU/ml which, at 95% confidence, could really be a value as low as 7 or as high as 129. The USP indicates that the method should be considered for samples that, by their nature, make colony counting difficult using pour plates, but since the MPN result is determined by recording the number of tubes showing growth (turbidity) in a series receiving different volumes of inoculum, any sample that makes colony counting difficult will probably also necessitate subculturing of turbid MPN tubes.The method is not recommended in either pharmacopoeia as a means of enumerating surviving organisms in a preservative efficacy test. It has, however, been described as the basis for an automated preservative efficacy test that may be used for screening large numbers of candidate preservative formulations.13
The lower limit of detection for a counting method must be specified on regulatory submissions and it is important that bioburden values lower than the stated detection limit are not recorded. It is sometimes possible for the observation of a single colony on a single plate to be inadvertently reported in this way. These detection limits, however, are difficult, if not impossible, to reconcile with the USP recommendations that bioburdens should, ideally, be based upon plate counts in excess of 25 to 30 (the USP is inconsistent: 25 is the value stated in <1227> and 30 in <61>). A count of 30 colonies on a pour plate that received the standard inoculum of 1 ml would normally correspond to 300 CFU/ml or gram of sample (assuming the normal sample preparation of 10 g dissolved in 100-ml diluent). A count this high would exceed the compendial permitted levels for certain product categories anyway, and would be well above the specifications for many raw materials. A count of zero colonies would commonly be recorded for many samples, and bioburden levels sufficiently high to conform to the USP minimum plate count would normally be expected only for materials of "natural" origin and herbal products. It is worth noting that while the EP identifies 300 colonies per plate as the upper limit consistent with good evaluation, it specifies no lower limit. Replication is another issue. A count performed in accordance with a compendial method must be plated in duplicate, and while it is useful to extend this to triplicate plates in order to achieve greater reliability for certain products that may be expected to give relatively high counts, triplication is simply a waste of effort when the material consistently gives zero colonies.
Contact (Rodac) plates and swabs that are employed for surface sampling of large medical devices are covered in Chapter 2.6 Their validation should confirm an ability to recover all, or a consistent known percentage of, organisms artificially inoculated and dried onto the surface in question. One point worth emphasizing is that when cotton swabs are used, the organisms removed from the sampled surface and attached to the cotton fibres are not necessarily all transferred to the suspending fluid in which the swab is placed; this problem might be avoided if alginate swabs are used, since these dissolve completely in the presence of 0.1% sodium hexametaphosphate.
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