A range of methods is available for the enumeration of microorganisms in food. The choice of method will depend on a number of factors.
• Type of sample.
• Characteristics, including the physiological state, of specific organisms sought.
• Characteristics of specific media. • Lower limit of enumeration required. • Purpose of the examination.
• Time available.
Legislation sometimes prescribes a specific counting method for the enu- meration of microorganisms in a particular product, for example the pour plate method is specified in European Union (EU) milk legislation. For environmental samples such as surfaces, utensils and equipment a surface contact technique may be the most useful method to choose.
Any of a number of methods given in this section may be selected for enu- meration of microorganisms in food. Whilst the pour plate method using plate count agar is regarded as the standard international method of enumeration for a total aerobic colony count, it is common for laboratories to use surface methods such as the surface drop and spiral plate. Apart from the obvious con- venience of using pre-poured plates, these surface methods have the advantages that they eliminate possible heat stress to the organisms from the molten agar, provide fully aerobic conditions of growth and facilitate identification of the organism types present.
Pour plate methods require the use of a clear growth medium to allow count- ing of colonies that have grown below the surface of the medium. This also applies to counts performed by automated colony counters using transmitted light.
In most instances surface methods are preferable when selective media are used for enumeration of specific groups of organisms because they allow full manifestation of colonial properties such as morphology, pigmentation, haemolysis, haloes of precipitation around the colonies or changes in colour around the surrounding medium. However, some organisms with particular atmospheric requirements, such as anaerobes, may be best enumerated by a pour plate method where the depth of medium helps maintain an anaerobic environment.
The use of a liquid method such as a multiple tube method for enumeration of organisms that are highly stressed, due to drying or high salt content for example, may allow better recovery and growth of the target organism and thus result in a more accurate assessment of the level of the target organism in the food sample. Multiple tube methods are also useful for enumeration of low numbers of organisms (below 100/g) but are less suitable when high numbers are expected.
If an enumeration is performed in order to determine compliance with limits set in microbiological standards, guidelines or specifications the choice of enu- meration method may also be affected by the required lower limit of detec- tion. Pour plate methods, membrane filtration and multiple tube methods are capable of detecting lower counts than surface methods of enumeration because a larger quantity of the sample can be examined.
Where large numbers of similar samples are to be checked for a microbial load within a defined range, such as in production runs within a factory, increasing use is being made of sophisticated equipment that detects bacterial growth elec- tronically by impedance or conductance within the growth medium. For any given product it is first necessary to produce a calibration curve for growth in a defined medium under carefully controlled test conditions. The advantage of such methods is that batch rejection can be triggered as soon as a predefined point on the calibration curve is reached and means that the samples with the highest bacterial count will be detected in the minimum period of time, some- times within 6 h. These methods are not included in this manual because of the diversity of foods which most non-industrial laboratories are required to examine.
Factors affecting the results
[1]The successful performance of the pour plate technique depends heavily on adequate and appropriate tempering of the molten agar. Bottles of molten agar should be placed in a water bath set at 44–47°C. The length of time required for tempering to that temperature will depend on the volume of agar in each bottle and should be determined on an individual basis. The number of bottles placed in the water bath will also affect the rate of cooling. Extended storage of the molten agar will reduce the gelling properties. Molten agar should be used with- in 8 h of melting and preferably within 3 h, and should not be remelted once it has set. For some particularly sensitive media such as agars containing bile, the
duration of holding in the molten state should not exceed 3 h. Even if adequate tempering of the molten agar has been ensured, heat stress of organisms may still occur, particularly in chilled and frozen foods.
Many of the organisms found in foods are obligate aerobes, for example some species of Pseudomonas and Bacillus. The relatively anaerobic conditions found in the depths of the agar in a pour plate may result in under-recovery of these or- ganisms. Use of surface methods utilizing pre-poured plates will remove these variables and may result in a more accurate determination of the levels of these organisms. Pre-poured plates usually require some drying before use, so that the inoculum used in the test is absorbed within 15 min of application. Over-drying must be avoided as this can result in concentration of inhibitory components at the surface of the plate with subsequent inhibition of growth.
Inoculated plates should be placed in the incubator as soon as possible after the agar has set or the inoculum absorbed. International standards recommend that plates should be stacked no more than three high to ensure good heat penetration. This may be difficult to achieve in practice and studies have shown that plates stacked six high are not subject to significant variation in heat penetration [1].
At the end of the incubation period it is not always possible to perform the colony counting, for example due to lack of time or work of a higher priority. In most cases it is acceptable to refrigerate the plates until counting can be per- formed. ISO 7218 [2] permits refrigerated storage of plates for up to 24 h after the incubation period unless otherwise specified in the method. For media contain- ing pH indicators such as violet red bile agars the plates must be allowed to regain ambient temperature before attempting to count the colonies to ensure accurate identification of suspect colonies.
It is good practice to monitor the microbial contamination of the laboratory environment, and this should be performed at regular intervals determined by the level of activity in the laboratory. Settle plates may be used to monitor the level of aerial environmental contamination in areas of sample processing by exposing the agar surface for a defined length of time, e.g. 15 min. The number of organisms are then counted after incubation. An action level should be estab- lished above which remedial action should be taken, for example thorough cleaning of the laboratory. Surface swabs may also be taken to monitor general levels of hygiene and to ensure the absence of pathogens.
Preparation of dilutions
[3]In order to enumerate fully the number of organisms in a food sample it may be necessary to prepare dilutions of the food homogenate. Commonly serial deci- mal dilutions in peptone saline solution (maximum recovery diluent, MRD) are prepared from the sample homogenate by adding 1 mL of sample homogenate to 9 mL of diluent etc. to the required endpoint. The accuracy of the volumes of diluent used should be ±2% and the accuracy of the sample volume dispensed should be ±5%. The use of automatic pipettors and associated sterile tips is advo-
cated to help ensure accuracy when preparing dilutions. Precision of ±1% is achievable with automatic pipettors compared with ±5% with volumetric graduated pipettes. All automatic pipettors should be checked regularly to ensure that the desired volume is being delivered. For dispensing volumes of 0.1 mL or more, the pipettor should be used in total delivery mode, that is the plunger is depressed only to the first stop when drawing up the liquid, but fully depressed when discharging the liquid. If the volume to be dispensed is less than 0.1 mL, the reverse pipetting technique should be used whereby the plunger is fully depressed when aspirating the liquid but only depressed to the first stop when discharging. In all cases care must be taken to prevent jump back of the liquid inoculum that may result in contamination of the pipettor, as this may also result in contamination of the sample inocula; regular sanitizing of the pipettor is recommended.
If total delivery volumetric pipettes are used, correct delivery is ensured by touching the tip of the pipette on an inside wall of the container when emptying.