ENUMERATION OF MICROORGANISM IN FOODS

SAMPLING Sampling Plans Sampling Methods Sample Suspension

Sampling Plans Decisions have to be made, prior the counting, as to how many sample of the product should be examined, what size sample should be taken for analysis, and what method should be used for sampling the product. Total numbers and uniformity of distribution of microorganisms in the food will influence the number of samples to be taken and the size of those samples. It must be stressed, however, that for any sampling plan the selection of lots for examination must avoid bias. it is essential to have random sampling.

Sampling Methods Samples should be examined as soon as possible after collection to minimize the possibility of any changes in microbial levels in the intervening period. This applies, in particular, to perishable products such as meat, fish and milk. If samples cannot be tested within 2-3 hours of collection then they should be stored at 4oC prior to examination – but storage should never be for longer than 10-12 hours.

Sample Suspension The final step in sampling is the release of microorganisms from the sample. Swabbing, rinsing and blending are three procedures commonly used for this purpose. The principles and techniques of each procedure are described in detail in the Short Course laboratoty notes.

COUNTING PROCEDURES Total Cell Counts Viable Cell Counts

Total Cell Counts The total number of cells in a sample suspension is most quickly and directly measured by counting the number of cells seen on examination under the microscope.

The drawbacks of total microsopic counts are : dead cells are not distinguished from living (viable) cells. small cells are difficult to see and possibly overlooked to give an underestimation of counts. lack of sensitivity since simple suspenssion must contain at least 106 cells/ml before any cell can be seen in a single microscopic field, which means that a cell suspension containing less than 106 cells/ml is not countable. high accuracy is hard to achieve continual microscopic examination is tedious, and sample suspensions must be reasonably free of other particulate matter since these tend to obscure the counting of microbial cells. For this reason food homogenates would not lend themselves to such microscopic analysis, although fluid foods may be examined in this way.

Viable Cell Counts Agar plating procedures Membrane filtration procedures Most Probable Number (MPN) techniques

Agar Plating Procedures In its simplicity a sample of cell suspension or food homogenate is inoculated in or on to a nutrient agar medium and after incubation the number of colonies are counted. This procedure counts only viable cells and is very sensitive. Other advantages include the ability to recognize the diversity of organisms in a sample from the different colony morphologies, as well as to isolate predominant colony types for taxonomic identification.

Some negatives points of the viable agar plate count are : The occasional possibility of a colony arising from more than one cells as occurs with organisms which form cell pairs, chains or in clumps. The possibility that some of the cell types present in the sample will not grow on the agar medium used or under the conditions of temperature, pH and oxygen tension of the incubator adopted. Colonies of some organisms, especially in food samples, occasionally spread over the surface of the agar medium thus obscuring the growth and counting of other microbial types. Cell suspension, including food homogenates, require appropriate aseptic dilution so that the colony numbers developing on any one plate fall between 30 and 300. There is a delay obtaining information due to the requirement for plate incubation, usually of the order of 24 hours or more.

Plate counts can be done by either of three procedures, namely the pour plate method, the spread plate method or the drop plate method. Pour Plate Method : In the pour plate procedure 1.0 ml of sample is transferred to the base of a petri dish and 15-20 ml of molten agar medium cooled to 40-50oC, is then poured into the plate and uniformly mixed. After incubation colonies, both inside and on the surface of the agar, are counted.

Spread plate method: With the spread plate procedure 0.1 ml of sample suspension is merely spread over the surface of a pre-prepared plate of agar medium with a sterile bent glass rod. After incubation, colonies which develop on the surface of the medium are counted. Drop plate method : In the drop plate procedure the plate of previously prepared agar medium is sectored into thirds or quarters and a sample drop of 0.02 ml volume is delivered to each sector. After allowing the inoculum drop to dry in to the agar plate is incubated. Sample dilutions are arranged so that between 5 and 20 colonies per drop are formed on the agar surface. Samples containing as little as 250 cells per ml may be counted and the technique is readily adapted to field trials.

Membrane Filtration Procedures The sample suspension under test is filtered through a presterilized filtration apparatus using a sterile membrane or filter containing pores of a sufficiently small size, so that microorganisms in the suspension are retained in the membrane. Membranes with pore sizes of 0.45 µ and will pass through the membrane. After filtration, the membrane, containing captures microbial cells on the surface, are aseptically removed and deposited, cell surface up, into a sterile dish containing an absorbent filter paper and saturated with liquid nutrient medium. The dish with filter is then incubated and checked for colony development upon the surface of the filter. Alternatively, membranes may be transfered to the surface of a nutrient agar medium in a petri dish. Nutrients diffuse through the membrane, thereby permitting growth of the microbial cells trapped on the surface.

The most serious limitation of the technique is that membranes easily clog with particulate matter such as food in food homogenates, so that the method is essentially limited to use with relatively clear liquid samples such as waters, carbonated beverages and alcohilic beverages.

Most Probable Number (MPN) techniques This procedure is used widely in public health food microbiology for enumerating the number of bacteria present in foods. The principle of the method is based on dilution. If a suspension of microbial cells is continually diluted, a dilution is eventually reached which contains no cells, that is, it is sterile.

Most Probable Number (MPN) techniques The assumptions of the technique are that : Cells are distributed randomly throughout the sample and that clumping, attracting and repelling forces between cells do not exist. Each aliquot from a dilution wil exhibit growth when inoculated into a nutrient broth whenever such an aliquot contains 1 or more viable cells, and Freedom from contamination of supplies and equipment.

Practice In practice, serial the-fold dilutions of the sample suspension are first prepared. Three or five replicate 1.0ml volumes are pipetted from each dilution into separate tubes of an appropriate growth medium. The tube are incubated and examined for a growth response. The highest dilution in which all three or five tubes give a possitive growth response is recorded along with the growth reponse in the tubes of the next two higher dilutions.

Advantages of the method are that : It can be made very sensitive through the use of large sample inocula of volumes greater than 1.0 ml/tube. Materials may be prepared in advance for fields use Selective growth media may be used enabling the enumeration of a desired microbial species while in the presence of many other species.

Disadvantages include the requirements for many replicate tubes for occurate results and the associated time and cost of preparing these materials. It should be emphasized that this method is widely used for counting the low levels of pathogenic bacteria often associated with foods.