A primer on sampling for biological contaminants--part 3: surface sampling equipment and techniques. (Tools for Environmental Health).

Introduction

There are certain topics and technologies relating to our profession that everyone seems inherently to know everything about; surface sampling is one of them. Still, without being too harsh on ourselves, we find that, when faced with the task of performing a microbiological or particulate surface survey, and, given the choice between the right and wrong way of doing things, a lot of sanitarians somehow gravitate toward the latter. If that's the case with you, you are not alone. We know that all too well, having learned from experience.

This part of our practice has never been more important. Microbial contamination has recently taken on a new significance for two reasons. Immediately after 9/11, the demands on those of us who work at local health departments to sample white powders, which might or might not have contained Bacillus anthracis spores, had us scrambling. The second reason is that for those of us who work in food quality control, the attempt to comply with the zero Listeria tolerance has had us searching for an easy method with good repeatability. So, with commercial kits freshly designed for the task, we take our samples and rely on our laboratorian to sort things out-hoping for the best.

Sampling Rationale

To help put a rational spin on this topic, let us first say that surface sampling for microbial contaminants is as diverse and as straightforward as air sampling. The method and equipment we choose should be predicated on the specific situation under evaluation. For instance, the methodology should take into account the types of microbial contaminants expected, predicted conditions of survival and dormancy, and their estimated concentration. The factors that influence the selection of the sampling tool are primarily the geometry (curved, flat, grainy, smooth, etc.) and the chemical condition (disinfectant residues, oils, salts, etc.) of the surface to be sampled. Also, somewhere in this process, we need to provide guidance to the laboratory so that the technician may select an analytical method that complements the objectives of the sampling test.

As with any sampling, some structured protocol should be followed. Ideally, random sampling eliminates the greatest bias. With surface sampling, a random scheme consists of laying out a grid pattern and following the statistical outline given in ANSI/ANSQC Z 1.4-1993. If, however, the contamination site is known, or the purpose of the sampling is to determine the degree of decontamination achieved by a cleaning and disinfection, either judgmental sampling methods (clean versus soiled) or stratified sampling methods (timed sequence or measuring the degree of cleanliness) may be considered, with appropriate controls.

Sampling Methodology

Surface sampling consists of four basic methods for enumerating microorganisms: contact plates, swabs and wipes, direct surface agar plating, and rinsing and vacuum collection.

Contact plates and swab/wipe techniques are both the easiest and the most frequently used. Contact plates work best on smooth, flat surfaces, but with some practice, they also can be used on surfaces with a slight curvature. Swabbing with a sterile cotton-tipped applicator or sponge is most useful for sampling small inaccessible areas to estimate gross contamination. Direct plating, rinsing, and vacuum recovery have limited application except where the geometry of the surface precludes the use of the other methodologies or whole-surface testing is necessary.

Contact Plates

Contact-plate sampling involves pressing a solidified nutrient agar surface against the sample surface. The plates are then incubated and a direct microbial count taken. The most widely used contact samplers are Rodac(r) and Hycon(r) plates. These surface samplers are designed so that the meniscus of the agar is raised slightly above the container, enabling ease of contact with the surface. Generally, five seconds' contact is sufficient for optimal microorganism recovery. Since microorganisms are unevenly distributed on surfaces, a specific sampling area should be delineated. A common size (for ease of arithmetic calculation) is a 10-inch-by-10-inch, or 100-square-inch (100 [in.sup.2]), area in which five Rodac or Hycon plates are randomly arranged; this size is usually sufficient for a relatively accurate picture of the bioburden. If high contamination or confluent growth does not prevent accurate counts of the contact plates, the total number of microorganisms is estimated as follows:

Total microorganisms/100 [in.sup.2] = Total microorganisms / 5 plates x 100 [in.sup.2]/3.9 [in.sup.2] x 5

For curved or uneven surfaces, contact slides (from Hycon) consisting of a flexible culture-medium-coated film board may be used. They are commercially available with several different media and have a larger surface area than the conventional Rodac plates. Another variant of the contact method is the use of sterile velvet pads or membrane filters to make an imprint on a surface and then imprint it onto agar.

A variant of the contact plate is a cellophane-tape imprint. Cellophane-tape

imprints are ideally suited for collecting spores and other larger nonviable biological contaminants. After the tape is pressed against the surface, the exposed area can be placed in a petri dish and covered with a suitable agar for culturing, or the exposed side of the tape is stained, inverted on a slide, and observed microscopically.

Swabs and Wipes

Two similar swab techniques are generally used. The first consists of a cotton-tipped wooden applicator or sterile gauze pad (which can be wrapped around the end of a sterile tongue depressor) that is used to wipe a surface of a given dimension. The swab is then either wiped directly across an agar surface or immersed in a solution to remove the collected microorganisms. Next, aliquots from the solution are plated with an appropriate culture medium. The second technique uses calcium-alginate swabs. After the surface is wiped, the swab is dissolved in Ringer's solution containing sodium hexametaphosphate to free the microorganisms collected in the strands. Although the alginate and eluants used in the dissolving medium may inhibit growth of some organisms, these swabs have been shown to have a higher recovery rate than regular cottontipped swabs. Another advantage to this method is the ability to capture the organisms suspended in the eluate on a membrane filter, which then can be placed on a nutrient broth-im pregnated pad or an agar plate for incubation.

To use this sampling technique, aseptically remove a sterile swab from its wrapper and insert it into a vial containing 4.5 milliliters (mL) of a buffered solution. After moistening the swab, deposit excess liquid against the inside of the vial. Next, move the swab across the testing surface, reversing direction between successive strokes while rotating the swab between the fingers. To quantify the microbes in the area sampled, a sterile paper template with an opening of up to 1 square foot can be used. Recovery may be enhanced by adding 0.01 percent Tween (a surfactant) to the eluant.

We have found that wipe techniques are also quite handy, particularly in food-processing areas where a larger sampling area is required. Whirl-Pak [R] manufactures Sterile Speci-Sponge [R] bags that contain a pair of sterile gloves, a desiccated sponge, and a neutralizing broth.

If the purpose of surface sampling is to detect a target organism, use the recommended specific temperature. If, however, the objective is to determine the total surface bioburden, consider incubating the samples at 22[degrees]C for 24, 48, or 72 hours, or all three, to recover psychrophilic organisms, and follow this step with incubation at 36[degrees]C to recover the mesophiles. We found it wise to check the sequentially incubated samples frequently (at least every six to 12 hours) to recognize changes in growth before the formation of confluent colonies by molds and other rapid-growth organisms.

Conclusion

Given the imprecise nature of this science, there is no universal methodology that will recover all the organisms from a surface being sampled. Nevertheless, by using a combination of techniques, we can provide a reasonably accurate picture of the bioburden under similar environmental conditions. The accuracy of these initial bioload determinations will provide the basis for establishing an ongoing sampling program and selecting the most efficient method for recovery Remember, as with any environmental sampling, success and efficiency of recovery depend on an understanding of bacterial adhesion, survival, and dormancy and on the recovery of stressed organisms. In a future column, we will review the Whirl-Pak Sterile Speci-Sponge [R] and the Biotest Hycon plates in greater detail.

Inspection Tip of the Month

When traveling by car during the summer months, place all your testing and sampling equipment, including temperature-measuring instruments, meters, and anything that is battery operated, in an insulated tote. The tote will protect the equipment against temperature extremes and loss of accuracy Use ice packs to maintain temperature.