Microbiological Testing

June 2001

By Bruce Floyd
Contributing Editor

Microbiological testing must be considered a system, not just a laboratory procedure. Is the reason for testing to measure the cleaning procedures, the quality of incoming raw materials, the quality of a process or finished product, or the presence of pathogenic contamination? The sampling plan and protocol are as important as the testing method — no level of laboratory sophistication will overcome an inadequate sampling plan and protocol.

Pesky pathogens
According to Dean Cliver, Ph.D., professor of food safety, University of California-Davis, testing a finished product will not increase safety past a certain level no matter what methods are used. Pathogenic contamination is usually a random event. If the organism is present, but not in the sample, the detection method isn’t relevant.

Another problem is the recovery of damaged organisms. According to Michael P. Doyle, Ph.D., regent’s professor, director of the Center for Food Safety, University of Georgia, Athens, any type of food processing causes damage to microorganisms. This includes freezing, drying, heating and other processing methods. Most pathogenic-organism detection methods require at least 1,000 cells/ml for detection; some rapid methods, such as the “dip sticks,” require at least 105 cells/ml for detection. Getting to these concentration levels requires an enrichment step. To be useful, this step must encourage the recovery of damaged cells.

Many non-pathogens have commercial importance and the emphasis on pathogenic organisms and their rapid-detection methods often overlooks some traditional methods. Before choosing a method, it is wise to know the official methods used by the various regulatory agencies to examine each product.

Method madness
For microorganisms without public-health significance, the method used is not important as long as it works in a particular situation and accurately reflects the existing bacterial load. For raw materials, use the same method that the customer uses or the method generally used in the industry. (Most industry associations publish analytical methods specific to their products.) If sterility is the basic issue, use an approved method for the specific product being examined. Someone knowledgeable about the microbiological problems specific to each product and process is invaluable when setting up a program, adding a new product/process or changing sanitation procedures.

Selecting a proper sampling plan that uses aseptic sampling methods is one step that cannot be overstressed. Assume that you really want to find any target bacteria that are present. By taking one 25-gram sample from a 44,000-lb. lot of product, the chances of finding Salmonella in the sample are virtually the same with or without the analysis. Unfortunately, this level of testing is very common. A sampling program must have a statistical opportunity to detect the problem.

Sample preparation also is important. This includes the obvious, such as avoiding cross-contamination, and less obvious factors, such as sample dispersion. The bacteria must be liberated, but not everything disperses or dissolves in water, like sugar or salt, and sample preparation should reflect this. If not, it can show up as inconsistent results. If this problem occurs, one way to find the cause is to have someone skilled in preparing samples of similar products observe the entire micro process under normal conditions. This means observing the operation for a long-enough period to ensure the technician uses the same techniques under observation as when working alone.

Remember, when evaluating any analytical procedure, new or old, that real-life situations have an impact, too. Test the procedure under actual conditions in the facility used and include the normal number of samples to be tested at any one time. The time pressure to prepare 100 samples is different than when preparing only 10 samples. If the procedure requires 20 ft. of bench space, special lighting and ventilation, don’t attempt to implement it at a plant that has a 10 ft. by 10 ft. micro area with no ventilation and normal ceiling-mounted light fixtures.

Testing troubles
Environmental and equipment swabbing can determine equipment cleanliness and contamination potential. For pathogenic surveillance programs, commercial laboratories, such as Chicago-based Silliker Laboratories, can provide environmental swabbing kits that are returned to the laboratory for analysis.

To evaluate if equipment is clean, several methods are available. These include the Lightning Cleaning Validation System offered by BioControl Systems, Inc., Bellevue, WA, which detects ATP (adenosine triphosphate) residues on equipment and is effective down to 100 ppm. This speedy system can be used by plant personnel to determine equipment cleanliness in real-time. This instrument can be used only on thoroughly washed equipment; because it detects all organic residues, improperly washed equipment will give a positive result. Processes where residues are normal, such as peanut butter or chocolate, can’t use this instrument. However, remember it’s still possible to have bacteria clinging to the surface of stainless steel at less than a 100-ppm level.

Environmental swabs can be taken using swabs prepared in-house or commercially. For example, 3M Microbiology Products, St. Paul, MN, makes a swab that can be used in conjunction with its Petrifilm™ System or with conventional plating methods. The product was developed specifically for the food, dairy and beverage industries, according to Kevin Habas, market development manager, and can be used either wet or dry; it will effectively neutralize iodine, chlorine, acid sanitizers and other residual sanitizers when used at manufacturer’s recommended strengths. The self-contained all-in-one swab device eliminates the need for pipetting and saves approximately 50% of the time it takes to prepare swab devices, and collect and plate samples. The 5-1/2-in. swab provides access to hard-to-reach locations, and the plastic design eliminates glass on the production floor.

A word of caution: equipment construction and maintenance may not only make it difficult to clean, but also to swab. Because easily accessed areas normally don’t cause cleaning problems, swabs must be taken in the areas hardest to clean to find if a potential problem exists. Much time is spent swabbing floors and drains, but attention should be given to elevated areas from which condensate or collected dust could drip or fall onto equipment, raw materials or packaging materials.

If monitoring air quality is a concern, Bioscience International Inc., Rockford, MD, offers the SAS Air Sampler, which can monitor yeast, molds, aerophiles and viruses. The device is a collection tool and the impaction disk is analyzed by polymerase chain reaction (PCR) or contact plate methods.

Testing types
Non-pathogenic organisms, including yeast and mold, coliforms and standard-plate count microbes are used as indicators of quality, shelf life and cleaning efficiency. Some companies offer simplified methods for aerobic plate counts, coliforms, E. coli, yeast and mold. These include 3M’s Petrifilm™ plates, which eliminate the need to prepare pour plates and are easy to read and require much less incubator space than conventional plating methods. It is AOAC approved and also is approved in other countries by product and/or organism.

Another system of prepared plates, the SimPlate system from BioControl Systems Inc., South San Francisco, CA, operate on a slightly different model, but also are simple to use and interpret. Both systems have different plate types for different organisms.

The advent of mandatory HACCP in several industries and the increased emphasis on testing ready-to-eat (RTE) foods prior to their release for L. monocytogenes, Salmonella and E. coli 0157H7 creates a need for validated, rapid pathogen-detection methods. In fact, FSIS/USDA is proposing that all RTE meat and poultry processing establishments conduct environmental testing for generic Listeria to verify that they are controlling the presence of L. monocytogenes. (FSIS’ definition of environmental testing is set down at www.fsis.usda.gov/.)

Traditional methods take time to enrich (grow out) microflora for detection, so large inventories can be awaiting test results. Tremendous strides have been made in decreasing the time required to determine certain bacteria’s presence. Some methods involve reduced enrichment time and others eliminate enrichment altogether.

According to Daniel Y. C. Fung, Ph.D., professor of food science, Kansas State University, Manhattan, one of the promising rapid methods is cell recovery using immunomagnetic separation. This method essentially adds microscopic magnetic beads coated with specific antibodies that attach to the cells of interest. According to Fung, this typically requires 106 cells for detection. Immunomagnetic separation offers a method of accelerating the enrichment step, making it possible to get a 10- to 100-fold concentration of cells. Combining this with the PCR method further amplifies the DNA, and gets results in six hours. The PCR method has drawbacks, such as distinguishing between live and dead cells, but Fung and his colleagues are working to perfect the procedure. Immunomagnetic separation has not been perfected for all types of food products and is recommended for use only by trained, experienced microbiologist.

Some companies that offer the magnetic beads commercially are Dynal Biotech Inc., Lake Success, NY, and Vicam, Watertown, MA. Vicam’s Lister Test™ Lift kit can detect strains of Listeria, including L. monocytogenes, on metal surfaces. According to Nancy Zabe, technical services manager, this microbiological testing system can produce accurate results within 24 hours without enrichment. This test kit detects injured Listeria cells that are common in environmental and processed-food samples. Vicam also makes a Salmonella Screen/Salmonella Verify microbiological testing system; one test is for the detection of undetermined species of Salmonella and the other is for identifying contamination by Salmonella enteritidis within 24 hours.

Enzyme-linked immunosorbent assays (ELISAs) were the first rapid methods in use. They allowed rapid screening of Salmonella directly from the enrichment broth and reduced the time to determine a negative result from five days to two. Positive screening results are verified by traditional methods.

The PCR system has received a great deal of attention from the research community. According to DuPont Qualicon, Wilmington, DE, PCR is an analytical tool that quickly replicates (amplifies) a targeted fragment of DNA into millions of copies. A laboratory can process a large number of samples in a reduced period of time with more selective results than other rapid methods. It can be used in conjunction with various enrichment methods as mentioned previously. An AOAC Performance Tested BAX® system is available for screening Salmonella, E. coli 0157:H7 and L. monocytogenes.

All of the systems that require enrichment require pathogens to be grown in the laboratory in large numbers. Some systems are safer than others, but the risk of accidental contamination is present whenever viable organisms are enriched. Many companies have separate pathogen testing areas physically isolated from production. This isolation includes lab personnel, air and all common areas. If a plant has a risk of cross contamination, it’s a good idea to send pathogen test samples to a qualified, third-party testing laboratory.

Rapid-detection methods are improving constantly. Steve Knabel, Ph.D., professor of food science, Penn State University, University Park, PA, mentions that the industry will see increased usage of nucleic-acid methods. This allows messenger RNA through reverse transcription to be taken to DNA and then analyzed by PCR. Knabel and associates have been working on an improved enrichment procedure for PCR analyses. They have developed a one-tube recovery/enrichment system for L. monocytogenes using oPSU (optimized Penn State University) broth. The one-tube system results in a high pathogen-to-background ratio that makes detection by PCR possible. The DNA is then detected using PicoGreen®, from Molecular Probes Inc., Eugene, OR, thereby eliminating gel electrophoresis.

Knabel mentions that technologies from the electronics industry now are being applied to microbiology. One of these is DNA microrays. In the future, it may be possible to accurately and quickly test for all of the different RNA messengers of interest on one microscope slide. Automation, miniaturization and computerization are being applied to the methods of the future in microbiological analysis.

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