By Bruce Floyd
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.
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 isnt relevant.
Another problem is the recovery of damaged organisms. According to Michael
P. Doyle, Ph.D., regents 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.
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
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, dont
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.
traditional methods for microbial analysis can be found
in such publications as FDAs Bacteriological
Analytical Manual (BAM, now available online); The
American Public Health Associations Standard
Methods for the Examination of Dairy Products (16th
edition); Official Methods of Analysis of AOAC International
(OMA, 17th edition); The Compendium of Analytical
Methods, Health Protection Branch (HPB) of Health
Canada; and The Microbiology Laboratory Guidebook
(3rd edition) FSIS/USDA. To find all of these methods plus
others, AOAC International offers Compendium of Microbiological
Methods for the Analysis of Food and Agricultural Products,
which is offered only in CD-ROM format, and may be ordered
at its website: http://184.108.40.206/pubs/microcompendium.htm.
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
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, cant use this instrument.
However, remember its 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 manufacturers 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 dont 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
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 3Ms
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
bacterias 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. Vicams
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
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.
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, its 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.
Bruce Floyd established Process Systems Consulting,
Iowa City, IA, after working more than 30 years in the food processing industry.
He has had extensive experience in sanitation, quality control, regulatory
relations, and product and process development (both domestic and international),
and specializes in integrating ingredient and manufacturing specifications
into total process systems. A graduate of Georgia State University, he has
successfully completed all areas of the Better Process Control School at
the University of Minnesota, and has been qualified by the International
HACCP Alliance as an instructor. He can be reached via e-mail at firstname.lastname@example.org.
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