MICROBIAL
TESTING PROCEDURES
1. Examination of foods for the presence, types and numbers
of MO and/or their metabolites is basic to food microbiology.
2. When using these techniques, it is important to remember
that, due to specific growth requirements or limits among different MO, none
of the methods commonly used will give an exact count of the MO in a food.
3. In the case of some pathogenic organisms or their
toxins, you simply want to know whether or not they are present.
First decision to make during the microbiological testing of foods is:
a. A surface sample.
b. A
homogenized sample of the food.
Although most food samples will be homogenized, in
some foods such as whole meat cuts where the interior is essentially sterile, a
surface sample may be more significant because homogenization will include
portions of food that contain no MO and dilute results.
Other applications where surface measurements are
important in MO testing include food contact surfaces in a plant.
A. SURFACE SAMPLING
Although
recovery of all microorganisms from a surface may not always be possible, the
consistent monitoring of specific areas in a food plant by surface testing does
provide valuable insight into the relative cleanliness of that area.
1. Swabbing – MO collected from
a surface with sterile cotton or calcium alginate swabs (alginate swabs are the
best since the alginate can be readily dissolved in hexametaphosphate),
transferred to broth where they are dislodged, then diluted and used with
further tests to determine total numbers.
Sponges can be used to swab larger areas then placed in a buffer-filled
bag.
Advantages:
a. Easy
to perform
b. Inexpensive
c. Well
suited to flexible, uneven and heavily contaminated surfaces
Disadvantage:
1. MO recovery may be poor (10% in some studies, but even
that is still acceptable for many applications)
2. Contact plates
(Rodac
plates) – raised agar plate that is pressed against a surface and
then incubated.
Advantages:
1.
Method of choice for smooth, firm and nonporous surfaces (e.g. vat in a
cheese plant)
2.
Any type of media can be used
Disadvantages:
1. Colony overgrowth makes enumeration difficult on heavily
contaminated surfaces
2. Only removes about 0.1% of contact flora - much less
than swabs
A
modified version of this technique is the agar syringe or Ņsausage.Ó Tube full of agar, samples are pressed
against a surface and then sliced off into a Petri plate for incubation.
3. Excision method – a plug of know
surface area is taken from the food and then 1-3 mm is taken from the surface
end, homogenized and plated to determine total numbers. Commonly used in whole meat cuts.
B. HOMOGENIZATION METHODS
1. Blender – not used as
extensively now (see reasons listed below)
2. Colwell stomacher is the method of choice.
Food is placed in a
sterile plastic bag with diluent and then inserted into the machine. The stomacher has two paddles that
vigorously disrupt the food and give a nice homogenous fluid for sampling.
Advantages over blenders include:
1. No
cleaning since food is in a bag
2. No
heat buildup that will injure Mo (better survival in the sample)
3. Homogenates
can be frozen in the bags if necessary for further use
4. Decreased
noise level
5. Decrease
in aerosols
C. METHODS TO DETERMINE TOTAL
MICROBIAL NUMBERS
Once the sample is
prepared, there are several methods available to determine total cell numbers
in the food. Many of these methods
also allow for the enumeration of important groups of bacteria, such as
coliforms. A few can allow you to
count the number of microbes from a specific genus like Salmonella.
1. Standard Plate Count
(SPC)
By far the most widely used method for determining the number of viable colony
forming units (CFU) in a food. Use
a spread or pour plate (psychrotrophs may not survive as well in pour plates)
that includes homogenized food sample.
Incubation is aerobic at 35oC for 48± 2 h.
APC–aerobic
plate count – cells spread over agar surface and incubated aerobically.
Advantage:
1. Easy
to perform
2. AOAC
(Assoc. of Official Analytical Chemists) approved for many foods
Disadvantages:
1. Results
are not available for at least 16-18 h (often much longer)
2. Counts
in a food are influenced by several factors including:
a. Sampling method and the
distribution of MO in the food
b. Nature of the food flora (mostly gets mesophilic aerobes
and fac. aerobes)
c. Nature of the food
d. Intrinsic parameters of the growth medium reflect the MO
which are detected
e. Incubation time and temp (extrinsic parameters)
f. Microbial antagonism among species on the food
2. Spiral Plate
Counter – An automated version of SPC is the spiral plater, a device that
distributes a continuously decreasing volume of liquid over a single rotating
agar plate (the dispensing arm moves like a needle on a turntable, only backward). The agar is then incubated and counts
are made.
-It can effectively deliver up to a 105
concentration range on one plate, but enumeration requires a special counting
grid.
Advantages over SPC:
1. Easy to perform (little training required)
2. Fewer materials are used (agar plates, dilution blanks,
pipettes)
3. 3-4X more samples can be run per hour
4. Spiral plating does agree well with SPC
values and is an AOAC method.
Disadvantage:
1. Food particles may clog the dispensing arm – more
suited to fluids like milk
3. Dry Petrifilms
Two plastic films held
together on one side and coated like a sandwich with
culture media ingredients,
tetrazolium dye (a reducing dye) and a water
soluble gelling agent. 1 ml of sample in diluent is placed between the films and
gently spread around by pressing the 2 sides of film. After incubation, cell growth reduces the dye and gives red
colonies.
Advantages:
1. nonselective and selective media are available in the
films. Films are available to perform total plate counts of bacteria or fungi,
coliform counts, and specific tests for hemorrhagic E. coli 0157:H7
2. the product has AOAC approval
3. store for long periods, no autoclave required
Disadvantage:
1. expensive
2. difficult to read without
training
4. Most Probable
Numbers: A method based upon statistical
probability. Food samples are
prepared like SPC. Three serial
dilutions are prepared and then transferred to 9 or 15 tubes (3- or 5-tube
method). The numbers of
organisms/g are then estimated using standard MPN tables.
Advantages:
1. easy
to perform
2. results from one lab more likely than SPC to agree with
those from another lab
3. specific groups of organisms (e.g. coliforms-it is the
method of choice) can be enumerated using different selective medias
4. AOAC for coliforms, S.
aureus & B. cereus (many
others) in various foods
Disadvantages:
1. lots of tubes required (clean up intensive)
2. lack of precision, generally higher than SPC results
5. Membrane filters:
1. Most useful for microscopic examination of water
supplies (e.g. coliform counts) since most foods will clog the filters. 0.45 µm-pore filters allow water to
pass but trap bacteria.
2. Filters are especially useful for samples with low
numbers of bacteria since large volumes of fluid can be passed through.
3. Polycarbonate filters are better than the cellulose one
because bacteria are caught on the filter surface more efficiently.
4. A given volume of fluid is passed through the filter:
a. the
membrane is placed on an agar plate and incubated
b. Direct Microscopic Count (DMC) can be made on the filter
DMC on filters has been
simplified by the use of fluorescent dyes that bind to bacteria.
1. Called the Direct
Epifluorescent Filter Technique (DEFT), this method uses dyes and
fluorescent microscopy to rapidly enumerate bacteria on a filter.
2. This procedure includes a pre-filtering step to remove
large food particles (-if necessary-5µm filter used) and then the filtrate is
treated with a detergent and a protease to degrade somatic cells that will clog
the small 0.6µm polycarbonate filter used next.
3. The filter is stained with acridine orange, dried and
then counted by epifluorescent microscopy.
4. Results
are available in less than 30 minutes.
DMC
(including DEFT):
Advantages;
1. rapid
and simple
2. morph
can be determined
3. repeatability
is better than SPC
4. solid
foods can be examined using the prefiltration step
Disadvantages:
1. tiring
to analysts
2. canÕt
tell live from dead cells
3. food
parts sometimes look like cells
4. cells
are usually not distributed evenly
5. different
MO donÕt stain equally
Hydrophobic Grid Membrane Filters (HGMF)
1. A special filter that contains 1,600 wax grids that
restrict colony growth to single grids.
2. 10 to 9 x 104 cells can be enumerated on a
single filter using a Most Probable Numbers statistical procedure.
3. Homogenized food is passed through the filter and then
it is placed on an agar plate and incubated overnight.
4. The colonies are counted and then MPN is calculated.
Advantages:
1. can detect as few as 10 CFU/g
2. can enumerate total CFU or specific groups (coliforms,
fungi, pseudomonads, salmonellae)
3. AOAC approval for total coliforms, fecal coliforms and
salmonellae
Disadvantage:
1. expensive
6. Dye Reduction: Supernatants of food are added to standard solutions of
methylene blue or resazurin:
a. Blue --> White for reduction of methylene blue
b. Slate
blue --> pink or white for resazurin
The
time required for dye reduction is inversely proportional to the total number
of microorganisms.
Technique lends itself to
automation and has a long history of use in dairy foods.
Developed
an automated colorimeter that has AOAC approval for determination of total
counts and coliforms.
Advantages:
1. simple,
rapid and inexpensive
2. only
viable cells reduce dye
3. automation
available
Disadvantages:
1. not
all MO reduce dye equally well
2. Some foods like meat contain high levels of natural
reductants that cloud results. Use
of a stomacher instead of a blender to homogenize meat has been shown to reduce
this problem. Apparently the
stomacher doesnÕt release as much NADH or other reductants from the tissue.
3. expensive
equipment
7. Impedance: Impedance is a measurement
based on the resistance in an electric circuit to the flow of alternating
current. Microorganisms metabolize
substrates of low conductivity into products of higher conductivity. As a result, resistance decreases with
growing cell numbers. With proper instruments,
the change in impedance can be followed and used to detect as few as 10 to 100
cells in a sample within about 6 h (impedance detection time or IDT). The technique is faster than SPC and
gives results that are within 90-95% agreement with the former technique but is
not AOAC approved.
Special
concern; metabolically injured cells:
1. Processing conditions often include treatments such as
mild heating or cooling steps (and many others) that injure but do not kill
microorganisms.
2. Metabolically injured cells (MI)
may not grow well on plate count agar and thus leave you with the impression
that your processing step is killing more MO than it really does and that the
food contains fewer MO than it actually does (since these cells will eventually
recover and grow in the food if conditions are favorable).
3. If you work for a company that is using a food process
where MI cells may be present, then it is useful to include a recovery step for
MI cells in your microbial testing procedure.
4. In general, rich media, sometimes supplemented with pyruvate
or catalase, favor the recovery of MI cells.
Pyruvate
and catalase degrade peroxidases and it is thought that MI cells lack the
ability to degrade these toxic oxygen derivatives.
Technique to enumerate MI
cells:
1. plate food samples on normal (minimal and rich media)
2. add an extra day of incubation time
3. comparison between media (plates)
– Rich media will include MI and uninjured cells, minimal will only have
uninjured cells. This will give
you an idea of how effective your processing step is on killing MO.
D. Molecular Methods to Detect Bacteria or Metabolites:
1. Except for those applications that involve coliform or Salmonella enumeration, the methods we
have examined thus far are primarily intended to predict product shelf life and
prevent spoilage.
2. The next group of testing procedures is instead designed
to detect specific genera or species of food-borne pathogens (or their
metabolites).
3. Unfortunately, many of these tests employ expensive
reagents and because of the expense, their use is generally (but not always)
more diagnostic than preventative (i.e. used only after an outbreak).
1. Oligonucleotide
DNA Probes - This test is based on DNA
or DNA-RNA complementary base pairing or hybridization.
-Go through a hybridization procedure;
include colony hyb (can run up to 69 filter w/48 CFU per filter in one Rxn),
dot blots or blots of restriction digests.
-can use radioactive or non-isotopic
labels on probes. Reactions can
require anywhere from 10 h - 2 days.
This
technique requires that the sequence of the target nucleic acid (frequently a
gene for toxin production or a species-specific 16S rRNA sequence) be
determined and unique to the organisms sought. As we discussed earlier in the lectures on taxonomy, 16S
rRNA contains widely conserved as well as species (and sometimes subspecies)
-specific regions. Probes to the
species-specific regions are used to detect particular bacteria such as Salmonella. One advantage to targeting the 16S rRNA versus a DNA
sequence in the chromosome is that there are several copies of the 16S rRNA per
cell. The same advantage applies
to genes located on multicopy plasmids.
Gene probes may be based on the DNA sequence of particular toxins such
as Clostridium perfringens or
staphylococcal enterotoxins. At
lease one test for Salmonella has
AOAC approval.
2. Polymerase Chain
Reaction (PCR-DNA Amplification).
This
technique is more useful than DNA probing when small numbers of a particular
microbe are present. It can
amplify a single copy of any DNA sequence 107 times within a few
hours. Amplified DNA can then be
detected by agarose gel electrophoresis or hybridization.
-PCR requires primers based on the
known nucleic acid sequence of the amplification target.
PCR has been used to detect 1-5 CFU of E. coli in 100 ml of water, useful for
any organism where a unique nucleic acid sequence is known, also for diagnosis
by amplification of 16s rRNA (ASM handout Ņnew vistas for bacteriologists).
-Very powerful tool with many, many
applications; the latest clinical manual for diagnostic microbiology is almost
completely based on PCR assays.
3. Enzyme-Linked
Immunosorbant Assay (ELISA). Another very powerful
technique that uses mono- or polyclonal antibodies to detect specific antigens
in a sample.
Commercial ELISA kits are available
for a variety of applications (e.g. home pregnancy tests) including the
detection of various microbes including Salmonella,
S. aureus and its enterotoxins, molds and mycotoxins, botulinal toxins and E. coli strains and toxins.
4. Immunoprecipitation (Immunodiffusion) - Another immunological technique
commonly used for toxin identification.
Several variations but the basic principle involves a reaction between
antigen (toxin) and antibody that forms a precipitate in an agar gel.
Immunodiffusion methods can detect as
little as 0.1-0.01 µg of toxin.
Incubation times increase with smaller amounts of antigen (1-6 d range).
In summary, when it comes to selecting a microbial
testing procedure, remember the following: QUICK, ACCURATE, CHEAP (choose any
two)