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 “sausage.” 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 35^oC 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. can’t tell live from dead cells

3. food parts sometimes look like cells

4. cells are usually not distributed evenly

5. different MO don’t 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 doesn’t 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 “new 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)