Microbial Testing Procedures

Question 1

First decision to make during the microbiological testing of foods is:

Answer 1

a. a surface sample

b. a homogenized sample of the food

Question 2

Types of surface sampling:

Answer 2

Swabbing
contact plates
excision method

Question 3

Swabbing method

Answer 3

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)

Question 4

Contact Plates (rodac plates) method

Answer 4

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

Question 5

modified version of Rodac plates:

Answer 5

agar syringe or “sausage.” Tube full of agar, samples are pressed against a surface and then sliced off into a Petri plate for incubation.

Question 6

excision method

Answer 6

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.

Question 7

Homogenation methods

Answer 7

blender

colwell stomacher

Question 8

Blender method

Answer 8

Not used extensively now

Question 9

Stomacher

Answer 9

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

Question 10

Methods to determine total microbial numbers:

Answer 10

• Standard plate count
• Spiral plate counter
• Dry Petrifilms
• Most Probable Numbers
• Membrane filters
• Hydrophobic Grid Membrane Filters
• Dye reduction
• impedance

Question 11

Standard Plate Count (SPC)

Answer 11

the most widely used method for viable numbers.
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

Question 12

Spiral Plate Counter

Answer 12

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

Question 13

Dry Petrifilms

Answer 13

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

Question 14

MPN

Answer 14

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

Question 15

Membrane filters

Answer 15

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

Question 16

Direct Microscopic count

Answer 16

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

Question 17

hydrophobic grid membrane filters (HGMF)

Answer 17

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:
5. can detect as few as 10 CFU/g
6. can enumerate total CFU or specific groups (coliforms, fungi, pseudomonads, salmonellae)
7. AOAC approval for total coliforms, fecal coliforms and salmonellae
   Disadvantage:
8. expensive

Question 18

Dye Reduction

Answer 18

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

Question 19

Impedance

Answer 19

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.

Question 20

Technique to enumerate MI cells

Answer 20

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.

Question 21

Molecular methods to detect bacteria or metabolites

Answer 21

-Oligonucleotide DNA probes
-Polymerase Chain Reaction (PCR-DNA Amplification)
ELISA
Immunoprecipitation

Question 22

Oligonucleotide DNA probes

Answer 22

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.

Question 23

Polymerase Chain Reaction (PCR-DNA Amplification)

Answer 23

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.

Question 24

Enzyme-Linked Immunosorbant Assay (ELISA)

Answer 24

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.

Question 25

Immunoprecipitation

Answer 25

(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).

Question 26

In summary, when it comes to selecting a microbial testing procedure, remember the following:

Answer 26

QUICK, ACCURATE, CHEAP (choose any two)