Laboratory Procedure MFLP-56
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Health Products and Food Branch
Canadian Food Inspection Agency
P.O. Box 1060,
1992 Agency Drive
Calgary Laboratory, CFIA
3650 36th St. N.W.
Bureau of Microbial Hazards
Health Products and Food Branch
Postal locator: 2204A2
Ottawa, ON K1A 0L2
The method may be used for the determination of the aerobic colony count (ACC) in foods and environmental samples in accordance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This revised method replaces MFLP-56, dated September 1998.
The method has been shown to produce satisfactory results with meat and poultry, seafood, dairy products and vegetables (11.1) as well as environmental samples (D. Douey, CFIA, Calgary; unpublished data).
The HGMF analysis takes 24-48 h and yields counts that are higher than those obtained by the AOAC standard aerobic plate count in 4 product categories, and equivalent in the remainder (11.1). A single dilution accommodates a wide range of contamination levels. Counting precision may be better than on conventional plates or membrane filters because the HGMF reduces the effect of individual visual acuity on the count (11.2). If a low count is expected, the detection limit can be lowered by filtering more of the suspension.
The HGMF method described gives a choice of 2 agars, tryptic soy agar with the growth being stained with triphenyltetrazolium chloride after 24-48 h of incubation (11.1) [or with TSA containing TTC] and tryptic soy agar supplemented with the dye Fast Green FCF. In the latter case, the stain is used at a concentration producing no evidence of toxicity (11.4). Colonies are coloured various intensities of green.
See Appendix A of Volume 3 (11.3).
See Appendix B of Volume 3 (11.3).
The following media (3, 4 and 6) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 11.3 for the formula of individual media.
Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.
1) HGMF (1600 grid-cell, 0.45 :m pore size) or equivalent. (Available as ISO-GRID from Oxoid, Ottawa or Neogen, Lansing MI)
2) Membrane filter forceps (Millipore Corp.)
3) Peptone water, 0.1% (PEP) or Peptone/Tween 80 diluent (PT)
4) Tryptic soy agar (TSA) plates (or TSA containing TTC (12.2))
5) Triphenyltetrazolium chloride (TTC) solution
6) Trypic soy-fast green agar (TSFA) plates
7) Enzyme solutions (Appendix E of Volume 3; 11.3) as required for some food products
8) Stomacher, blender or equivalent
9) Spreadfilter with funnel plus pipet-tip prefilters (Filtaflex, Almonte, Ont.), or ISO-GRID filtration unit (Oxoid)
10) HGMF Interpreter (Filtaflex)
11) Incubators capable of maintaining 35°C
12) HGMF Linecounter are not commercially available
Analyze each sample unit individually. Carry out the test in accordance with the following instructions:
7.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5°C) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.
7.2.1 Have ready sterile peptone water (PEP) or peptone/Tween 80 diluent (PT), tryptic soy agar (TSA), TSA containing TTC or Tryptic soy-fast green agar (TSFA) plates and enzyme solutions needed for food product category if required. Use PT diluent if the spreadfilter is to be used.
7.2.2 Clean the surface of the working area with a suitable disinfectant.
7.2.3 Clearly label duplicate Petri dishes with appropriate identifying information.
7.3.1 To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.
7.3.2 Prepare a 1:10 dilution of the food by aseptically adding 10 g or mL (the analytical unit) into 90 mL of the PEP or PT diluent. If the spreadfilter is to be used to inoculate the HGMF, use PT diluent. Stomaching is the preferred method of suspending the organisms, since it minimizes the quantity of suspended food debris. Transfer representative portion from 1:10 dilution for higher serial dilutions as needed. Consult Appendix E of Volume 3 to see if enzyme treatment is required. Enzyme treatment is not necessary for samples filtered at a dilution of 1:100 or greater.
7.3.3 Stomach for 2 minutes.
7.3.4 If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc in approximately 7 seconds.
7.3.5 Filtration through the HGMF will remove acids or other inhibitors; there is no need to check and adjust the pH of the suspension.
7.3.6 The HGMF will allow counts to be made from suspensions containing up to 5,000 organisms/mL. There normally should be no need to prepare further dilutions. If this is necessary, prepare succeeding decimal dilutions as required, using a separate sterile pipette for each transfer. Record the dilution (C) used for analysis.
Helpful Hint: You can filter and plate every other dilution, i.e., 10-1, 10-3 and 10-5, and thus cover all possible counts.
7.3.7 If a low count is expected, filter more than 1.0 mL of the suspension. Filter the total volume to be filtered in one operation; do not attempt to filter successive 1 mL aliquots. Record the volume (V) filtered.
7.3.8 Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.
7.4.1 Agitate each stomacher bag or dilution bottle to resuspend material that may have settled out.
7.4.2 Handle HGMF with sterile forceps.
7.4.3 Following the manufacturer's instruction for use of the filtration apparatus, aseptically pipette 1.0 mL of the required dilution and inoculate the HGMF. Open the filter valve until all liquid has passed through and aseptically remove the HGMF. Do in duplicate.
7.4.4 Follow the manufacturer's instructions for cleaning the filtration apparatus.
7.4.5 Repeat with subsequent dilutions as required.
7.4.6 Continue plating following the instructions in either Section 8 or section 9.
8.1.1 Transfer the HGMF to the surface of a TSA (or TSA with TTC) plate by rolling it onto the agar to avoid trapping air bubbles. Incubate plates at 35°C in an inverted position for 24- 48 h for all samples. Incubate dairy products for 48 h.
8.2.1 Lift the corner of the HGMF using filter forceps, apply 1.5 ▒ 0.5 mL of 0.1% TTC solution to the agar, and relay the HGMF so that all of its undersurface is wetted.
8.2.2 Leave at least 15 min on the bench before counting.
8.3.1 The pink to red growth inside HGMF grid-cells is caused by aerobic organisms, and is counted as such.
8.3.2 For automated counting, use an HGMF Interpreter. Follow manufacturer's instructions for its use.
8.3.3 For manual counting, use a Linecounter. HGMF will give accurate counts over a wider range than is possible with plates. If possible, count only those HGMF containing 20-1580 occupied grid-cells (11.2). Counts outside this range may be counted, but reported as an "estimated" count.
188.8.131.52 Count 1 (one) for each grid-cell showing any pink to red growth. (DO NOT count the individual colonies if a grid-cell contains more than one red colony). If a rough estimate indicates fewer than 200 occupied grid-cells, count the whole HGMF.
184.108.40.206 For higher densities (up to 50% occupied grid-cells), rotate the HGMF so that the centre indicator lies either to the left or right. Count positive grid-cells in the 4 rows immediately below the centre and in the 4 rows immediately above the centre (8 rows). Multiply this partial HGMF count by 5 to estimate the score.
220.127.116.11 If the HGMF is so full that counting negative grid-cells appears easier, then do so as in (18.104.22.168) and (22.214.171.124). Subtract the HGMF negative count from 1600 or 320, depending on whether the whole or 1/5 of the HGMF was counted. Multiply by 5 to obtain the score if only one-fifth of the HGMF was counted.
126.96.36.199 Record as too numerous to count (TNTC) any HGMF for which all grid-cells contain pink to red growth.
8.3.4 Record the scores of both of the duplicate HGMF. If there are no pink to red grid-cells, record the score as zero.
See Appendix C of Volume 3 (11.3).
9.1.1 Transfer the HGMF to the surface of a TSFA plate by rolling it onto the agar to avoid trapping air bubbles. Incubate plates at 35°C in an inverted position for 48h.
9.2.1 The green growth inside HGMF grid-cells is caused by aerobic organisms, and is counted as such.
9.2.2 For automated counting, use an HGMF Interpreter. Follow manufacturer's instructions for its use.
9.2.3 For manual counting, use a Linecounter. HGMF will give accurate counts over a wider range than is possible with plates. If possible, count only those HGMF containing 20-1580 occupied grid-cells (11.2). Counts outside this range may be counted, but reported as an "estimated" count.
188.8.131.52 Count 1 (one) for each grid-cell showing any green growth. (DO NOT count the individual colonies if a grid-cell contains more than one green colony). If a rough estimate indicates fewer than 200 occupied grid-cells, count the whole HGMF.
184.108.40.206 For higher densities (up to 50% occupied grid-cells), rotate the HGMF so that the centre indicator lies either to the left or right. Count positive grid-cells in the 4 rows immediately below the centre and in the 4 rows immediately above the centre (8 rows). Multiply this partial HGMF count by 5 to estimate the score.
220.127.116.11 If the HGMF is so full that counting negative grid-cells appears easier, then do so as in (18.104.22.168) and (22.214.171.124). Subtract the HGMF negative count from 1600 or 320, depending on whether the whole or 1/5 of the HGMF was counted. Multiply by 5 to obtain the score if only one-fifth of the HGMF was counted.
126.96.36.199 Record as too numerous to count (TNTC) any HGMF for which all grid-cells contain green growth.
9.2.4 Record the scores of both of the duplicate HGMF. If there are no green grid-cells, record the score as zero.
See Appendix C of Volume 3 (11.3).
10.1 Report average MPNGU as calculated in 8.4 or 9.3 rounded-off to two significant figures (e.g., record 2850 as 2.9 x 103).
10.2 If the lowest dilution plated shows no positive grid-cells, the recorded value will be the lowest average obtainable with a given volume plated onto a given set of replicate HGMF, preceeded by a "less than" (<) sign, e.g., for 1.0 mL and a set of duplicate HGMF (1 mL per HGMF) the value is < 0.5. This figure should be multiplied by the dilution factor of the inoculum on the HGMF.
11.1 Parrington, L.J., A.N. Sharpe and P.I. Peterkin. 1993. Improved aerobic colony count technique for hydrophobic grid membrane filters. Appl. Environ. Microbiol. 59:2784-2789.
11.2 Sharpe, A.N. and P.I. Peterkin. 1988. Membrane filter food microbiology. Research Studies Press Ltd., Taunton, Somerset, U.K.
11.3 Appendices A, B, C, E, and G. Vol. 3, Compendium of Analytical Methods.
11.4 Entis, P. and P. Boleszczuk. 1986. Use of Fast Green FCF with tryptic soy agar for aerobic plate count by the hydrophobic grid membrane filter. J. Food Prot. 49:278-279.
11.5 Entis, P. 1986. Hdrophobic Grid Membrane Filter Method for Aerobic Plate Count in Foods. J. Assoc. Offic. Anal. Chem. 69:671-676.
12.1 When steam sterilization is used it is essential to allow sufficient time for the load to reach the required temperature before the actual sterilizing period commences. This varies with the nature and size of the load. Thus, proper exposure times should be followed to ensure sterilization of solutions in flasks and heat stable culture media. Refer to the sterilizer manual.
Trypticase Soy Agar (BBL) or Tryptic Soy Agar (Difco)
1% TTC stock solution
Suspend dehydrated medium in distilled water. Sterilize at 121/C for 15 minutes. Cool to 47/C. Aseptically add 10.0 mL TTC solution per litre of medium. Allow to mix thoroughly. Dispense 20 mL into petri plates. Final pH 7.3 ▒ 0.1.
Note: TTC solution is heat sensitive. Cool agar to 47/C before adding the solution. Do not use if medium turns pink.
This solution is used for making 0.1% TTC working solution and Tryptic Soy Agar (TSA) with
2,3,5-triphenyltetrazolium chloride (TTC)
Dissolve TTC in purified water. Sterilize by filtration. Dispense 10.0 mL into Sarstedt tubes, for addition to TSA media, or in bulk in an erlenmeyer flask, for preparing 0.1% TTC. Store the 10.0 mL aliquots at -20/C, and flasks at 4/C.
To prepare a 0.1% TTC working solution, in a 250 mL erlenmeyer flask add 90.0 mL of purified water and 10.0 mL of the 1% TTC solution. Gently swirl to mix. Store at 22/C for up to 1 month.