Chapter 9: Aerobic Plate Count

Updated: 9/20/00



Contents

Potential Food Safety Hazard

The aerobic plate count (APC) indicates the level of microorganisms in a product (Maturin and Peeler, 1998). Aerobic plate counts on fish and fishery products generally do not relate to food safety hazards, but sometimes can be useful to indicate quality, shelf life and post heat-processing contamination. Fresh fish and fishery products often have an APC of 104-105/g, although there are examples of seafoods with an APC of 106-108/g without objectionable quality changes (Nickelson and Finne, 1992).

The plating medium (nutrient source) used in an APC can affect the number and types of bacteria isolated because of differences in nutrient and salt requirements of the various microorganisms. For many fish and fishery products, a plate incubation temperature of 25C (77F) produces significantly higher numbers of bacteria than incubation at 35C (95F) (Nickelson and Finne, 1992).

Contents

FDA Guidelines

Table 9-1. FDA guidelines for APC in fish and fishery products.

Product Guideline Reference
Raw breaded shrimp Aerobic plate counts (35C [95F]) – The mean log of 16 units of finished product breaded shrimp collected prior to freezing is greater than 5.00 (i.e., geometric mean greater than 100,000/g) and exceeds the mean log of 16 units of stock shrimp by more than twice the standard error of their difference (2 SED) FDA, 1996a
Clams oysters, and mussels, fresh or frozen--domestic APC - 1 or more of 5 subs exceeding 1,500,000/g or 2 or more exceeding 500,000/g FDA, 1998
Clams, oysters, and mussels fresh or frozen--imports APC - 500,000/g (average of subs of 3 or more of 5 subs) FDA, 1998

Contents

State Guidelines

Table 9-2. State APC Guidelines.

State Product Maximum APC
Alabama Oysters, fresh or frozen 5 105/g
Alaska Oysters, clams, and mussels 5 105/g
Oysters, clams, and mussels: in shell or shucked, but not eviscerated 5 105/g
Oysters, clams, and mussels: eviscerated 1 105/g
Arizona Clams, mussels, and oysters 5 105/g
Arkansas -  
California Oysters, clams, and mussels 5 105/g
Colorado Oysters, clams, mussels, and scallops 5 105/g
Connecticut Oysters, clams, and mussels 5 105/g
Delaware Clams, mussels, or other mollusks, fresh or frozen 5 105/g
Florida Blue crab 105/g
Shellfish 5 105/g
Georgia Clams, mussels, and oysters - fresh or frozen 5 105/g
Fried clams, frozen 104/g
Scallops, fried, frozen 104/g
Scallops, breaded, frozen 104/g
Crabmeat, fresh cooked 104/g
Deviled crab, frozen, cooked 104/g
Deviled crab, fresh, uncooked 106/g
Shrimp, peeled, cooked 105/g
Shrimp, breaded, frozen, raw 106/g
Fish, frozen, breaded, fried 2.5 104/g
Fish, frozen, breaded, raw 105/g
Fried fish cakes, frozen 104/g
Hawaii Oysters, clams, mussels, fresh or frozen 5 105/g
Idaho

-

-

Illinois

-

-

Indiana

-

-

Iowa

-

-

Kansas

-

-

Kentucky Oysters, clams, scallops, shrimp, fresh or frozen 5 105/g
Louisiana

-

-

Maine

-

-

Maryland Fresh crabmeat 105/g
Pasteurized crabmeat 2.5 104/g
Oysters, clams, mussels, fresh or frozen 5 105/g
Massachusetts Oysters, clams, mussels, fresh or frozen 5 105/ml
Michigan

-

-

Minnesota

-

-

Mississippi Oysters, clams, mussels, fresh or frozen 5 105/g
Missouri Oysters, clams, mussels, fresh or frozen 5 105/100ml
Foods 1.5 106/g
Montana

-

-

Nebraska Oysters, clams, mussels, fresh or frozen 5 105/g
Deli foods (shrimp salad, etc.) 105/g
Nevada

-

-

New Hampshire Oysters, softshell clams, fresh or frozen 5 105/g
New Jersey Oysters, clams, mussels, fresh or frozen 5 105/g
"Potentially hazardous" (tuna, shrimp salad) 104/g
New Mexico

-

-

New York

-

-

North Carolina Shellfish 5 105/g
Crustacea, fresh 104/g
Crustacea, pasteurized 3 103/g
North Dakota

-

-

Ohio

-

-

Oklahoma

-

-

Oregon Oysters, clams, mussels, fresh or frozen 5 105/100g
Pennsylvania

-

-

Rhode Island Oysters, clams, mussels, fresh or frozen 5 105/g
Fresh seafood 106/g
Smoked fish 105/g
South Carolina Fresh cooked blue crabmeat 105/g
Pasteurized blue crabmeat 2.5 x 104/g
Oysters, clams, mussels, fresh or frozen 5 105/g
South Dakota

-

-

Tennessee

-

-

Texas Crabmeat 105/g
Oysters, clams, mussels, fresh or frozen 5 105/g
Utah

-

-

Vermont

-

-

Virginia Fresh blue crabmeat 105/g
Pasteurized blue crabmeat 3 103/g
Shellfish - shucked or in the shell 5 105/g
Washington Molluscan shellfish (Oysters, clams, mussels, fresh or frozen) 5 105/g
West Virginia -

-

Wisconsin -

-

Wyoming -

-

(AFDO, 1998)

Contents

Recommended Microbiological Limits

Contents

ICMSF

Table 9-3. Recommended microbiological limits for fish and fishery products (ICMSF, 1986).

Product

n1

c2

Bacteria/g or cm2

m3

M4

Fresh and frozen fish and cold-smoked fish

5

3

5 105

107

Precooked breaded fish

5

2

5 105

107

Frozen raw crustaceans

5

3

106

107

Frozen cooked crustaceans

5

2

5 105

107

Cooked, chilled, and frozen crabmeat

5

2

105

106

Fresh and frozen bivalve molluscs

5

0

5 105

-

1 Number of representative sample units.
2 Maximum number of acceptable sample units with bacterial counts between m and M.
3 Maximum recommended bacterial counts for good quality products.
4 Maximum recommended bacterial counts for marginally acceptable quality products.

Plate counts below "m" are considered good quality. Plate counts between "m" and "M" are considered marginally acceptable quality, but can be accepted if the number of samples does not exceed "c." Plate counts at or above "M" are considered unacceptable quality (ICMSF, 1986).

Contents

Analytical Procedures

Contents

Food Sampling and Preparation of Sample Homogenate (Andrews and June, 1998)

The adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard.

The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the collector at the time of sampling by using the Investigations Operation Manual (FDA, 1993). Sampling and sample plans are discussed in detail in ICMSF (1986).

Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment.

Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. Whenever possible, avoid glass containers, which may break and contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Take care not to overfill bags or permit puncture by wire closure. Identify each sample unit (defined later) with a properly marked strip of masking tape. Do not use a felt pen on plastic because the ink might penetrate the container. Whenever possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile containers with the sample.

Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in pre-chilled containers.

Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4C and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4C until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection. Special conditions apply to the collection and storage of shucked, unfrozen shellfish and shell stock (APHA, 1985). Pack samples of shucked shellfish immediately in crushed ice (no temperature specified) until analyzed; keep shell stock above freezing but below 10C. Examine refrigerated shellfish and shell stock within 6 h of collection but in no case more than 24 h after collection. Further details on sample handling and shipment may be found in the Investigations Operation Manual (FDA, 1993) and the Laboratory Procedures Manual (FDA, 1989). The Investigations Operation Manual (FDA, 1993) contains sampling plans for various microorganisms. Some of those commonly used are presented here.

  1. Sampling plans
    1. Aerobic plate counts, total coliforms, fecal coliforms, Escherichia coli (including enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp., Campylobacter spp., Yersinia spp., Bacillus cereus, and Clostridium perfringens
      1. Sample collection. From any lot of food, collect ten 8 ounce (227 g) subsamples (or retail packages) at random. Do not break or cut larger retail packages to obtain an 8 ounce (227 g) subsample. Collect the intact retail unit as the subsample even if it is larger than 8 ounce (227 g).
      2. Sample analysis. Analyze samples as indicated in current compliance programs.
  2. Equipment and materials
    1. Mechanical blender. Several types are available. Use blender that has several operating speeds or rheostat. The term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at 10,000-12,000 rpm or with equivalent shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring blender, or equivalent, meets these requirements.
    2. Sterile glass or metal high-speed blender jar. 1000 ml, with cover, resistant to autoclaving for 60 min at 121C.
    3. Balance, with weights. 2000 g capacity, sensitivity of 0.1 g.
    4. Sterile beakers. 250 ml, low-form, covered with aluminum foil.
    5. Sterile graduated pipets. 1.0 and 10.0 ml.
    6. Butterfield's phosphate-buffered dilution water (R11). Sterilized in bottles to yield final volume of 90 1 ml.
    7. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue depressors. For sample handling.
  3. Receipt of samples
    1. The official food sample is collected by the FDA inspector or investigator. As soon as the sample arrives at the laboratory, the analyst should note its general physical condition. If the sample cannot be analyzed immediately, it should be stored as described later. Whether the sample is to be analyzed for regulatory purposes, for investigation of a foodborne illness outbreak, or for a bacteriological survey, strict adherence to the recommendations described here is essential.
    2. Condition of sampling container. Check sampling containers for gross physical defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture marks. If sample units were collected in plastic bottles, check bottles for fractures and loose lids. If plastic bags were used for sampling, be certain that twist wires did not puncture surrounding bags. Any cross-contamination resulting from one or more of above defects would invalidate the sample, and the collecting district should be notified (see 3-e, below).
    3. Labeling and records. Be certain that each sample is accompanied by a completed copy of the Collection Report (Form FD-464) and officially sealed with tape (FD-415a) bearing the sample number, collecting official's name, and date. Assign each sample unit an individual unit number and analyze as a discrete unit unless the sample is composited as described previously in this chapter.
    4. Adherence to sampling plan. Most foods are collected under a specifically designed sampling plan in one of several ongoing compliance programs. Foods to be examined for Salmonella, however, are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. Depending on the food and the type of analysis to be performed, determine whether the food has been sampled according to the most appropriate sampling plan.
    5. Storage. If possible, examine samples immediately upon receipt. If analysis must be postponed, however, store frozen samples at -20C until examination. Refrigerate unfrozen perishable samples at 0-4C not longer than 36 h. Store nonperishable, canned, or low-moisture foods at room temperature until analysis.
    6. Notification of collecting district. If a sample fails to meet the above criteria and is therefore not analyzed, notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced.

  4. Thawing
  5. Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5C within 18 h. If rapid thawing is desired, thaw the sample at less than 45C for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath.

  6. Mixing
  7. Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. If contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test.

  8. Weighing
  9. Tare high-speed blender jar; then aseptically and accurately ( 0.1 g) weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Total volume in blender must completely cover blades.

  10. Blending and diluting of samples requiring enumeration of microorganisms
    1. All foods other than nut meat halves and larger pieces, and nut meal. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and blend 2 min. This results in a dilution of 10-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25 times in 30 cm (1 foot) arc in 7 s. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media.
    2. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into sterile screw-cap jar. Add 50 ml diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 100 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.
    3. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add 90 ml of diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.

    Contents

    Aerobic plate count (Maturin and Peeler, 1998)

    The aerobic plate count (APC) is intended to indicate the level of microorganisms in a product. Detailed procedures for determining the APC of foods have been developed by the Association of Official Analytical Chemists (AOAC, 1990) and the American Public Health Association (APHA, 1984). The conventional plate count method for examining frozen, chilled, precooked, or prepared foods, outlined below, conforms to AOAC Official Methods of Analysis, sec. 966.23, with one procedural change (966.23C). The suitable colony counting range is 25-250 (Tomasiewicz et al., 1980). The automated spiral plate count method for the examination of foods and cosmetics, outlined below, conforms to AOAC Official Methods of Analysis, sec. 977.27 (Gilchrist et al., 1977). For procedural details of the standard plate count, see APHA (1993).

    Guidelines for calculating and reporting plate counts have been changed to conform with the anticipated changes in the 16th edition of Standard Methods for the Examination of Dairy Products (APHA, 1993) and the International Dairy Federation (IDF) procedures (IDF, 1987).

    Contents

    Conventional Plate Count Method
    1. Equipment and materials
      1. Work area, level table with ample surface in room that is clean, well-lighted (100 foot-candles at working surface) and well-ventilated, and reasonably free of dust and drafts. The microbial density of air in working area, measured in fallout pour plates taken during plating, should not exceed 15 colonies/plate during 15 min exposure.
      2. Storage space, free of dust and insects and adequate for protection of equipment and supplies
      3. Petri dishes, glass or plastic (at least 15 90 mm)
      4. Pipets with pipet aids (no mouth pipetting) or pipettors, 1, 5, and 10 ml, graduated in 0.1 ml units
      5. Dilution bottles, 6 ounces (160 ml), borosilicate-resistant glass, with rubber stoppers or plastic screw caps
      6. Pipet and petri dish containers, adequate for protection
      7. Circulating water bath, for tempering agar, thermostatically controlled to 45 1C
      8. Incubator, 35 1C; milk, 32 1C
      9. Colony counter, dark-field, Quebec, or equivalent, with suitable light source and grid plate
      10. Tally register
      11. Dilution blanks, 90 1 ml Butterfield's phosphate-buffered dilution water (R11); milk, 99 2 ml
      12. Plate count agar (standard methods) (M124)
      13. Refrigerator, to cool and maintain samples at 0-5C; milk, 0-4.4C
      14. Freezer, to maintain frozen samples from -15 to –20C
      15. Thermometers (mercury) appropriate range; accuracy checked with a thermometer certified by the National Institute of Standards and Technology (NIST)
    2. Procedure for analysis of frozen, chilled, precooked, or prepared foods
    3. Using separate sterile pipets, prepare decimal dilutions of 10-2, 10-3, 10-4, and others as appropriate, of food homogenate (see "Food Sampling and Preparation of Sample Homogenate" for sample preparation) by transferring 10 ml of previous dilution to 90 ml of diluent. Avoid sampling foam. Shake all dilutions 25 times in 30 cm (1 foot) arc within 7 s. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. Reshake dilution bottle 25 times in 30 cm arc within 7 s if it stands more than 3 min before it is pipetted into petri dish. Add 12-15 ml plate count agar (cooled to 45 1C) to each plate within 15 min of original dilution. For milk samples, pour an agar control, pour a dilution water control and pipet water for a pipet control. Add agar to the latter two for each series of samples. Add agar immediately to petri dishes when sample diluent contains hygroscopic materials, e.g., flour and starch. Pour agar and dilution water control plates for each series of samples. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on flat level surface. Let agar solidify. Invert solidified petri dishes, and incubate promptly for 48 2 h at 35C. Do not stack plates when pouring agar or when agar is solidifying.

    4. Guidelines for calculating and reporting APCs in uncommon cases
    5. Official Methods of Analysis (AOAC, 1990) does not provide guidelines for counting and reporting plate counts, whereas Standard Methods for the Examination of Dairy Products, 16th ed. (APHA, 1993) presents detailed guidelines; for uniformity, therefore, use APHA guidelines as modified (IDF, 1987; Niemela, 1983). Report all aerobic plate counts (APHA, 1993) computed from duplicate plates. For milk samples, report all aerobic plate (APHA, 1992) counts computed from duplicate plates containing less than 25 colonies as less than 25 estimated count. Report all aerobic plate counts (APHA, 1993) computed from duplicate plates containing more than 250 colonies as estimated counts. Counts outside the normal 25-250 range may give erroneous indications of the actual bacterial composition of the sample. Dilution factors may exaggerate low counts (less than 25), and crowded plates (greater than 250) may be difficult to count or may inhibit the growth of some bacteria, resulting in a low count. Report counts less than 25 or more than 250 colonies as estimated aerobic plate counts (EAPC). Use the following guide:

      1. Normal plates (25-250). Select spreader-free plate(s). Count all colony forming units (CFU), including those of pinpoint size, on selected plate(s). Record dilution(s) used and total number of colonies counted.
      2. Plates with more than 250 colonies. When number of CFU per plate exceeds 250, for all dilutions, record the counts as too numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those portions of plate that are representative of colony distribution. See APHA (1993) for detailed guidelines. Mark calculated APC with EAPC to denote that it was estimated from counts outside 25-250 per plate range (see 4-3).
      3. Spreaders. Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2) one that develops in film of water between agar and bottom of dish; and 3) one that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader growth so that (a) area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area, or (b) area of repressed growth exceeds 25% of plate area, report plates as spreaders. When it is necessary to count plates containing spreaders not eliminated by (a) or (b) above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony. Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC.
      4. Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the corresponding lowest dilution used. Mark calculated APC with asterisk to denote that it was estimated from counts outside the 25-250 per plate range. When plate(s) from a sample are known to be contaminated or otherwise unsatisfactory, record the result(s) as laboratory accident (LA).
    6. Computing and recording counts (see IDF, 1987; Niemela, 1983)

    To avoid creating a fictitious impression of precision and accuracy when computing APC, report only the first two significant digits. Round off to two significant figures only at the time of conversion to SPC. For milk samples, when plates for all dilutions have no colonies, report APC as less than 25 colonies estimated count. Round by raising the second digit to the next highest number when the third digit is 6, 7, 8, or 9 and use zeros for each successive digit toward the right from the second digit. Round down when the third digit is 1, 2, 3, or 4. When the third digit is 5, round up when the second digit is odd and round down when the second digit is even.

    Examples

    Calculated Count

    APC

    12,700

    13,000

    12,400

    12,000

    15,500

    16,000

    14,500

    14,000

    1. Plates with 25-250 CFU
      1. Calculate the APC as follows:
      2. where

        N = Number of colonies per ml or g of product
        c = Sum of all colonies on all plates counted
        n1 = Number of plates in first dilution counted
        n2 = Number of plates in second dilution counted
        d = Dilution from which the first counts were obtained

        Example

        1:100

        1:1,000

        232, 244

        33,28

        N = 24,409 = 24,000

      3. When counts of duplicate plates fall within and without the 25-250 colony range, use only those counts that fall within this range.

    2. All plates with fewer than 25 CFU. When plates from both dilutions yield fewer than 25 CFU each, record actual plate count but record the count as less than 25 x 1/d when d is the dilution factor for the dilution from which the first counts were obtained.
    3. Example

      Colonies

       

      1:100

      1:1,000

      EAPC/ml (g)

      18

      2

      <2,500

      0

      0

      <2,500

      EAPC, estimated aerobic plate count.

    4. All plates with more than 250 CFU. When plates from both 2 dilutions yield more than 250 CFU each (but fewer than 100/cm2), estimate the aerobic counts from the plates (EAPC) nearest 250 and multiply by the dilution.
    5. Example

      Colonies

       

      1:100

      1:1,000

      EAPC/ml (g)

      TNTC

      640

      640,000

      TNTC, too numerous to count.
      EAPC, estimated aerobic plate count.

    6. All plates with spreaders and/or laboratory accident. Report respectively as Spreader (SPR), or Laboratory Accident (LA).
    7. All plates with more than an average of 100 CFU/cm2. Estimate the APC as greater than 100 times the highest dilution plated, times the area of the plate. The examples below have an average count of 110/cm2.
    8. Example

      Colonies/Dilution

       

      1:100

      1:1,000

      EAPCa/ml (g)

      TNTC

      7,150b

      >6,500,000

      TNTC

      6,490c

      >5,900,000

      aEstimated aerobic plate count.
      bBased on plate area of 65 cm2
      cBased on plate area of 59 cm2

    Contents

    Spiral Plate Method

    The spiral plate count (SPLC) method for microorganisms in milk, foods, and cosmetics is an official method of the APHA (1993) and the AOAC (1990). In this method, a mechanical plater inoculates a rotating agar plate with liquid sample. The sample volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating plate. The microbial concentration is determined by counting the colonies on a part of the petri dish where they are easily countable and dividing this count by the appropriate volume. One inoculation determines microbial densities between 500 and 500,000 microorganisms/ml. Additional dilutions may be made for suspected high microbial concentrations.

    1. Equipment and materials
      1. Spiral plater (Spiral Systems Instruments, Inc., 7830 Old Georgetown Road, Bethesda, MD 20814)
      2. Spiral colony counter (Spiral Systems) with special grid for relating deposited sample volumes to specific portions of petri dishes
      3. Vacuum trap for disposal of liquids (2-4 liter vacuum bottle to act as vacuum reservoir and vacuum source of 50-60 cm Hg)
      4. Disposable micro beakers, 5 ml
      5. Petri dishes, plastic or glass, 150 x 15 mm or 100 x 15 mm
      6. Plate count agar (standard methods) (M124)
      7. Calculator (optional), inexpensive electronic hand calculator is recommended
      8. Polyethylene bags for storing prepared plates
      9. Commercial sodium hypochlorite solution, about 5% NaOCl (bleach)
      10. Sterile dilution water
      11. Syringe, with Luer tip for obstructions in stylus; capacity not critical
      12. Work area, storage space, refrigerator, thermometers, tally, and incubator, as described for Conventional Plate Count Method, above.
      13. Sodium hypochlorite solution (5.25%). Available commercially.

    2. Preparation of agar plates
    3. Automatic dispenser with sterile delivery system is recommended to prepare agar plates. Agar volume dispensed into plates is reproducible and contamination rate is low compared to hand pouring of agar in open laboratory. When possible, use laminar airflow hood along with automated dispenser. Pour same quantity of agar into all plates so that same height of agar will be presented to spiral plater stylus tip to maintain contact angle. Agar plates should be level during cooling.

      The following method is suggested for prepouring agar plates: Use automatic dispenser or pour constant amount (about 15 ml/100 mm plate; 50 ml/150 mm plate) of sterile agar at 60-70C into each petri dish. Let agar solidify on level surface with poured plates stacked no higher than 10 dishes. Place solidified agar plates in polyethylene bags, close with ties or heat-sealer, and store inverted at 0-4.4C. Bring prepoured plates to room temperature before inoculation.

    4. Preparation of samples
    5. As described in "Food Sampling and Preparation of Sample Homogenate," select that part of sample with smallest amount of connective tissues or fat globules.

    6. Description of spiral plater
    7. Spiral plater inoculates surface of prepared agar plate to permit enumeration of microorganisms in solutions containing between 500 and 500,000 microorganisms per ml. Operator with minimum training can inoculate 50 plates per h. Within range stated, dilution bottles or pipets and other auxiliary equipment are not required. Required bench space is minimal, and time to check instrument alignment is less than 2 min. Plater deposits decreasing amount of sample in Archimedean spiral on surface of prepoured agar plate. Volume of sample on any portion of plate is known. After incubation, colonies appear along line of spiral. If colonies on a portion of plate are sufficiently spaced from each other, count them on special grid which associates a calibrated volume with each area. Estimate number of microorganisms in sample by dividing number of colonies in a defined area by volume contained in same area. Studies have shown the method to be proficient not only with milk (Donnelly et al., 1976) but also with other foods (Jarvis et al., 1977; Zipkes et al., 1981.

    8. Plating procedure
    9. Check stylus tip angle daily and adjust if necessary. (Use vacuum to hold microscope cover slip against face of stylus tip; if cover slip plane is parallel at about l mm from surface of platform, tip is properly oriented.) Liquids are moved through system by vacuum. Clean stylus tip by rinsing for 1 second with sodium hypochlorite solution followed by sterile dilution water for 1 second before sample introduction. This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops. Remove inoculated plate from platform and cover it. Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new sample. Invert plates and promptly place them in incubator for 48 3 h at 35 1C.

    10. Sterility controls
    11. Check sterility of spiral plater for each series of samples by plating sterile dilution water. CAUTION: Prepoured plates should not be contaminated by a surface colony or be below room temperature (water can well up from agar). They should not be excessively dry, as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater than 2 mm in agar depth, and they should not be stored at 0-4.4C for longer than l month. Reduced flow rate through tubing indicates obstructions or material in system. To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve accumulated residue with chromic acid. Rinse well after cleaning.

    12. Counting grid
      1. Description. Use same counting grid for both 100 and 150 mm petri dishes. A mask is supplied for use with 100 mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid edge. Other lines within these arcs are added for ease of counting. A segment is the area between 2 arc lines within a wedge. Number of areas counted (e.g., 3) means number of segments counted within a wedge. Spiral plater deposits sample on agar plate in the same way each time. The grid relates colonies on spiral plate to the volume in which they were contained. When colonies are counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward center of plate.
      2. Calibration. The volume of sample represented by various parts of the counting grid is shown in operator's manual that accompanies spiral plater. Grid area constants have been checked by the manufacturer and are accurate. To verify these values, prepare 11 bacterial concentrations in range of 106-103cells/ml by making 1:1 dilutions of bacterial suspension (use a nonspreader). Plate all Incubate both sets of plates for 48 3 h at 35 1C. Calculate concentrations for each dilution. Count spiral plates over grid surface, using counting rule of 20 (described in H, below), and record number of colonies counted and grid area over which they were counted. Each spiral colony count for a particular grid area, divided by aerobic count/ml for corresponding spirally plated bacterial concentrations, indicates volume deposited on that particular grid area. Use the following formula:
      3. Volume (ml) for grid area = Spiral colonies counted in area/Bacterial count/ml (APC)

        Example:

        Volume (ml) = 0.0015 ml

        To check total volume dispensed by spiral plater, weigh amount dispensed from stylus tip. Collect in tared 5 ml plastic beaker and weigh on analytical balance ( 0.2 mg).

    13. Examination and reporting of spiral plate counts
    14. Counting rule of 20. After incubation, center spiral plate over grid by adjusting holding arms on viewer. Choose any wedge and begin counting colonies from outer edge of first segment toward center until 20 colonies have been counted. Complete by counting remaining colonies in segment where 20th colony occurs. In this counting procedure, numbers such as 3b, 4c (see Figure 9-1) refer to area segments from outer edge of wedge to designated arc line. Any count irregularities in sample composition are controlled by counting the same segments in the opposite wedge and recording results. Example of spirally inoculated plate (see Figure 9-1) demonstrates method for determining microbial count. Two segments of each wedge were counted on opposite sides of plate with 31 and 30 colonies, respectively. The sample volume contained in the darkened segments is 0.0015 ml. To estimate number of microorganisms, divide count by volume contained in all segments counted. See example under Figure 9-1.

      If 20 CFU are not within the 4 segments of the wedge, count CFU on entire plate. If the number of colonies exceeds 75 in second, third, or fourth segment, which also contains the 20th colony, the estimated number of microorganisms will generally be low because of coincidence error associated with crowding of colonies. In this case, count each circumferentially adjacent segment in all 8 wedges, counting at least 50 colonies, e.g., if the first 2 segments of a wedge contain 19 colonies and the third segment contains the 20th and 76th (or more), count colonies in all circumferentially adjacent first and second segments in all 8 wedges. Calculate contained volume in counted segments of wedges and divide into number of colonies.

      When fewer than 20 colonies are counted on the total plate, report results as "less than 500 estimated SPLC per ml." If colony count exceeds 75 in first segment of wedge, report results as "greater than 500,000 estimated SPLC per ml." Do not count spiral plates with irregular distribution of colonies caused by dispensing errors. Report results of such plates as laboratory accident (LA). If spreader covers entire plate, discard plate. If spreader covers half of plate area, count only those colonies that are well distributed in spreader-free areas.

      Compute SPLC unless restricted by detection of inhibitory substances in sample, excessive spreader growth, or laboratory accidents. Round off counts as described in 4, above. Report counts as SPLC or estimated SPLC per ml.

    Contents

    Other APC methods

Contents

Commercial Test Products

Table 9-4. Commercial APC test products.

  Test 

Analytical Technique

Approx. Total Test Time1

Supplier

3M PetrifilmTM
Aerobic Count Plate2
Dry rehydratable film method 48 h 3M Microbiology Products 
3M Center, Building 275-5W-05 
St. Paul, MN  55144-1000 
Phone: 800/228-3957 
E-mail: microbiology@mmm.com
bio-spoTM Solar Cult Flex Paddles Gellified agar slide 24 h Applied Research Institute 
Contact: Trevor Hopkins
3N Simm Ln. 
Newton, CT 06470 
Phone: 888/324-7900
E-mail: sales@arillc.com
Web: www.arillc.com
BioProbe ATP Detection System  ATP bioluminescence 1 min Contamination Sciences LLC  
Contact: Robert Steinhauser 
4230 East Towne Blvd., Suite 191 
Madison, WI  53704 
Phone: 608/825-6125 
E-mail: info@contam-sci.com
Web: www.contam-sci.com
HYGI-PRO Chemical Analysis visual color change 10 min Contamination Sciences LLC  
Contact: Robert Steinhauser 
4230 East Towne Blvd., Suite 191 
Madison, WI  53704 
Phone: 608/825-6125 
E-mail: info@contam-sci.com
Web: www.contam-sci.com
HygicultTPC (Total Plate Count) Gellified agar slide 24 h Neogen Corporation 
620 Lesher Pl. 
Lansing, MI 48912 
Phone: 517/372-9200 
E-mail: NeogenCorp@aol.com 
Web: www.neogen.com
ISO-GRID Method for Aerobic Plate Count using TSAF Agar2 Membrane filtration using culture medium with non-inhibitory dye to enhance colony appearance 48 h QA Life Sciences, Inc. 
6645 Nancy Ridge Dr. 
San Diego, CA  92121 
Phone: 800/788-4446; 858/622-0560 
E-mail: bugsy@qalife.com
SimPlateTM for Total Plate Count2 84/198 MPN well plates/UV fluorescence 
 
24 h IDEXX Laboratories, Inc. 
Contact: Greg Getchell 
One Idexx Dr. 
Westbrook, ME  04092 
Phone: 800-321-0207; 207/856-0580
E-mail: greg-getchell@idexx.com 
Web: www.idexx.com/fed/home/start.asp
Total Microbe Hunter Selective media with color indicator that changes based on approximate TPC 30 min for 108 organisms 
10 h for 101 organisms
Contamination Sciences LLC 
Contact: Robert Steinhauser 
4230 East Towne Blvd., Suite 191 
Madison, WI  53704 
Phone: 608/825-6125 
E-mail: bsteinha@contam-sci.com
1Includes enrichment
2AOAC Approved

Table 9-5. Sanitation test kits.

Test 

Used to Identify

Analytical

Technique

Approx. Total Test Time

Supplier

AssureSwab visual swab test Protein residue Visual chemical assay 10 min Biocontrol Systems, Inc.
Contact: Robin Forgey
12822 SE 32nd St.
Bellevue, WA 98005
Phone: 425/603-1123
E-mail: info@rapidmethods.com
Web: rapidmethods.com
BIOPROBE  Bacteria/food residue ATP Bioluminescence 1 min Contamination Sciences LLC
Contact: Robert Steinhauser
4230 East Towne Blvd., Ste. 191
Madison, WI 53704
Phone: 608/825-6125
E-mail: bsteinha@contam-sci.com
Hy-LiteATP Hygiene Monitoring System Bacteria/food residue ATP Bioluminescence 2 min Neogen Corporation
620 Lesher Pl.
Lansing, MI 48912
Phone: 517/372-9200
E-mail: NeogenCorp@aol.com
Web: www.neogen.com
LighteningTM Bacteria/food residue ATP Bioluminescence 11 s IDEXX Laboratories, Inc.
Contact: Greg Getchell
One Idexx Dr.
Westbrook, ME 04092
Phone: 207/856-0580
E-mail: greg-getchell@idexx.com
Web: www.idexx.com/fed/home/start.asp
PocketSwab with Charm LUM-T Meter Bacteria/food residue ATP Bioluminescence 30 s Charm Sciences, Inc.
36 Franklin St.
Malden, MA 02148-4120
Phone: 781/322-1523
E-mail: charm1@charm.com
Web: www.charm.com
systemSure Bacteria/food residue ATP Bioluminescence 2 min Becton Dickinson Microbiology Systems
7 Loveton Circle
Sparks, MD 21152
Phone: 410/316-4472

Contents

References

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Andrews, W.H., Hammack, T.S., and Amaguana, R.M. 1998. Salmonella. Ch. 5. In Food and Drug Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). R.L Merker (Ed.). AOAC International, Gaithersburg, MD.

AOAC. 1990. Official Methods of Analysis of AOAC International, 15th ed. AOAC International, Gaithersburg, MD.

AOAC. 1995a. Aerobic plate count in foods: Dry rehydratable film. Sec. 17.02.07, Method 990.12. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), 10-11. AOAC International, Gaithersburg, MD.

AOAC. 1995b. Aerobic plate count in foods: Hydrophobic grid filter method. Sec. 17.2.05, Method 986.32. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), 8-10. AOAC International, Gaithersburg, MD.

AOAC. 1995c. Aerobic plate count: Pectin gel method. Sec. 17.02.06, Method 988.18. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), 10. AOAC International, Gaithersburg, MD.

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Donnelly, C.B., J.E. Gilchrist, J.T. Peeler, and J.E. Campbell. 1976. Spiral plate count method for the examination of raw and pasteurized milk. Appl. Environ. Microbiol. 32:21-27.

FDA. 1978. Product code: Human foods. In EDRO Data Codes Manual. Food and Drug Administration, Rockville, MD.

FDA. 1989. Laboratory Procedures Manual. Food and Drug Administration, Rockville, MD.

FDA. 1993. Investigations Operations Manual. Food and Drug Administration, Rockville, MD.

FDA. 1996a. Raw breaded shrimp – Microbiological criteria for evaluating compliance with current good manufacturing practice regulations (CPG 7108.25). Section 540.420. Compliance Policy Guides, (August 1996 ed.), updated through October 31, 1996. Department of Health and Human Services, Public Health Service, Food and Drug Administration.

FDA. 1998. FDA & EPA guidance levels. Appendix 5. In Fish and Fishery Products Hazards and Controls Guide, 2nd ed., p. 245-248. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC.

Gilchrist, J.E., Donnelly, C.B., Peeler, J.T., and Campbell, J.E. 1977. Collaborative study comparing the spiral plate and aerobic plate count methods. J. Assoc. Off. Anal. Chem. 60:807-812.

ICMSF. 1986. Microorganisms in Foods. 2. Sampling For Microbiological Analysis: Principles and Specific Applications, 2nd ed. University of Toronto Press, Buffalo, NY.

IDF. 1987. Milk and milk products: Enumeration of microorganisms—Colony count at 3C. Provisional IDF Standard 100A. International Dairy Federation, Brussels, Belgium.

Jarvis, B., V.H. Lach, and J.M. Wood. 1977. Evaluation of the spiral plate maker for the enumeration of microorganisms in foods. J. Appl. Bacteriol. 43:149-157.

Maturin, L.J. and Peeler, J.T. 1998. Aerobic plate count. Ch. 3. In Food and Drug Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). R.L. Merker (Ed.). AOAC International, Gaithersburg, MD.

NAS. 1969. An evaluation of the Salmonella problem. National Academy of Sciences, Washington, DC.

Nickelson, R. and Finne, G. 1992. Fish, crustaceans, and precooked seafoods. Ch. 47. In Compendium of Methods for the Microbiological Examination of Foods, 3rd ed., C. Vanderzant and D. F. Splittstoesser (Ed.), p. 875-895. American Public Health Association, Washington, DC.

Niemela, S. 1983. Statistical evaluation of results from the quantitative microbiological examinations. Report No. 1, 2nd ed. Nordic Committee on Food Analysis, Uppsala, Sweden.

Tomasiewicz, M.R., Hotchkiss, D.K., Reinbold, G.W., Read, R.B. Jr., and Hartman, P.A. 1980. The most suitable number of colonies on plates for counting. J. Food Protect. 43:282-286.

Zipkes, M.R., J.E. Gilchrist, and J.T. Peeler. 1981. Comparison of yeast and mold counts by spiral, pour, and streak plate methods. J. Assoc. Off. Anal. Chem. 64:1465-1469.