Miscellaneous Food Products 213 49 Powers, E.M., R Lawyer, and Y Masuoka 1975 Microbiology of processed spices J Milk Food Technol 38:683–687 50 Rehberger, T.G., L.A Wilson, and B.A Glatz 1984 Microbiological quality of commercial tofu J Food Protect 47:177– 181 51 Sagoo, S.K., C.L Little, and R.T Mitchell 2003 Microbiological quality of open ready-to-eat salad vegetables: Effectiveness of food hygiene training of management.J Food Protect 66:1581–1586 52 Schmidl, M.K., and T.P Labuza 1992 Medical foods Food Technol 46:87–96 53 Smittle, R.B 2000 Microbiological safety of mayonnaise, salad dressings, and sauces produced in the United States: A review J Food Protect 63:1144–1153 54 Snyder, H.E 1970 Microbial sources of protein Adv Food Res 18:85–140 55 Soriano, J.M., H Rico, J.C Molto, and J Ma˜nes 2001 Incidence of microbial flora in lettuce, meat and Spanish potato omelette from restaurants Food Microbiol 18:159–163 56 Surkiewicz, B.F 1966 Bacteriological survey of the frozen prepared foods industry Appl Microbiol 14:21–26 57 Surkiewicz, B.F., R.J Groomes, and A.P Padron 1967 Bacteriological survey of the frozen prepared foods industry III Potato products Appl Microbiol 15:1324–1331 58 Swartzentruber, A., A.H Schwab, B.A Wentz, A.P Duran, and R.B Read, Jr 1984 Microbiological quality of biscuit dough, snack cakes and soy protein meat extender J Food Protect 47:467–470 59 Szewzyk, U., R Szewzyk, W Manx, and K.-H Schleifer 2000 Microbiological safety of drinking water Ann Rev Microbiol 54:81–127 60 Todd, E.C.D., G.A Jarvis, K.F Weiss, and S Charbonneau 1983 Microbiological quality of frozen cream-type pies sold in Canada J Food Protect 46:34–40 61 Tranter, H.S., and R.G Board 1984 The influence of incubation temperature and pH on the antimicrobial properties of hen egg albumen J Appl Bacteriol 56:53–61 62 Villari, P., M Crispino, P Montuori, and S Stanzione 2000 Prevalence and molecular characterization of Aeromonas spp in ready-to-eat foods in Italy J Food Protect 63:1734–1757 63 Warburton, D.W., J.W Austin, B.H Harrison, and G Sanders 1998 Survival and recovery of Escherichia coli 0157:H7 in inoculated bottled water.J Food Protect 61:948–952 64 Waslien, C.I 1976 Unusual sources of proteins for man CRC Crit Rev Food Sci Nutr 6:77–151 65 Watt, B.K., and A.L Merrill 1950 Composition of foods—Raw, processed, prepared Agricultural Handbook No Washington, DC: USDA 66 Wentz, B.A., A.P Duran, A Swartzentruber, A.B Schwab, and R.B Read, Jr 1984 Microbiological quality of frozen breaded onion rings and tuna pot pies J Food Protect 47:58–60 67 Xiong, R., G Xie, and A.S Edmondson 1999 The fate of Salmonella Enteritidis PT4 in home-made mayonnaise prepared with citric acid Lett Appl Microbiol 28:36–40 Chapter 10 Culture, Microscopic, and Sampling Methods The examination of foods for the presence, types, and numbers of microorganisms and/or their products is basic to food microbiology In spite of the importance of this, none of the methods in common use permits the determination of exact numbers of microorganisms in a food product Although some methods of analysis are better than others, every method has certain inherent limitations associated with its use The four basic methods employed for “total” numbers are as follows: Standard plate counts (SPC) or aerobic plate counts (APC) for viable cells or colony forming units (cfu) The most probable numbers (MPN) method as a statistical determination of viable cells Dye reduction techniques to estimate numbers of viable cells that possess reducing capacities Direct microscopic counts (DMC) for both viable and nonviable cells All of these are discussed in this chapter, along with their uses in determining microorganisms from various sources Detailed procedures for their use can be obtained from references in Table 10–1 In addition, variations of these basic methods for examining the microbiology of surfaces are presented along with a summary of methods and attempts to improve their overall efficiency CONVENTIONAL STANDARD PLATE COUNT By the conventional SPC method, portions of food samples are blended or homogenized, serially diluted in an appropriate diluent, plated in or onto a suitable agar medium, and incubated at an appropriate temperature for a given time, after which all visible colonies are counted by use of a Quebec or electronic counter The SPC is by far the most widely used method for determining the numbers of viable cells or colony-forming units (cfu) in a food product When total viable counts are reported for a product, the counts/numbers should be viewed as a function of at least some of the following factors: Sampling methods employed Distribution of the organisms in the food sample 217 218 Modern Food Microbiology Table 10–1 Some Standard References for Methods of Microbiological Analysis of Foods Reference 72 Direct microscopic counts Standard plate counts Most probable numbers Dye reductions Coliforms Fungi Fluorescent antibodies Sampling plans Parasites 10 11 12 79 X X X X X 73 31 80 36 89 X X X X X X X X X X X X X X X X X X X X X X X X X X X X Nature of the food biota Nature of the food material The preexamination history of the food product Nutritional adequacy of the plating medium employed Incubation temperature and time used pH, water activity (aw ), and oxidation–reduction potential (Eh) of the plating medium Type of diluent used Relative number of organisms in food sample Existence of other competing or antagonistic organisms In addition to the limitations noted, plating procedures for selected groups are further limited by the degree of inhibition and effectiveness of the selective and/or differential agents employed Although the SPC is often determined by pour plating, comparable results can be obtained by surface plating By the latter method, prepoured and hardened agar plates with dry surfaces are employed The diluted specimens are planted onto the surface of replicate plates, and, with the aid of bent glass rods (“hockey sticks”), the 0.1-mm inoculum per plate is carefully and evenly distributed over the entire surface Surface plating offers advantages in determining the numbers of heat-sensitive psychrotrophs in a food product because the organisms not come in contact with melted agar It is the method of choice when the colonial features of a colony are important to its presumptive identification and for most selective media Strict aerobes are obviously favored by surface plating, but microaerophilic organisms tend to grow slower Among the disadvantages of surface plating are the problem of spreaders (especially when the agar surface is not adequately dry prior to plating) and the crowding of colonies, which makes enumeration more difficult See Spiral Plater section below Homogenization of Food Samples Prior to the mid- to late 1970s, microorganisms were extracted from food specimens for plating almost universally by use of mechanical blenders (Waring type) Around 1971, the Colwell Stomacher was developed in England by Sharpe and Jackson114 and this device is now the method of choice in many laboratories for homogenizing foods for counts The Stomacher, a relatively simple device, homogenizes specimens in a special plastic bag by the vigorous pounding of two paddles The pounding effects the shearing of food specimens, and microorganisms are released into the diluent Several Culture, Microscopic, and Sampling Methods 219 models of the instrument are available, but model 400 is most widely used in food microbiology laboratories It can handle samples (diluent and specimen) of 40–400 ml The Stomacher has been compared to a high-speed blender for food analysis by a large number of investigators Plate counts from Stomacher-treated samples are similar to those treated by blender The instrument is generally preferred over blending for the following reasons: The need to clean and store blender containers is obviated Heat buildup does not occur during normal operational times (usually minutes) The homogenates can be stored in the Stomacher bags in a freezer for further use The noise level is not as unpleasant as that of mechanical blenders In a study by Sharpe and Harshman113 the Stomacher was shown to be less lethal than a blender to Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli One investigator reported that counts using a Stomacher were significantly higher than when a blender was used129 whereas other investigators obtained higher overall counts by blender than by Stomacher.5 The latter investigators showed that the Stomacher is food specific; it is better than high-speed blending for some types of foods but not for others In another study, SPC determinations made by Stomacher, blender, and shaking were not significantly different, although significantly higher counts of Gram-negative bacteria were obtained by Stomacher than by either of the other two methods.63 Another advantage of the Stomacher over blending is the homogenization of meats for dye reduction tests Holley et al.54 showed that the extraction of bacteria from meat by using a Stomacher does not cause extensive disruption of meat tissue, and, consequently, fewer reductive compounds were present to interfere with resazurin reduction; whereas with blending, the level of reductive compounds released made resazurin reduction results meaningless Another device, the Pulsifier r , is somewhat similar to the Stomacher It creates a high level of turbulence on food samples resulting in the release of microorganisms from the sample The Spiral Plater The spiral plater is a mechanical device that distributes the liquid inoculum on the surface of a rotating plate containing a suitable poured and hardened agar medium The dispensing arm moves from the near center of the plate toward the outside, depositing the sample in an Archimedes spiral The attached special syringe dispenses a continuously decreasing volume of sample so that a concentration range of up to 10,000:1 is effected on a single plate Following incubation at an appropriate temperature, colony development reveals a higher density of deposited cells near the center of the plate, with progressively fewer toward the edge The enumeration of colonies on plates prepared with a spiral plater is achieved by use of a special counting grid (Figure 10–1A) Depending on the relative density of colonies, colonies that appear in one or more specific areas of the superimposed grid are counted An agar plate prepared by a spiral plater is shown in Figure 10–1B, and the corresponding grid area counted is shown in Figure 10–1C In this example, a total sample volume of 0.0018 ml was deposited, and the two grid areas counted contained 44 and 63 colonies, respectively, resulting in a total count of 6.1 × 104 bacteria per milliliter The spiral plating device described here was devised by Gilchrist et al.44 although some of its principles were presented by earlier investigators, among whom were Reyniers105 and Trotman.128 The method has been studied by a rather large number of investigators and compared to other methods of enumerating viable organisms It was compared to the SPC method by using 201 samples of raw and pasteurized milk; overall good agreement was obtained.30 A collaborative study from six analysts 220 Modern Food Microbiology Figure 10–1 Special counting grid for spiral plater (A); growth of organisms on an inoculated spiral plate (B); and areas of plate enumerated (C) In this example, the inoculum volume was 0.0018 ml counts for the two areas shown were 44 and 63, and the averaged count was 6.1 × 104 bacteria per milliliter Courtesy of Spiral System Instruments, Bethesda, Maryland on milk samples showed that the spiral plater compared favorably with the SPC A standard deviation of 0.109 was obtained by using the spiral plater compared to 0.110 for the SPC.94 In another study, the spiral plater was compared with three other methods (pour, surface plating, and drop count), and no difference was found among the methods at the 5% level of significance.62 In yet another study, the spiral plate maker yielded counts as good as those by the droplet method.51 Spiral plating is an official Association of Official Analytical Chemists (AOAC) method Among the advantages of the spiral plater over standard plating are the following: less agar is used; fewer plates, dilution blanks, and pipettes are required; and three to four times more samples per hour can be examined.69 Also, 50–60 plates per hour can be prepared, and little training is required for its operation.62 Among the disadvantages is the problem that food particles may cause blocking in the dispensing stylus It is more suited for use with liquid foods such as milk A laser-beam counter has been developed for use with the plater Because of the expense of the device, it is not likely to be available in laboratories that not analyze large numbers of plates The method is further described in reference 36 MEMBRANE FILTERS Membranes with a pore size that will retain bacteria (generally 0.45 µm) but allow water or diluent to pass are used Following the collection of bacteria upon filtering a given volume, the membrane is placed on an agar plate or an absorbent pad saturated with the culture medium of choice and incubated ... conventional SPC method, portions of food samples are blended or homogenized, serially diluted in an appropriate diluent, plated in or onto a suitable agar medium, and incubated at an appropriate temperature... often determined by pour plating, comparable results can be obtained by surface plating By the latter method, prepoured and hardened agar plates with dry surfaces are employed The diluted specimens... 400 is most widely used in food microbiology laboratories It can handle samples (diluent and specimen) of 40–400 ml The Stomacher has been compared to a high-speed blender for food analysis by a