a Optical density measurement. b Total soluble solids.
From Wu, V.C.H., Jitareerat, P., and Fung, D.Y.C., J. Rapid Methods Automat. Microbiol., 11, 145, 2003. With permission from Food Nutrition Press, Trumbull, CT.
incubating the plates at 35°C, and counting the colonies after 48 hours. There is a great variety of factors to be considered. These include plating media, incubation time and temperature and incubation environment, and volumes to be plated. The operation of the conventional standard plate count method, although simple, is time-consuming both in terms of execution and data collection. Also, this method utilizes a large number of test tubes, pipettes, dilution bottles, dilution buffer, sterile plates, incubator space, and related disposable materials and requires resterilizing and clean up of reusable materials for further use.
Several methods have been developed, tested, and used effectively in the past 20 years as alternatives to the standard plate count method. Most of these methods were first designed to perform viable cell counts and relate the counts to standard plate counts. Later, coliform count, fecal coliform count, and yeast and mold counts were introduced. Further developments in these systems include differential counts, pathogen counts, and even pathogen detection after further manipulations. Many of these methods have been extensively tested in many laboratories throughout the world and went through AOAC (Association of Official Analytical Chemists) International collaborative study approvals. The aim of these methods is to provide reliable viable cell counts of food and water in more convenient, rapid, simple, and cost effective alternative formats, compared to the cumbersome standard plate count method.
The spiral plating method is an automated system to obtain viable cell count (Spiral Biotech, Bethesda, MD). By use of a stylus, this instrument can spread a liquid sample on the surface of a prepoured agar plate (selective or nonselective) in a spiral shape (the Archimedes spiral) with a concentration gradient starting from the center and decreasing as the spiral progresses outward on the rotating plate. The volume of the liquid deposited at any segment of the agar plate is known. After the liquid containing microorganisms is spread, the agar plate is incubated overnight at an appropriate temperature for the colonies to develop; the colonies appearing along the spiral pathway can be counted either manually or electronically. The time for plating a sample is only several seconds compared to minutes used in the conventional method. Also, using a laser counter an analyst can obtain an accurate count in a few second as compared with a few minutes in the tiring procedure of counting colonies by the naked eye. The system has been used extensively in the past 20 years with satisfactory microbiological results from many products including meat, poultry, seafood, vegetables, fruits, dairy products, and spices. Manninen et al.  evaluated the spiral plating system against the conventional pour plate method using both manual count and laser count and found that the counts were essentially the same for bacteria and yeast. Newer versions of the spiral plater were introduced as Autoplater (Spiral Biotech, Bethesda, MD) and Whitley Automatic Spiral Plater (Microbiology International, Rockville, MD). With these automatic instruments an analyst needs only to present the liquid sample, and the instrument completely and automatically processes the sample, including resterilizing the unit for the next sample.
The Isogrid system (Neogen, Lansing, MI) consists of a square filter with hydrophobic grids printed on the filter to form 1600 squares for each filter. A food sample is first weighed, homogenized, diluted, and enzymatically treated, then passed through the filter assisted by vacuum. Microbes are trapped in the squares on the filter. The filter is then placed on prepoured nonselective or selective agar and then incubated for a specific time and temperature. Since a growing microbial colony cannot migrate over the hydrophobic material, all colonies are confined to a square shape. The analyst can then count the squares as individual colonies. Since there is a chance that more than one bacterium is trapped in one square, the system has a most probable number (MPN) conversion table to provide statistically accurate viable cell counts. Automatic instruments are also available to count these square colonies in seconds. This method also has been used to test a great variety of foods in the past 20 years.
Petrifilm (3M Co., St. Paul, MN) is an ingenious system involves appropriate rehydratable nutrients embedded in a series of films in the unit. The unit is little larger than the size of a credit card. To obtain viable cell count, the protective top layer is lifted, and 1 ml of liquid sample is introduced to the center of the unit, and then the cover is replaced. A plastic template is placed on the cover to make a round mold. The rehydrated medium will support the growth of microorganisms after suitable incubation time and temperature. The colonies are counted directly in the unit. This system has a shelf life of over one year in cold storage. The attractiveness of this system is that it is simple to use, small in size, has a long shelf-life, does not require agar preparation, and provides easy-to-read results. Recently the company also introduced a Petrifilm counter so that an analyst only needs to place the Petrifilm with colonies into the unit, and the unit will automatically count and record the viable cell count in the computer. The manual form of the Petrifilm has been used for many food systems and is gaining international acceptance as an alternative to the standard plate count method.
Redigel system (3M Co., St. Paul, MN) consists of tubes of sterile nutrient with a pectin gel in the tube but no conventional agar. This liquid system is ready for use, and no heat is needed to "melt" the medium since there is no agar in the liquid. After an analyst mixes 1 ml of liquid sample with the liquid in the tube, the resultant contents are poured into a special Petri dish coated with calcium. The pectin and calcium will react and form a gel which will solidify in about 20 minutes. The plate is then incubated at the proper time and temperature, and the colonies can be counted the same way as the conventional standard plate count method.
The four methods described above have been in use for approximately 20 years. Chain and Fung  made a comprehensive evaluation of all four methods against the conventional standard plate count method on 7 different foods, 20 samples each, and found that the alternative systems and the conventional method were highly comparable at an agreement of r = 0.95. In the same study these researchers also found that the alternative systems cost less than the conventional standard plate count method.
A newer alternative method, the SimPlate system (BioControl, Bellevue, WA), has 84 wells imprinted in a round plastic plate. After the lid is removed, a diluted food sample (1 ml) is dispensed onto the center landing pad, and 10 ml of rehydrated nutrient liquid, provided by the manufacturer, is poured onto the landing pad. The mixture (food and nutrient liquid) is distributed evenly into the wells by swirling the SimPlate in a gentle, circular motion.
Excessive liquid is absorbed by a pad housed in the unit. After 24 hours of incubation at 35°C, the plate is placed under ultraviolet (UV) light. Positive fluorescent wells are counted and the number is converted in the MPN table to determine the number of bacteria present in the SimPlate. The method is simple to use with minimum amount of preparation. A 198-well unit is also available for samples with high counts. Using different media, the unit can also make counts of total coliforms and E. coli counts, as well as yeast and mold counts.
The above methods are designed to count aerobic microorganisms. To count anaerobic microorganisms, one has to introduce the sample into the melted agar, and after solidification the plates need to be incubated in an enclosed anaerobic jar. In the anaerobic jar, oxygen is removed by the hydrogen generated by the gas pack in the jar to create an anaerobic environment. After incubation, the colonies can be counted and reported as anaerobic count of the food. The method is simple but requires expensive anaerobic jars and disposable gas packs. It is of concern that the interior of the jar needs almost an hour to become anaerobic. Some strict anaerobic microorganisms may die during this one-hour period of reduction of oxygen. Fung and Lee  developed a simple anaerobic double-tube system which is easy to use and provides instant anaerobic condition for the cultivation of anaerobes from foods. In this system, the desired agar (~23 ml) is first autoclaved in a large test tube (OD 25 x 150 mm). When needed, the agar is melted and tempered at 48°C. A liquid food sample (1 ml) is added into the melted agar. A smaller sterile test tube (OD 16 x 150 mm) is inserted into the large tube with the food sample and the melted agar. By so doing, a thin film is formed between the two test tubes. The unit is tightly closed by a screw cap. The entire unit is placed into an incubator for the colonies to develop. No anaerobic jar is needed for this simple anaerobic system. After incubation, the colonies developing in the agar film can be counted and provide an anaerobic count of the food being tested. The Fung double-tube system has been used extensively for applied anaerobic microbiology in the author's laboratory for more than 20 years [8,9]. Recently, the author tested the double-tube method for Clostridium perfringens in recreational waters, and he was able to obtain anaerobic C. perfringens counts in about 6 to 8 hours from the time of sampling to the time of reading the results. By combining the Isogrid system with the double-tube method, the author can test volumes of waters ranging from 1 to 100 ml or more.
The above-mentioned methods are designed to grow colonies to visible sizes for enumeration and report the data as CFU per gram, milliliter, or square centimeter of the food being tested.
A few ''real time'' viable cell count methods have been developed and tested in recent years. These methods rely on using ''vital'' stains to stain ''live'' cells or ATP detection of live cells. All these methods need careful sample preparation, filtration, selection of dyes and reagents and instrumentation. Usually the entire systems are quite costly. However, they can provide results in one shift (8 hours) and can handle a large number of samples.
The direct epifluorescent filter techniques (DFET) method has been tested for many years and is in use in the U.K. for raw milk quality assurance programs. In this method, the microorganisms are first trapped on a filter and then the filter is stained with acridine orange dye. The slide is then observed by UV microscopy. "Live" cells usually fluoresce orange-red, orange-yellow, or orange-brown whereas "dead" cells fluoresce green. The slide can be read manually or by a semiautomated counting system marketed by Bio-Foss, which can provide a viable cell count in less than an hour.
The Chemunex Scan RDI system (Monmouth Junction, NJ) involves filtering cells on a membrane and staining cells with vital dyes (Fluorassure). After approximately 90 minutes of incubation (for bacteria), the membrane with stained cells is read in a scanning chamber that can scan and count fluorescing viable cells. This system has been used to test disinfecting solutions against such organisms as Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, and Staphylococcus aureus with satisfactory results.
The MicroStar system (Millipore Corporation, Billerica, MA) utilizes ATP bioluminescence technology by trapping bacteria in a specialized membrane (Milliflex). Individual live cells are trapped in the matrix of the filter and grow into microcolonies. The filter is then sprayed with permeabilizing reagent in a reaction chamber to release ATP. The bioluminescence reagent is sprayed onto the filter. Live cells will give off light due to the presence of ATP, the light is measured using a CCD camera, and thus the fluorescent particles (live cells) are counted.
These are new developments in staining technology, ATP technology, and instrumentation for viable cell counts. The application of these methods for the food industry is still in the evaluation stage. The future looks promising.
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