Anavella Gaitan Herrera
Microbiological sampling of utensils, tableware, and kitchenware, in addition to equipment, permits objective evaluation of sanitation practices and procedures used for these items from food service operations (1).
Rinse Solution Method
1. Sterile Petri dishes.
2. Sterile pipets.
3. Stock phosphate buffer solution.
5. Plate count agar (PCA), Violet Red Bile agar (VRBA), MF Endo broth.
6. Buffered rinse solution: add 1.25 mL of stock phosphate buffer solution, 5 mL of 10% aqueous sodium thiosulfate (Na2S2O3-5H2O), 4 g azolectin (see Note 1), and 10 g Tween-20 (or Tween-80) to distilled water, and make up to 1 L. Dispense in screw-capped vials made up to contain, after sterilization, 20 mL, 100 mL, or other volumes needed. Weigh the powder and dissolve rapidly by heating over boiling water (see Note 2).
7. Sodium thiosulfate solution 10%: dissolve 100 g of Na2S2O3-5H2O in distilled water, and make up to 1 L, filter, and store in the refrigerator or dark place (or in an amber bottle).
1. Remove container cartons or containers from the conveyor line.
2. Add 20 mL of sterile buffered rinse solution to each container and aseptically reap the container (see Note 3).
3. Shake each container vigorously 10 times through a 20-cm arc.
4. Turn the container 90° and repeat the horizontal shaking treatment. Turn the containers 90° twice more and repeat the horizontal shaking.
5. Swirl the container vigorously 20 times in a small circle with the long axis in the vertical position; then invert and repeat.
6. Rinse solutions for container samples are analyzed by distributing a total of 10 mL of the rinse among three sterile Petri dishes. In addition, seed two other Petri dishes with 1-mL portions of the rinse.
7. Pour the plates with 15-18 mL of the desired medium (PCA, VRBA) using appropriate incubation conditions for each (see Note 4).
8. After incubation, count the colonies. If 20 mL of rinse solution was used, then the total number of colonies on the three plates that received 10 mL of rinse is multiplied by 2, or the total number of colonies on the two plates that received 1 mL each, multiplied by 10, yields the number of colonies per container. When the plates that received 1 mL each show more than 20 colonies, report the counts as more than 5000 per container.
For interpretations of results, take into consideration the numbers and types of microorganism (1,2).
1. Types of microorganism. The type is important in terms of potential to cause spoilage. The number should be very low; however, under certain conditions (such as aseptic packaging), the microbiological condition of the container is a critical control point.
2. Bacterial standards have been published for multiuse and single containers used for:
a. Packaging pasteurized milk or milk products. Four containers from any given day are sampled, and three of four samples must meet this standard.
b. Bottled water. State that at least once each 3 mo, bacteriological swab or rinse samples are to be taken from at least four containers and closures selected just prior to filling and sealing.
The standards require that such containers have a residual bacteria count of <1 colony/mL of capacity or not >1 colony/cm2 of product contact surface. No coliform organisms may be present.
3. Processing assemblies (tanks, pipelines, fillers, and so on). Rinse water for large volume rising of equipment may be sterilized or may be sanitized by chlorinating to a residual concentration of 25 mg/L, holding for 10 min, and then neutralizing by adding an excess of sterile 10% sodium thiosulfate solution. Tap water may be used after sterilizing by membrane filtration followed by the addition of sterile 10% w/v sodium thiosulfate to inactivate residual disinfectant (see Note 5 and 6).
A sufficient volume of treated rinse water is added to the system at the upstream end of the assembly and then pumped or allowed to flow by gravity through the assembly. A control sample (1 L) of the treated water rinse is taken before using the water for rising. Samples of water rinse are collected from the discharge end of the assembly from the first, middle, and final portions of the rinse water. Average the number of colonies obtained from rinse samples taken at the beginning, middle, and end of drainage and subtract the number of colonies obtained from the control samples. Calculate the ratio of sample volume to rinse volume and multiply by the corrected yield to obtain an indication of numbers of organisms present in the entire system (3).
1. Azolectin is hygroscopic and should be stored in a desiccator.
2. Nutrient broth may also be applied as a rinse since it effectively neutralizes residual active chlorine or quaternary ammonium compound. It may be used instead of buffered rinse solution.
3. For containers larger than 1 gallon, use 100 mL of rinse solution.
4. The presence of molds and yeast, proteolytic bacteria, and other microorganisms may be determined by the use of appropriate differential media and incubation temperatures and times.
5. When 100 mL or greater portions of rinse solutions are used, membrane filtration procedures for analysis should be followed (if low levels of contamination are expected)
6. Membrane filtration may be used for analysis of the 20-mL rinse samples.
1. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products, 14th ed. (Marth, E. H., ed.). Washington DC, American Public Health Association.
2. American Public Health Association (1984) Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. (Speck, M. L., ed.). Compiled by the APHA Technical Commitee on Microbiological Methods for Foods. Washington DC, American Public Health Association.
3. Code of Federal Regulations (1983) Title 21, Food and Drugs. Par 129, Processing and Bottling of Bottled Drinking Water. Washington, DC, US Govt. Printing Office.
Surface Contact Methods
Swab and replicate organism direct agar contact (RODAC) is the customary and appropriate method for microbiological examination of surfaces.
Swab procedures should be used for areas on equipment such as cracks, corners, or services and for sampling utensils, tableware, kiltchenware, and so on. RODAC should only be utilized on flat, impervious surfaces that are easy to clean and disinfect.
The levels of microorganisms should not exceed some number of colonies per sampling site; the type of microorganism may be more important than the number in terms of potential spoilage of the finished product. For example, the presence of very low numbers (1 or <10 CFU) of Pediococcus spp. or Xantomonas spp. in a brewery may be highly significant with respect to potential spoilage of the finished product.
The United States Public Health Service recommends standards for utensils and equipment of not more than 100 colonies per utensil or surface area of equipment swabbed.
This method is used for sampling by the cellulose (1) sponge swab technique. Large areas, such as cleaned and sanitized floors and other areas (where high residual micro-bial levels are expected), may be sampled with a cellulose sponge, a method useful for detecting Salmonella and other pathogens on food plant equipment and in the environment (see Note 1). This technique can be used to evaluate the efficacy of cleaning and sanitizing procedures for environment when pathogens microorganisms such as Salmonella exist (2-4). A 4-5 log cycle reduction in the residual microbial level should be obtained on surfaces after cleaning and sanitizing. The results should be negative after the correct application of sanitation procedures (see Note 2).
1. Cellulose sponges cut into 5 x 5-cm pieces placed in individual bags and autoclaved.
2. Sterile buffered solution for the rinsing agent (see Note 3).
3. Sterile plastic bags suitable to contain the sponge sample.
4. Sterile crucible tongs, and sterile gloves to hold the sponge aseptically during sampling.
1. Hold the sponge aseptically with sterile gloves and swab the surface to be sampled by vigorously rubbing the sponge over the specific area.
2. Area of several square meters may be effectively sampled with the swabbed method (see Note 4).
3. Aseptically place the sponge into a sterile plastic bag.
4. Transport under refrigeration.
5. Moisten the sponge with 10 mL of the chosen rinsing agent.
6. Quantitative analysis: take 50-100 mL of diluent, and add it to the bag containing the sponge. Mix vigorously for 1 min, plate, and incubate under specific conditions and appropriate selective differential media.
7. Calculation. Calculate the number of microorganisms per unit of surface (on the basis of the size of aliquot plated, the amount of diluent used, and the area swabbed). Example: 50 colonies are obtained from a 1-mL aliquot derived from the sponge in 100 mL of the rising agent.
Swabbed area: 1 m2 The result is 5000 CFU/m2.
1. If the surface to be sampled is flat, the rinse solution may be applied directly to the surface and then taken up into the sponge by the rubbing action.
2. This method would be used for development of a program to eradicate pathogenic microorganisms from the environment of the food factory.
3. Nutrient broth or 0.1% peptone water should be used as the rinsing agent.
4. When the surface to be sampled contains fatty materials, use as the rinsing agent 0.5% tergitol anionic or 0.5-1.0% Tween-80.
1. Silliker, J. H. and Gabis, D. S. (1975) A cellulose sponge sampling technic for surfaces. J. Milk Food Technol. 38, 504.
2. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products, 14th ed. (Marth, E. H., ed.). Washington DC, American Public Health Association.
3. American Public Health Association (1984) Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. (Speck, M. L., ed.). Compiled by the APHA Technical Commitee on Microbiological Methods for Foods. Washington DC, American Public Health Association.
4. Code of Federal Regulations (1983) Title 21, Food and Drugs. Par 129, Processing and Bottling of Bottled Drinking Water. Washington, DC, US Govt. Printing Office.
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