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Introduction
Pathogenic microorganisms cause diseases by infecting the body through various ways, such as by entering through cuts on the skin, through the pulmonary route by inhalation, and through the digestive tract by ingestion. Because we consume food on a regular basis, it is important to determine that whatever we eat is free from pathogenic microbes. This is a particularly serious issue especially in the food industry, whose components must strictly abide by regulations set up by regulatory bodies, especially for microbiological counts (i.e. quantitation) in food products (Jay, 2000; Alcamo and Warner, 2010).
Population numbers of microorganisms are usually recorded as the number of cells in a milliliter (mL) of liquid or in a gram (g) of solid material. Bacterial populations are usually very large, so they are counted through direct (microscopic cell count) or indirect means (plate count) using very small samples which can then be used to calculate the total microbial population. However, it is quite impractical to directly measure out a very small amount of sample, so the procedure is done indirectly by diluting the original inoculum several times in a process called serial dilution (Tortora et al., 2010; Madigan et al., 2012).
A total viable count (a.k.a plate count) is the most common method of quantifying bacteria in a sample. It is done either by the pour-plate method or the spread-plate method and is based on the assumption that one cell can grow to a single colony on a plate. However this is not necessarily the case, because bacteria frequently grow linked in chains or clumps, therefore a colony often results not from a single bacterium but from short segments of a chain or from a bacterial clump. Taking this into consideration, plate counts are often reported as colony-forming units (CFU), as opposed to number of cells (Tortora et al., 2010; Madigan et al., 2012).
In this experiment, the total viable count of two food samples, bean sprouts and cheese, was determined using serial dilution and spread plate technique. The study aimed to familiarize the students with the basic bacterial quantitation techniques, as well as to impart the necessity of microbiological safety of food.
Methods
Serial Dilution
Ten grams of the food sample was placed in a Stomacher(r) (Seward Ltd.) bag with 90 mL of sterile 0.1% peptone water as diluent. The sample was homogenized for two minutes and transferred to sterile universal containers. Serial dilution was performed on the suspension (which served as the 10-1 dilution of the food sample) to obtain dilutions up to 10-3. This was done by diluting 1 mL of the sample in 9 mL diluent twice in a sequential manner.
Total Viable Count (TVC)
One hundred microliters (100 µL=0.1 mL) of the 10-3 dilution was aseptically pipetted unto the surface of a nutrient agar plate. The inoculum was spread out evenly using a sterile plastic spreader. The 10-2 dilution was also spread plated in a similar manner. Preparation of spread plates was performed for both the bean sprouts and cheese samples. The plates were incubated at 30°C for 48 hours. Bacterial colonies on plates were counted and the dilution with 30 to 300 colonies was selected for calculation of the CFU/g of the food sample. To calculate the CFU/g, the following formula was used:
CFU/g= Nvx df [Eq. 1]
where N is the number of colonies/CFUs, v is the volume plated, and df is the dilution factor (the reciprocal of the dilution), and 1 g of food is assumed to be equivalent to 1 mL in volume.
Results
Discussion
Bacterial quantification of food samples was performed using total viable count. This method was used because it is a conventional technique for studying microbial counts in food products. Its advantages include its simplicity, adaptability, and being a straightforward technique. Direct quantitation such as direct microscopic count, which involves counting the cells of a sample smear which have been stained using an appropriate dye as viewed under a microscope, is a tedious task and does not distinguish between viable and nonviable cells. Moreover, it is prone to inaccurate results, as food particles might sometimes be indistinguishable from bacteria and some cells that did not take up the dye well might not be included. Other techniques for quantifying bacteria also exist but total viable count remains the standard and most versatile technique, as it can be applied to almost all types of bacteria and can be jointly used with specialized types of media (Jay 2000; Tortora et al. 2010).
In general, food safety guidelines are set in order to avoid outbreaks of deadly diseases and infections as well to reduce the risk of poisoning by toxic microbial byproducts. Bacterial quantitation is a common technique for surveillance of proper practice and eliminating the spread of pathogenic microorganisms. According to the food safety guidelines by the European Commission (EC), Salmonella must not be detected at all in 25 g of ready-to-eat sprouted seeds as even small amounts of pathogens indicate a public health risk, while cheeses have varying limits depending on their type and production. Escherichia coli and coagulase-positive staphylococci values in cheeses made from pasteurized milk or whey or milk with lower heat treatment, and ripened cheeses made from pasteurized (or stronger heat treatment) milk or whey may be within 100 to 1000 CFU/g. Furthermore, bacterial count in cheeses made from raw (unpasteurized) milk has higher limits of 104 to 105 CFU/g (EC, 2005; European Food Safety Authority [EFSA], 2011).
The experimental results showed significantly higher amounts of bacterial counts (bean sprouts: 5.12x106; cheese: 4.7x105) than the recommended values. However, because the guidelines are quite specific for bacteria with potential health risks and our bacterial isolates were not characterized, it is hard to compare the values. There is a published value in Jay (2000) which shows that as much as 1.8x107 CFU/g bacteria was observed in aerobic plate counts of fresh bean sprouts. Also, the colony count of the bean sprout sample was over 300 but was used anyway because it is the lower value of the two dilutions. Nonetheless, the experimental viable count for bean sprouts is still within the observed value as that published by Jay (2000). As for cheeses, certain types are actually ripened with microorganisms such as bacteria and fungi (e.g. some Penicillium spp.). Raw milk prior to pasteurization also has high counts of many types of bacteria that are present in the milk’s udder, the milking device, and storage containers.
The high bacterial count of the food samples obtained in this experiment by itself is not indicative of potential safety hazards. The isolated bacteria are most probably non-pathogenic that could have come from the packaging or exposure to air if produce was not packaged (such as if the bean sprouts were homegrown). This experiment was also limited by the lack of values for comparison, as the recommended standards are specifically tailored for pathogenic and potentially harmful (e.g. opportunistic pathogens) organisms. Moreover, published values selective for certain types of microbes and even total microbial count are more empirical in nature.
Total viable count would be more useful if selected for specific microorganisms such as coliforms and staphylococci in order to draw out valid conclusions using recommended guidelines. Also, the experiment would be more successful with the inclusion of more samples in order to have a baseline and to effectively compare values. For example would be the comparison of total microbial counts of food samples of varying types or originating from varying sources (e.g. soft, semi-hard, and hard cheeses; bean sprouts samples that are commercially produced by different manufacturers, home- or laboratory-grown using different types of growth media such as soil or simply water). Overall this experiment was successful in acquainting the students with food safety and microbial quantitation techniques but the experimental design was too simple to yield results that may be subjected to deeper analysis.
References
Alcamo, I. E. and Warner, J. M., 2010. Schaum’s Outlines Microbiology. 2nd ed. New York: The McGraw-Hill Companies, Inc.
European Food Safety Authority, 2011. ‘Scientific Opinion on the risk posed by Shiga toxin-producing Escherichia coli (STEC) and other pathogenic bacteria in seeds and sprouted seeds.’ EFSA Journal, 9(11), pp. 2424-2324.
European Commission 2005. Commission Regulation on microbiological criteria for foodstuffs, EC 2073/2005.
Madigan, M. T., et al., 2012. Brock Biology of Microorganisms. 13th ed. San Francisco: Pearson Education, Inc.
Tortora, G.J., Funke, B.R. and Case, C.L., 2010. Microbiology: an Introduction. 10th ed. San Francisco: Pearson Education, Inc.
