Total and Viable Counts of Microorganisms - Term Paper Example

Name : xxxxxx Tutor : xxxxxxx Title : TOTAL AND VIABLE COUNTS OF MICROORGANISMS Institution : xxxxxxx @2009 . 1. Total and viable counts of microorganisms. 2. Introduction to microorganisms. The term microorganisms also called microbes refer to small tiny single celled organisms usually not visible to the naked eye. Microorganisms require magnification for proper visualization and resolution of their structure. These involve use of microscopes or magnifying lens with appropriate level of magnification (Tortora, Funke and Case 1995). Microorganisms are found in all living things all over the world i.e. plants and animals. They exist in a variety of habitats. They can live in air, on land or in water both fresh and salty. The three main classes of microorganisms are bacteria, fungi and viruses. Different microorganisms have different effects. Some have negative effects and are said to be harmful while others have positive effects. Pathogenic and spoilage microbes cause diseases and food spoilage respectively thus referred to as harmful microorganisms. We also have others that are needed by living things to survive and they are termed as beneficial microbes (Tortora, Funke and Case 1995). Microbes are classified either by placing them in prokaryote or eukaryote group or by classifying them according to the temperature in their surrounding environment where they are classified as thermophilic microbes (thermophiles), mesophilic microbes (mesophiles) or psychrophilic microbes (psychrophiles). Thermophilic microbes are those microbes that grow well in high temperatures above the human body temperature. Mesophilic microbes are those that grow well in temperatures equal to that of human body while psychrophililic microbes are those that grow well in low temperatures below that of human body (Jaggi 1985). Like any other living organism, microbes reproduce for enhancement of their species. They reproduce by means of either sexual or asexual reproduction. Sexual reproduction occurs by mating of a male and a female while asexual reproduction is by cell division either by mitosis, meiosis or binary fission. 3. Counting of microorganisms. Micro-organisms can be counted. They need to be detected first before being counted. There are several techniques involved when counting microbes. When counting microbes, one can apply one or several of the following techniques. Direct counting techniques can be used. The oldest of these is microscopy which involves magnification of individual cells to become visible to the naked eye. Direct counting techniques do not depend much on the population of the cultured cells. A more recent of the direct counting techniques involves use of immunofluorescence and epifluorescence adaptations of cell labeling used in conjunction with cytometry. In both technologies, the trigger for a count is derived from single cells (Diaper et al. 1992). We also have culture techniques that rely on growth of microbes to a level where they are visible. This is done under specific conditions of temperature, oxygen, time and nutrients among others. The last technique employed is that of reporter assays which asses the population of microbes through their metabolic activities. In this technique, the cell population does not necessarily have to display a progressive growth. Examples of such techniques include conductance, colorimetry, adenosine triphosphate (ATP) and turbidometry (Bowden 1977). Counting of microorganisms can be done by different methods. In most cases the sample to be counted is first diluted to avoid overwhelming the whole counting procedure. However in some cases, the sample may be too dilute to give the required minimum count to be able to estimate the microbial population of that sample. In such cases, concentration of the sample is carried out. Counting of microbes can employ use of microbial slides. A drop of the diluted sample is put on the slide with a suitable agent for proper visualization of the sample. It is then covered wit a cover slip and put under a microscope and observed at a suitable magnification. The centre area can be dimensioned with etched grids. The number of microbes in the grids is multiplied by the dilution factor to get the number in the original sample (Black 1996). The Petri dish count is where the sample is diluted to a point where the colonies will be statistically significant to be counted but not so many to overgrow each other. This method takes time for the individual cells to grow into colonies. The colonies counted are multiplied by the dilution factor to get the number in the original sample. The results here are expressed in colony forming units per milliliter i.e. CFU/ML. The time taken for the cells to grow into individual colonies is called the incubation period. Counting of microbes is important as it enables us estimate the microbial population in a variety of products (Breeuwer et al. 1994). 4. Total count. Total count is also termed as standard plate count or colony count. It gives the total number of microbes both viable and non-viable. All cells are counted. These include bacteria, yeasts and moulds. It is usually done by pour plate method. Total count generally requires employment of a microscope. For instance, when determining total microbial count in water by pour plate, a known volume of water is mixed with molten yeast-malt extract agar and given time to solidify. This is done on several plates. A set of the plates are put under incubation at 37°C for about a day and the other set incubated for about 3 days at 20-22°C. You will find that most bacteria capable of growth in water do so well at 22°C than at higher temperatures. While the microbes that grow well at 37°C will not grow very well in water. This means that the two types of microorganisms need to be counted differently since they differ in their growth pattern. In this case, carrying out of total count on water is beneficial in several ways (Paulse, Jackson and Khan 2007). It assists in evaluation of the efficiency of certain water treatment processes like coagulation, flocculation and disinfection. It also gives an indication of the level of cleanliness of the water distribution system. It can also be used it determine the suitability of water supply to firms where food and drinks are prepared on large scale. Total count is achieved either by use of direct or indirect counts. One method of direct count is the use of a haemocytometer. A haemocytometer is a specialized microscope slide important in cell counting. The central part of this slide has etched grids with precisely spaced lines to enable accurate counting. In order to get an accurate count in this method, the cell number should range between 40 to 70 cells in a one mm square. If this requirement is not met, necessary adjustments by either dilution or concentration are done as necessary (Rapposch, Zangerl and Ginzinger 2000). In indirect counts, one method is by use of a colorimeter. As the microorganisms grow with time, they make the agar more and more turbid. This turbidity can be measured by use of a colorimeter where optical density is measured. The greater the optical density the greater the number of microbes (Breeuwer et al. 1995). There is also a measure of dry weight. This method involves centrifugation followed by weighing to get the dry weight. The limitation of this method is that cells are destroyed The other indirect count method involves the use of a coulter counter. A coulter counter is a probe which measures variation in conductivity of a solution as a bacteria passes through a narrow gap (Daley 1979). The advantage of direct and indirect counts is that the process can be automated but the disadvantage is that they can not differentiate dead cells from living ones. 5. Viable count. Viable cont involves counting of colonies produced by only viable cells under favorable growth conditions. This can be accomplished by techniques like pour plating, spread plating and most probable number with an assumption that each and every viable cell gives rise to a pure colony. (Black 1996). In pour plating, the liquid media and the diluted sample are poured together in Petri dishes while still in liquid form and left to solidify. After solidifying, the Petri dishes are incubated at appropriate temperature for the required period of time during which the growth is realized. The plates are then removed and distinct colonies counted and expressed in colony forming units per ml. In spread plating, the media is prepared separately and poured in Petri dishes while still in liquid form. It is then left to solidify. After solidifying, a small known volume from the diluted sample is put to each Petri dish and with the help of a sterilized spreading rod, the sample is evenly spread over the media. The plates are then put under incubation for an appropriate period of time at recommended temperature during which growth is realized (Black 1996, p. 140). The plates are then taken for counting of colonies using magnifying lens. Colonies grown in Petri dishes by various methods excluding streaking method may be used to estimate the count of viable microbes since plate counts assume that every colony is founded by a single cell and that the cell must have been alive to grow and form that colony. Problems with plate counts are several. They require a long time of incubation to be able to visualize the colonies. Clumping of cells can lead to undercounting of viable cells. Cases of too many or too few colonies on a plate to accurately estimate viable count are common. Serial dilution is often required to prevent cases of overcrowding of cells. Too few cells require concentration by either centrifugation or filtration. In a case where too few colonies are present then the original culture must be concentrated before determining the plate count. Filtration is a method used to concentrate microorganisms by sieving microbes out of the medium. Centrifugation is also a separation method based on the density. This helps separate the microbes from the medium since both have different densities (Pettipher, Mansell, McKinnon and Cousins 1980). . In serial dilution, increments are made in 1000, 100, or 10. The number of dilutions to be done depends on the concentration of the original solution and the required concentration. The volume of the solution needed is also vital. In situations where lower volumes of solutions are required then more numbers of dilutions will be needed. Serial dilution allows small aliquots to be diluted instead of unnecessary big volumes of materials. When carrying out serial dilution, a small amount of original sample is removed to another container and its volume adjusted to original volume using a suitable buffer or distilled water e.g. If 1ml of the original solution is taken and 10µL removed and put in 990µL of media or water then we will have made a 1:100 dilution. If the originally prepared solution contained 6 x 106 cells/mL, then we will now a new concentration of 6 x 104cells/mL. this is simply because we have divided the original concentration by 100 (Pettipher, Mansell, McKinnon and Cousins 1980). Another method of determining approximate viable count is by use of most probable number. This method involves diluting the growth cultures and then growing the dilution cultures in broth tubes. This method is useful where it is beneficial to use broth other than solid media especially for highly motile organisms which are poor in forming colonies. The most probable number method is mostly used when the organism in question is not able to grow on solid agar or in situations where the microorganisms are too few to give reliable measure of population size by the standard plate count method (Black 1996). The advantage of viable count is that the method can be made very sensitive and that one can be able to count subsets of population. However the disadvantage is that sometimes the colony forming units may underestimate the number of cells because of clumping or chains of cells. It is also time consuming as the counts require at least few hours or overnight for incubation (Bowden 1977). Viable count of microbes is mostly applicable in food processing industries mostly dairy and meat processing plants where microbiology is most applicable. It helps in estimation of shelf life of processed food products as well as evaluation of sanitary conditions under which the products were manufactured. The efficiency of certain treatment processes like pasteurization, sterilization and cold storage done during production is also evaluated by viable microbial count. 6. Conclusion. Total and viable counts of microorganisms are important practices in microbiology applicable in fields of medicine, food among others. The main difference between the two is that total count determines the count of all cells both dead and alive while viable count estimate the number of viable or live cells only capable of growing into distinct colonies. References Black, J G 1996, Microbiology. Principles and Applications, Third Edition, Prentice Hall Upper Saddle River, New Jersey. pp. 140-144. Breeuwer et al., 1994, ‘Rapid Assessment of Cell Viability by Flow Cytometry, Applied and Environmental Microbiology’, American association of microbiologist centre, US. Breeuwer et al. 1995, ‘Characterization of Uptake and Hydrolysis of Fluorescent Diacetate and Carboxyfluorescein Diacetate by Intracellular Estrases in Saccharomyces cerevisiae, Which Result in Accumulation of Fluorescent Product’, Applied and Environmental Microbiology, US. Bowden, W B 1977, ‘Comparison of two direct-count techniques for enumerating aquatic bacteria’, Applied Environmental, Microbiology, US. Daley, R J 1979, ‘Direct epifluorescence enumeration of native aquatic bacteria: uses, limitations and comparative accuracy’, American Society for Testing and Materials, Philadelphia. Diaper et al. 1992, Rapid Assessment of Bacterial Viability by Flow Cytometry, Applied Microbiology Biotechnology, US, PP 38, 268-272. Jaggi et al. 1985, ‘The Design and Development of an Optical Scanner for Cell Biology’, Proceedings of the Seventh Annual Conference of the IEEE/Engineering in Medicine and Biology Society, pp 2, 980-985. Paulse, A., Jackson, V A and Khan, W 2007, Comparison of enumeration techniques for the investigation of bacterial pollution in the Berg River, Western Cape, South Africa. Pettipher, G L., Mansell, R., McKinnon, C H. and Cousins, C M 1980, Rapid membrane filtration-epifluorescent microscopy technique for direct enumeration of bacteria in raw milk, Applied and Environmental Microbiology, US. Rapposch, S, Zangerl, P and Ginzinger, W 2000, Influence of fluorescence of bacteria stained with acridine orange on the enumeration of microorganisms in Raw Milk, dairy sciences publishers Cape town. Tortora, G J, Funke, B R, and Case, C L 1995, Intoduction to Microbiology. Fifth Edition, The Benjamin/Cummings Publishing, Company, Redwood City, CA, pp. 147-154, 158-166. Read More