The Determination Of The Presence Of Coliform Bacteria From Selected Sources By Means Of The Most Probable Number (Mpn) Method Abstract Coliform bacteria are good indicator organisms for the presence of pathogenic bacteria due to their realtionship with these pathogenic bacteria, their relative ease of determination by simple methods, and by their occurrence in large quantities in human feces. The MPN method used in this experiment is one of the prescribed techniques for the determination of these coliform bacteria from the Standard Methods for the Examination of Water and Wastewater as prescribed by the EPA. It consists of three stages, each of which necessitates a positive result for the previous stage. The first stage (presumptive test) determines the gas-producing coliform characteristic during lactose-fermentation. The second stage (confirmed test), determines the gram-reaction and also the lactose fermentation abilities of the organism, while the last stage (completed test) determines the endospore presence to determine if the organisms in the sample indeed are coliforms.
The number of coliforms or bacteria present is readily seen with the use of a special table and then the statistically estimated numbers are determined. The samples, however, did not produce positive results for the presence of coliforms. Enventhough there was a large MPN value for one of the samples, about 1100 MPN per 100 ml, the sample still tested negative in the last stage. It is therefore suffice to say that the samples did not present any health risks for humans. Future researchers should, however, device or perform other more specific procedures due to the fact that there might have been still coliforms present but these may have been negated by possible endospore-forming relatives. Introduction Human health has always been a hard condition to preserve and the detection and control of pathogens in the environment have been the very key to the success of the human race. Although microbial pathogens are relatively few in comparison to the total number of microorganisms, their detection have been made easy with the use of indicator organisms.
Indicator organisms give researchers the benefit of making good assumptions on the presence of pathogens before the pathogens multiply in distressing numbers. For a microbe to be accepted as an indicator organism, it must be present in human feces in large amounts so much so that the presence of these bacteria in a given sample would already point to human fecal contamination. It was reasoned that the largest amount of pathogens was present in human feces, and thus, the indication of the entry of large amounts of human waste, from healthy persons or not, already indicate a great risk (NCSU). Also, indicator organisms must be present wherever and whenever the pathogen organisms are present. More importantly, these indicator organisms must be easily detectable in samples and tests for the measurement of their numbers must be simple enough (Tortora et al.
1995). Coliform bacteria fit all the requirements and are even safe to handle in the laboratory. Coliform bacteria are gram-negative and non-spore/endospore forming bacteria, which include aerobes and facultative anaerobes, and when incubated at 35C with lactose in the media, will evolve gas (CO2) within 48 hrs, like Escherichia, Klebsiella, Citrobacter and Enterobacter (NCSU). They are also prevalent in the colon and intestinal tract (but not all groups are present) of warm-blooded mammals, including man (Anderson et al. 1998).
They are also related to pathogenic bacteria in that a large number of these coliform bacteria usually imply the presence of some pathogenic bacteria (Frank). These characteristics of coliform bacteria already suffice the conditions outlined for these organisms to be classified as indicator organisms. They occur in large amounts in human feces, in fact, humans excrete billions of these coliforms (called fecal coliforms). They are present whenever and wherever the pathogen organisms are present. More importantly, their presence is easily detected as their characteristics are easily tested with the use of simple procedures like gram-staining, endospore-staining and lactose fermentation tests. These principles and procedures now form the basis and the rationale for the methods by which this experiment was conducted.
Actually, the use of coliform bacteria as indicators of the presence of pathogenic bacteria is not new already. It as been established since 1880, and because of their reliability as indicator organisms, the procedures have not changed much and have only geared on specifically measuring the amount of fecal coliforms by use of special growth media and techniques. Today, the basis of the Standard Methods for the Examination of Water and Wastewater that are being used (also in this experiment) have been specified by the Environmental Protection Agency (EPA) (NCSU). There are several methods prescribed by the EPA and although the Most Probable Number (MPN) method is not the most frequently used, it still provides adequate proof for the presence of coliform bacteria. Better and more simple methods are being used, like the Colilert methods that is done by just adding special powdered media to a sample water and then observing color changes within 24 hrs after incubation at 35C (yellow = coliform, and if the yellow-colored solution fluoresces under UV light, the fecal coliforms are present) (Frank). The MPN method operates on a somewhat deductive manner, providing stages by which each step builds up or confirms for the manifestation of the coliform characteristics and thus, would readily separate coliform from non-coliform bacteria based on cytological (gram reaction and endospore formation) and lactose fermentation reactions.
Thus, one can expect sterile water to already be given a negative result on the first stage while sewage water would be expected to test positive for all stages. The number of the coliforms are determined by the use of a special table if coliforms are indeed present, based on the last stage. In this experiment, all mentioned coliform cytological characteristics as well as the ability to produce gas during lactose fermentation are done in stages by which, the colonies left at the end (if any) have coliform characteristics. Methodology The procedures were grouped into three stages, each of which necessitates a positive result from the previous stage, otherwise, the process is stopped at the particular stage and the sample gets a negative result on the presence of coliform bacteria. The samples tested in this experiment were from drinking water, tap water, AS pond, and from the UP lagoon but this paper concentrates more on the sample obtained from the AS pond.
Presumptive Test 10-ml portions of the water samples were inoculated into three large test tubes containing 10ml lactose broth and an inverted Durhan tube each, per sample (note that the Durham tubes must be rid of air inside before inoculation). Then, 1-ml portions were inoculated into three test tubes containing each an inverted Durham tube and 10ml lactose broth. Afterwards, 0.1-ml portions were inoculated into three test tubes containing 10ml lactose broth and an inverted Durham tube, each. These were inoculated for 24 hrs then the presence of air in each of the Durham tubes was observed. For the test tubes with gas inside the Durham tubes, these were called the positive presumptive test and were then subjected to the confirmed test.
The other test tubes were then incubated for another 24 hrs and after which, were also observed for the presence of gas inside the Durham tubes. If gas were present, these were then called the doubtful test and were subjected to the confirmed test. The other test tubes with no gas inside the Durham tubes were then set aside and labeled negative tests. Confirmed Test All test tubes that were either positive presumptive or doubtful tests from the first part were subjected to this test. The test tube/s with the largest dilution from these test tubes was then chosen for the next processes (priority = 0.1-ml sample test tubes*1-ml sample test tubes*10-ml sample test tubes).
Two each of pre-poured EMB and MacConkey agar plates were then inoculated, using streak plating technique for isolation, with samples from the test tube chosen. These plates were then incubated for 48 hrs at 37C. For the EMB plates, the presence of colonies with green-metallic shades or colonies that were dark purple were detected. For the MacConkey agar plates, the presence of red colonies was observed. These colonies were possible coliform bacteria and were subjected to the last stage, the completed test. Completed Test Portions were picked up and inoculated onto a lactose broth and a nutrient agar slant, individually, from the possible coliform bacterial colonies from the previous stage.
These were then incubated for 48 hrs at 37C. The lactose broth tubes were observed for gas production from lactose fermentation while the colonies inside the nutrient agar tubes were subjected to the gram-staining and endospore staining procedures (see Appendix). Results Fortunately or unfortunately, there were no coliform bacteria observed from the samples. The samples from tap and drinking water already did not give positive results in the confirmed test (no green-metallic or purple colored colonies in the EMB plates nor red colonies on the MacConkey agar plates). The samples from the …