Pathogenic Microorganisms - Term Paper Example

Microbiology Introduction: The food ingredients that are used in the domestic kitchen are found to contain potential pathogenic micro organisms. They are found to cause large outbreaks of food borne diseases. The foods are prepared either by the untrained people or trained people without following the food safety methods. Of the foods, poultry was identified as the major source for pathogenic micro organisms mainly concentrated on the raw chicken that are mainly concentrated with Escherichia Coli, Campylobacter, Salmonella and Staphylococcus. The raw chicken wash waters are found to contain a large number of coliform and the hygiene of the washing up clothes is also generally poor. An evaluation of the total viable count and the presence of pathogens in the uncooked chicken were taken into account. The kitchen sponge and the hand wash water of the chicken were considered for the analysis. The analysis for the presence of the pathogenic micro organisms will help us to overcome the toxicity levels. (Mattick et al. 2003; Mattick et al. 2003a). Results: A lot of tests were done to determine the presence of the bacterial load in the micro organisms. Some of them include Gram staining, Catalase test, Oxidase test, coagulase test, API Strip test, the effect of disinfectant hypochloride, cleaning agents and morphological tests. The experiments and their results are discussed below. API strip test: The presence of the pathogens is found using the biochemical tests and the API strip test. The differential staining of the bacteria helps to identify the unknown bacteria present in the sample. Before all the tests the samples are serially diluted and the 4th (10-4) and 5th diluted (10-5) sample are used for the analysis. The API 20E strip consists of 20 individual and miniature tests that contain the reagents for the identification of the metabolic capabilities and specifically the Enterobacteraceae family. (Butler et al. 1975). Culture O N P G A D H L D C O D C C I T H 2 S U R E T D A I N D V P G E L G L U M A N I N O S O R R H A S A C M E L A M Y A R A identification Sponge isolate + – + + – – – – + – – + + – + + + + – + Escherichia coli From the results, it was identified as containing Escherichia coli, a known pathogen in the wash water. Disc Diffusion Method for Antimicrobial Susceptibility Testing: For the antimicrobial susceptibility testing, the Iso-Sensitest Agar (ISA) media was used. To test the presence o f enterobacteriaceae, the ISA media is enough according to the BSAC. The Molten agar was poured into the sterile Petri plates and they are dried to remove the excess moisture. To these plates, the antimicrobials of choice are used to such that it diffuses into the medium and they interact very well with the micro organism incorporated. The disc diffusion method of AST is the most commonest and effective method for the detection of the microorganisms in the sample. The inoculums was prepared and inoculated in the medium. The plates were incubated at 35-37 degree Celsius for at least 24 hours. The zone of inhibition was observed to the nearest millimeter. The test was used for the Sponge isolates and the Chicken isolate. It was found that the sponge isolate had the maximum inhibition diameter of 3.8 cm. ( 38mm). Osmotic pressure inhibition test: To check the ability of the micro organisms to grow in the saline medium, the effect of NaCl was tested. To the 10 ml of nutrient broth a stock solution, NaCl was added at a concentration of 0%, 2%,4%,6%, 8% and 10% to the test tubes. The isolates from the kitchen sponge and chicken waste water were added to the broths and then they were allowed to grow in these concentrations. After 18 hours of incubation, the growth of the micro organisms in the broth was determined. It was found that the rate of growth increased with the increase in the concentration of NaCl in the nutrient broth. At 0% concentration, the growth was at the bottom and the appearance was cloudy. At 2% concentration, there were some floating small colonies and the cloudiness was found to be less when compared to the 0% concentration. At 4% concentration, the presence of filamentous organisms at the bottom with mild cloudiness was observed in the test tube. At 6% NaCl concentration, the filamentous cells were found more at the medium and the cloudiness was found to be less when compared to other concentrations. At 8% and 10% NaCl concentration, there was no growth in the test tubes. Thus if the salt concentration is made high in the chicken during preservation, the growth of the microbes can be inhibited. S.No Salt Concentration (%) Observation Inference 1 0 Clusters of colonies No growth inhibition 2 2 Floating small colonies Less growth inhibition 3 4 Filamentous cells at the bottom, less in suspension Comparative growth inhibition 4 6 Filamentous cells more in suspension and less at the bottom More growth inhibition 5 8 No Growth Complete growth inhibition 6 10 No growth Complete growth inhibition Determining the efficiency of the Disinfectants: After confirming that the disinfectants can be used to reduce the bacterial load in the chicken, the concentration of the disinfectant required must be found out. This can be done by varying the concentration of the disinfectant in different parts of the same Petri dish by dividing them into different regions like a wheel. The total time at which the bacterial activity is completely reduced must be found out for the given disinfectants. It was found that when hypochlorite was used, the bacterial count got reduced in 60 seconds. This was confirmed by the zone of clearing that occurred after the addition of the disinfectant to the Petri dish containing that medium. Thus we can conclude that the microbial load control can be done by using the disinfectants of desired concentration. Sodium hypochlorite is the most common disinfectant used in the food industries. The disinfection level depends upon the concentration of the available chlorine and the pH of the solution. (Fukuzaki 2006). The Hypocholorous acid is a weak acid and it dissociates in the low pH into hypochlorite ion (HOCl) and proton (The hydrogen ion H+). This dissociation helps it to act as the germicide. The concentration of OCl helps to determine the cleaning efficiency of the hypochlorus acid. Catalase test: The catalase test is one of the three important tests that are done to determine whether the bacteria are able to decompose hydrogen peroxide in to water and oxygen. (Chelikani et al. 2004). The catalase test is used to differentiate the bacteria. If the bubbles are formed when a drop of hydrogen peroxide is added to the test tube, then the test is positive indicating the presence of staphylocci and if no bubbles are formed then it indicates the presence streptococci. Oxidase test: This test is used to determine twhether the bacteria are able to produce cytochrome C oxidases.  N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine , the redox indicator is added to the medium. If there occurs a color change to pink, followed by maroon and finally into black, then it indicates the test positive. If there is no color change or pink color formation then it indicates that the test is negative. This test is mainly used to identify the Enterobacteriacea, which are oxidase negative. (Farmer et al. 1981). Discussion: Name of the Test Sponge Isolate Chicken Isolate Gram Stain Negative rod Positive coccus Catalase Positive Positive API E.coli (99.3%) No CIP 35/36/39/38 37/40 Disinfectant ( hypochloride) 0 7/10 Cleaning Agent (Anionic surfactants ) 0 0 Coagulase No Negative/negative Identified Micro organisms Escherichia Coli Staphylococcus epidermis This study investigated the factors that were responsible for the reduction of the microbial load on the washing up process and the cleaning and storage of the chicken in the domestic kitchens. The disinfectant level, the effect of the anionic detergents and the concentration of the microbes, the most common microbes present in the sponge and the chicken in the domestic kitchen were identified and analyzed. The presence of the coli form bacteria in the chicken isolates were identified by the API strip test. It was confirmed from the API Strip test that the micro organism Escherichia Coli and Staphylococcus epidermis are present in the sample. Apart from the strip test, catalase test, coagulase test, oxidase test were also done. The catalase test was found to be positive for both the sponge isolate and the chicken isolate. The coagulase test was done for the isolates and was found to be negative. When the disinfectants were used, the growth of the bacteria was found to be controlled very much. As the concentration of the salt was increased, the increase in the osmotic pressure inside the cells resulted in the inhibition of the growth of the microbes. The cell wall, which is a semi permeable membrane, could not withstand the concentration of salt outside the cell in the environment so at greater concentrations it busted the cells. Thus it is concluded that the bacterial load is present in both the sponge and chicken isolates and the most predominant ones are Escherichia Coli and Staphylococcus epidermis. References: Butler, DA., Lobregat, CM and Gavan, TL 1975, “Reproducibility of the Analytab (API 20E) System”, Journal of Clinical Microbiology, vol. 2, no. 4, pp. 322-326. Chelikani, P., Fita, I. and Loewen, PC 2004, “Diversity of structures and properties among catalases,” Cellular and Molecular Life Sciences, vol. 61, no. 2, pp.192– 208. Farmer, JJ., Fanning, GR., Huntley-Carter, GP., Holmes, B., Hickman, FW., Richard, C and Brenner DJ 1981, “Kluyvera, a New (Redefined) Genus in the Family Enterobacteriaceae: Identification of Kluyvera ascorbata sp. nov. and Kluyvera cryocrescens sp. Nov in Clinical Specimens”, Journal Of Clinical Microbiology, vol. 13, no.5, pp.919-933. Fukuzaki, S 2006, “Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes”, Biocontrol Science, vol. 11, no.4, pp.147-57. Mattick, K., Durham, K., Domingue, G., Jørgensena, F., Senb, M., Schaffner, DW and Humphrey, T 2003, “The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges, kitchen surfaces and food”, International Journal of Food Microbiology, vol. 85, pp213– 226. Mattick, K., Durham, K., Hendrix, M., Slader, J., Griffith, C., Sen, M and Humphrey, H 2003a, “The microbiological quality of washing-up water and the environment in domestic and commercial kitchens”, Journal of Applied Microbiology, vol. 94, pp.842–848 Read More