Identification of Micro-Organisms - Term Paper Example

 «Identification of Micro-Organisms» Microorganisms, which are the most abundant inhabitants on earth, work and interact with humans in many ways resulting in either positive or negative consequences. Some of the commonly encountered interactions include diseases which are caused by microbes or the process of decomposition of leaves in the yard (Tortora, 2006, p2). The human body plays host to millions of bacteria some of which are harmless and there is a constant battle for supremacy between these microbes, also referred to as normal flora, when there is an imbalance created by external factors such as immune suppression or injury (Tortora, 2006, p2). During such conditions the human body becomes vulnerable to attack by pathogenic bacteria, which are only few in numbers (Tortora, 2006, p2). Given the fewer number of disease causing microbes it has become critically essential for health professionals to be able to isolate and identify disease causing bacteria through experimental techniques. Hence the aim of this experiment is to identify microorganisms found in samples obtained from various locations. Testing the antibiotic susceptibility and resistance of microbes are crucial for development of drugs to stop infection. The first method used is the simple gram staining technique followed by differential staining in which the stain reacts differently with the cell wall components of each bacterium and hence it can be used to differentiate bacteria based on their cell wall formation (Tortora, 2006, p69). The bacteria isolated from a single purified colony in the agar plates are used for subsequent differential testing which include the Catalase test, Microbact test and growth in differential media. The catalase test is used to determine whether the bacteria is aerobic or anaerobic which is determined by its reaction with hydrogen peroxide and the microbact test is used to identify gram negative bacteria in isolation tubes. Specific bacteria are identified by using selective and differential media. The growth of bacteria in a lab requires an abundant supply of nutrients for the adequate growth of micro-organisms. In order to achieve this selective and differential media were used which would encourage growth of specific bacteria while suppressing the growth of other micro-organisms (Tortora, 2006, p168). Additionally the efficacy of the treatment depends largely on the sensitivity of the causative organism to the antibiotic being used and that the antimicrobial drug exhibits selective toxicity, which in turn would be used to treat the infection or disease (Lab manual, 2007, p589). The samples tested include peanut butter, a dish cloth and water from a dam .The peanut butter, which was considered to have caused abdominal pain, nausea, vomiting and diarrhoea in the children, was taken from a child care centre. According to the Tasmanian Department of Health and Human Services Streptococcus pyogenes ,Salmonella enterica, Shigella spp, Escherichia coli and Streptococcus pyogenes were considered to be the likely organisms causing these symptoms. Contamination with these microbes occurs through personal contact or by touching contaminated surfaces with hands and food prior to ingestion. The second sample was a house hold dish cloth. As dish cloths are frequently exposed to different surfaces and media and are usually kept in moist and warm environments dish cloths could be effective vectors and reserves for microbial growth and transmission. Possible bacteria that could be present in dish cloths are the normal flora Staphylococcus aureus and Staphylococcus epidermidis both of which are Gram positive cocci. Another microbe that could be present is Escherichia coli, which is a Gram negative bacillus. Both the above groups of microbes are capable of contaminating food (Johnson, 1989) which could be transported to a dish cloth during handling of food. The third sample taken for the study was obtained from dam water. Staining Techniques As micro-organisms have a colourless natural state when viewed through a light microscope, the organisms must be stained in order to be visible. Staining imparts a color to the micro-organisms’ by the reaction of the dye with certain structures of the microorganism (Tortora, 2006, p69) 1.2.1 Simple staining A simple stain is an alcohol or aqueous based solution of a singular basic dye and its main purpose is to highlight an entire micro-organism in order for its cellular shapes and basic structure to be observed under a microscope (Tortora, 2006, p69). In this experiment methyl blue was used as the simple stain to view microbes obtained from cheek and tooth scraping. The illustrations on page 3 under the heading ‘Scientific Drawing 2’ shows the images that were observed. Figures 1.5 at x400 magnification and 1.6 at x1000 are scientific drawings of the eukaryotic epithelium cells obtained from a human cheek. When viewed at x1000 magnification most of the internal structures of the cells got from the cheek could be seen under a light microscope. Figures 2.3 and 2.4 are scientific drawings of prokaryotic bacterial cells obtained from human teeth scrapings. The bacterial cells, which are approx 1µm in diameter, are much smaller in comparison to the human cheek cell and hence the internal structures could not be observed even at x1000 magnification. 1.2.2 Differential staining Unlike simple stains differential stains react differently with each bacteria and therefore can be used to differentiate between each type of bacteria (Tortora, 2006, p69) The most commonly used differential stains are Gram stain and Acid fast stains and the staining procedures involves several steps. In this experiment Gram stain was used to distinguish between gram positive and gram negative bacteria, taken from a vaginal and a urine specimen. Figures 3.1and 3.2 are scientific drawings of gram positive and gram negative bacteria respectively as viewed under a light microscope at x1000 magnification. The vaginal swab obtained contained gram-positive microbes present with a cellular arrangement consisting primarily of staphylococcus. The urine sample was found to contain gram negative organisms with a cellular arrangement predominantly of diplobacillus with the presence of some cocci. 1.3 Identification of bacteria isolated from patient samples 1.3.1 Isolation techniques The purpose of this technique was to obtain a pure bacterial culture and observe the colony morphology (Tortora, 2006, p173). Often microbial colonies have distinctive characteristics which would help to distinguish one from the other (Tortora, 2006 p173). In this experiment agar pates were inoculated with the urine and vaginal samples. When the plates were observed after incubation similar colony morphology, height, and colony margins, were seen in both the samples thereby indicating that similar micro-organisms could be present in the two samples. The pigment colour of the colonies differed in the two specimens as the vaginal specimen culture had a creamy yellow colour and the urine culture had white creamy coloured colonies. The size of the culture also differed as the vaginal sample had small pinpoint colonies while the urine sample had medium sized colonies. 1.3.2 Microbial growth on differential and selective media The growth of bacteria in a laboratory is dependent on adequate supply of nutrients which are sufficient for the growth of micro-organisms. This is achieved by using selective and differential media which would encourage the growth of the required bacteria while suppressing the growth of other micro-organisms (Tortora, 2006, p168). In this experiment three types of media were used namely Blood Agar, MacConkey Agar and Mannitol Salt Agar. Both the vaginal and urine samples were inoculated onto each of these media. After incubation, it was observed that the vaginal and urine specimens where non-haemolytic on the Blood Agar media and the vaginal specimen showed no growth on the MacConkey Agar plate. The MacConkey agar plate inoculated with the urine sample had red pigment colonies which indicated that the bacteria present were lactose fermenting, and that the sample contained gram negative bacteria. A positive result for the Mannitol Salt media could be inferred by a colour change from pink to yellow in the agar surrounding the colonies, which would indicate that the bacteria are mannitol fermenting. Both the vaginal and urinary plates showed a negative result in the Mannitol salt agar plate, and so were neither mannitol fermenting nor salt tolerant. The finding for the vaginal sample did not match the expected results as the test was meant to select salt tolerant organisms such as Staphylococcus species which were earlier determined to be present in the vagina samples during the gram staining procedure. Hence the negative result for the vagina sample could possibly be due to contamination. 1.3.3 Catalase test This test is indicative of whether the organisms being tested are capable of hydrolysing hydrogen peroxide by producing the enzyme catalase. (Mc Fadden, 1980). This test is useful in differentiating staphylococci and micrococci, both of which are catalase-positive, from streptococci and enterococci, which are catalase-negative. Many organisms could be catalase-positive and if the unknown gives catalase-positive results it need not necessarily be a staphylococcus (Mc Fadden, 1980). In the experiment, three drops of a 3% hydrogen peroxide solution was applied to both the urine and vaginal specimens. A positive result is inferred by the formation of small bubbles. The test showed a positive result in the vaginal and urine specimen as bubbles were produced in both the tubes. The positive result for the catalase test in the vaginal sample confirmed the presence of staphylococci in sample, as identified by the gram staining method. 1.3.4 Oxidase test The oxidase test is used to differentiate between the families of Pseudomonadaceae (ox+) and Enterobacteriaceae (ox-).  This test indicates the presence of cytochrome oxidase in bacteria, a characteristic of aerobic bacteria. A bacterium which has cytocrome oxidase will test positive as it will turn the reagent to purple or blue and if it does not have the enzyme they would test negative as the reagent will remain colourless. In the experiment the vaginal and urine specimens did not possess the enzyme and hence could not oxidise the reagent thus leaving the test strip colourless and giving an ox- reading. This is indicative of the presence of aerobic microbes in both the specimens. 1.3.5 Biochemical analysis The Microbact Gram-negative system is designed to simulate the conventional biochemical substrates which are used to identify the different types of Enterobacteriaceae and common Gram-negative bacilli (Oxoid, 2007). The organism identification is based on pH change and utilization of the substrates. The results of the Microbact System are then compared against those of a known list of organisms. This test is only used for the identification of gram negative bacteria and the urine sample gave a positive result for the Microbact test which is detailed in Laboratory Report Form 2. After analysis was done the identification strip read a Code Number of 4740 which identified the following bacteria based on the probability scores: Escherichia Vulneris, with a probability of 74%, K.ozaenae with a 20% probability, Escherichia coli with a 2% probability and Escherichia coli inactive and S. kubidaea with a probability of 1%. Previous tests showed the presence of Escherichia coli bacteria in the urine sample, and according to the journal of clinical microbiology Escherichia Vulneris has similar characteristics as that of E.coli. Hence only one of the 12 microbact reactions needs to be wrong to obtain a false reading; however, this test has revealed that the microbe is an Enterobacteriaceae. 2. Microbial Control 2.1 Normal flora Those microorganisms which thrive in various regions of the human body but do not cause any disease under normal conditions are referred to as the normal microbial flora (Tortora, 2006, p 421). These organisms establish a symbiotic relationship, with the human body thus protecting it from the attack of other harmful microbes (Tortora, 2006, p421). In this experiment normal microbes of the skin such as from behind the ear, and from the mouth were collected. These microbes were then inoculated onto a Nutrient Agar plate, and incubated. The ‘Laboratory Report Form 4’ on page 8 of the laboratory manual show the results obtained for the sample taken from the skin behind the ear. The microbial flora that was predominantly present was gram positive bacteria with a majority in staphylococcal cellular arrangement. In contrast, the oral sample slide showed the presence of gram negative bacteria in a streptococcal cellular arrangement. The colony morphology was very similar, which indicates that the bacteria are from similar species. Cellular morphology and Gram staining was used to confirm the presence of distinct bacterial colonies from different regions of the body. 2.2 Chemical agent control According to a report conducted by the Ministry of Agriculture in the United States 36% of food borne disease outbreaks caused by an outside influence, were due to poor personal hygiene. Hand-washing is a very important process in preventing the spread of infection (Tortora, 2006, p430). The purpose of this experiment was to assess the efficacy of various hand washing practices for controlling microbial growth. The class results have been documented in the Laboratory Report Form 5 on page 8 of the lab manual for results. In this experiment, swabs taken from the inter-digital space of unwashed hands were transferred to a Nutrient Agar plate. The hands were then washed with soap, swabbed again in the same manner and transferred to another agar plate. The same procedure was repeated using a hand wash instead of soap and the swabs were used to inoculate in the agar plates. The results showed that washing hands with both soap and the hand wash significantly reduced the amount of microbes present compared to the un-washed hands. In fact, the results with hand wash were slightly better with lesser number of microbes present than those obtained with soap. 2.3 Antimicrobial agents Antimicrobial drugs are useful in the treatment of infectious diseases and effective treatment depends, to a large extent, on the sensitivity of the causative organism to the antibiotic that is being used and whether the antimicrobial drug exhibits selective toxicity (Lab manual, 2007, p583). In order to determine whether the bacteria in the vaginal and urine sample are susceptible or resistant to the specific antibiotics, four separate antibiotic discs were placed evenly over the lawns of the specimens. The results have been documented in ‘Laboratory Report Form 3’ on page 7 of the laboratory manual. The results indicate that bacteria in both the vaginal and urine specimens were susceptible to all the four antibiotics. This implies that any of the antibiotics used in the experiment can be used to treat diseases caused by the bacteria present in the vagina and urine sample. Those antibiotics which had a larger special zone size would be the more effective compared to the others. The Vaginal sample showed a special distance of 34mm for Tetracycline and the urinal swab had a special distance of 28mm for the antibiotic Amphicillin. 2.4 Chemical agents of control Minimum Inhibitory Concentration is a technique used to assess the concentration at which a chemical will have an effect on a microbe. For the procedure test tubes are filled with nutrient broth and inoculated with serial dilutions of Staphylococcus aureus. The results have been recorded on ‘Laboratory Report Form 6’ on page 9 of the laboratory manual. By viewing the tubes against a white background, the amount of turbidity present was evaluated. The turbidity of the solution is directly proportional to the microbial growth. The results shown for surface spray reveal that the microbial growth in the tubes increased as the concentration of surface spray decreased. However, there was no growth on the agar plates. The class results showed similar results when the bacterial growth increased as the solution used became more diluted, as in Bleach, Ethanol, and floor cleaner. Vinegar was found to be better compared to the other chemical agents with an efficacy of 90% at all microbial concentrations and was therefore considered to be a good bactericidal agent. The other chemical agents where only effective at half the dilution as when the concentration of these agents decreased the bacterial growth subsequently slowed down. 3.0 Identification of Microbes The aim of this experiment was to determine the bacteria present in both the vaginal and urine specimens. A series of experimentations was carried out to determine the cellular morphology, the colony morphology, and colony characteristics on selective and differential media, along with specific biochemical tests and the Microbact system. A Vaginal Specimen was taken from an 18 year old female who had symptoms of high fever, vomiting, diarrhoea, hypotension and signs of septic shock. The gram stain of the sample taken showed the presence of gram-positive bacteria which predominantly had a staphylococcal cellular arrangement. Results from the Blood Agar media indicated that the bacteria were non-haemolytic and those from the MacConkey Agar showed no growth, which highlighted the fact that the bacteria were neither rods nor gram negative. The Mannitol Salt Agar reaction gave a negative result while the Catalase test gave a positive reaction which was useful in differentiating the staphylococci. These results lead to the final conclusion that the bacteria present in the vaginal specimen were Staphylococcus aureus. The Urine Specimen was collected from a 26 year old male who had drunk local water while during a holiday in Bali. As a result of an infection he showed symptoms of diarrhoea, fever, nausea, vomiting and abdominal cramping. The gram stain reaction for the urine sample showed that the organism was gram-negative. The cellular arrangement was dominantly diplobacillus with the presence of some cocci. The selective and differential media used to identify the bacteria in the urine sample, showed the following results: non-haemolytic result from the Blood Agar and a positive result in the MacConkey Agar. The tests showed the presence of lactose fermenting bacteria which were identified as gram negative rod-shaped bacteria. The Mannitol Salt Agar provided inconclusive results. The Microbact system helped to ascertain the identity of the bacteria present in the urine as it showed a 74 % probability of it being Escherichia Vulneris. Escherichia Vulneris is a gram-negative, oxidase-negative, fermenting, motile rod-shaped bacterium with the characteristics of the family Enterobacteriaceae (Brenner, 1982). The Departments of Microbiology and Infectious Diseases have revealed that Escherichia Vulneris could cause symptoms of fever, nausea, vomiting and abdominal cramping. However, the organism is more of an opportunistic bacterium and infections can occur due to open wounds during surgery, from infected intravenous catheters, osteomyelitis from a foreign body, and urinary sepsis (Awsare&Lillo, 1991). Other tests which will positively identify Escherichia vulneris include: a positive test for methyl red, malonate, and lysine decarboxylase; a delayed positive test for arginine dihydrolase; acid production from D-mannitol, L-arabinose, raffinose, L-rhamnose, D-xylose, trehalose, cellobiose, and melibiose; a negative test for Voges-Proskauer, indole, urea, H2S, citrate, ornithine decarboxylase, phenylalanine deaminase, and DNase; and no acid production from dulcitol, adonitol, myo-inositol, and D-sorbitol. To ascertain all the above tests is out of the scope of this experiment (Awsare, 1991). There for it is unlikely that Escherichia vulneris is the cause of the infection and Escherichia coli is a more likely source of infection especially due to the fact that it is the most common cause of urinary tract infection. The Microbact system identified Escherichia coli at 2% probability and it would have also shown the same results as with gram-negative, oxidase-negative, fermenting, motile rod with the characteristics of the family Enterobacteriaceae, commonly inhabiting the gastrointestinal tract of humans (Brenner, 1982). References 1. Austin community college, Catalase test http://www2.austin.cc.tx.us/microbugz/html/catalase_test.html (Accessed 29 September 2007) 2. Awsare, S. V., and M. Lillo. 1991. A case report of Escherichia vulneris urosepis. Rev. Infect. Dis. 13:1247-1248. [PubMed]. 3. Don J. Brenner, Alma C. McWhorter, Jean K. Leete Knutson, and Arnold G. Steigerwalt, Escherichia vulneris: a New Species of Enterobacteriaceae Associated with Human Wounds. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=272265#reference-sec. (Accessed 2 October 2007) 4. Food Safety & Quality Unit BC Ministry of Agriculture, Food and Fisheries http://www.agf.gov.bc.ca/foodsafety/factsheets/washing_hands.pdf (Accessed 29 September 2007) 5. Oxoid Microbiology, http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=MB1130&c=UK&lang=EN (Accessed 29 September 2007) 6. Schaechter M, Engleberg N.C, Eisestine B.I, & Medoff G; Mechanisms of Microbial disease 3rd Ed,1998 Published Williams& Wilkins Maryland USA. 7. Tortora, G.T., Funke,B.R.,Case,C.L.(2006) Microbiology:An Introduction.9th Ed. Pearson Benjimin Cummings. 8. Wylie, C. (2006). Microbiology Laboritory Class Manual. Griffith University Gold Coast Antibiotic resistance The discovery of penicillin in 1928 by Alexander Flemming was seen as a crucial development of modern medicine, as it realized the potential of penicillin as an antibiotic which has since then been used to effectively reduce the numbers of deaths due to disease and infection (Marton, 1999). Antibiotics are substances which are derived from other microorganisms with an ability to inhibit the growth of bacteria and destroy a wide range of bacteria.  The mode of action varies between different microbial species, for example in some organisms it could act on the cell wall, cell membrane, interfere with enzyme system, and inhibit nucleic acid and protein synthesis thereby increasing the effectiveness of the antibiotic. An important quality for an antibiotic is its selective toxicity, which enables the antibiotic to target foreign micro-organisms without causing any harm to the host (Rang, Dale, Ritter, Moor, 2007, p620) In the past 80 years since the discovery of the first antibiotics, the fight against diseases has been quite successful. However, in the recent years findings have shown that some of the front line defenses against diseases which worked earlier are not working anymore. Bacteria have managed to develop ways to protect themselves from antibiotics as it is a natural process in the course of evolution. Therefore as long as antibiotics continue to be used microbial resistance will continue to develop. Antibiotic resistance can occur in many ways one of which is through spontaneous DNA mutation which occurs due to changes in the DNA of the micro-organism by normal processes. Such changes in bacterial genome can occur either through mutation and selection, which is also known as vertical evolution and the other is by the exchange of genes between strains and species, a process referred to as horizontal evolution. The five general mechanisms of antibiotic resistance include: decreased cell permeability, active efflux – greater exit, altered target by modification of the drug receptor site and synthesis of resistant metabolic pathway. Some bacteria may be naturally resistant to an antibiotic as they posse’s genes that confer antibiotic resistance and which can eventually degrade the drug. An example for such an organism is the streptomycetes bacteria such as Pseudomonas, which is a gram negative bacterium. They are not affected by certain antibiotics as their cell capsule is impermeable to the beta-lactam aminoglycocides, which will not have an effect on an anaerobic organism as the drug enters the bacteria by an oxygen dependant mechanism (Row-Magnus, 2002). The gram-negative bacterium has an outer membrane that establishes a permeability barrier against the antibiotic, or the organism could lack the transport system for the antibiotic, or lack the target on which the antibiotic needs to act on. Staphylococcus aureus which is a gram positive bacterium produces an extra cellular enzyme, beta-lactamase which inactivates penicillin by breaking the beta-lactam ring of the drug. Escherichia coli, Heamophiles influenzae and Pseudomonas also employ a similar process. Efflux mechanisms can result in increasing drug elimination, as seen in drug resistant cancer cells. Escherichia coli may become resistant to tetracyclines by the formation of an inner protein which actively pumps the antibiotic out of the cell. A streptococcus bacterium which uses an efflux pump may become resistant to macrolides. The synthesis of resistant metabolic pathway crosses the metabolic block used by the antibiotic; for example Staphylococcus aureus can become resistant to methicillin or flucloxacillin when it acquires the gene mecA (Schaechter, 1998). This code is a substitute for the penicillin binding protein which methicillin does not inhibit, which then enables the organism to multiply as shown by the organism Staphylococcus pneumonia. A method of enzyme inactivation with an antibiotic occurs by altering the enzyme thus preventing it from performing its normal function within the microorganism. Also some microorganisms can alter the target site as in the case of Rifampicin, which acts by inhibiting the beta-subunit of the RNA polymerase and resistance develops when the RNA polymerase is altered by a point mutation, insertion or deletion. The Rifampicin can no longer inhibit the enzyme due to the altered structure of the target site. In vertical gene transfer, mutation occurs as an error in the replication or DNA repair process whereby the same mutation is transmitted to all subsequent replicates and to future generations (Starr, 1992). Vertical gene transfer involves spontaneous occurrence while horizontal gene transfer involves physical contact. Horizontal gene transfer can occur through the following three methods (Schaechter, 1996): conjugation, transduction and Transformation. Conjugation involves cell to cell contact as here the DNA moves from a donor to a recipient via a sex pilus (Starr, 1992). This is the main mechanism for transfer of resistance (Rang 1995). The ability to conjugate is encoded in the conjugative plasmids which are produced by the host bacteria (Rang, 1995). This process occurs generally within the same species as also in gram positive and gram negative conjugate. During Transduction a virus transfers the genes between the mating bacteria. Plasmid DNA is enclosed in a bacterial virus and transferred to another bacterium of the same species. It is not an effective means of genetic transfer of resistance genes particularly between strains of staphylococci and streptococci. Transformation occurs when naked DNA is acquired from the external environment, when released from another organism and this transformation process involves changes in the hereditary system of the bacteria (Starr, 1992). Genetic recombination can occur following the transfer of DNA from one cell to another leading to a totally new genotype. It is very common for DNA to be transferred as plasmids between bacteria, this usually occurs from the host bacteria or from the same species. Since bacteria usually develop their genes for drug resistance on plasmids they are able to transfer this resistance to other strains and species during such the genetic exchange processes. Due to the increased growth rates, high concentrations of cells, genetic processes of mutation, the ability to exchange genes account for the increased rate of adaptation observed in bacteria (Champe, 1992) References 1.Champe, P.C; Gertner S.B; Harvey, R.A; Mycek, M.J; Perper, M.M Pharmacology. Philadelphia: Lippincott, c1992. 2. Jhonson, A.G; Microbiology and Immunology. Boltimore, Md:Williams & Wilkins,1989 3. Lowrie, P; wells,S; Microbiology Biotechnology. Cambridge: Cambridge University Press, 1994. 4. Marton, T. (1999). WWWII and the Miracle Of Penicillin. (Accessed 2 October 2007) http://www.mcatmaster.com/medicine&war/penicillin.htm 5. Rang,H.P;Dale M.M; Ritter J.M; Moor P.K; Pharmacology 3rd Ed. 1995. Edinbourgh: Churchill Livingston. 6. Rowe-Magnus,D.A; Davies, J; Mazel, D; (2002). Impact of integrons and transposons on the evolution of resistance and viralence. In (Hacker, J & Kaper,J.B.eds), Pathogenicity Islands and the Evolution of Pathogenic Microbes. Vol 2. p.161-188. Springer-Verlag. 7. Schaechter M, Engleberg N.C, Eisestine B.I, & Medoff G; Mechanisms of Microbial disease 3rd Ed,1998 Published Williams& Wilkins Maryland USA. 8. Starr,C; Starr,L; Taggart, Ralph: Biology: The unity and diversity of life 6th Ed.1992. Australia; Pacific Grove, CA; Brooks/Cole. 9. Theoharides,T.C; Essentials of Pharmacology 2nd ed. Boston, MA: Little, Brown,1996. 10. Tortora, G.T., Funke, B.R.,Case,C.L.(2006) Microbiology: An Introduction.9th Ed. Pearson Benjamin Cummings. 12. Wylie, C. (2006). Microbiology Laboratory Class Manual. Griffith University Gold Coast Read More