Identification of Microorganism - Lab Report Example

 «Identification of Microorganism» Part One: Identification of Microorganism Identification of microorganism is the foremost step to accomplish further investigation procedures. Bacterial isolation was done from the different samples collected patients. The three preliminary steps for the analysis procedure comprise principal isolation, staining and biochemical reactions It is evident that sample contains various kinds of microorganism belonging to different species, although the disease causing organism pre-dominates other micro flora present in the sample. The three samples that were collected consist of - vaginal swab 1, virginal swab 2 and fecal sample. Identification is based on the kind of Gram reaction shown by the organism. This is the most pragmatic staining also called differential staining performed to categorize microbial population into two groups the Gram positive and Gram negative organisms. The reaction displays the characteristic of the cell wall of the bacterial species. If the bacterial species possess thick peptidoglycan layer it displays Gm +ve differential staining and if the microorganism possess thin or single layer of peptidoglycan then it takes up the counter stain Safranin of the Gram reaction. Thus, G +ve organisms display purple staining whereas the G –ve organisms take up the counter stain and display pink staining. Counter stain Safranin is added to the reaction after washing the Gram stain with alcohol. This step is most imperative as Gram positive organisms possessing thick peptidoglycan retains the stain while Gram negative organisms possessing thin peptidoglycan cell wall becomes colorless and therefore takes up the counter stain when stained with Safranin. It is therefore this staining procedure aids as a diagnostic device in medical as well as research pursuits. Staining also helps in identifying the morphology of the microbial population. When the vaginal swab 1 specimen was gram stained, it took the counter stain and stained pink indicating the predominance of G –ve organisms in the sample. On observing the morphology of cells under microscope the cells display single rod shaped structures. The observations both Gram staining and the colony morphology were recorded in Report Form 1 (included). Thus the bacterial species observed in the virginal swab 1 specimen were identified as gram-negative single bacillus bacteria. In the similar manner Gram staining was also performed for virginal swab 2. In this case the purple color of the Gram reaction was retained by the bacterial cell wall indicating that Gram positive species predominated in this sample. On observing the microorganisms under microscope, the morphology displayed by this set of microbial population was cocci, round cluster of bacterial microflora. The results were recorded in Report Form 1 (included). Thus the bacterial species observed in the virginal swab 2, specimen were identified as gram-positive cluster or bunch of round or spherical bacteria. Although vaginal swab 1 and virginal swab 2 came from the same patient but two different microbial species predominated these two samples, signifying the need of isolation of these organisms in pure culture. The third sample was the fecal specimen. Gram staining was performed with this sample too. The microbial population present in fecal sample was predominated by gram-negative microorganisms. The morphological features encompass single bacilli. The results procured were recorded in Report Form 1. The staining procedure as followed by the differentiation step where the pathogenesis of the microbes was tested. In order to perform this, differential and selective media were used. The selective and differential media used in this experiment were Blood Agar (BA), MacConkey Agar (MAC) and Mannitol salt Agar (MSA). The Blood Agar (BA) identifies those groups of bacteria capable of secreting haemolytic enzymes lysing the red blood cells in Blood Agar. Blood Agar contains 5% suspension of red blood cells; therefore, these enzymes create a colorless zone around the colonies (Korolik and Beacham, 2008). Furthermore, Blood Agar is an enrichment medium that classifies organisms such as Streptococci (Korolik and Beacham, 2008). The vaginal swab 1, 2 samples and fecal sample were found to be non-haemolytic, therefore no obvious marks of haemolysis around the colonies were observed on the plates. The results indicate that the samples are devoid of Streptococci. Another selective differential media is MacConkey Agar that comprises lactose, bile salts, crystal violet and neutral red (pH indicator). Cristal violet inhibits the growth of most gram-positive bacteria colony (Korolik and Beacham, 2008). Thus it was anticipated that the vaginal swab (2) specimen would contain no growth (non-lactose fermenting) on this media. This provides the evidence that supports gram-positive reaction which was recorded previously for the vaginal swap (2) in Report Form 1. Conversely, the vaginal swab (1) and fecal specimens produced results coherent with a lactose-fermenting organism. MAC is used for the selection and differentiation of the enteric gram-negative rods associated with the intestinal tract such as Escherichia coli (Tortora et al, 2007). Since the results of vaginal swab (1) and fecal specimens were negative bacilli, it can be seen from their lactose fermentation that the unknown microorganisms are those belonging to the enteric group Escherichia coli or Salmonella. Mannitol Salt Agar is the next selective differential media for fecal and vaginal specimens (1) and (2). MSA encompasses 7.5% sodium chloride that inhibits growth of most bacterial species, other additives that are present in this media are phenol red and mannitol. MSA is also considered as a selection and differentiation media for Staphylococcus species. The colonies of Staphylococcus aureus develop a yellow zone around them. This reaction is shown because of the presence of phenol red with acid formed from mannitol fermentation (Korolik and Beacham, 2008). The vaginal swab (2) specimen was the only sample which showed a visible yellow zone around the colony (mannitol fermenting) suggesting that the microbes presented were Staphylococcus aureus while fecal and vaginal swab (2) do not show the presence of this organism. The results for the colony characteristic on selective and differential media such as Blood Agar, MacConkey Agar and Mannitol Salt agar were recorded for all specimens on Report Form 1. For further confirmation of the microbial population, various biochemical tests were performed for these three samples. For vaginal and fecal samples catalases and oxidase test were executed. Catalase test determines the ability of the organism to produce catalase enzyme metabolizing hydrogen peroxide (Tortora et al, 2007). On the other hand, oxidase test is aimed for differentiating oxidase positive bacteria of the genera Neisseria (cytochrome oxidase bacteria). This test differentiates Neisseria from other gram-negative organisms. It can be seen from the results in Report Form 1 that vaginal sample (1) and fecal sample catalase negative, which means lacking Neisseria or cytochrome oxidase bacteria. Biochemical tests also determine and identify the microorganism present in the vaginal and fecal specimen. Biochemical test contains multiple diagnosing systems aids in identification of the organism (Korolik and Beacham, 2008). A Microbact System was used in the experiments which contains a large number of differential and selective media. This system conducts 12 different biochemical tests. The tests are conducted only for the gram-negative bacteria and therefore it is used for fecal and vaginal (1) samples. Colonies from fecal and vaginal (1) samples were suspended in sterile saline and then inoculated them in Microbact strip for observation. Results were recorded in Report Form 2 and the values associated with a positive result for the series of biochemical tests were also recorded in Report Form 2. The code number produced from the addition of the values resulting from positive testes for the vaginal swab (1) was 4760. This code is associated to a series of probabilities of different microbes such as 70% E. coli, 29% E. coli (inactive). This result is accepted along with the results obtained previously from MacConkey Agar plate, because the Microbact Test was also applied for unknown A code number 6760 and gave 95% E. coli, 4% inactive E. coli as well. These results safeguard that the microbe in vaginal swab (1) was E. coli. Besides, the reading from Microbact unknown specimen 1 code number 7600 gave 97% Salmonella sp. This result was also associated with previous test of MacConkey Agar for gram-negative test. Vaginal swab 1, 2 and fecal specimen were also tested for antibiotic sensitivity. The tests are crucial for the treatment of infectious diseases. The results were recorded by measuring the observed zone of inhibition, an area around the antibiotic disc that is placed over the agar. Then, it was measured with the zone diameter for each antibiotic disc which belonged to the specimens. The results are compared with the standard table that indicates whether the organisms were sensitive, intermediate or resistant to that particular group of antibiotic. Four antibiotic discs were applied to the agar surface – Ampicillin, Gentamycin, Tetracycline and Chloramphenicol. The results obtained for Ampicillin indicated its bactericidal action against gram-negative bacteria. Therefore fecal and vaginal (1) produced a zone of inhibition of 25mm and 20mm for vaginal swab (1), which is classified according to the standard table as susceptible to the antibiotic. These results ensured that the fecal sample and vaginal (1) are gram-negative while vaginal (2) sample is gram-positive as it shows fair degree of resistance for Ampicillin. The results procured after testing the antibiotic sensitivity implies that, Gentamycin was the most effective antibiotic found in the experiment against the vaginal (2) specimen, while Ampicillin was the most effective antibiotic against the fecal specimen. Despite the fact that there must be a mistake in measurement of inhibition area around the vaginal swab (2) because it showed that the diameter of Ampicillin around the colony was 40mm, it was supposed to be lower than that, and Gentamycin was supposed to be clinically the selective antibiotic in gram-positive bacteria. (Rang et al, 2003) According to Rang Pharmacology, 2003, Ampicillin was considered as broad-spectrum antibiotic hence it is found to be effective against gram-negative or gram-positive microorganisms. Second part: Microbial Control The skin is the plethora of microbial population. The population varies at different parts, from one side of the body to another. This variation is due to the moisture content of the skin that varies throughout the body. High moisture content displays higher population of normal microflora (Wylie et al, 2008). The oral cavity also, considered as suitable location for the growth of normal microflora. The second part of the experiment examines the characteristics of bacteria taken from surface of human skin and inner side of the oral cavity especially from the surface of cheeks. The skin specimen was taken from behind of the ear and found to be gram positive, coccus shaped and staphylo or cluster arrangement. This result was recorded in Report Form 4. While the specimen from buccal cavity also display the presence of gram positive organisms. However, it has different cell arrangement called as sterpto or chain. These results reported in Report Form 4 with colony morphology as well. This section of the experiment approved that human body have several microorganisms growing on the surface of the body and even inside our mouth. However, the availability of microorganisms is different from one site to another. For example, the temperature of the oral cavity and the presence of the food particle supports a different group of microbial population as compared to the population present outside the oral cavity. Next aspect determines the effectiveness of some disinfectants on microbial growth. The test was applied for unwashed and washed hands. However the unwashed hand proved to have some microorganism and this was expected. These results applied for individual and for all class average and recorded in Report Form 5. It is observed that microorganisms that appeared in unwashed hand were removed during hand washing with both soap and Antibacterial hand-wash. While the result obtained from using normal hand soap was significant because even after washing the hand with normal soap the growth of colonies were identified. These results plus the class average observation was recorded in Report Form 5. The critical reason for this observation could be due to the presence of bacteria on the soap used previously by other student, led partial prevention of soap from acting for complete removal of colonies during washing. Usually certain types of skin normal flora are capable of surviving and multiplying and most of them were Gram-positive bacteria of the genera Staphylococcus, Micrococcus and Propionibacterium. (Tortora et al, 2007) Last test of the experiment was to determine the minimum inhibition concentration of unknown microbe inculcated in five tubes with different concentration. Antimicrobial agents are divided into two classes: Bactericidal and bacteriostatic (Tortora et al, 2007). The chemical agent used for this test is Surface Spray. It can be seen that individual results have growth of unknown microbes that is increased (bacteriostatic) with each dilution and growth was decreased in tube with concentration level of surface spray. When compared with class results, it was noticed that the Bleach appeared to be the best chemical agent have a bactericidal mode of action against the unknown microbe. Reference Korolik, V., Beacham, I.R. (2008). Microbiology study guide, 2007HSC Microbiology, Griffith Uneversity, Gold Coast, Australia. Rang, H.P., Dale, M.M., Ritter, J.M. and Moore, P.K. (2003). Pharmacology 5th ed. Churchill Livingston:UK. Tortora, G.L., Funke, B.R., Case, C.L. (2007). Microbiology: An introduction. 9th ed. Benjamin Cummings, California, USA Wylie, C., Korolik, V., Louge, C., Shewell, L. (2008). 2007 MSC Microbiology laboratory class manual, Griffith Uneversity, Gold Coast, Australia Read More