Advanced Medical Microbiology: Identification of Unknown Mixture - Lab Report Example

Advanced Medical Microbiology: Identification of Unknown Mixture Name Institutional Affiliation Identification of Unknown Mixture Introduction In a microbiology laboratory, the first step in conducting the experiments is the preparation of samples of unknown bacteria for the purpose of biochemical and serological identification. Various methods exist for the identification of bacteria. Biochemical and observational approaches have traditionally been the commonly used. Simple methods like the observation and smelling of a colony of bacteria that grow on a plate of agar can be used to provide clues to the identity of a bacteria by only experienced researchers. Bacteria are broadly classified into gram negative or gram positive according to their ability to be stained by a dye of chemical, which is the most popular biochemical approach. Differential gram stain basis occurs in the construction of the cell wall. The various sugars fermented by a bacteria as well as the different antimicrobials bacteria that express resistance to and the enzymes produced by bacteria are all fundamental approaches used for the identification of features that are reasonable and easy to test. In addition to the use of biochemical tests, the molecular analysis has also remained crucial for identification of bacteria. Such approaches involve the examination of Deoxyribose Nucleic Acid (DNA) of the bacteria that is under study.1 Such can be done by the use of techniques of mapping some essential characteristics of the genome of an organism or through a body portion sequencing. Comparison of the outcomes to the known microorganism database is later made which, could lead to a match allowing for the identification. The aim of the report was to identify unknown samples using various biochemical. Materials and methods. a) Aerobic and Aerobic Culturing of Bacteria. The materials needed to include three thioglycollate broth, plates of TSA, which included GasPak container, a sachet of GasPak and a strip of methylene blue indicator. Various samples of bacteria suspected to be anaerobes or aerobes were also used. The procedure involved inoculation of thioglycollate broth tube with the unknown sample then incubated at 37 degrees Celsius. The three TSA plates were then labeled to include the ambient air, GasPak jar, and a candle jar. The plates were then divided into various section, for every organism, that is the strict aerobe, the unknown, and the strict aerobe. The section was inoculated by streaking straight lines. The presence of methylene blue in the jar was confirmed. The indicator remains blue when oxidized and colorless following reduction. The set up was then incubated at 370C.2 b) Gram staining The materials included glass slides, inoculation loop, a bunsen burner, distilled water bibulous paper a cultured organism and a microscope. For observation, immersion oil and a lens cleaner were used. The gram staining procedure was done as outlined in the RMIT Techniques Manual 2013.3 c) Motility test The experiment used the hanging drop method for testing motility. Materials include a glass slide, distilled water, glass pipette, normal saline, cover slip and soft paraffin. The procedure involved placement of a suspension of bacteria on the cover slip center, application of a soft paraffin on coverslip corners then gently placing a glass slide on top of a coverslip being held on an upside down position. The preparation was then observed on a microscope initially under 10X then 40X magnification.4 d) Lactose Fermentation The procedure is majorly aimed at testing for the ability of a bacterial microorganism in fermenting lactose. The material involved include a lactose broth which has extracts of beef, lactose, and peptone of gelatin. An indicator, phenol red is added to show production of acid due to fermentation. The general procedure involves inoculation of a broth of lactose using a loop of inoculation. e) Mannitol fermentation.' The materials and reagents used included the phenol red broth of mannitol, a nutrient broth containing 0.5% mannitol. The procedure of the test includes aseptically transferring of an inoculum to a mannitol broth of phenol red. The already inoculated tube is then followed by a period of incubation for 24 hours at 37 degrees Celsius, and then the outcome is determined. When the color turns from red to yellow, the test is regarded as positive, and it indicates that the pH changed to an acidic condition. f) Ortho-nitro phenyl-beta-D-galactoside (ONPG) Test The ONPG test was mainly performed to determine the lactose non-fermenting microbial capability of producing beta-galactosidase. The material used included conical flasks, test tubes, inoculation loop, cotton plugs, the needle of inoculation, Bunsen burner, a chamber of laminar flow, incubator, toluene, TSI agar, dispose jar, the solution of ONPG and bacterial sample cultures. In the procedure, TSI medium agar ingredients of iron and three sugars was weighed then dissolved in an approximately 110 ml of distilled water into a conical flask through swirling and shaking. The pH was ascertained through the use of a pH meter then the flask was heated to dissolve a medium of agar completely.5 Before solidification, the medium that existed in a molten state was distributed to five test tubes. The tubes were plugged with cotton; then a craft paper was used to cover them and finally tied using a band of rubber. Sterilization of the tubes at 121 degrees Celsius for fifteen minutes followed after which they were taken from the autoclave and slanted to cool-solidified to obtain slants of TSI agar. The bacteria to be tested were aseptically inoculated in a laminar flow by streaking. The slants already inoculated were then incubated for 24 hours at 37 degrees Celsius. When the bacteria was a fermenter of lactose, it was not taken for the test of ONPG, but if it was indicated by a red slant and butt, then it was made for an ONPG test. A bacterial culture was put into a test tube of 0.25 ml saline using a sterilized loop then a single drop of toluene was added, mixing done followed by a five-minute incubation at a room temperature. Finally, an ONPG solution was added followed by a room temperature incubation for close to 30 minutes. The change of color was observed. g) Hydrogen sulfide production test The test assesses whether a bacteria can reduce compounds that contain sulfur into sulfides during metabolism processes. The test used Kliger Iron Agar (KIA) for the detection of hydrogen sulfide. The H2S production test procedure was done as outlined in the RMIT Techniques, 2013.6 h) Citrate Utilization Test The test operates on a principle that in case an organic acid like citrate is used as a source of energy and carbon, the production of alkaline bicarbonates and carbonates occurs. Ammonium hydroxide is additionally produced in case salts of ammonium are the primary source of nitrogen. The procedure includes light inoculation of Simmons citrate agar on the slant by touching a needle tip to a 24-hour old colony. Incubation then follows at 37 degrees Celsius for 24 hours. Finally, a blue color development denotes alkalinization. I) Nitrate Reduction Test The material required include a broth of nitrate containing inverted Durham tubes, sulphonic acid, alpha-naphthylamine, burner, dropper and an inoculation loop. The procedure involves the injection of the broth using a huge organism test growth, an incubation of 24 hours, and an addition of sulphuric acid dropwise and a full drop addition of alpha-naphthylamine to every drop. A small quantity of zinc is finally added to the own broth. Zinc catalyzes the reduction of nitrate reduction to nitrite. Table 1: Responses of Microbes to various Tests Test E1 E2 E3 Growth on MCA positive positive negative Growth on Air positive positive positive Gram stain positive positive Positive bacilli Growth on HBA positive positive positive Growth in anaerobic condition positive positive negative Xylose negative NA Mannitol positive NA NA Catalase NA positive negative oxidase negative NA NA Coagulase NA Negative negative Lactose Negative NA positive Motility positive NA Negative PYR NA positive NA ONPG Negative NA NA Lysine Dercarboxylase Negative NA NA Nitrate Reduction Test positive NA NA Indole positive Na NA In the experiment, the first organism, E1 indicated a positive growth in both aerobic and anaerobic conditions. As shown in the above table, E1 grew in the Macconkey (MCA) as demonstrated by the pinkish color, a small size of 2 mm diameter, and the smooth surface. In the Horse Blood Agar (HBA), the organism indicated a growth of gray color, was round, flat and elevated. E1 was a gram-negative bacillus, oxidase negative, Indole, motility and Mannitol Test positive. The sample was positive for glucose as confirmed by the production of H2S by the formation of yellow color at the bottom of the tube. The sample was negative for lactose as indicated by a red color of the top tube. As shown in figure 1, for citrate utilization test, E1 turned blue showing positive results. Further, the sample tested negative for Ornithine Decarboxylase test and positive for nitrate reduction test as shown in figure 3. Figure 1 Figure 2: Observation for Motility test Figure 3; Observation for Nitrate reduction test Sample E2, which was unknown mixture grew aerobically and anaerobically, increasing on both MCA and HBA. The sample tested positive for gram stain as indicated by gram-positive cocci in clusters, tested positive for catalase and PYR. Both coagulase and tube coagulase tested negative. Sample E3 only grew on anaerobic condition and HBA but not in MCA. The colony morphology was gray, round with smooth surfaces and had clear zones. E3 was gram positive Bacilli, catalase and coagulase negative. Both lactose, glucose and sucrose were present, but the sample tested negative for motility test as represented in figure 2 and Figure 4. Figure 4: Flow chart Representations of Identifying Unknown Organisms Discussion. Organism E1 was identified to be Providencia Rettgeri which is in the family of Enterobacteriaceae. P. Rettgeri is always mannitol positive. The bacteria turned blue in citrate utilization test indicating a positive reaction. The microorganism is a gram negative microbe commonly occurring on land and water. P. rettgeri is often negative for lactose, xylose, lysine Decarboxylase and Ornithine Decarboxylase Test. The microbe includes bacilli that produce urease and are pathogens which cause various infections in human. Multiple infections caused by Providencia often engage the urinary tract and are additionally related to bacteremia and gastroenteritis. P. rettgeri also cause infections of the eyes and the travelers’ diarrhea. Organism E2 was Staphylococcus hemolytic. E2 is a common bacteria in the aerobic and anaerobic environment often growing in subtle colonies when cultured on MCA or HBA. Staphylococcus are gram positive microorganisms which appear in cocci. The stated bacteria are commensals that occur abundantly in the skin microbiome of the human being S. epidermis and S. aureus are particularly opportunistic pathogens causing various diseases. As pathogens, S. aureus remains a huge cause of infections of soft tissues and skins such as furuncles, cellulitis, and boils.7 Some severe infections from the microbe can entail infection of the blood stream, joint and bone infection and pneumonia. S. hemolytic was clearly susceptible to various antibiotics. The antibiotics which, indicated a significant strength against the microbe was novobiocin and Polymycin B. E3 was Clostridium perfringens. The bacteria produce harmful toxins to human. C. perfringens and the associated toxins are ubiquitous in the environment.8 However, infection of human occurs mostly from the consumption of food containing the microbe. The microorganism cause food poisoning which is quite frequent but less severe and may be mistaken for the flu. Most outbreaks of the infection are related to inefficiently prepared meat, always in massive quantities for a big group of consumers. The symptom of the disease occurs between 6 to 24 hours following the consumption of toxins. The bacteria causes stomach cramps and pain in the abdomen and diarrhea. References 1. Lowy, F. D. Staphylococcus aureus infections. New England journal of medicine, 339(8), 520-532; 1998. 2. McClane, B. A. (2003). Clostridium perfringens. FOOD SCIENCE AND TECHNOLOGY-NEW YORK-MARCEL DEKKER-, 91-104; 2003 3. Walduck AK, Andersen LP, Raghavan S. Inflammation, Immunity, and Vaccines for Helicobacter pylori Infection. Helicobacter. 2015 Sep; 20 Suppl 1(S1):17–25. 4. Williams H, Walduck AK, Deighton M, Lawrie A. RMIT Microbiology Techniques Manual 2016, 3rd Ed. RMIT University; 2016. 5. Goldman E, Green LH, editors. Practical handbook of microbiology. CRC Press; 2015 Jun 15. Read More