The identification of pathogens is critical in a clinical setting. Proper identification allows medical professionals to decide the appropriate course of treatment for infected individuals. Additionally, knowledge of characteristics of microbes that are present in everyday life is essential in protecting oneself from infection. A series of differential and biochemical tests was performed in order to identify an assigned unknown specimen. The bacterium to be identified was sample B from wounds.
Materials and Methods
Prior to the performance of biochemical tests, a Gram stain was performed in order to label the unknown bacterium as either Gram positive or negative. Crystal violet and iodine are the first stains used, and cause the cell wall to possess a purple color. Ethanol is then used to clean off the purple dye. In Gram positive bacteria, the ethanol is unable to successfully wash out the purple color because of the thick cell walls that these bacteria possess. When performing this stain on Gram negative bacteria, the ethanol will be successful and the final counterstain will result in a pink color.
First, a catalase test was performed by placing the specimen on a microscope slide. 3% hydrogen peroxide was then added to the sample on the slide, and an immediate reaction was observed. During a citrate test, the bacterium was inoculated onto Simmon’s citrate slants, which were then incubated at 35 C for approximately 24-48 hours. In order to test for carbohydrate fermentation, a Brem Cresol Purple (BCP) lactose broth was inoculated with the unknown specimen. The broth was then inoculated at 35C for approximately 24-48 hours, after which period, results were observed. Next, a Peptone Iron Agar (PIA) lactose broth was inocul...
... middle of paper ...
... colon and intestinal tract, it can be extremely detrimental to the human lungs. Infections in the lung involving this bacterium often result in “necrosis, inflammation or hemorrhage within the lung tissue” (Umeh). K. oxytoca is an opportunistic pathogen, meaning that it takes advantage and targets individuals with a weakened immune system. For this reason, it is a source of 8% of nosocomial, or hospital-acquired, infections (Umeh). Furthermore, older men who have “alcoholism, diabetes, or chronic bronchopulmonary disease” are especially at risk for infection by this bacteria (Umeh).
References
DeMers, Marlene. Fundamentals of Microbiology Laboratory Manual. Dubuque, IA:
Kendall Hunt
Umeh, Obiamiwe, Geffen, David & Berkowitz, Leonard B. (2011, August 31).
Klebsiella Infections. Retrieved from http://www.emedicine.medscape.com/article/219907-overview
Table 6 shows the results of the biochemical tests. The isolate can obtain its energy by means of aerobic respiration but not fermentation. In the Oxidation-Fermentation test, a yellow color change was produced only under both aerobic conditions, indicating that the EI can oxidize glucose to produce acidic products. In addition to glucose, the EI can also utilize lactose and sucrose, and this deduction is based on the fact that the color of the test medium broth changed to yellow in all three Phenol Red Broth tests. These results are further supported by the results of the Triple Sugar Iron Agar test. Although the EI does perform fermentation of these three carbohydrates, it appears that this bacterium cannot perform mixed acid fermentation nor 2,3-butanediol fermentation due to the lack of color change in Methyl Red and Vogues-Proskauer
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
These labels indicated the lactose solution that was be placed into the mini-microfuge tubes. The varying lactose ph solutions were obtained. The four miniature pipets were then used, (one per solution,) to add 1mL of the solution to the corresponding mini-microfuge tubes. When this step is completed there were two mini-microfuge tubes that matched the paper towel. Then, once all of the solutions contained their respective lactose solutions, 0.5mL of the lactase enzyme suspension was added to the first mini-microfuge tube labeled LPH4 on the paper towel, and 4 on the microfuge tube. As soon as the lactase enzyme suspension was added to the mini-microfuge tube, the timer was started in stopwatch mode (increasing.) When the timer reached 7 minutes and 30 seconds, the glucose test strip was dipped into the created solution in the mini-microfuge tube for 2 seconds (keep timer going, as the timer is also needed for the glucose strip. Once the two seconds had elapsed, the test strip was immediately removed, and the excess solution was wiped gently on the side of the mini-microfuge tube. The timer was continued for 30 addition seconds. Once the timer reached 7:32 (the extra two seconds accounting for the glucose dip), the test strip was then compared the glucose test strip color chart that is found on the side of the glucose test strip
I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
If the oxidase test was to be negative then the bacteria would have to be S. flexnery. S. flexneri is a bacterium that causes diarrheal disease, and it’s a “facultative anaerobe belonging to the family Enterobacteriaceae” (“Shigella flexneri”, n.d). However, if the oxidase test came as positive then the bacteria would have to either P. aeruginosa or A. faecalis (“Microbiology 20 Biochemical Unknown, 2009). Then an indole test will have to be done. An indole test is to identify if the bacteria could produce the enzyme tryptophase (SIM Medium”, n.d). If the test came as positive then unknown bacteria 2 would be P. aeruginosa (“Microbiology 20 Biochemical Unknown, 2009). P. aeruginosa is a bacterium that is the number one leading infections in humans. The bacterium is Gram negative that can lead to “endocarditis, meningitis, etc. (Friedrich.M, Dec 5, 2016). In the other hand if the bacteria came as indole negative then bacteria 2 would be A faecalis. A faecalis is a Gram-negative, rod-shape bacterium with flagella, and that belongs to the family Alcaligenaceas”, and it’s an opportunistic pathogen that induces infection (“Alcaligenes faecalis”, n.d). All in all, skills were practices to determine two unknown
Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative, rod-shaped aerobic bacterium. It is a primary cause of hospital-acquired infections. P. aeruginosa is primarily a nosocomial pathogen. It also acts as an opportunistic pathogen, which can only infect a host that is immunocompromised, due to an underlying disease or medication. Although, P. aeruginosa can cause damage to virtually any tissue in the body, it almost never affects the tissues of healthy individuals. It is a problematic pathogen in hospitals; infecting individuals with cancer, burn wound, catheters and cystic fibrosis. P. aeruginosa is most recognized for its resistance to a wide range of antibiotics. In its planktonic form, P. aeruginosa has been found to have many virulence factors. However, P. aeruginosa within biofilms have been found to have a resistance to antibiotics 1,000 times greater than that of its planktonic counterparts [4]. Infections that are caused by bacterial biofilms are very persistent and very difficult to treat.
mutans was problematic due to its difference with Bergey’s Manual result for the catalase test. However, after comparing it with a peers results, it seems very possible that the strain we are working with varies from the strain used in Bergey’s. Bacteria possess the ability to develop varying phenotypes within the same species due to frequent mutation and horizontal gene transfer. Therefore, it is possible that the results obtained in our lab may vary from those provided in Bergey’s Manual. Arriving to the conclusion that the Gram negative bacteria was Klebsiella pneumoniae was much more direct. Using Bergey’s Flowchart for identification, the bacteria shared the test results and had a similar shape and
Bloodborne Pathogens are pathogenic microorganisms that can eventually cause disease. They are found in human blood and other bodily fluids such as synovial fluid, semen, vaginal secretions, cerebrospinal fluid and any other fluid that mixes or has contact with blood. The bloodborne pathogens are pathogenic, which means they are disease causing, and they are also microorganisms, which means that they are very small so the human eye cannot see them.
"Bloodborne Pathogens : MedlinePlus Medical Encyclopedia." U.S National Library of Medicine. U.S. National Library of Medicine, n.d. Web. 09 Feb. 2014.
Upon receiving the unknown Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes. After ten minutes had passed, I collected the ingredients needed to perform a gram stain. I got the primary stain, crystal violet, and flooded my smear for sixty seconds, and then rinsed the color off with water until the water ran clear. I then flooded the smear with the mordant, grams iodine, and let that sit on the slide for sixty seconds as well. I then rinsed the grams iodine off with water and applied alcohol to the smear to decolorize the cells; however I made sure not to over decolorize and only put enough drops on the smear till the purple ran clear. I then rinsed the slide with water and flooded the smear with safranin the counter stain and let it sit for sixty seconds and then rinsed the color off with water. I blo...
The principle sites of nosocomial infections in patients, in order from most common to least common are: urinary tract, surgical wounds, respiratory tract, skin, blood, gastrointestinal tract, and central nervous system (Abedon). According to the CDC, the most common pathogens that cause nosocomial infections are Staphylococcus aureus, Pseudomonas aeurginosa, and Escherichia coli (EHA). Methicillin resistant Staphylococcal aureus (MRSA) is a strain of bacteria that is commonly...
VP test (a.k.a. butanediol fermentation test) contains peptone, glucose, and a phosphate buffer in its media, which is the same media as the MR test (Stout et al, 47). To perform the VP test, I used the stabbing technique to inoculate the VP media. I sterilized the stabbing utensil under an open flame, obtained a small amount of unknown bacteria, and stabbed the VP media. Once the VP media was inoculated, I let it incubate for 24 hours in the 37°C hot room. After the 24-hour incubation, I added 15 drops of both Barrits Reagent A and Barrits Reagent B to the VP media, for a total of 30 drops.