Identification of mystery microbe based on its morphology and biochemical properties
The experiment was conducted to detect the species of a given mystery microbe. The microbe was subjected to a battery of tests in order to identify its physical and biochemical characteristics. Based on the tests, different possible microbe species were eliminated to finally arrive at the right conclusion. The possible microbes included Escherichia coli, Providencia alcalifaciens, Enterobacter aerogenes, Staphylococcus epidermidis, Klebsiella pneumonia, Micrococcus luteus, Serratia marcescens, Mycobacterium smegmatis, Sarcina aurantiaca, Pseudomonas aeruginosa and Bacillus licheniformis.
The first test conducted was to detect the Gram staining properties of the given microbe. This helped in identifying whether the given microbe had a thin or thick peptidoglycan layer (Alderson, 2011). The stain also helped in identifying the shape of the microbe in question. For this test, a smear was prepared using the NA broth culture followed by heat fixation. The slide was covered with crystal violet for 30 to 60 seconds and then rinsed with water. This was followed by treatment with Gram’s iodine for another 30 to 60 seconds. This acted as a mordant to help the stain to be more tightly bound to the cells. The slide was then treated with ethanol, which helps in removing the stain if the microbe has a thin peptidoglycan layer. This was followed by a counterstain, safranin. Safranin helped in dyeing any colorless cells red in color. The safranin dye was used for 30 seconds. The slide was then observed under a bright field microscope in 1000x magnification with oil immersion.
The next tests performed were to determine whether the microbe was encapsulated and had spores. To check for a capsule, a drop of congo red was taken on a clean slide and a sample of the bacteria from the streak culture was added to it. After the smear dried, the slide was covered with acid alcohol for 15 seconds. This was followed by treatment with carbol fuschin for 1 minute. To check for endospores, a smear was prepared from the NA slant tube. The smear was covered with a paper and malachite green was added as a stain. The slide was then steamed for 5 minutes with care taken to ensure that the slide did not dry out. After the slide cooled down, the paper was removed; the slide was then rinsed and counterstained using safranin. The slide was rinsed and dried for observation. Both the spore and capsule slides were observed under a bright field microscope in 1000x magnification with oil impression.
The next test conducted was to check the acid-fastness of the microbe. This helped in detecting the presence of any mycobacteria, as they have a mycolic layer surrounding the cell wall (Alderson, 2011). For this test, the process used was similar to the test for spores but the smear was prepared using the streak plate culture. Also, carbol fuschin was used for staining and methylene blue for counterstaining. Before counterstaining, the slide was decolorized using acid alcohol. The slide was then observed under a bright field microscope in 1000x magnification with oil immersion.
The final test, for the microbe’s physical properties, was the test for motility. For this test a depression slide was used in which a few drops of the NA broth culture were placed and covered with a cover slip (Alderson, 2011). The slide was then observed under a dark field microscope in 10x magnification.
After completing the tests for physical properties of the microbe, a series of tests were conducted to evaluate its biochemical properties. The first test in the series was the catalase test. A sample of the bacteria was transferred from the nutrient agar on to a slide and some drops of hydrogen peroxide were added to the slide. Catalase is detected by the formation of bubbles. This was followed by the test for oxidase. The test was performed by placing an oxidase paper strip in an empty petri plate and moistening a part of it. A sample of the given bacteria was transferred on to the strip with a pipette and observed for any change in the color of the strip. After this, the citrate agar and urease tests were conducted for the given microbe. For the first test, a citrate agar slant was inoculated with a small amount of the bacteria given and allowed to incubate for 48 hours. The urease test was conducted by taking 5 ml of sterile urea in a test tube inoculated with the microbe. Even this was allowed to incubate for 48 hours (Alderson, 2011).
While the citrate agar and urease tubes were incubating, four tests were performed to determine the sugar fermentation ability of the microbe. The first of these was growing a culture of the bacteria on an EMB plate. The EMB plate helped in, firstly, to isolate any Gram negative bacteria, and also to detect whether the bacteria could ferment lactose. A culture of the bacteria was also prepared on MacConkey agar which further helped in detecting any lactose fermenting ability. For both EMB and MacConkey plates, the bacteria were obtained from the streak plate culture. Both the plates were allowed to incubate for 48 hours. Next, glucose and lactose Durham tubes were inoculated with the microbe using the NA broth. The tubes were allowed to incubate for 24 hours and then observed for gas formation and color change. The final test was performed using TSIA slants. The TSIA slant was inoculated using bacteria from the streak plate culture. The test helped in differentiating bacteria which can ferment only glucose from those which can ferment both glucose and lactose. The test also helped to detect formation of hydrogen sulfide, if any. The TSIA slant was also allowed to incubate for 48 hours. The tube was then observed for any changes in color (Alderson, 2011).
Results
The various test results for the mystery microbe have been enumerated in Table 1. In the Gram staining test red rods were observed under the microscope. The simple stain also showed presence of rods indicating that the given microbe is a bacillus. In the capsule test, a clear halo was seen surrounding the bacteria cell indicating that the bacteria were encapsulated. The spore test and motility test were not clear and hence, no definite inference could be drawn from them.
Amongst the biochemical reactions, the microbe was positive for catalase as it showed presence of air bubbles within one minute of performing the test, as seen in figure 1. The citrate agar test showed a definite change in color, as seen in figure 1, indicating that the microbe can survive on citrate alone. However, tests for oxidase and urease showed no changes in color.
The fermentation tests showed some contrasting results. The EMB plate showed bacterial growth, but, the colonies were pinkish purple in color indicating that the bacteria were not lactose fermenting. However, observations of the MacConkey Agar, Durham tubes and TSIA slants indicated the presence of lactose-fermenting gas-producing bacteria as seen in figure 2.
The mystery microbe is, therefore, a Gram negative bacillus with a capsule, which is catalase and citrate positive, but oxidase and urease negative, and it can ferment glucose as well as lactose, producing gas in the process.
Test results of mystery microbe
Figure 1. Results of the biochemical reactions with positive catalase and citrate tests
Figure 2. Fermentation tests indicating the presence of gas-producing sugar fermenting bacteria
Discussion and Conclusion
Based on the tests conducted, the given microbe can be categorized as a Gram negative bacillus. Out of the possible microbial choices this rules out Staphylococcus epidermidis, Micrococcus luteus, Sarcina aurantiaca and Bacillus licheniformis which are Gram positive bacteria. Mycobacterium smegmatis is an acid-fast bacterium; however, the given microbe is not acid-fast, hence, ruling out M. smegmatis (Winn et al., 2006). Out of the remaining bacteria, none can be ruled out based on their gram staining properties, as all of them are Gram negative bacilli. All of the remaining bacteria also have capsules, so they have to be differentiated based on the biochemical tests.
The mystery microbe is catalase positive and oxidase negative. This helps us to rule out Pseudomonas aeruginosa which is oxidase positive (Winn et al., 2006), leaving us with five bacteria which are members of the family Enterobacteriaceae. For the lactose fermenting tests, three out of four tests showed the presence of lactose fermenting bacteria. On this basis, it can be concluded that the mystery microbe is lactose fermenting. Of the five remaining bacteria, Serratia marcescens and Providencia alcalifaciens are non lactose-fermenting (Winn et al., 2006). Hence, they are eliminated as possible choices.
The last two tests performed were citrate agar test and urease test. The mystery microbe was identified as citrate + and urease -. The last three remaining bacteria are Eschericia coli (citrate -, urease -), Enterobacter aerogenes (citrate +, urease -) and Klebsiella pneumonia (citrate +, urease +) (Winn et al., 2006). Thus, the only bacterial species with properties similar to the mystery microbe is Enterobacter aerogenes.
The experimental process presented some flaws, specifically in the physical tests, as no definite conclusion could be drawn regarding the microbe’s motility and spore production. Even the EMB plate test presented contrasting results by indicating that the microbe was non-lactose fermenting. It is essential to take utmost care while performing the tests as even a single error could lead to an erroneous identification of the microbe.
References
Alderson, G. (2011). Microbiology: Experiments and Lab Techniques. (13th ed., p. 36). S.l.: Fountainhead Press.
Alderson, G. (2011). Microbiology: Experiments and Lab Techniques. (13th ed., pp. 85-115). S.l.: Fountainhead Press.
Alderson, G. (2011). Microbiology: Experiments and Lab Techniques. (13th ed., pp. 249-255). S.l.: Fountainhead Press.
Winn, W., Allen, S., Janda, W., Koneman, E., Procop, G., Schrekenberger, P., & Woods, G. (2006). Koneman's color atlas and textbook of diagnostic microbiology (6th ed.). Philadelphia: Lippincott Williams & Wilkins.
