Example of How to Write an Unknown Report in Microbiology
*NOTE: Includes Introduction, Materials/Methods, Results and Discussion sections. Flowcharts in Results sections are omitted due to formatting issues.
There are a multitude of benefits in discovering, and further exploring, the origin and characterization of omnipresent microorganisms. This includes but is not limited to, identifying the virulence and course of effective treatment of various microbes that cause disease in patients, as well as providing further research on bacteria useful in developing certain foods and antibiotics. More recently, such findings have played an important role in combatting the emergence of antibiotic drug resistance. This study was conducted by putting into practice all of the procedures and techniques that have been learned thus far in the microbiology lab curriculum for the recognition of an unknown bacterium.
MATERIALS AND METHODS:
A broth tube that contained two unknown bacteria labeled as number 125 was given out by the lab instructor on the 2nd of April, 2015. The methods that have been learned thus far for identifying microbes have been applied to this unknown. Procedures and aseptic techniques were followed and executed accordingly, as instructed in the course laboratory manual by McDonald etc. (2), unless otherwise noted.
The first test that needed to be completed was to streak the unknown out on a nutrient agar plate, using the quadrant streak method detailed in the lab manual. This was a critical first test in order to isolate and grow a pure culture from the mixed unknown. After, the streak plate was incubated and grown at 37* C, the analysis was observed. It should be noted that pure cultures labeled as ALT 4 and ALT 8, were provided by the designated instructor after multiple days and attempts made to isolate and grow pure cultures of the unknown using the appropriate streak plate technique. The three separate streak plates performed all resulted in overgrowth and thus the inability to isolate. These samples were then used to carry out all further biochemical tests and will be referenced as Unknown A and Unknown B hence forward.
A Gram stain was then performed to determine the Gram reaction of Unknown A and Unknown B. After completion of the Gram stains, the reactions were checked and compared with supplied data and information both in the course lab manual (2) and Microbiology textbook (4) to ensure accuracy. Once both unknown were identified as Gram positive or Gram negative they were returned to the incubator set at 37*C. Further detailed biochemical tests were then executed.
All of the following tests were performed on this unknown:
- Urea test
- Casein test
- Methyl Red test
- Simmons Citrate test
- Indole test
The next day, a Urea test was conducted on Unknown A, as stated in the lab manual, to test for the unknown’s ability to hydrolyze urea using the enzyme urease. A Urea broth tube was inoculated with Unknown A using a sterile, flamed loop. The tube was then placed in the 37*C incubator. Simultaneously, a Casein test was also obtained using a milk agar plate that was then streaked with Unknown A and placed upside-down in the incubator. 48 hours later, results were observed and recorded for both tests. At that time, a Methyl Red test was performed on Unknown A as outlined in the lab manual. The MR-VP broth was inoculated with the bacterium using a flamed, sterile loop and placed into the 37*C incubator. It should be noted that a control tube of MR-VP broth was also used during this test to ensure precision and was not inoculated with the unknown. Two days later, five to ten drops of methyl red, a pH indicator, was added to both the inoculated and control tube. Results were then determined.
For Unknown B, a Urea test was also performed as described above. Following the 48 hours of incubation, results were obtained and recorded. After, a Methyl Red test was conducted on Unknown B as outlined above, which determined if acid was produced as a result of glucose fermentation. It was at this time that a Simmons Citrate test was performed, according to lab manual protocol in which a sterile, flamed loop was used to streak the surface of the slant with Unknown B. A SIM tube was used to test for the unknown’s ability to produce Indole. The SIM tube was stab inoculated, as instructed in the lab manual, with unknown B using a sterile, flamed needle; All tests were placed in the 37* incubator for 48 hours and results were then determined upon observation of the urea broth, as well as the Simmons Citrate slant. The appropriate 5-10 drops of methyl red were added and the results of the test were finalized. Several drops of Indole were squeezed onto the surface of the inoculated SIM tube and the outcome was concluded.
Table 1 outlines the test, purpose, reagents or media, observations, and results in list format for unknown A, numbered 125. Table 2 also illustrates the test, purpose, reagents or media, observations, and results for unknown B, numbered 125.
The following flowcharts illustrate the path taken along with the various tests, processes, and measures used to narrow down and eliminate other potential microorganisms from previous test results, in order to conclude with the identity of Unknown A and Unknown B. The results are presented using a simple and easy to follow chart, highlighting the process of elimination technique.
Table 1: Biochemical Test Results (Unknown A)
|Test||Purpose||Reagents or Media||Observations||Results|
|Gram Stain||To determine the Gram reaction of the bacterium||Crystal violet, Iodine, Alcohol, Safranin||Purple rods||Gram positive rods|
|Urea test||To determine if bacterium can breakdown urea as indicated by a color change from tan to pinkish red||Urea broth tube||No color change; broth remained tan||Negative Urea test; organism is not able to hydrolyze urea|
|Casein test||To determine if bacterium can breakdown the milk protein casein||Milk agar plate||Clearing of the white cloudiness around area of bacterial growth||Positive Casein test; organism can hydrolyze casein|
|Methyl Red test||To determine if bacterium produces acid in glucose fermentation as indicated by a color change from yellow to red||MRVP broth tube|
Methyl Red (pH indicator)
|Color change from yellow to red when reagent added||Positive Methyl Red test; organism is able to produce large amounts of acid from glucose fermentation|
Table 2: Biochemical Test Results (Unknown B)
|Test||Purpose||Reagents or Media||Observations||Results|
|Gram Stain||To determine the Gram reaction of the bacterium||Crystal violet, Iodine, Alcohol, Safranin||Pink rods||Gram negative rods|
|Urea test||To determine if bacterium can breakdown urea as indicated by a color change from tan to pinkish red||Urea broth tube||No color change as broth remained same color||Negative Urea test; organism is not able to hydrolyze urea|
|Simmons Citrate test||To determine if the bacterium can breakdown citrate as indicated by a color change of green to blue||Citrate slant (green)||No color change; inoculated test tube remained green||Negative Simmons Citrate test; organism is not able to utilize citrate as a carbon source|
|Methyl Red test||To determine if the bacterium produces acid in glucose fermentation as indicated by a color change from yellow to red||MRVP broth tube|
Methyl Red (pH indicator)
|Color change to red||Positive Methyl Red test; organism is able to produce large amounts of acid from glucose fermentation|
|Indole test||To determine if the bacterium in the SIM tube produces Indole|| SIM tube|
|Appearance of red ring at top of broth within 5 seconds||Positive Indole test|
Upon proper completion of various tests and thorough process of elimination techniques, it was finalized and thus determined that Unknown A, a Gram positive, rod-shaped bacterium, was Bacillus cereus and Unknown B, a Gram negative, rod-shaped bacterium, was Escherichia Coli. The identification of both unknowns were checked and confirmed as correct by the lab professor. Luckily, only minimal problems were encountered throughout the testing and research duration. Initially, there was difficulty with several of the streak plates performed, all displaying mass overgrowth. This could have resulted from possible contamination, the plates remaining in the incubator for too long or incorrect performance of the correct quadrant streak plate technique.
B. cereus, more commonly referenced by its origin of “fried rice syndrome” that results from rice sitting out at room temperature for too long (i.e. buffets), is an endemic, soil-dwelling, Gram-positive, rod shaped and motile bacterium. Though regularly associated with food poisoning or two types of food-borne illness in humans, many cases go undiagnosed and are not reported because of the few strains that are pathogenic and pose a threat to humans. Some strains can even be constructive and used as a probiotic for animals. As a member of the genus Bacillus, B. cereus bacteria are facultative anaerobes capable of producing protective endospores that are highly resistant.
An article that was recently released, however, warned citizens of Hong Kong to avoid consuming and get rid of previously bought preserved bean curd found to be contaminated with Bacillus cereus. In a follow-up investigation, The Center for Food Safety found yet another batch to have excessive B. cereus, far exceeding safe levels for consumption and resulting in strict reminders to both consumers and distributors alike. A recall had previously been conducted, but the CFS re-iterated the importance of the trade market to discontinue selling the contaminated product that has resulted in either a diarrheal or vomiting type food poisoning. It was noted that such types of food poisoning are easily avoidable and can be prevented by ensuring food is cooked and stored properly (5).
E. coli, a predominant leader and member of the Enterobacteriaceae family, is a facultatively anaerobic, Gram-negative, rod shaped bacterium found in the lower intestinal tract of warm blooded animals. E. coli constitutes approximately .1% of normal gut flora in humans, strains proven harmless and even beneficial, contributing to vitamin production and the breakdown of food otherwise not digestible. Certain serotypes of E.coli are identified as causing severe food poisoning, on occasion leading to recall of certain contaminated food products. Other strains can become pathogenic, causing potential disease and infection, when normal flora is disturbed. Historically, this bacterium is versatile, growing in the presence or absence of oxygen and is easy to adapt to its environment or habitat.
Recent international news has directed much attention to scientists in China as further research is gathered, potentially pointing to the benefits of using E.coli in biofuel production. This is achieved “by bombarding sample of Escherichia coli bacteria with deadly heat.” (1) This unusual but effective method could set off an “evolutionary explosion” and more news is surely to come as the experiments continue.
- Chen, Stephen. “Chinese Scientists Create E.coli Superbacteria for Possible Use in Biofuels.” South China Morning Post, 26 Apr. 2015. Web. <http://www.scmp.com/tech/science-research/article/1776449/chinese-scientists-create-e-coli-superbacteria-possible-use>.
- McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology BIO 203 Louis Community College at Meremac (2011): pg. 10, 18-19, 28-29, 36-39.
- Todar, Kenneth. “B. Cereus Food Posioning.” Todar’s Online Textbook of Bacteriology. Madison: Kenneth Todar, PhD, 2012.
- Tortora, Gerald J., Berdell R. Funke, and Christine L. Case. Microbiology: An Introduction. Tenth ed. Pearson Benjamin Cummings, 2010. 20, 69-70, 88, 317.
- “Hong Kong Officials Warn of Bacillus Cereus Tainted Bean Curd.” Outbreak News Today. The Global Dispatch, Inc., 10 Apr. 2015. Web. <http://outbreaknewstoday.com/hong-kong-officials-warn-of-bacillus-cereus-tainted-bean-curd-24328/>.