UNKNOWN LAB REPORT, Microbiology
Unknown Number 114
Rhea Becnel
INTRODUCTION
Knowing the identity of different microorganisms is important for various reasons. Different microbes can be bacteria, viruses, fungi, and helminths. It is important to know the differences and identities between these microbes to treat them in a patient. It is also useful to know the identity of different microbes when trying to treat a patient because microbes can have cell walls that differentiate them. Knowing this information can help determine the best antibiotic to use. To identify the unknown bacteria in this study, testing methods were used that have been discussed and learned in the microbiology class and laboratory.
MATERIALS AND METHODS
An unknown test tube labeled 114 was given by the lab professor. The methods and tests that have been learned in class and lab for identifying different bacteria were used to identify the unknown. Tests completed were done as shown in the laboratory manual by McDonald et al. (1) unless stated elsewhere.
The test tube contained two different bacteria, a Gram-positive bacterium, and a Gram-negative bacterium. The first procedure done was a streak technique using a Nutrient Agar plate. This was done to help isolate the bacteria, so a pure, isolated colony could be gathered. After the plates were streaked they were incubated at 37 degrees Celsius. After 48 hours of incubation, the streak plate was observed. The streak plate had a noticeable color change of blue/green. To determine if this was contamination or the bacteria causing the color change, another streak plate was done. One isolated colony was observed on the first streak plate. The colony was carefully removed and streaked onto another Nutrient Agar plate to get pure isolation and both the isolation streak and new streak were incubated at 37 degrees Celsius. After incubation it was confirmed that the color change was from the bacteria and not contamination. Also, a Gram stain, as described in the lab manual, was performed on the isolated Nutrient Agar plate. This procedure was done to determine whether the bacteria were Gram-positive or Gram-negative. The results from the Gram stain indicated the isolated bacterium was gram-negative rods. This isolated bacterium was used for future tests to determine the exact bacteria.
After isolating the Gram-negative bacteria, the Gram-positive bacteria needed to be isolated. To isolate the Gram-positive bacteria, a Mannitol Salt Agar plate was streaked from the original test tube. The MSA was incubated at 37 degrees Celsius. After incubation, no growth was observed on the MSA plate. Since this was the only test that could be performed to promote gram-positive growth, the isolated Gram-positive bacteria was obtained from the instructor. A Gram stain was performed to determine the shape. The results indicated Gram-positive rods. After having both bacteria isolated, tests, as listed below, were performed following the methods in the lab manual by McDonald et al. (1).
The following tests were performed:
- Mannitol test
- Casein test
- Maltose
- Glycerol
- Methyl Red
RESULTS
The unknown, labeled 114, had the following characteristics on the Nutrient Agar plate: medium-sized, circular, and cream-colored colony. After a Gram stain indicated it was a Gram-negative rod, a mannitol test was performed using a mannitol tube. The result of this test showed no mannitol fermentation. This narrowed the list of bacterium down to Proteus vulgaris and Pseudomonas aeruginosa. The second test performed was a Casein test using a Milk Agar plate. This test resulted in a clearing around the bacteria indicating a positive result for casein hydrolysis. Lastly, a Maltose test was done just to make sure the results matched the other test results which it did. It was a negative result. After this test, it was determined that the Gram-negative bacterium was Pseudomonas aeruginosa.
After a Gram stain indicated the Gram-positive bacteria were rods, a mannitol test was performed using a mannitol tube. The results indicated no mannitol fermentation. A Maltose test was performed giving a positive result for maltose fermentation. Next, a Casein test was done giving a positive result for casein hydrolysis. A Glycerol test was done, but the results were negative for glycerol production which did not match up to the results of the two previous tests. Therefore, a Methyl Red test was done. After the pH indicator was added, it turned red indicating a positive reaction. After multiple tests, it was determined the Gram-positive bacteria was Bacillus cereus. The results are also noted in a table format and flow chart format.
TABLE 1. Unknown 114 Gram Negative Biochemical Results
| TEST | PURPOSE | REAGENTS | OBSERVATIONS | RESULTS |
| Gram stain | Determine the Gram reaction | Crystal violet, Iodine, Alcohol, Safranin | Pink rods | Gram-negative rods |
| Mannitol test | Determine fermentation of a specific carbohydrate, mannitol | Phenol red mannitol broth | No color change, the color of the broth remained red | Negative mannitol fermenter |
| Casein test | Determine casein hydrolysis | Milk Agar plate | Clearing around bacteria | Positive for case production |
| Maltose test | Determine fermentation of specific carbohydrate, maltose | Phenol red maltose broth | No color change, the color remained red | Negative for maltose fermentation |
TABLE 2. Unknown 114 Gram Positive Biochemical Results
| TESTS | PURPOSE | REAGENTS | OBSERVATIONS | RESULTS |
| Gram stain | Determine the Gram reaction | Crystal violet, Iodine, Alcohol, Safranin | Purple rods | Gram-positive rods |
| Mannitol test | Determine fermentation of a specific carbohydrate, mannitol | Phenol red mannitol broth | No color change, the color of the broth remained red | Negative mannitol fermenter |
| Casein test | Determine casein hydrolysis | Milk Agar plate | Clearing around bacteria | Positive for case production |
| Maltose test | Determine fermentation of specific carbohydrate, maltose | Phenol red maltose broth | The color changes to yellow | Positive for maltose fermentation |
| Glycerol test | Determine fermentation of specific carbohydrate, glycerol | Phenol red glycerol broth | No color change, the color remained red | Inconclusive, results did not match other tests |
| Methyl Red test | Determine if acid is produced from glucose fermentation | MRVP | After the pH indicator was added, the color changed to red | Positive, acid produced from glucose fermentation |
NOTE: Flowcharts were removed due to problems with the formatting.
DISCUSSION/CONCLUSION
The following bacteria were identified by following the steps and procedures in the microbiology lab manual: Pseudomonas aeruginosa and Bacillus cereus. Determining the Gram-negative rod bacteria required three tests, the Mannitol test, Casein test, and Maltose test. The Mannitol test narrowed the options down to Proteus vulgaris and Pseudomonas aeruginosa. After the Casein test, it determined the unknown bacterium was Pseudomonas aeruginosa. One more test, the Maltose test, was performed to make sure the result was accurate. The result of the Maltose test confirmed the unknown identity was correct. Determining the Gram-positive rod bacteria required five tests. First, a Mannitol Salt Agar test was done to see if the bacteria would grow. The result was no growth and a Gram-positive isolation was provided from the instructor which was then Gram-stained to determine the shape. The Gram stain led to Gram-positive rods. Knowing the bacteria was a rod it narrowed the bacteria down to Bacillus cereus and Bacillus subtilis. The tests performed needed to make sure they led to those two bacteria. A Casein and Maltose test were performed both of which came back positive. Then, a Glycerol test was performed but the result came back negative when it should have been positive to match the two previous tests. Therefore, a Methyl Red test was done and came back with a positive result. This result matched the results from the Casein and Maltose test and confirmed the Gram-positive rod to be Bacillus cereus.
Bacillus cereus is a bacterium which produces toxins. These toxins can cause two types of illness: one type is characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting (2). Typically, this bacterium is known as food poisoning and can be obtained through various foods. The symptoms of B. cereus diarrheal-type food poisoning mimic those of Clostridium perfringens food poisoning (3). A wide variety of foods including meats, milk, vegetables, and fish have been associated with diarrheal-type food poisoning (3). Rice-based foods are attributed to vomiting and nausea. Fried rice is a leading cause of B. cereus emetic-type food poisoning in the United States (4). The typical duration of Bacillus cereus food poisoning can last up to 24 hours. If a person contracts Bacillus cereus food poisoning, it is helpful to get plenty of fluids to prevent dehydration and get plenty of rest. This type of food poisoning can also be prevented. If food is to be stored longer than two hours, keep hot foods hot (over 140°F) and cold foods cold (40°F or under) (2). If fluids cannot be kept down or if symptoms persist, it is advised to call a doctor.
REFERENCES
(1) McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General
Microbiology. St. Louis Community College at Meramec, 2011. Print.
(2) Foodsafety.gov. Bacillus cereus. Web 1 December 2013. http://www.foodsafety.gov/poisoning/causes/bacteriaviruses/bcereus/
(3) U. S. Food and Drug Administration. BBB-Bacillus cereus and other Bacillus spp. Web 1 December 2013. http://www.fda.gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm070492.htm
(4) Todar, Kenneth. Todar’s Online Textbook of Bacteriology. Bacillus cereus Food Poisioning. http://textbookofbacteriology.net/B.cereus_2.html
