Example of an Unknown Lab Report in Microbiology

             Unknown Lab Report 

Liwei Jiao

12/03/13

General Microbiology

Fall/2013

INTRODUCTION

Microbiology is the study of microorganisms. Microorganisms have much longer history than that of mankind. It is fact that human beings survive in microorganisms. Identifying the unknown organisms is significant meaning for human beings to maintain a healthy environment, prevent infectious diseases, conduct biomedical researches, and develop new antibiotic drugs. This semester, several biochemical tests and techniques were provided and could be used to identify two unknown bacteria from #101 test tube.

MATERIAL AND METHODS

The number 101 tube containing two unknown bacteria was given out by the microbiology instructor. The methods learned from previous labs were applied to identify the unknown bacteria. Procedures were followed as provided in the general microbiology lab manual by McDonald et, unless otherwise noted.

The first procedure did was streaking the unknown out on a Nutrient Agar and Mannitol Salt Agar, using the quadrant streak method provided in the lab manual. To do this was to get two distinct bacteria colonies (gram + and gram -). After the plates were incubated, put the plates into the 37-degree incubator; and then, isolated those colonies on Nutrient Agars to let them grow. After that, gram stain method was needed to determine the shape and the type of those two distinct bacteria. For Gram negative bacteria, EMB (EOSIN-METHYENE BLUE AGAR), MR (METHYL RED) and UREA tests were used. The organism was incubated on EMB agar, MR tube and UREA broth tube. For gram-positive bacteria, MR (METHYL RED) and Caselin testes were used. The Gram positive bacterium was incubated into VP tube and Milk agar. Note all of the biochemical tests were provided in the lab manual by McDonald et al. (1).

Table 1 Gram Negative Bacterium includes the test, purpose, reagents, observations and results.

All of the following tests were performed on the unknown:

1. Nutrient Agar
2. Gram stain
3. Eosin-Methyene Blue (EMB)
4. Methyl Red

Table 2 Gram Positive Bacterium displays the test, purpose, reagents, observations and results.

All of the following tests were conducted on the Gram Positive unknown:

1. Mannitol Salt Agar (MSA)
2. Nutrient Agar
3. Gram stain
4. Urea
5. Casein
6. Oxidase

RESULTS:

The unknown #101 was determined having Gram positive bacillus and gram-negative rods. Morphology on Nutrient Agar: Gram positive bacillus had large raised opaque colonies with irregular margins; and Gram negative rods had white colonies. VP and Caselin tests were used for testing gram positive bacteria. EMB (EOSIN-METHYENE BLUE AGAR), MR (METHYL RED) and UREA tests were used testing Gram negative bacteria. MR (METHYL RED) and Caselin testes were used testing Gram positive bacteria. Table 1 & table 2 lists observations, results and purposes of all the tests, as well as shown in the flowchart.

Table 1: Gram Negative Bacterium

Table 2: Gram Positive Bacterium

DISCUSSION/CONCLUSION

The isolation only presented one type of bacteria colony. After doing Gram stain on this bacterium and concluding it was gram negative rods, the EMB test was used to narrow the spectrum. The result was positive and pinkish-purple color showed on the agar which means bacteria fermented lactose producing weak acids as the end product; so Escherichia coli (green color) and Pseudomonas aeruginosa (no reaction) were eliminated. Urea was the next step to narrow the spectrum. It showed negative result. Only Enterobacter aerogenes was negative, while the other two Klebsiella pneumonia and Proteus vulgaris were positive for urease. Additional Methyl Red test was conducted to verify the result. After adding methyl red indicator into the broth, there was no color change, negative result; and it confirmed E. aerogenes was the Gram-negative unknown.

The bacteria isolated in nutrient agar from the unknown #101 tube didn’t have Gram-positive colonies. So the sample was streaked onto a MSA agar that only selected Gram-positive bacterium. MSA agar turned yellow from red-pink color surrounding the white bacterium and that was a positive result. It’s the growth of a Gram-positive bacterium. Isolating the Gram positive bacterium into nutrient agar let it grow. After doing Gram stain on this bacterium, the purple rods appeared concluding it was a Gram-positive bacillus. Based on the bacillus shape, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus facecalis (round shape, cocci) were crossed out; and only Bacillus cereus and Bacillus subtilis were left. The Methyl red test was chose due to Bacillus cereus was a positive result while Bacillus subtilis was negative. Adding Methyl red indicator into broth yellow color appeared. That’s negative result so the bacterium was Bacillus subtilis. An additional Casein test was done to verify the result. Incubating the bacterium onto milk agar, a clearing area appeared around the bacterium growth, which is positive for casein. That means the bacterium was Bacillus cereus. Due to the results of the Casien test and Methyl red being opposite to each other, a further test, the oxidase test, was conducted. The bacterium made the color of the paper having oxidase agent turn purple, blue. It was a positive result. Therefore, the bacterium must be Bacillus cereus. 

The identifications for Gram-negative and Gram-positive bacteria were verified by additional tests.

The problem encountered was that the contamination of isolated Gram-positive nutrient agar from MSA. One large contaminated colony appeared that is distinct from the Gram positive bacterium colony shown on the nutrient agar. The most possible reason is that when doing the isolation from MSA to nutrient agar, the inoculating loop mixed two types of bacteria or is not completely sterilized by flame. New isolation was conducted. There is only Gram-positive bacterium grew on the nutrient agar. The problem was solved.

Another problem met was the contamination of isolated Gram positive nutrient agar from MSA. One large contaminated colony distinct from the Gram positive bacterium colony showed on the nutrient agar. The most possible reason is that when doing the isolation from MSA to nutrient agar, the inoculating loop mixed two types of bacteria or is not completely sterilized by flame. A new isolation was conducted. There is only Gram positive bacterium grew on the nutrient agar. Problem was solved.

Enterobacter aerogenes is a Gram-negative rod-shaped bacterium belonging to the Enterobacteriacease family. It commonly resides in the human gastrointestinal tract and does not cause infections/diseases in healthy people but inthose who have weakened immune system. E. aerogenes is an intractable bacterium and is resistant to most antibiotics like cephalosporins, which is overused in hospitals due to it containing β- lactamase enzyme can break B-lactam ring in the antibiotics; so the antibiotics doesn’t work anymore. Therefore, there are limited choices to inhibit E. aeurogenes infection.  It mainly has three mechanisms to counteract the effects of antibiotics: inactivating enzymes, misleading antibiotic drug targets and preventing the drug from entering the body cells. Hospitals have high rates of E. arogenes infections. The reason is that healthcare workers use contaminated medical devices during surgical procedures leading to cross-infection, which facilitates the transmission of Enterobacter aerogenes ( “Enterbactor aerogenes” 2013).

References

1. McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology.
2. “Enterobacter aerogenes.” Bioquell. N.p., n.d. Web. 25 Apr. 2013.
<http://www.bioquell.com/technology/microbiology/enterobacter-aerogenes/>.