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Example of a Microbiology Unknown Lab Paper | Unknown Bacteria Lab

By at July 2, 2014 | 8:31 am | Print

UNKNOWN LAB REPORT

Unknown  Number 109

Tyler Wolfangel

April 29, 2014

BIO 203-604

 

Introduction

The study of microbiology requires not only an academic understanding of the microscopic world but also a practical understanding of lab techniques and procedures used to identify, control, and manipulate microorganisms. The proper identification of a microorganism is not only important in a microbiology lab but also in the medical, industrial, and pharmaceutical fields. In this lab report, lab techniques and procedures learned during this course were performed to assess each students’ practical knowledge in microbiology.

The goal of this lab report is 1) to demonstrate comprehension of the methods and lab techniques learned during the semester 2) to explain the tests performed on each isolated unknown that led to the identification of each unknown 3) and to give a background on the characteristics, pathogenicity and some uses of one of the identified unknowns.




Materials and Methods

At the beginning of this project, the professor handed each student a numbered test tube containing two unknown bacteria (one Gram positive and one Gram negative). The test tube used in this study was tube 109. The instructor asked that each student isolate the two unknown bacteria and then identify each. The lab techniques and procedures used throughout this experiment were from McDonald’s laboratory manual (4).

To start, the two unknown bacteria had to be successfully isolated. Using a wire loop, a small sample of the numbered test tube was plated on a nutrient agar using the quadrant streak method (4). The numbered test tube was then placed in a refrigerator to slow down any continued growth. The nutrient agar was labeled “Isolation 1” and was placed in an incubator for 48 hours at 37 degrees Celsius. After 48 hours, the Isolation 1 plate was observed. There were 34 isolated colonies present, all of which showed the same growth pattern. Four were chosen at random and Gram stains were performed on each (procedure for the Gram stain was used according to McDonald’s laboratory manual). Each of the four Gram stains showed positive rods. Seeing that no negative Gram stains were observed, a different approach was taken. Instead, a small sample of the numbered test tube was streaked using the quadrant isolation method on an EMB (eosin-methylene blue) plate and then on an MSA (mannitol salt agar) plate (4). The EMB plate inhibits Gram positive bacteria and the MSA plate inhibits gram negative bacteria (4). So, growth on each plate should favor only one of the unknowns. The EMB plate was labeled “EMB 1” and the MSA plate was labeled “MSA 1.” Each was placed in the incubator for 48 hours at 37 degrees Celsius. After 48 hours, each plate was observed. EMB 1 had very little growth so it was returned to the incubator and allowed to grow for another 48 hours. MSA 1 had significant overgrowth and was discarded. A new MSA plate, labeled MSA 2, was created using the same quadrant isolation streak method. It was only let to incubate for 24 hours at 37 degrees Celsius instead of 48 hours. After 24 hours, MSA 2 was observed. It had many well isolated colonies. One of the colonies was transferred to another MSA plate, labeled MSA 3, using the quadrant isolation streak. (This was performed to ensure that the Gram positive bacteria were well isolated from the Gram negative.) MSA 3 was placed in the incubator at 37 degrees Celsius for 24 hours. After another 24 hours, both MSA 3 and EMB 1 were observed. Both EMB 1 and MSA 3 had well isolated colonies. One of the colonies from the MSA 3 plate was Gram stained. It showed Gram positive rods. A loopful of bacteria from that same colony from MSA 3 was transferred to a nutrient agar plate labeled “NA positive stock.” This nutrient agar plate became the stock plate for Gram positive bacteria for the rest of the experiment. One of the well isolated colonies on the EMB 1 plate was transferred to a new EMB plate, labeled EMB 2, using the quadrant isolation streak method. (This was to ensure that the Gram negative bacteria were well isolated from the Gram positive on the plate.) Both the EMB 2 plate and the NA positive stock plate were placed in the incubator at 37 degrees Celsius. After 72 hours, one of the well isolated colonies on EMB 2 was Gram stained. Gram negative rods were observed. A loopful of bacteria from that same colony was transferred to a new nutrient agar plate and labeled “NA negative stock.” It was placed in the incubator at 37 degrees Celsius. At this point, both bacteria were successfully isolated and plated on stock nutrient agar plates.

Various tests were conducted on each the unknown bacterial cultures. An explanation of each test and the results are in Table 1 (for the Gram positive bacteria) and Table 2 (for the Gram negative bacteria). The tests were performed in such a way that each test eliminated a possible unknown candidate. Chart 1 details the sequence of tests performed on the Gram positive bacteria and Chart 2 details the sequence of test performed on the Gram negative bacteria.

 

For the gram positive bacteria, the following tests were performed:

1)      Urea

2)      Catalase

3)      Casein

4)      Methyl Red

5)      Glycerol

6)      Simmons Citrate

For the gram negative bacteria, the following tests were performed:

1)      Nitrate

2)      Simmons Citrate

3)      Urea

4)      Casein

5)      Voges-Proskauer (V-P)

6)      Sorbitol

*All of the tests were performed and explained as described in the McDonald Laboratory Manual

 

Results

 

Table 1. Results of Gram Positive Unknown

TEST REAGENTS or MEDIA OBSERVATIONS RESULTS INTERPRETATIONS
Gram Stain Crystal violet, Gram’s Iodine, alcohol, safranin Purple Rods Gram positive rods
Urea Urea tube No color change; remained yellow Negative The Gram positive bacteria is unable to produce urease, which hydrolyzes urea to carbon dioxide and ammonia
Catalase Hydrogen peroxide Bubbling Positive The Gram positive bacteria possesses the enzyme catalase, which breaks down hydrogen peroxide into oxygen gas and water
Casein Milk Agar No clearing around bacteria Negative The Gram positive bacteria is unable to produce casease, which degrades the casein protein in milk
Methyl Red MR-VP tubes, methyl red dye After adding methyl red to test tube, color changed from light yellow to a darker yellow; no red was present Negative The Gram positive bacteria does not produce acid during glucose catabolism
Glycerol Glycerol tube After inoculation and incubation, the tube remained red Negative The Gram positive bacteria does not ferment glycerol
Citrate Simmons Citrate agar Agar changed color from green to blue Positive The Gram positive bacteria produces the enzyme citrase, which breaks down citrate

 

 

Table 2. Results for Gram Negative Unknown

TEST REAGENTS or MEDIA OBSERVATIONS RESULTS INTERPRETATIONS
Gram Stain Crystal violet, Gram’s iodine, alcohol, safranin Pink Rods Gram negative rods
Nitrate Nitrate broth tubes, nitrate reagent A, nitrate reagent B, powered zinc After addition of reagents A and B, color was deep red Positive Gram negative bacteria can reduce nitrate to nitrite
Citrate Simmons Citrate agar Agar changed color from green to blue Positive The Gram negative bacteria produces the enzyme citrase, which breaks down citrate
Urea Urea tube No color change; remained yellow Negative The Gram negative bacteria is unable to produce urease, which hydrolyzes urea to carbon dioxide and ammonia
Casein Milk Agar Clearing around bacterial streak Positive The Gram negative bacteria is able to produce casease
Voges-Proskauer (VP) MR-VP tubes, VP reagent A, VP reagent B After addition of reagents A and B, the tube turned yellow Negative The Gram negative bacteria does not produce acetoin as an end product of glucose fermentation
Sorbitol Sorbitol tube Color change from red to yellow Positive Gram negative bacteria ferments sorbitol

 

 




Discussion/Conclusion

The Gram positive unknown was identified to be Bacillus subtilis. The instructor verified this to be correct. This deduction was reached with several bits of evidence. Firstly, the Gram positive unknown was rod shape. This narrowed it down to both of the Bacillus species. However, the researcher felt it was important to perform a battery of tests in order to confidently conclude that the Gram positive unknown was indeed B. subtilis. So, the pre-constructed sequence of tests was still performed as if all five of the Gram positive options were still possible. Secondly, the series of tests performed on the Gram positive unknown led to B. subtilis as the only possible candidate. A negative urea test removed S. epidermidis as a possibility. A positive catalase test removed E. faecalis as a possibility. A negative casein test removed S. aureus as a possibility. And, a negative methyl red test removed B. cereus as a possibility. This left only B. subtilis. The citrate test and the glycerol test were both done to assure accuracy of the identification of B. subtilis as the Gram positive unknown. The citrate test was positive and the glycerol test was negative. Both of these tests concurred with the expected results of B. subtilis metabolism.

The Gram negative unknown was identified to be Pseudomonas aeruginosa. The instructor verified this to be correct. This deduction was reached for a couple reasons. Firstly, each of the tests in the series ruled out a possible candidate until P. aeruginosa was the only possibility left. A negative nitrate test ruled out E. coli. A positive citrate test ruled out P. vulgaris (and reinforced the decision that E. coli was not the unknown). A negative Urea test removed K. pneumoniae (and reinforced the decision that P. vulgaris was not the unknown). A negative casein test ruled out E. aerogenes as a possibility. This left only P. aeruginosa as the unknown. A VP test and a sorbitol test were performed to validate that P. aeruginosa was the unknown. A negative VP test and a positive sorbitol test concurred with the expected results of P. aeruginosa metabolism.

No difficult problems occurred with the identification of B. subtilis. The only slight issue was that B. subtilis grew extremely quickly and overgrowth was a common problem. For example, a nutrient agar stock plate for B. subtilis was inoculated almost daily to ensure the cultures used for each of the identification tests were young.

The Gram negative unknown, however, was very difficult to identify. Firstly, all of the Gram negative unknown possibilities were rods. This rendered the Gram stain useless in identifying P. aeruginosa based on shape. Secondly, the culture was very difficult to isolate because it grew extremely slowly on the EMB plates and a pure culture took two weeks to achieve. Thirdly, several of the tests had to be repeated because the first stock plate of P. aeruginosa was found to be contaminated. All of the tests had to be thrown away and repeated using a newly grown stock plate of P. aeruginosa. After a second round of tests, all of the results matched up with the expected results of P. aeruginosa except for the VP test. It turned up positive. It was performed for a third time and the result was negative. The second VP test was probably contaminated and was not a reliable test result.

 

Background Information on Bacillus subtilis

Bacillus subtilis is a very well-studied microbe. It is considered the “type” species of the Bacillus genus (2). B. subtilis, of the Family Bacillaceae, was named in 1872 by Ferdinand Cohn (3). This bacterium is also known by the names hay bacillus or grass bacillus (1). Like other bacteria in the Bacillaceae family, B. subtilis can grow in the presence of oxygen (with the help of its catalase enzyme) and is endospore-forming (1). It is predicted that it spends most of its time inactive and in spore form. B. subtilis is commonly recovered from soil, water, air, and decomposing plant material (1). Different strains of B. subtilis can be used as biological control agents under differentsituations. One of the B. subtilis strains produces a chemical called iturin (1). This chemical acts as a growth inhibitor for many other bacteria and some fungi, which allows B. subtilis to out-compete several other soil-living microorganisms (1).  Because of this strains’ inhibitory affect on many other bacteria, it is sometimes added to fertilizer to help delicate plants to grow (1). Furthermore, some probiotic companies are using B. subtilis in their probiotics because of their inhibitory affect on many pathogenic enteric bacteria (2). Some research, however, questions whether it is wise to put an endospore-producing bacteria in a human’s gut even if it currently is not pathogenic to humans. Contrary to this, studies have found that B. subtilis sometimes naturally inhabits the intestinal tracts of humans without causing any harm (2). As it currently stands, B. subtilis is believed not to be pathogenic to humans. Some other commercial applications of B. subtilis include: cleaning agents in detergents, leather processing, preparing special Japanese and Korean dishes, starch modification, and several other specialized chemicals (3).

 

References

  1. Bacillus subtilis.” Organic Resource Guide: Material Fact Sheet. http://www.cefs.ncsu.edu/newsevents/events/2010/sosa2010/20101013tomato/product01-bacillussubtilis.pdf. Accessed April 27, 2014.
  2. Cartwright, Peter. “Bacillus subtilis: Identification & Safety.” Protexin: 2009. http://www.protexin.com/attachments/Probiotic%20News%20Issue%201.pdf. Accessed April 27, 2014.
  3. O’Keeffe, Jillian. Environmental and Industrial Use of Bacillus Subtilis. Global Post. http://everydaylife.globalpost.com/environmental-industrial-use-bacillus-subtilis-30152.html. Accessed April 27, 2014.
  4. McDonald, Virginia et al. Lab Manual for General Microbiology. Saint Louis  Community College at Meramec, 2011.

 

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