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Serratia marcescens & Bacillus cereus Report Essay

Introduction
The purpose of this study is to differentiate and identify two unknown organisms provided by the instructor in a nutrient broth. It is only known that the two organisms are from vomit; one is gram-positive and the other is gram-negative. It is necessary to first separate the two organisms by inoculating a nutrient agar plate using the streak-plate method. The initial streak-plate procedure was performed and placed in the incubator at 37◦C for 24-48 hours. Upon observing the growth on this plate, it is fairly obvious that one of the organisms is Serratia marcescens. However, it is necessary to perform the requisite tests in order to be certain that this is accurate. The growth on this streak plate is bright red and white. With the color contrast, the separation of organisms is certain and pure cultures can be grown. Each organism was used to inoculate a liquid nutrient broth which was then incubated at 37◦C for 18-24 hours. The red growth is labeled #1 and the white growth #2. Subsequent tests and procedures are described elsewhere in this report.

Procedures

Gram Stain: The organisms must first be identified as gram-negative or gram-positive. This is determined using a Gram Stain process. (Cappuccino & Sherman, 2008, p. 73) Using the fresh pure cultures grown in nutrient broth, a sample is placed on a slide and allowed to dry completely before heat setting. The staining process is accomplished using first Crystal Violet, rinsing, then Gram’s Iodine for a mordant, rinsing with alcohol, then water, and finally staining with Safranin and rinsing with water. After blotting carefully, the slide is placed on a microscope and focused on the organism at the lowest magnification, progressing to the highest magnification with oil immersion. Using this process, the results were inconclusive for specimen #1, but #2 is definitely gram-positive rods. Specimen #1 initially appeared to be gram-positive diplococci. Further testing is needed. Phenylethyl alcohol agar (PEA): PEA is a selective medium which is used to identify gram-positive bacterium. Both organisms were inoculated onto opposite sides of the PEA plate and incubated at 37◦C for 18-24 hours. Observation shows that for #1, there is no growth and no change in the appearance of the medium, while #2 does have growth, but with no change in the appearance of the medium. This is further indication that #2 is gram-positive, while #1 is most likely gram-negative.

MacConkey agar: MacConkey agar is both a differential and selective medium used to select for gram-negative bacteria. It also differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria. Only #1 was used to inoculate this medium. After inoculation, the plate was incubated at 37◦C for 18-24 hours. Observation shows there is a bright red growth but no change in the appearance of the medium. This indicates no lactose fermentation, however since there is growth, this means the bacterium in gram-negative. Mannitol salt agar: This medium was inoculated with both specimens and incubated at 37◦C for 18-24 hours. There being no growth and no change in appearance of the medium for either organism, this indicates there was no mannitol fermentation and they do not tolerate salt.

Methyl Red/Voges-Proskauer tests: The Methyl Red-Voges Proskauer medium was used to perform two tests. The methyl red test is used to identify enteric bacteria based on glucose metabolism. The Voges-Proskauer test is used to determine if an enteric bacterium produces acidic or neutral end products. First, the medium was inoculated with isolate #1, and incubated at 37◦C for 48 hours. After incubation, 2.5 ml of the medium was transferred to another tube to be used for the Voges-Proskauer test. For the methyl red test, five drops of pH indicator methyl red is added to one of the tubes of medium. After being gently mixed by rolling the tube between the palms of the hands, the results are observed. The MR-VP broth has changed to a yellow-orange color. This is a negative reaction and indicates that pyruvic acid was metabolized to neutral end products. For the Voges-Proskauer test, 10 drops of Barritt’s reagent A is added to the second tube of medium.

The culture was shaken, then immediately 10 drops of Barritt’s reagent B was added and it was shaken again. For the next 15 minutes, the culture was shaken every 3-4 minutes. Observations were made at 15 minutes. There was no change in the medium which indicates a negative reaction and also indicates that #1 does not ferment glucose. This is another contradiction of expected results and is contradicted in the next test. Triple Sugar-Iron (TSI) agar test: This test was used for both isolates, #1 and #2, and is designed to differentiate based on differences in carbohydrate fermentation patterns and hydrogen sulfide production. The medium used is a TSI slant. Each isolate was inoculated into a TSI slant by using a sterile needle with a stab-and-streak technique. After inoculation, the slant tubes were incubated at 37◦C for 18-24 hours. Observation showed #1 was red on the slant but yellow in the butt of the tube. In addition, the stab pathway revealed some motility of the organism. This indicates the organism is a motile, anaerobe that ferments glucose with no gas. Upon observing #2, the entire medium was yellow with a small amount of liquid at the bottom of the slant, and slight motility in the stab pathway. This indicates the organism is a lactose/ sucrose/glucose fermenter, an anerobe and has motility. Catalase test: This test is to determine whether the isolates can produce catalase in order to degrade toxic hydrogen peroxide. To do this, a few drops of 3% hydrogen peroxide were placed on a smear of each of the isolates. As expected, isolate #1 bubbled nicely, indicating the presence of catalase and allowing the conclusion that #1 is a facultative anaerobe instead of a strict anaerobe. Isolate #2 had no reaction so the result is, it does not produce catalase.

Oxidase test: This test is designed to distinguish among groups of bacteria based on cytochrome oxidase activity. Oxidase enzymes play an important role in electron transport during aerobic respiration. Both isolates were tested for oxidase by adding p-aminodi-methlyaniline oxalate to colonies grown on a medium, and neither had a reaction, indicating that they do not produce cytochrome oxidase.

Chocolate agar: This medium was used to grow colonies of isolate #2 to be used in the oxidase test. After inoculation, the plate was incubated at 37◦C for 18-24 hours, before the test. There was heavy gray growth on this enriched medium.

Blood agar: Blood agar can be used to cultivate fastidious organisms and also shows the hemolytic properties of some microbes. Bacterium #2 was inoculated onto a blood agar plate using a sterile loop. The plate was incubated at 37◦C for 24 hours. It was then observed that there was heavy growth on the plate with a clear ring around the colonies. This indicates lysis of red blood cells occurred, which is beta-hemolysis. Starch hydrolysis test: Starch agar is used to determine if bacteria produce the enzyme amylase which breaks down starch, causing hydrolysis. The starch agar plate was inoculated with isolates #1 and #2 using a sterile loop. After inoculation, the plate was incubated at 37◦C for 24 hours. The plate was then flooded with Gram’s iodine which reacts with starch to turn purple-blue. Both bacteria had growth on the starch, however, only isolate #2 had a ring around the colonies. This indicates that the bacterium is positive for starch hydrolysis and the presence on amylase. #1 was negative for starch hydrolysis, not producing amylase. The following tables show the physiological and biochemical tests performed for this study, as well as the student’s observations and interpretations.

Table 1: Physiological & Biochemical Test Results – #1 Serratia marcescens Test
Reagent or Media
Temperature
Observations
Results
Interpretations
Gram Stain
Crystal violet, iodine, alcohol, safranin

—————–
Reddish-purple small rods
Gram negative
Slightly inconclusive. Looks like coccobacilli.
Selective media

Phenylethyl alcohol agar

37◦C
No growth
No change in medium
Gram negative
As a selective medium, PEA identifies this as a gram-negative bacterium. Differential/ Selective media
MacConkey agar

37◦C
Red growth
No change in medium
Gram negative
Indicates this is a gram-negative bacterium
No lactose fermentation

Mannitol salt agar
37◦C
No growth
No change in medium

No mannitol fermentation
Does not tolerate salt
Methyl Red
MR-VP broth
Methyl red indicator

37◦C
Yellow-orange color in broth
Negative
Indicates that pyruvic acid was metabolized to neutral end products Voges- Proskauer
Barritt’s reagents A & B

37◦C
No change in medium
Negative-should be positive
Does not ferment glucose
(contradicted below)
Triple Sugar-Iron agar

TSI slant

37◦C
Red color on slant, yellow in bottom, movement away from stab site Motile
Anaerobic
Glucose fermentation, no gas
Catalase
3% hydrogen peroxide

————–
Bubbles present
Positive for catalase
Indicates presence of catalase; facultative anaerobe
Oxidase
p-amino-dimethylaniline oxalate

————–
No change in medium
Oxidase
Negative
Does not produce cytochrome oxidase
Starch hydrolysis
Starch agar
Gram’s iodine
37◦C
Growth
No change in medium

Negative
Does not produce amylase

Table 2: Physiological & Biochemical Test Results – #2 Bacillus cereus Test
Reagent or Media
Temperature
Observations
Results
Interpretations
Gram Stain
Crystal violet, iodine, alcohol, safranin

—————–
Purple rods
Gram positive
Definitely gram positive rods
Selective media
Phenylethyl alcohol agar

37◦C
Growth
No change in medium
Gram positive
PEA selects for gram positive organisms
Differential/ Selective media
Mannitol salt agar

37◦C
No growth
No change in medium

——————–
No mannitol fermentation
Does not tolerate salt
Enriched agar
Chocolate agar
37◦C
Heavy gray growth

Grows well on this medium

Blood agar
37◦C
Growth with clear area around colonies
Positive for beta-hemolysis
An expected result for B. cereus
Triple Sugar-Iron agar

TSI slant

37◦C
Medium turned yellow, small amount of liquid, motile
Positive for lactose/sucrose/ glucose fermentation
Fermentation occurred creating an acidic pH in the medium
Catalase
3% hydrogen peroxide
————–
No reaction
Negative for catalase
No catalase is present; anaerobic
Oxidase
p-amino-dimethylaniline oxalate

————–
No change in medium
Oxidase negative
Does not produce cytochrome oxidase
Starch hydrolysis
Starch agar
Gram’s iodine
37◦C
Growth with ring around colonies
Positive for starch hydrolysis
Indicates the presence of amylase

Discussion
The identification of bacteria #1 was very easy since there are few organisms which have a red growth, and Serratia marcescens is the only one that is on our unknowns list. When the Gram stain was inconclusive for this bacterium, it was sensible to use other tests to positively identify it. By inoculating PEA and MacConkey agar, it was possible to conclusively identify #1 as a gram-negative organism. Observation of the bacterium with oil immersion, seemed to indicate a diplococcus, however after completing the aforementioned physiological and biochemical tests, it must be concluded that isolate #1 is probably a short rod. Once it is determined to be a gram-negative rod, the oxidase test being negative leads to doing a methyl red test. This test is also negative. The MacConkey agar indicates there is no lactose fermentation, leading to the final conclusion that this organism is Serratia marcescens. Organism #2 was obviously a gram-positive rod from the very first Gram stain. A spore stain was not done, however positive result for the starch hydrolysis test leads to the final conclusion that this unknown is Bacillus cereus. Several other tests were done, such as for detection of hemolysis and glucose fermentation. Each result was consistent with expected results for B. cereus.

Epidemiology

The patient presenting with vomiting likely has food poisoning due to the contamination of a starchy dish such as pasta salad or rice. Bacillus cereus can be characterized by nausea and vomiting within ½ – 6 hours after ingesting the contaminated food. Other symptoms may include abdominal pain and headache. There is also a diarrheal type of food poisoning due to B. cereus, however in this instance the illness presents only with vomiting. The second organism, Serratia marcescens, might be present in the vomit due to the same contaminated food; however it could also have been in the intestines of the patient, without causing a problem. (General Background, 2009) B. cereus food poisoning is usually self limiting and not very severe. It has a very quick onset and usually resolves within several hours. Because of this, the illness often goes unreported. This emetic food poisoning is common year round and can affect everyone. Most people are unaware that uncooked rice is potentially a hazardous food since B. cereus can be present and actually survive cooking. Prevention includes refrigerating rice products right away instead of cooling before putting in the refrigerator. Good hygiene is always important, but be aware that Bacillus cereus can be in uncooked rice, dried beans, cereals and spices, and this bacterium is resistant to heat.

BIBLIOGRAPHY
(n.d.). Retrieved May 10, 2009, from Wikipedia: The Free Encyclopedia: http://en.wikipedia.org/ Alexander, S., & Strete, D. (2001). Microbiology: A Photographic Atlas for the Laboratory. San Francisco, et al: Daryl Fox. Black, J., & Black, L. (2008). Microbiology: Principles and Explorations (7th ed.). Jefferson City: John Wiley & Sons, Inc. Breed, R. S. (1957). Bergey’s Manual of Determinative Bacteriology (Seventh ed.). Baltimore: The Williams & Wikins Company. Cappuccino, J. G., & Sherman, N. (2008). Microbiology: A Laboratory Manual (Eighth ed.). San Francisco, CA: Pearson Benjamin Cummings. General Background. (2009). (T. W. Firm, Producer) Retrieved May 10, 2009, from Serratia- Marcescens.org: http://www.serratia-marcescens.org/index.cfm Katelijne Dierick, e. a. (2005, Aug). Journal of General Microbiology. Retrieved May 10, 2009, from Pub Med Central: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1233987 Nutrition, C. f. (2009, April 9). Bad Bug Book: Foodborne Pathogenic Microorganisms. Retrieved May 10, 2009, from United States Food and Drug Administration: http://www.foodsafety.gov/~mow/chap12.html


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