Tika Ghimirey Unknown Number 58 Staphylococcus epidermidis Proteus mirabilis

Abstract: The first unknown organism was identified as Staphylococcus epidermidis. Several tests were conducted on this organism. First the organism was inoculated using the streak plate method and then incubated on a nutrient agar slant. A gram strain was next completed. Following the gram strain several other tests were run on this organism including the Catalase test. Then a glucose + bromcresol Purple slant was inoculated, and finally a mannitol salt agar plate was inoculated. The second unknown organism was identified as Proteus mirabilis. This organism was also first inoculated using the steak plate method, then a nutrient agar slant, and finally a gram strain was performed. Then a first round of biochemical fermentation tests were completed including a lactose, glucose, and sucrose test. Following the first round of biochemical test, a second set of biochemical tests were conducted. Theses test included a urea test and indole ring test .

Introduction: The purpose of this experiment was to identity two unknown organisms by both genus and species. Experiments such as these are done many times in microbiology labs and in doctors offices when identifying what the cause of a patients sickness maybe. Identifying unknown organisms and pathogens is important in all medically related fields to be able to determine the severity of a patients condition. It is also important to determine what an unknown organism is to be able to stop its spread if it is pathogenic. Furthermore, this type of testing is important to the Department of Homeland Security when dealing with potential terroristic threats and in accessing their severity and what harm maybe done by the organism.

Materials and tests available include: Stain Name Gram Stain Schaeffer-Fulton Endospore Stain Ziehl-Neelsen Acid-Fast Stain Positive Purple Green (structure) Red Negative Red Pink Blue Purpose Detects thick peptidoglycan layer in cell wall, determine cell shape Detects presence of endospores Detects mycolic acid in cell wall

Reagent Catalase Methyl Red Indicator Voges- Proskauer Indicator Kovacs Reagent Indicator

Positive Bubbles Red Red Ring Red Ring at top

Negative No Bubbles Wheat color or yellow Wheat color or yellow Yellow or green ring at bottom, no red ring

Purpose Presence of catalase Detects use of mixed pathways in fermentation Detects use of butanediol fermentation pathway Detects production of enzyme tryptophan, producing indol

Media / Test Fluid Thioglycollate Glucose + bromcresol purple Mannitol salt agar Glucose Lactose Sucrose MR- VP (Methyl Red) MR-VP (VogesProskauer)

Positive Growth toward top or in middle Yellow Yellow Yellow Yellow Yellow Red Gas Gas Gas

Negative Growth on bottom Purple No change / red Red Red Red No Gas No Gas No Gas

Wheat / Yellow

Red Ring

Wheat / Yellow

Purpose Determine oxygen requirements of bacteria Differentiates between staph and non-staph Differentiates between types of staph Determines if the organism can ferment the glucose Determines if the organism can ferment the lactose Determines if the organism can ferment the sucrose Identifies organisms that use mixed pathways for fermentation Identifies production of acetoin, in butanediol pathway

Tryptone Simmons Citrate Urea Sims Semi-Solid Motility with Sims Semi-Solid EMB

Red Ring

Detects production of tryptophan producing indol Blue Green Tests for utilization of citrate as carbon source Pink Wheat / no color Measures the production of change urease Black precipitates Wheat / no color Tests for the production of change iron 2 sulfide (FeS) Motile / Non-motile / little to Tests whether bacteria is movement no movement motile or non-motile throughout media Color on growth No color on growth Differentiates gram-negative and enteric rod bacteria, selective for lactose fermenters

Wheat / Yellow

Procedure: First both organisms were inoculated using the steak plate method for isolation, two plates were done for each, one plate of each was incubated at 37 degrees Celsius in the incubator, while the other two plates were left out at room temperature. Then the isolated colonies from the streak plates were used to inoculate a nutrient agar slant of each organism for maximum growth and future inoculation of other media. Next gram stains were performed on both organisms to determine whether they are gram-positive or gram-negative and also to determine their shape. Organism A was identified as a gram-positive coccus. The next test ran for this organism was the catalase test to test for the presence of catalase. A small sample of the organism was place on a slide and hydrogen hydroxide was added. Then a glucose + bromcresol purple slant was inoculated using organism A, to determine if the organism was a staph or non-staph. The bacteria was transferred from the nutrient agar slant onto the glucose+bromcresol purple slant and then incubated at 37 degrees Celsius. After determining that organism A was a staph, a mannitol salt agar plate was inoculated to determine the type of staph, the inoculated plate was placed into the incubator at 37 degrees Celsius. The tests ran for organism B were slightly different, since organism B was identified as a gram-negative rod. The first set of tests ran were the fermentation tests: glucose, lactose, and sucrose, to determine if the organism could ferment the particular sugar. The organism was simply placed into the test tube with the different sugars using the inoculation loop and incubated at 37 degrees Celsius. After collecting the results for the fermentation tests the next set of tests were run. These tests included urea, to tests for the production of the enzyme urease, in this test the organism was placed into the media using the inoculation loops. Also an indole ring test was done.

Results: Organism A (Staphylococcus epidermidis) Structural Characteristic Gram Stain: gram positive / purple Cell Shape: Coccus Cell Arrangement: grape- like clusters Motility: N/A Special Stains: N/A only gram stain Colony Appearance: yellow colonies, small dots of growth, smear like appearance Growth Temperature: Equal growth at room and in incubator at 37 degrees Celsius Pigmentation: Yellow

Biochemical Characteristics Test Performed Catalase Glucose+bromcresol purple slant Mannitol Salt Agar Plate Results Bubbles produced Slant turned yellow No color change Reaction Positive for catalase production Positive for staph Negative for S. aureus (Positive for S. epidermidis)

Results: Organism B (Proteus mirabilis) Structural Characteristics: Gram Stain: Gram-negative / Red Cell Shape: Rod Cell Arrangement: individual cells, in small Motility: Yes groups Special Stains: Capsule stain (not performed) Colony Appearance: White colonies, large rounded individual spots of growth Growth Temperature: grows well at both room temperature and in incubator at 37 degrees Celsius Pigmentation: White

Biochemical Characteristics: Test Performed Glucose - fermentation Lactose - fermentation Sucrose - fermentation Urea Indole Test Results Positive yellow with gas Production Negative red with no gas production Negative red with no gas production Positive pink colored Negative- wheat colored, no red ring formation Reaction Organism can ferment glucose Organism cannot ferment lactose Organism cannot ferment sucrose Production of urease Inability to split indole from amino acid tryptophan

Conclusion: After completing all the necessary tests for each organism, organism A was identified as Staphylococcus epidermidis and organism B was identified as Proteus mirabilis. Organism A was identified as Staphylococcus epidermidis, due to the gram-positive cell wall, coccus shape, yellow colonies, positive catalase test, positive glucose + bromcresol purple slant, and negative mannitol salt agar plate, which are all characteristics of Staphylococcus epidermidis. There was almost same growth obtained using the incubator at 37 degrees Celsius and at room temperature after incubating for 48 hours. Organism B was identified as Proteus mirabilis due to the gram-negative cell wall, rod shape, positive fermentation tests for glucose, with gas production, positive urea and negative indole tests, and negative EMB test. There were no problems encountered with these organisms.

Research: Staphylococcus epidermidis is a gram-positive coccus found in the family Staphylococcaceae. When grown S. epidermidis exhibits yellow colonies and under a microscope slide produces grape like clusters. It is a facultative anaerobes, which can live through either aerobic respiration or fermentation. The optimum growth temperature for S. epidermidis is between 15 and 45 degree Celsius, and it can also survive in relatively high salt (NaCl) concentrations. Furthermore, S. epidermidis is found in the normal flora of mainly the nose, but can also be found in the skin, oral cavity and gastrointestinal tract. However, S. epidermidis can be pathogenic to humans since it is an opportunistic pathogen. Its pathogenicity can range from very mild such as skin infections to very severe if it becomes systemic. It can cause skin problems such as boils, impetigo and carbuncles. Moreover, if S epidermidis becomes systemic it can become more of a problem causing pneumonia, bacteremia, and osteomyelitis. It can also cause nosocomial infections often acquired during hospitals stays, especially ones involving surgical wounds and implanted medical devices such as catheters Proteus mirabilis is a gram-negative rod or bacillus. It belongs to the Enterobacteriaceae family, which also contains Escherichia coli and Salmonella shigella. Proteus mirabilis is a facultative anaerobe and lives well without oxygen and its optimum growth is obtained near 30 to 37 degree Celsius. Proteus species are most commonly found in the human intestinal tract as part of
normal human intestinal flora, along with Escherichia coli and Klebsiella species, of which E coli is the predominant resident. Proteus is also found in multiple environmental habitats, including long-term care facilities and hospitals.It

can be found in fresh water, soil, sewage plants, vegetables, and animal

and human. P. mirabilis can be commonly present in healthy individuals as part of the normal mucosa.
The bacterium becomes a significant problem mostly in individuals that have vulnerable immune systems and are in danger of nosocomial transmission, such as hospital patients Patients with recurrent infections, those with structural abnormalities of the urinary tract, those who have had urethral instrumentation, and

those whose infections were acquired in the hospital have an increased frequency of infection caused byProteus. The first step in the infectious process is adherence of the microbe to host tissue. Fimbriae facilitate adherence and thus enhance the capacity of the organism to produce disease. E coli, P mirabilis, and other gram-negative bacteria contain fimbriae (ie, pili), which are tiny projections on the surface of the bacterium. Specific chemicals located on the tips of pili enable organisms to attach to selected host tissue sites (eg, urinary tract endothelium). The presence of these fimbriae has been demonstrated to be important for the attachment of P mirabilis to host tissue. P.mirabilis can be treated with broad-specrum penicillins or cephalosporins except in the severe cases. Current studies show that there are a number of antibiotics that were once effective against P. mirabilis that are now useless due to extended spectrum beta lactamases.

References: Fraser, MD, Susan L. "Enterobacter Infections Treatment & Management." www.medscape.com. 07 Jan. 2010. Web. 05 Nov. 2012. <http://emedicine.medscape.com/article/216845-overview>. Kelly, Cowen. Biol 2420 Tarrant County College South Campus. 3rd ed. Boston: McGraw-Hill Companies, 2012. Print. Todar, PhD, Kenneth. "Online Textbook of Bacteriology." Online Textbook of Bacteriology. web. 05 Oct. 2012. <http://textbookofbacteriology.net/staph.html>. Murphy, Pam.Student presentation on Proteus mirabilis. University of Connecticut Web. 13 November 2012<http://web.uconn.edu>