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Microbiology Lab Unknowns Presentation

Presentation of the Identification of Two Unknowns

Kyle Black

on 20 November 2012

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Transcript of Microbiology Lab Unknowns Presentation

Bacterial Unknowns Kyle Black Mixed Culture #4 Plating and Gram Staining Nutrient Agar
37 Celsius * translucent
* small, circular colonies
* undulate margin
* larger individual colonies
* "wet" in appearance * White
* dry, "scale-like" appearance
* irregular form
* umbonate elevation w/ crystal in center using a sterile loop, pick an isolated colony from the mixed culture, streak for isolation on nutrient agar, and incubate at 37 degrees Celsius "A" "B" Four Simple Steps to Gram Staining:

1. add crystal violet, wait for 1 minute, rinse with DI water
2. add iodine, wait for 1 minute
3. rinse iodine using decolorizer
4. add safranin, wait for one minute, rinse with DI water Bacteria "A" * Gram-positive
* long rods
* attached end-to-end
* motile
* spores (?) Bacteria "B" * Gram-negative
* short, irregular rods
* no spores
* clusters So what do I have? Gram-Positive Rods from Class: Gelatin Hydrolysis Litmus Milk Blood Agar Bacillus cereus
Bacillus subtilis
Mycobacterium smegmatis * results: positive
* eliminates M. smegmatis results: no change (blue)
eliminates B. cereus results: alpha-hemolysis -- discoloration of media without clearing Catalase Test results: positive Phenol Red Mannitol results: positive for acid production (yellow in appearance) tests for the presence of gelatinase, an enzyme that allows for the breakdown of gelatin
all species of Bacillus produce gelatinase, M. smegmatis does not produce this enzyme four reactions: lactose fermentation, reduction of litmus, casein coagulation, and casein hydrolysis
B. cereus: peptonization, alkaline reaction
B. subtilis: no reaction tests for the presence of hemolysins, enzymes that breaks down red blood cells and hemoglobin; three types of results used to differentiate several bacteria types B. cereus: beta-hemolysis
B. subtilis: alpha-hemolysis Methyl Red (MR) & Voges Prokauer (VP) results: positive for MR, negative for VP (+/-) Conclusion:
Bacteria "A" is Bacillus subtilis tests for the ability of organism to utilize mannitol for fermentation, acidic byproducts are often produced in these pathways; phenol red turns yellow at a pH below 6.8 B. cereus: cannot ferment mannitol
B. subtilis: ferments mannitol methyl red is a pH indicator used to test the presence of acidic byproducts of fermentation, will turn red at pH below 4.4
VP tests for the production of acetoin through butandiol fermentation production, Kovak's reagent turns red in presence of acetoin B. cereus: MR-/VP+
B. subtilis: MR+/VP- ”Biol 465 General Microbiology Lab: Laboratory Manual Fall 2012”, Dr. Jack Kennell, St. Louis University, 2012. Bacillus spp.: positive
M. smegmatis: positive or negative tests for the presence of catalase, an enzyme that detoxifies hydrogen peroxide, breaking it down into water and gaseous oxygen; most bacteria species have this enzyme Holt, J. G. (1994). Bergey’s Manual of Determinative Bacteriology. Lippincott Williams & Wilkins, Philadelphia, 787 pages. ROUND TWO!!! Bacteria "B" mannitol lactose glucose sucrose Carbohydrate Fermentation four carbohydrate broths were inoculated with bacteria sample; each broth contained phenol red, which changes from red to yellow at pH below 5.2; color change indicates the ability of the bacteria to utilize that carbohydrate type in fermentation pathways Carbohydrate Fermentation Results mannitol: positive (acidic)
lactose: no change
glucose: positive (acidic)
sucrose: no change mannitol lactose glucose sucrose Comparison of Unknown "B" to Known Bacteria only S. typhimurium was similar in results acid production
acid/gas production
no change SIM Deep Indole Production Motility Hydrogen Sulfide Production stands for "sulfer indole motility": 1. enzyme thiosulfate reductase reduces sulfur to hydrogen sulfide gas, which reacts with ferrous sulfate to produce black precipitate
2. enzyme tryptophanase breaks down tryptophan into indole and other products, which turns red in the presence of Kovak's reagent
3. motile organisms will show diffuse growth throughout media S. typhimurium

positive Bacteria "B"

negative S. typhimurium

negative Bacteria "B"

negative S. typhimurium

positive Bacteria "B"

negative Simmon's Citrate Agar enteric bacteria?? many Gram-negative rods are found inside of the intestines, appearing as both pathogenic and non-pathogenic
examples: Salmonella species, Shigella species, E. coli, Enterobacter species, Klebsiella species, Salmonella species, and Proteus species agar containing citrate as sole carbohydrate source, as well as Brom thymol blue, which transitions from green to blue during alkaline citrate oxidation Bacteria "B": no growth (negative) Shigella?? S. flexneri S. boydii S. dysenteriae S. sonnei According to Bergey's... "Straight rods. Gram negative. Nonmotile. D-glucose...catabolized with the production of acid...methyl red positive, Voges-Proskauer and Simmon's citrate negative...[hydrogen sulfide] is not produced; urea is not hydrolyzed ...carbohydrates fermented include D-mannitol..." (Holt 187). based upon this description, I did MR-VP test and urea test, which produced the same results as expected Now for the species... ornithine decarboxylase ONPG (Beta-galactosidase test) tests for the presence of ornithine decarboxylase, an enzyme that catalyzes the decarboxylation of ornithine into putrescine; bromscresol purple turns purple in alkaline conditions of reaction S. boydii, S. dysenteriae, and S. flexneri: negative

S. sonnei: positive S. boydii, S. dysenteriae, and S. flexneri: negative

S. sonnei: positive tests for the presence of beta-galactosidase, an enzyme required for the breakdown of lactose, molecular decoy of lactose present in media called Orth-nitrophenyl-b-D-galactopyranoside (ONPG), enzyme will break down this to form the yellow indicator O-nitrophenol unable to conduct these tests, I concluded: Bacteria "B" must be from the genus Shigella Leboffe, M. J. and Pierce. B. E. (2011). A Photographic Atlas for the Microbiology Laboratory. Morton Publishing, Englewood: 256 pages. http://www.microbiologyinpictures.com/shigella.html
Provides information on characterization tests of Shigella species. http://0.tqn.com/d/webclipart/1/0/0/J/5/Test-Tubes.png http://0.tqn.com/d/webclipart/1/0/0/J/5/Test-Tubes.png http://postfiles3.naver.net/data32/2008/4/27/242/0005_llllllsan.jpg?type=w2 Bacillus subtilis Genus Bacillus (Bauman 330):
Gram-positive rods that form endospores
both pathogenic and non-pathogenic
commonly found in soil
certain species are response for antiobiotic production, including polymyxin and bacitracin
one (in)famous species of this genus is Bacillus anthracis, the bacteria responsible for anthrax
a gene for Bt toxin, a crystalline protein produced by B. thuringiensis, was introduced in genetically-modified corn

Bacillus subtilis
uniform-shaped rods with oval endospores
heavily flagellated
Gram-positive model organism (Bauman 330)
commonly thought to live only in the soil, but new research shows that this species is commonly found as a commensal in the human gut (Hong 2009).
only causes disease in severely immunocompromised patients, but used as a probiotic for healthy individuals (Gibson 2005). Gibson, G. R. (2005) Functional Foods: Perspectives on foods for specific health uses (FOSHU), Colette Shortt, Yakult UK. 1: 1-7.

Hong, H.A., et al. (2009). Bacillus subtilis isolated from the human gastrointestinal tract, Res. Microbial. 160: 134-143. http://en.wikipedia.org/wiki/File:Bacillus_subtilis.jpg

Gram-negative rods, enteric bacteria, nonmotile
causitive agent of disease Shigellosis; symptoms include fever, abdominal cramps, diarrhea, and bloody stool
disease caused by type III secretion systems (20 different polypeptides) and diarrhea-producing enterotoxins
Shiga-toxin -- produced by S. dysenteriae, interrupts host cell's protein synthesis
similar toxin as produced by E. coli O157:H7
(Bauman 119, 338, 713) Genus Shigella
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