BIOL 3200 Unknown lab report PDF

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Taylor Breech Tuesday 1:00pm Unknown #428 Bacillus megaterium

Identity of Unknown: Bacillus megaterium Gram stain: While using a Brightfield microscope, with a total magnification of 1000x, a Gram positive organism in a rod shaped formed was observed. This rod shaped Gram positive bacteria led to the Bacillaceae family.

Primary media: The primary media for this organism is a TSA plate. On November 1, 2016 a TSA plate was inoculated and incubated it at 37° C for 24 hours. After 24 hours isolated colonies in quadrant 4 were observed as seen below. Small, white, round colonies appeared on the TSA plate.

Biochemical Test: Test

Media/Reagents

KOH

Slide, 3% KOH

Oxidase

Swab, Oxidase

reagent (N,N,N′,N′-

tetramethyl-pphenylenediamine)

Enzyme/End Products Indicated by Test Enzyme tested for: None End products: Cell wall lysis and release of DNA

Interpretatio n of Test

Enzyme tested for: Cytochrome C oxidase End products: None

Negative

Gram Positive Strings absent

Photo

Catalase

Slide, 3% H2O2

Enzyme tested for: Catalase enzyme, End products: H2O2 → H 2O + O 2

Positive

VogesProskauer

MRVP tube, VP-A (alphanaphthol in absolute alcohol), VP-B (40% KOH)

Negative

Starch Hydrolysis

Starch agar plate, Gram’s iodine

Differentiates microorganisms based on their ability to produce a neutral end product, 2,3 butanediol from glucose fermentation. This end product is oxidized to form acetoin that is detected with the addition of the VP reagents A & B (alpha-naphthol and KOH). Differentiates bacteria on their ability to hydrolyze starch using the enzyme α-amalase starch → glucose

Positive

Nitrate Reduction

Nitrate broth, Nitrate reagent A, Nitrate reagent B, Zinc dust

Differentiates microorganisms based on their ability to reduce nitrate to nitrite or nitrogen gas (N2)

Negative, After the addition of Nitrate A and Nitrate B there was no color change. Upon addition if Zinc the reaction tube turned red

MSA (Growth/Acid)

Mannitol Salt Agar plate

Selects for organism that can grow in 7.5% NaCl. Differentiates bacteria based on fermentation of mannitol

Growth + Acid +

Phenyl Red Mannitol

Phenyl Red Mannitol tube

Differentiates bacteria on the ability to ferment a specific carbohydrate, mannitol. If fermentation occurred, acid or acid and gas are produced

Negative

Dichotomous Key: Negative Positive

Gram Stain

Enterobacteriaaceae

Streptococcaceae

Pseudomonadaceae

Staphylococcaceae Bacillaceae

Morphology

Rods

Cocci

Enterobacteriaaceae

Morphology

Rods

Cocci

Bacillaceae

Streptococcaceae

Pseudomonadaceae

Staphylococcaceae

Negative

Positive

VogesProskauer

Negative

Bacillus cereus Bacillus subtilis

Bacillus megaterium

Catalase Positive Pseudomonadaceae Enterobacteriaaceae

Catalase

Oxidase Negative

Negative

Positive

Streptococcaceae

Staphylococcaceae

Positive Pseudomonadaceae

Enterobacteriaaceae

Confirmation Tests:

Starch Hydrolysis + MSA (G+/A+) PR Mannitol – Nitrate Reduction –

Summary: On November 1, 2016 a Gram stain, KOH, Oxidase, and catalase test was performed. After preparing a smear and observing it using a Brighfield microscope with a total magnification of 1000x, a purple, rod shaped organism was observed. After observation of the Gram stain a KOH test was performed, which confirmed that the organism was Gram positive because it did not produce a string when the bacteria was mixed with 3% KOH. The confirmation of a Gram positive, rod shaped organism led to the Bacillaceae family. Just to further confirm the Bacillaceae family, a starch hydrolysis test was performed and was positive. To differentiate the three organisms in the Bacillaceae family, a Voges-Proskauer test was performed. This test appeared yellow/brown which indicated a negative result. Upon the completion of this test, the organism Bacillus megaterium was isolated. To confirm the organism a MSA (Growth/Acid) test was performed. After inoculation and 24 hour incubation yellow growth was observed which indicated growth positive, acid positive results. The only organism in the Bacillaceae family that would yield these results would be Bacillus megaterium. Two other confirmation tests were performed, PR mannitol and nitrate reduction. After 48 hours of 37°C incubation both tests were negative. The PR mannitol tube remained red and the Nitrate broth required the addition of nitrate A, nitrate B, and zinc dust before a red color change. These five tests indicated the organism Bacillus megaterium. Throughout this experiment, no problems occurred. As a Pre-Pharmacy major, I feel that it is important to know certain bacteria’s and their characteristics in order to properly prescribe specific medications. After further research of Bacillus megaterium, I found that “B. megaterium has economic importance because of its commercially important enzymes such as penicillin amidase and steroid hydrolases. It is the major aerobic producer of vitamin B12 and is one of the organisms involved in fish spoilage” (1). I also discovered that “During the 1980s, genetic techniques of transduction, plasmid transformation, protoplast fusion and transposition became developed enough B. megaterium to apply them to the study of many of its metabolic and developmental functions. Moreover, it is increasingly used as a host to produce foreign genes since it has been found to express, secrete and process foreign proteins without degradation” (1).

Works Cited

1) Vary Patricia S. 1994. Prime Time for Bacillus megaterium. Review Article 1001–1013....