Indentification of Unknown Microorganism

2785 words (11 pages) Essay

18th May 2020 Biology Reference this

Disclaimer: This work has been submitted by a university student. This is not an example of the work produced by our Essay Writing Service. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UKEssays.com.

Lab 5: Pure Culture Technique: Isolation of Bacteria

 This experiment has two techniques which are continuous and discontinuous streaking; our laboratory instructor gave us our unknown for this experiment. We sterilized our wire loop by holding until it turns bright orange-red. We wait for it to color then streak the bacteria with a loop from the unknown and made discontinuous and continuous streaking on both plates. We covered the labeled and covered the plates with tapes on the sides. Then we incubated them at 37 for 24-48  hours in an inverted position, then we observed the plates to check if there are any well- isolated colonies.

Lab 8 – Polymerase Chain Reaction ( PCR) for DNA Amplification and Sequence Analysis DNA  Extraction:

For this Lab, we turned in the heat to 100C and vortex 5% of Chelex for 60 seconds. Then we vortexed the solution for 30 more seconds and centrifuging it for 1 minute at maximum speed, which was 13,300 rpm/ 17000 g. While carefully removing the tube, we extracted 30 ul of the supernatant (contains DNA) pipette it into a 1.5 microfuge tube. Then we discard the tube with the remaining Chelex mixture. The purity was determined through a NanoDrop  ND- 1000 Spectrophotometer. This was done with the help of our Lab instructor and the remaining part in lab 8.

BLAST:

 Our lab Instructor, took our sequencing regarding our unknown and copied and pasted it into nucleotide option on the BLAST website. Once we blasted the sequence, it gave use many resulted for what genus our unknown belong to.

Lab 12- Physiological Characteristics: Biooxtidation, Hydrosis, and Others

For this experiment, a series of inoculations for various microorganisms was performed shown in the Table below. After the inoculations, all the cultures wee place into the incubator for 37C in preparation for the biochemical analysis. The nutrient agar plate was removed from the incubator after 24 hours and placed into a  4C cold room or refrigerator.

Inoculations

Biochemical Tests

Microorganism Inoculated

Test Reagent

1.      Glucose/ Dextrose broth with Durham Tubes

·         Escherichia coli

·         Staphylococcus epidermidis

·         Unknown #11

2.      Lactose broth with Durham Tubes

·         Escherichia coli

·         Staphylococcus epidermidis

·         Unknown #11

3.      MR-VP medium (mixed acid, methyl red test)

·         Escherichia coli

·         Enterobacter aerogenes

·         Unknown #11

Methyl Red

4.      MR-VP medium (butanediol Voges-Proskauer)

·         Escherichia coli

·         Enterobacter aerogenes

·         Unknown #11

Barritt’s Reagent A (alpha-naphthol) & Barritt’s Reagent B (KOH)

5.      Nutrient Agar Plates (Catalase Production)

·         Escherichia coli

·         Staphylococcus epidermidis

·         Unknown #11

3% Hydrogen Peroxide (H2O2)

6.      Nitrate broth (Nitrate Production)

·         Escherichia coli

·         Bacillus megaterium

·         Unknown #11

Nitrate Reagent A (sulfanilic acid) & Nitrate Reagent B (N, N-dimethyl-1-naphtthylamine)

7. Starch Hydrolysis

 Eschericha coli

 Bacillus Megaterium

Grams Iodine

7.      Indo-Nitrate broth (Tryptophan Hydrolysis)

·         Escherichia coli

·         Enterobacter aerogenes

·         Unknown #11

Kovac’s Reagent

8.      Urea Hydrolysis

·         Escherichia coli

·         Proteus mirabilis

·         Unknown #11

9.      Kligler’s Iron Sugar (Hydrogen Sulfide Production)

·         Enterobacter aerogenes

·         Proteus mirabilis

·         Unknown #11

10.  Simmons Citrate Agar Slant

·         Escherichia coli

·         Enterobacter aerogenes

·         Unknown #11

11.  Litmus milk Reactions

·         Escherichia coli

·         Staphylococcus epidermidis

·         Pseudomonas fluorescens

·         Yogurt

·         Unknown #11

12.  Nutrient Agar Plates (Oxidase Test)

·         Escherichia coli

     S. epidermidis

·         Pseudomonas fluorescens

       Yogurt

·         Unknown #11

Oxidase Reagent

13.  Gram Stain (Cell Wall Characteristics)

·         Unknown #11

14.  Thioglycolate (O2 Requirement)

·         Unknown #11

Table 1: Inoculations of microorganisms

Results:

Lab 12 & 13 – inoculations & Biochemical Analysis

Table 2: The IMViC Test

I

M

V

C

E.Coli

+

+

E. aerogenes

+

+

The table shows the combination of test used to differentiate E.coli from E.aerogenes

Table 3:

No.

Biochemical Test

Known Microorganism I

Known Microorganism II

Unknown #11

1.

Glucose/Dextrose

Fermentation

E.coli (+/-)

S.epidermis (+/-)

(+,-)

2.

Lactose Fermentation

E.coli (+/-)

S.epidermis (+,-)

(+/-)

3.

MR-VP (Methyl Red Test)

E.coli (-)

E.aerogenes (-)

 (+)

4.

MR-VP ( Voges-Proskauer Test)

E.coli (-)

E.aerogenes (-)

(-)

5.

Catalase Production

E.coli (+)

S.epidermis  (+)

(+)

6.

Nitrate Production

E.coli (-)

B.megaterium  (+)

(-)

7.

Starch Hydrolsis

E.coli (-)

B. megaterium (+)

(-)

8

Tryptophan Hydrolysis

E.coli (-)

E. aerogenes (-)

(-)

9

Urea Hydrolysis

E.coli (-)

P. mirabilis (+)

(-)

10

Hydrogen Sulfide production

E.aerogenes (-)

P. mirabilis (+)

(-)

11

Citrate Utilization

E.coli (+)

E.aerogenes (+)

(-)

12

Litmus milk Reduction

E.coli (B&C)

S.epidermidis (B)

P. fluorescens (B)

Yogurt (A, C&D)

B

13

Oxidase Test

E.aerogenes (-)

P.fluorescens (+)

(-)

14

Gram Stain

Refer to Lab 3 & 4

Cytological Techniques

G+

15

Oxygen Requirement

Factors Affecting Microbial Growth

Facultative Anaerobic

Discussion:

Identifying our Unknown has led us performing numerous biochemical test, comparing and contrasting results from known microorganisms that were already given to find our unknown. Lab 5 ( pure Culture), Lab 3&4 ( Cytological Techniques), Lab 8 ( PCR & Sequence Analysis), BLAST, Lab 12&13 (Inoculation & Biochemical Tests) were the experiments performed to help us find our unknown. Bergey’s Manual would also help us know the exact unknown we had but I am not using it I am only using the BLAST results and the other labs mention above to identify what the unknown maybe. In the test performed from the Biochemical knowns, our match with E.coli in Lactose Fermentation, Catalase production, Voges-Proskauer Test, Tryptophan Hydrolysis Starch Hydrolysis, Urea Hydrosis, Hydrogen Sulfide Production, Litmus Milk, Gram Stain were positive tests. I assumed our unknown was E.coli at first because the results of our unknown matched with the majority of the results of E.coli.

Find out how UKEssays.com can help you!

Our academic experts are ready and waiting to assist with any writing project you may have. From simple essay plans, through to full dissertations, you can guarantee we have a service perfectly matched to your needs.

View our services

 For the sequencing, we use BLAST to the sequence. While using the BLAST, it showed that our microorganism was from the genus Staphylococcus. If I used the Bergey’s manual, it would have given me not only the genus but also the specific species in which it belonged to. I think our unknown may be Staphylococcus epidermidis (S. epi).

 In conclusion, I came to the conclusion that our unknown was Staphylococcus epidermidis and not E.coli because of the blast and some of the results matched Staphylococcus. Lactose Fermentation, catalase production, litmus Milk Reaction, and the Gram stain were positive for Staphylococcus epidermidis. Even though, our biochemical test for our unknown coincidentally matched the majority of E.coli’s. For the unknown microorganism to not match the S.epi, then the unknown sample must have been contaminated during the course of completing the lab.

Work cited

Cite This Work

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Related Services

View all

DMCA / Removal Request

If you are the original writer of this essay and no longer wish to have your work published on the UKDiss.com website then please: