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Lab #2/Tube #3

Boyd Robert Maples

Advanced Microbiology

November 4, 2017
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The purpose of this lab was to be able to identify two separate types of bacteria and

identify them. The gram-negative bacteria were to be identified to the species level, and the

gram-positive to the genus level. To do this, the experiment required the following materials:

Gram-Staining chemicals

NA Agar

MSM Agar

MSMG Agar

BHI Agar

MAC-PEA-TSA triplate

An Enterotube

A freezer

Bile test tubes

Glucose test tubes

TSI tubes

OF-Glucose test tubes

Nitrate test tubes

MR-VP tubes

Anaerobe jar

Kovacs Reagent

To do this, the lab begins with streaking a mixed culture for isolation, (4) but this technique

never worked for me so after several tries, the ever so noble and gracious professor gave me

the bacteria in pre-isolated cultures. From that point, I streaked the cultures across all the
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types of agar listed above as well as three extra BHI plates for use in the cold-growth test and

the anaerobe/ CO2 tests. In addition, I gram stained both cultures. This revealed that there

were gram-negative rods (now known as A), and gram-positive cocci (now known as B). (2)

Next, I ran a wide spread of tests. I set an Enterotube for A and tested B for catalase, as

well as running B in a bile tube. After that I set up Glucose tubes, TSI tubes, OF-Glucose tubes,

Nitrate tubes, MR-VP tubes, anaerobe and CO2 jars, and ran oxidase tests on both A and B.

(1,3,5,6,7,8,9,10)

The Results of all if these tests are as follows

Media Tastes

Basic Tests

Enterotube

The results of these tests tell me that A is most likely Klebsiella pneumoniae and that B

is part of the genus Staphylococcus.

For Klebsiella, the tests showed that the bacteria could ferment glucose, used glycolosis,

produced gas, and could not reduce nitrates. The fact that MR-VP showed that it produces acids

but not alcohols combined with all prior tests indicated that the bacteria in question used

propionic fermentation, which narrowed my options down significantly.

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However, my greatest determinate was the Enterotube test. What the Enterotube told

me was that Klebsiella pneumonia, but there was still some doubt. What finally convinced me

was that the colonies were very mucoid, and that was consistent with other descriptions of

Klebsiella that I had been presented with, and all the other tests that I had run served to

support that conclusion.

To determine that B was Staphylococcus, the process was much simpler. The shape and

gram stain narrowed the search considerably, and the media and basic tests narrowed down

the possible genus to Streptococcus and Staphylococcus. The major determining factor

between those two genera is that Staphylococcus can tolerate bile and grow in a bile rich

environment. The results of the bile tube growth test showed that the bacteria grew

extensively, meaning that the bacteria in question are Staphylococcus.

Bile Tube showing black coloration

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Enterotube results

Oxidase tests and CO2/Anaerobe plates

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TSI tube results

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OF-Glucose Tube Results

Glucose tube results

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MR-VP test results

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References
1. GasPak System (The Anaerobic jar). Medical Laboratories, 2014, www.medical-
labs.net/gaspak-system-the-anaerobic-jar-2914/.

2. Rollins, D M. Gram Stain Technique,

www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/GramStain.htm.

3. Aryal, Sagar. Oxidase Test- Principle, Uses, Procedure, Types, Result Interpretation,
Examples and Limitations. Online Microbiology Notes, 8 May 2017,
microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-result-
interpretation-examples-and-limitations/.

4. Rollins, D M. Streaking for Isolation. BSCI 424 Streaking Technique,

www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/StreakingTechnique.htm.

5. METHYL RED-VOGES PROSKAUER (MR-VP) BROTH. Methyl Red Voges Proskauer (MR-
VP) Broth, catalog.hardydiagnostics.com/cp_prod/Content/hugo/MR-VPBroth.htm.

6. Reynolds, Jackie. Nitrate Test. Http://Delrio.dcccd.edu/, 2011,

delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/nitrate.pdf.

7. Reynolds, Jackie. O-F Glucose. Http://Delrio.dcccd.edu/,

delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/OF.pdf.

8. Acharya, Tankeshwar Triple Sugar Iron Agar (TSI): Principle, Procedure and
Interpretation. Microbeonline, 9 May 2017, microbeonline.com/triple-sugar-iron-agar-
tsi-principle-procedure-and-interpretation/.

9. Biol 230 Lab Manual: Interpretation of Enterotube II, Sept. 2017,

faculty.ccbcmd.edu/courses/bio141/labmanua/lab12/tab12a.html.

10. Reynolds, Jackie. O2 Requirements & culturing anerobic

bacteria. Http://Delrio.dcccd.edu/, 2011,
delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/oxygen_needs.pdf.