Microbiology Laboratory Report

Identification of Unknown Bacteria

4/19/05-4/27/05

Author: Richard Hendricks; NMU

Abstract:

Broth culture 21 was randomly selected and subjected to qualitative tests for taxonomic

identification. The culture did not appear homogenous throughout the testing period, but repetition

yielded consistent results. This culture is currently retained by Northern Michigan University’s

department of Microbiology. It is suggested that culture 21 is an example of Proteus vulgaris.

Background:

Techniques used were in accordance with NMU Professor Dr. D. Becker’s chosen lab manual (ISBN

0-390-53911-2; McGraw Hill).

Materials and Methods:

Microscope, incubator, and deionizer functioned correctly throughout testing period, with stains,

dishes, agars, and test reagents readily available.

4/19/05

A 24 hour old stock broth culture of the unknown specimen 21 was obtained. It appeared somewhat

turbid, reflecting some growth. To determine if the stock culture was pure, a streak was performed

using loops of the stock broth on a fresh dish of nutrient agar and incubated for 48 hours @ 32C.

A Gram stain was then carried out to differentiate the unknown sample from a broad class to a more

specific category of bacteria. The Gram stain produced satisfactory results with a clear indication of

negativity. Light pink staining was evident on the cells in the FOV and a search of the slide revealed

uniformity among the sample. From this result it was concluded that unknown 21 was Gram

negative in nature, therefore it could possible be any of the following bacteria: Alcaligenes faecalis,

Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, or Pseudomonas

fluorescens.

A negative stain was then conducted to reveal the shape of the cell and possibly capsule presence.

The first negative stain was successful yielding rod shaped cells occurring singly and in pairs. These

appeared somewhat sparsely distributed at 1000x oil immersion.

A simple stain was then performed using a sample of bacteria from the 24 hour old stock broth

culture. The appearance of the microbe was again rod in shape, and distribution as before.

The unknown bacteria displayed a capsule when stained with crystal violet.
4/21/05

After 48 hours of incubation, the results of the streak plate were successful and showed many moist

and colorless opaque streaks of colonies the size of pinpricks in four quadrants (See figure 1). All

colonies appeared to be of the same color, size, and shape. Therefore it was concluded that the stock

broth culture was pure.

A Potassium Hydroxyl (KOH) Gram test was then performed to confirm the results of our Gram

stain and to assure that this bacteria was Gram negative in nature. The KOH test revealed a fine

filament which retained adhesion to a toothpick for 50-75mm before breaking from tension. This

was first performed with 3 loops from the streak plate prepared on 4/19/05. Based on the results of

the Gram stain and the KOH Gram test it was concluded with greater certainty that the unknown

sample was a Gram negative bacterium, which limited 13 bacteria to 6.

Aerobic capacity was checked next with a shake tube test. (See figure 1)

A faulty spore test was performed at this time, producing no conclusive results.

4/22/05

Shake tube test revealed a facultative anaerobe, thus Pseudomonas flourescens and Alcaligenes

faecalis could be eliminated, leaving 4 bacteria possible.

It was also realized after more research that an ONPG test could now be performed, and this would

differentiate Enterobacter aerogenes and Escherichia coli from Proteus mirabilis and Proteus

vulgaris. This test being performed, an indole test would separate the Protei, and the Enterics from

their pairings, revealing the unknown bacteria’s identity.

ONPG tests were prepared in accordance with three separate protocols because this test was so

important to the rest of the procedures. One was a broth culture, one H20, and one was H2O with 1%

NaCl.

An indole production test was prepared with TSA, and a citrate test was performed for additional

cross checking. (See figure 1)

4/23/05

Positive indole production results were obtained at this time, which distinguished between the Protei.

Proteus vulgaris is tentatively chosen as unknown 21.

4/25/05

Results:

Unknown sample 21 is Proteus vulgaris. (See figure 2)

Acknowledgements:

Dr. Becker, Lopez, Rebers, and their staffs facilitated our efforts with their expertise. Many thanks

are owed to the lab techs for preparing samples, providing media, glassware, and other crucial

materials.

References:

Benson-Brown, Amy (200*) Microbiological Applications Lab Manual;Complete version, 9e

Kleyn, J. Bicknell, M. (2001) Microbiology Experiments: A Health Science Perspective, 4e

Harley, John (1996) Laboratory Exercises in Microbiology, Sixth Ed.

Reynolds, Jackie (2004) Lab Procedures Manual

J.G. Holt, Lippincott, Williams and Wilkins (1994) Bergey’s Manual of Determinative Bacteriology,

9th ed.
Figure 1. Results from shake tube, ONPG tests, citrate test, and indole test.
Figure 2. Test results

Gram stain -
KOH Gram test -
Colony formation moist, colorless, opaque
Negative stain rod
Simple stain rod
TGYA shake tube facultative anaerobe
ONPG -
Indole +
Citrate -
Microbiology Unknown Report, BI 303

Richard Hendricks, NMU

4/28/05