Microbiology:

Identification of Unknown Mixed Cultures

Sierra Medina

Biology&260

Professor Dr. Kristy Henscheid

March 15, 2015

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Introduction

In this exercise, various stains and differential tests are performed on isolated bacteria

originating from a mixed culture of unknowns in an attempt to identify each as a previously

discovered and known bacterium. The selection of possibilities originate from and are used to

compare to the results of a database comprised of 14 different species of class-tested bacteria. By

fixing salts of different metals onto a specimen (positive stain) or to the slide background

surrounding it (negative stain), added contrast and an increased visibility for observation can be

achieved as the first step to identification. The use of these stains have not only assisted scientists

in visibly examining bacteria, but now also provide information regarding various species. The

collection of this information allows for a comparison to distinguish each by class based on

structure through their interaction with each stain. Through differential tests, the biochemical

activity of bacteria can distinguish each species as the reactions differ with their interaction with

the media and any chemical reagents added.

Through the gradual process of elimination with each test, dependent on the results of the

one preceding, then in comparison to known (control) bacteria, the identification of the unknown

bacterium can be narrowed down and confirmed to the best available knowledge. The value of

each test is demonstrated by the process of recognizing bacteria by its structure and reaction to

each stain, and as a result, how the bacteria behaves in certain environments, and how it can be

eliminated, if so desired. These results provide useful information for a variety of settings, as the

cell structures’ observed in each stain help to determine the behaviors, capabilities, longevity &

resilience of each bacteria within the human body, and/or in various environments. The

differential tests provide information regarding the metabolism (chemical reactions) and

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therefore, additional information regarding the interaction between the bacteria with a given

environment.

Results

Test Result Flow Chart (To Eliminate Bacteria For Identification)

After the isolated cultures from mix #52 are incubated (grown) at 30º C in nutrient agar

plates for 2 days, a gram stain is performed to determine cell structures and the next appropriate

test for identification following the Test Result Flow Chart.

Fig. 1 Gram Stain Results For Culture A Rod-shaped bacteria measuring 1µm x 4µm at 1000x magnification, stained purple, indicating gram-positive results.

Fig. 2 Gram Stain Results For Culture B Coccobacillus (semi-round) bacteria measuring < 1µm x <1µm at 1000x magnification, stained pink, indicating gram-negative results

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Isolated results of Culture A as shown in figure 1 indicate gram-positive results, with

purple stain remaining on most of the bacteria in the sample. The structure of Culture A depicts

rod-shaped (long) bacterial cells measuring 1µm x 4µm in diameter and length at 1000x

magnification, the cells connected in long chains. Isolated results of Culture B in figure 2 show

coccus to coccobacillus (round, semi-round) bacteria measuring less than 1µm x < 1µm in

diameter at 1000x magnification, stained pink, indicating gram-negative results. The results of

Culture B determine the presence of an outer lipid membrane surrounding the existing cell walls

of this bacteria, which would provide a higher resistance to control methods than gram-positive

bacteria or viruses with lipid envelopes.

Following the Test Results Flow Chart, half of the possible candidates are eliminated for

culture A, and the next step for determining the gram-positive bacteria is dependent on the

morphology (cell shape) of culture A, which is rod-shaped, thereby reducing the possibilities to

only three different bacteria, Bacillus megaterium, Bacillus subtilis, and Mycobacterium phlei.

These are further distinguished by an endospore stain. A 4 day old sample of Culture A is

positive for blue elliptical endospores that show signs of separation from the pink vegetative

rods, eliminating the possibility of M. phlei. This result demonstrates the ability of these cells to

survive unfavorable conditions through the production of endospores, as with nutrient depletion

through longer incubation periods. These endospores also withstand heat and chemicals due to a

protein outer covering of keratin and leave the vegetative cell, thereby not increasing the number

of cells, but rather, preserving it.

The Nitrate Reduction Test then separates B. subtilis from B. megaterium, in the

anaerobic respiration (transfer of electrons to an inorganic molecule other than oxygen) as in B.

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subtilis through the production of a red broth after the reagents A and B are added and mixed.

Results for Culture A show no reduction of nitrate after reagents are mixed, but produce red

broth after the addition of zinc (negative result), indicating that the organism could be B.

megaterium.

A Casein Hydrolysis Test is performed

to confirm the results of B. megaterium, with

positive results shown in figure 3. A clear halo

surrounding the inoculated portion of the

Casein milk agar plate indicates the production

of Casease, which is the enzyme produced and

excreted by bacteria that hydrolyze casein

(milk protein)1, which is confirmed to be in

this case, B. megaterium.

Continuing from the gram-negative

stain result of Culture B (figure 2), and following the Test Result Flow Chart, Culture B is then

tested for capsules through a Capsule Stain to eliminate the possibility of Aeromonas sobria.

There is no capsule detected upon the administration of the stain, demonstrating a vulnerability

to phagocytosis as there is no added protection by a capsule such as that seen with A. sobria,

leaving 5 remaining possible bacteria.

The Methyl Red broth is then used to distinguish between mixed acid fermenting bacteria

and non-mixed acid fermenting bacteria. There is no color change indicating that there is no pH

Fig. 3 Casein Hydrolysis Test For Culture A Milk Agar plate inoculated with Culture A for 2 days at 30º C showing clearing around inoculum. Positive for Casease, the enzyme that breaks down milk protein casein.

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change, and therefore no mixed acid fermentation, reducing the possibilities to Enterobacter

aerogenes, and Pseudomonas flurescens.

A positive Bile Esculin Test following the Methyl

Red Test, indicates the production of an excreted

enzyme (exoenzyme) that hydrolyzes esculin in the

presence of bile (figure 4) as seen in E. aerogenes,

thereby eliminating the possibility of P. flurescens.

To confirm the possibility of E. aerogenes, a Citrate

Test is performed. Citrate media limits the essential

nutrients to citrate and ammonium ion, to distinguish

members of Enterobacteriaceae from other gram-

negative rods 4. Results are positive, as shown in

figure 5, confirming E. aerogenes as the identity of

Culture B.

Discussion

Culture A (figure 1), identified as Bacillus

megaterium, is a saprophyte, commonly found in soil,

and utilizes decaying organic matter as a source of

nutrients2. It is considered non-pathogenic, or not

disease causing, and is studied for medical

applications through its protein production of various

Fig. 4 Bile Esculin Test For Culture B Bile Esculin slant inoculated for 2 days at 30º C showing growth and positive hydrolysis of esculin (darkened medium)

Fig. 5 Citrate Test For Culture B Citrate slant inoculated for 2 days at 30º C showing citrate-positive (blue)

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enzymes (such as in the Casien Hydrolysis test, figure 3) and its industrial use in for

bioremediation clean up methods3. As seen in comparison to Culture B (figure 2) the bacteria is

considered extremely large, about 100 times the size of E. coli (another gram-negative bacteria)

as suggested by it’s name “mega” 3.

Culture B, identified as Enterobacter aerogenes, is a member of the bacteria belonging to

Enterobacteriaceae, which includes Escherichia, Shilgella, Salmonella, Enterobacter, Klebsiella,

Serratia, and Proteus among others. E. aerogenes is found as normal intestinal flora of humans

and animals, and is a nosocomial opportunistic pathogen 5, which is to say that it does not cause

disease in it’s normal environment, but may cause damage if introduced to a foreign environment

of a weakened immune system such as those of patients residing in clinical or hospital settings.

Such foreign environments include infections of the respiratory, gastrointestinal, and urinary

tracts more commonly, but may also be responsible for infections of the central nervous system

and adult meningitis 5. Resistant strains are increasingly common. Care to prevent the spread of

disease from E. aerogenes include utilizing aseptic techniques in medical settings and practicing

proper sanitary methods when coming into contact with patients.

Conclusions

The isolation of each organism from the mixture #52 presented as a challenge,

specifically when attempting to find the presence of gram-positive B. megaterium, as the E.

aerogenes became present in most samples. Initial tests provided sufficient evidence to continue,

until the Phenol Red Test was conducted from the previous Flow Chart draft (not shown) for the

gram-positive bacteria. Results were inconclusive and unexpected for the remaining possible

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bacteria, so an alternative route was established, using the Endospore Stain. It was during the

examination of this stain that it became apparent that both the gram-negative and gram-positive

bacteria were still present in the same culture, which lead to inconclusive results in the test

preceding, but also confirmed the presence of endospores forming from the visibly different rod-

shaped bacteria still remaining in the sample. Once a pure culture was obtained, the Nitrate Test

and confirmatory Casein Test results were conclusive for B. megaterium. The original route for

the confirmation of E. aerogenes provided sufficient evidence for the identification of Culture B,

however due to prior experience examining E. aerogenes, personal bias interfered with the

identification process. The culture appeared much smaller and rounder in visual inspection in

comparison to previous samples of E. aerogenes. The test results confirmed the identification

however, and revealed that sometimes the visible differences between samples of the same

organism cannot be conclusive on their own, revealing the necessity for various tests in the

identification process.

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Cited References

1. Leboffe MJ, Pierce BE. Brief Microbiology Laboratory Theory & Application 2nd ed.

Englewood (CO): Morton; 2012. p. 303

2. The American Heritage® Dictionary of Student Science, Second Edition. [Internet] S.v.

"saprophyte." [modified 2014; cited 2015 Mar 15.] Available from http://

www.thefreedictionary.com/saprophyte.

3. "Bacillus Megaterium." - MicrobeWiki. edited by student of Glogowski M of Loyola

University [Internet] 2010 Dec 8 [cited 2015 Mar 15] Available from: http://

microbewiki.kenyon.edu/index.php/Bacillus_megaterium

4. Leboffe MJ, Pierce BE. Brief Microbiology Laboratory Theory & Application 2nd ed.

Englewood (CO): Morton; 2012. p. 273-274.

5. "Enterobacter aerogenes" - MicrobeWiki. Larsen R of University of California, edited by

students of Glogowski M of Loyola University [Internet] 20110 April 22 [cited 2015 Mar 15]

Available from http://microbewiki.kenyon.edu/index.php/Enterobacter_aerogenes

Figure 1. Gram Stain Results For Culture A. Dr. Henscheid KL, photographer; Columbia Basin

College; 2015 Feb 26

Figure 2. Gram Stain Results For Culture B. Dr. Henscheid KL, photographer; Columbia Basin

College; 2015 Feb 26

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