Solving the Bacterial Unknown

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TITLE:(Lab#11) Identifying Bacterial Unknown

AUTHOR:Denisse Cuarteros

UNKNOWN TUBE #:10

ABSTRACT

Identifying unknown microbes through different types of tests and experiments is one of

the most essential steps in classifying the organisms background and characteristics. It is very

important to know what type, and which kind of organism is being dealt with, in order to be able

to enhance, modify, and create a drug that can inhibit its growth (if it is labeled as pathogenic),

through the application of several techniques learned and developed through exposure in the

laboratory when it comes to performing the experiment. Extreme care and precaution are needed

to be safe from the microbes because a single careless mistake might result in lifetime regret.

Some of the steps involved in identifying bacterial unknown are streaking for bacterial isolation

via different kinds of culture plates (TSA, MAC, EMB, and MSA), gram staining which divides

the bacteria into gram positive (purple) or gram negative (pink), and several types of biochemical

tests that can be done in order to determine the exact name of the two kinds of bacteria being

identified. After performing different kinds of laboratory techniques, the two bacterial unknowns

contained in the tube, were finally identified through legitimate test results and careful analysis.


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INTRODUCTION

Identifying bacterial unknowns have served as one of the most important keys in

microbiology, medicine, and research. By knowing the bacterias exact identity, it will be much

simpler to control and inhibit its growth, as well as prescribe certain medications. There are

many ways to determine the identity of the bacteria present in the tube given. These methods are

streaking for isolation, gram staining, and certain biochemical tests.

Streaking for isolation is a method used to obtain a pure culture of the bacteria. There

are two main kinds of culture media used in identifying which unknowns are contained in the

given tube; MacConkey (MAC) agar which is selective for the growth of gram negative bacteria

and Mannitol Salt agar (MSA) which is a selective plate that favors the growth of gram positive

bacteria. Both of the culture media are also differential, since they differentiate lactose

fermenting from non-lactose fermenting bacteria (Lab 5: Isolation by Dilution: Differential and

Selective Media, page 23).Another important procedure is gram staining. Gram stain is an

example of differential stain used to separate gram positive from gram negative bacteria and are

used to know if the bacteria are pure culture or not (Lab 2: Gram Stain, page 8).

One of the most important procedures practiced in identifying the unknown bacteria are

the use of different kinds of biochemical tests that are unique in various microbial metabolism

and microbial growth. Each type of microbial metabolism test uses a specific enzyme to perform

certain types of reactions. The two main types are catalase (for gram positive) and oxidase (for

gram negative). By determining if the unknown bacteria has positive or negative results on the

oxidase or catalase, it will be easier to determine which kind of biochemical tests will follow.

Example of biochemical tests are citrate test (for lactose positive) and carbohydrate fermentation

with the use of trehalose broth. Catalase is an enzyme that is used to convert the toxic hydrogen

peroxide into water and oxygen. If the bacteria are catalase positive, it will form bubbles and


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vice-versa. However, if it is negative, it is necessary to test the bacteria for carbohydrate

fermentation using the trehalose broth as medium. Carbohydrates can be broken down by

fermentation which results in acid production (Lab 9: Microbial Metabolism (part 1), page 37).

It is also important to perform a chemical control for microbial growth through a Novobiocin

(antibiotic) test. Oxidase is involved in the reduction of oxygen at the end of the electron

transport chain. The oxidase reacts with oxygen and the oxidase reagent to produce a purple

color change if the bacteria have positive result for the oxidase test (Lab 9: Microbial

Metabolism (part 1), page 39). After performing an oxidase test and the result is negative,

another biochemical test which can be used in identifying the unknown bacteria is a citrate test

and streaking for isolation using Eosin-Methylene Blue (EMB) plate. Citrate test uses the

Simmons citrate agar which tests for the ability of the organism to use citrase (an enzyme) as a

sole carbon source (Lab 9: Microbial Metabolism (part 1), page 44). It is very important to

follow the procedure in a stepwise and sophisticated manner to avoid making false conclusions.

MATERIALS AND METHODS

The first step in identifying which two kinds of bacteria were present in tube #10 was to

perform a streaking for isolation on the MSA plate and MAC plate (day 1) (Lab 5: Isolation by

Dilution: Differential and Selective Media, page 23).The next step performed was another

streaking for isolation on two TSA plates by taking one isolated colony on MAC and MSA (day

2). In order to know if the growths on the TSA plates were pure culture, it is important to

perform gram staining (one for gram positive and another on gram negative). After the

confirmation that they were both pure culture, a catalase test was performed on the gram positive

bacteria while an oxidase test was performed on the gram negative. The result for the gram

positive bacteria was negative for the catalase; therefore, a carbohydrate fermentation test was


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performed as well as Novobiocin test (Lab 8: Chemical Control of Microbial Growth page 33-

36). The result for the gram negative bacteria after performing an oxidase test was negative; the

color of the colony was pink, therefore it is lac positive and a citrate test was performed. On the

fourth day, it was confirmed that the gram positive bacteria has a negative result on carbohydrate

fermentation and is Novobiocin sensitive (33mm zone of inhibition) while the gram negative

bacteria is a citrate positive (Lab 9 and 10: Microbial Metabolism, page 33-45).

RESULTS for Unknown tube #10

Mannitol Salt agar (MSA) MacConkey agar (MAC)

Color of Colony Tiny yellow-like Color of Colony Pink-Purple

TSA plate 35 colonies TSA plate 40 colonies

Gram Staining Gram positive; purple;

cocci;

chainlike/clusters

Gram Staining Gram negative; pink;

rodlike; single

Catalase test Negative result; no

bubbles formed

Oxidase Test Negative result; no

purple color change

Trehalose No color change

EMB plate Purple color/Pink

Novobiocin test 33 mm zone ofinhibition; Susceptible

Citrate Test Positive Result; Blueand green color

Staphylococcus epidermidis Enterobacter aerogenes

MSA MAC

Gram staining (+) Gram staining (-)

Catalase (+)

Trehalose (-)

Novobiocin sensitive

Oxidase (-)

Dark Purple color

on EMB

Citrase (+)

Staphylococcus epidermidis

Enterobacter aerogenes

Streaking for Isolation on TSA Streaking for Isolation on TSA

See Appendix Page for

the Complete Flowchart

of Bacterial Unknown


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DISCUSSION

According to the different tests, the bacterial unknowns contained in tube#10 are

Staphylococcus epidermidis (gram positive)andEnterobacter aerogenes (gram negative). S.

epidermidis is a gram positive purple-colored bacterium which appears to be spherical in shape

and tend to be in chains and clusters. It has a negative result in catalase test therefore it does not

have the ability to convert peroxides into water and hydrogen. When it comes to the

carbohydrate fermentation test, the trehalose broth appeared to have no color change therefore; it

does not have the ability to utilize carbohydrate fermentation. The result of the Novobiocin test

makes S. epidermidissusceptible and sensitive to this type of antibiotic because of its zone of

inhibition thatsapproximately 33 mm.

Enterobacter aerogenesis a gram negative bacterium present in tube#10 together with

the gram positive bacteria S. epidermidis.E. aerogenesappears to be pinkish rod-like shape

bacteria under the microscope and is consist of single colonies. It appears to have a negative

result in the oxidase test because it didnt change its original color after the addition of the

oxidase reagent which is supposed to turn it into purple if it is positive. It has a pinkish/purplish

color on the EMB plate and resulted to be positive on the citrate test because of the bluish color.

This means, it has the ability to utilize the enzyme called citrase,as its sole carbon source.

Applying the knowledge learned on different kinds of tests and techniques to identify

bacterial unknowns will be very beneficial in the long run. In and out of the laboratory, it is very

important to know which kind of bacteria is present if it is causing harm. Not only will it help in

identifying the organisms identitybut it will help them prevent the bacteria in spreading as well.

It will be easier to prescribe medicine to kill the microbes and if there is someone who is sick,

you can use these tests to know the bacteria and from that point, youll know what to do.


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References

University of Arizona. 2013. Lab 2: Gram Stain, page 8-12.InMIC 205L Biology of

Microorganisms Laboratory: Laboratory Manual Guide to Laboratories 1-15. University

of Arizona, Tucson, AZ.

University of Arizona. 2013. Lab 5: Isolation by Dilution: Differential and Selective Media, page

23-25.InMIC 205L Biology of Microorganisms Laboratory: Laboratory Manual Guide

to Laboratories 1-15. University of Arizona, Tucson, AZ.

University of Arizona. 2013. Lab 8: Chemical Control of Microbial Growth page 33-36.InMIC

205L Biology of Microorganisms Laboratory: Laboratory Manual Guide to Laboratories

1-15. University of Arizona, Tucson, AZ.

University of Arizona. 2013. Lab 9: Microbial Metabolism (part 1), page 39-45.InMIC 205L

Biology of Microorganisms Laboratory: Laboratory Manual Guide to Laboratories 1-15.

University of Arizona, Tucson, AZ.

University of Arizona. 2013. Lab 10: Microbial Metabolism (part 2), page 46.InMIC 205L

Biology of Microorganisms Laboratory: Laboratory Manual Guide to Laboratories 1-15.

University of Arizona, Tucson, AZ.

University of Arizona. 2013. Lab 11: Bacterial Unknown, p. 54-58.InMIC 205L Biology of

Microorganisms Laboratory: Laboratory Manual Guide to Laboratories 1-15. University

of Arizona, Tucson, AZ.

University of Arizona. 2013. MIC 205L Biology of Microorganisms Laboratory: Laboratory

Manual Guide to Laboratories 1-15. University of Arizona, Tucson, AZ.


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APPENDIX 1


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APPENDIX 2


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APPENDIX 3: Pictures of Culture Media

Bacterial Isolation for TSA:


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Lab 2: Gram Stain, page 8-12:


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Lab 2: Gram Stain, page 8-12:


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Lab 5: Isolation by Dilution: Differential and Selective Media, page 23

Lab 8: Chemical Control of Microbial Growth page 33-36

Note: Novobiocin was the only antibiotic used on the Bacterial Unknown

Lab 9 & 10: Microbial Metabolism p. 37-45


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Lab 9 & 10: Microbial Metabolism p. 37-45


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Solving the Bacterial Unknown

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