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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 of
inhibition; Susceptible
Citrate Test Positive Result; Blue
and 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) and Enterobacter 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. epidermidis susceptible and sensitive to this type of antibiotic because of its zone of
inhibition thats approximately 33 mm.
Enterobacter aerogenes is a gram negative bacterium present in tube#10 together with
the gram positive bacteria S. epidermidis. E. aerogenes appears 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 identity but 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. In MIC 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. In MIC 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. In MIC
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. In MIC 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. In MIC 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. In MIC 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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