Rebecca Dover

Microbiology 210, Section 004

25 April 2009

Mixed Culture Unknown Identification Report

Introduction:

The objective of this experiment is to identify the organisms in a mixed culture of two

unknown bacteria. One organism is known to be a gram positive, and the other a gram

negative. It is important to isolate colonies of individual organisms and obtain a pure culture

of each in order to determine the identity of the organisms. With a pure culture, it is simple to

inoculate the culture into media that identify the biochemical properties of each organism.

Comparing the known results of particular organisms with that of the unknown culture can

identify the genus and species of the bacteria in the culture. Using this knowledge, the identity

of bacteria in unknown culture #1 will be revealed in this report, based on this information,

which directed the tests that were performed. The possible Gram positive species were

Clostridium perfringes, Corynebacterium xerosis, Enterococcus faecalis, Staphylococcus

aureus, or Staphylococcus epidermidis. The potential Gram negative species were

Enterobacter aerogenes, Moraxella catarrhalis, Neisseria flavescens, Pseudomonas

aeruginosa, or Proteus vulgaris.

Materials and Methods:

On the first day, after receiving the tube containing the two bacteria, a Gram stain was
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performed as directed from Exercise 7 in the lab manual (McPherson 27). This was important

to do initially because morphology of the organisms can change as the culture ages. Secondly,

the mixed culture unknown was streaked for isolation on two tryptic soy agar (TSA) plates as

directed in Exercise 8 in the lab manual (McPherson 32). One plate was incubated aerobically

and the other was incubated anaerobically, while both were incubated at 37° Celsius for 24-48

hours. Aseptic techniques were followed throughout all of the experiments as written in the

lab manual in Exercise 5 (McPherson 20). This specific test was used to determine the oxygen

requirements of each organism.

On the second day, I looked at the two TSA plates and found that the organisms had

grown on both of them. I preformed a Gram stain again to confirm my previous results. I then

streaked the organisms from my TSA aerobic plate onto a MacConkey agar plate (MAC) and

a Columbia CNA agar with 5% sheep’s blood (CNA). Both of these were incubated at 37°C

for 24-48 hours under aerobic conditions. The Columbia CNA agar was used for the isolation

and differentiation of the gram positive organism and to determine the hemolysis pattern. The

MacConkey agar was used because it is selective for gram negative organisms and differential

on the basis of lactose fermentation.

On the third day, I observed the MAC and CNA plates. There was growth on both of

them. I did a Gram stain to make sure that they were pure colonies. On the CNA plate, there

was clear β-hemolysis. In β-hemolysis there is a clear zone in which few red blood cells are

present. On the MAC plate, there was a hazy precipitate surrounding the organisms.

Results:

From the Gram stain test, the view under the microscope revealed both cocci and rod
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morphology, and I determined that I had a Gram positive cocci and a Gram negative rod. The

streaked cultures on the TSA plates revealed a positive result on the aerobic TSA plate and a

positive result on the anaerobic TSA plate, due to apparent growth on both. The Columbia

CNA agar test on the Gram positive cocci resulted in a β-hemolysis pattern because there was

a hazy precipitate surrounding the organisms on the CNA plate. The MacConkey agar test on

the Gram negative rod resulted in a positive indication for lactose fermentation due the pink

color of the organisms and a hazy precipitate surrounding the organisms.

Discussion:

The reason for my inoculations was to determine under which conditions my organism

could grow. The TSA was used to see if it grew aerobically or anaerobically. The MAC agar

was used to isolate for Gram negative organisms, and the CNA agar was used to isolate for

the Gram positive organisms. They were all incubated aerobically (except for the TSA

anaerobic plate) because I knew that both of my organisms were aerobic or facultatively

anaerobic. Also, growing the organisms under reduced concentration of oxygen, as in the

MAC agar test, decreased the quantity peroxidase produced and increased the visibility of this

reaction. They were all incubated at 37°C because this is the best condition for growth, and

After finding that my Gram positive organism was a cocci, I streaked it onto the CNA

plate to see if it had a hemolysis pattern. This medium was used for the Gram positive cocci

because it is selective due to the inclusion of the antimicrobials colistin and nalidixic acid.

Colistin disrupts the cytoplasmic membrane of Gram negative bacteria while the nalidixic acid

prevents DNA replication in susceptible gram negative organisms. The addition of 5% sheep

blood, which provides the nutrients hemin and vitamin K1, aids in the cultivation of more
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fastidious microbes. It also allows for the differentiation of organisms based upon their

hemolysis pattern. The colonies were surrounded by a white/clear zone, which indicates an

area in which few or no intact red blood cells are found, and is also signifying of β-hemolysis.

After I discovered the colony had β-hemolysis, I was certain that it was S. aureus, because it

is the only Gram positive cocci, TSA aerobic and TSA anaerobic positive organism out of the

options showing this hemolysis pattern.

To identify my Gram negative rod I streaked it on a MAC agar to determine if it

fermented lactose. There was a hazy precipitate around the organisms and they appeared a

pink color, signifying that it did ferment lactose. If an organism ferments lactose, acidic by-

products are released and the pH of the medium surrounding the colony drops. This drop in

pH causes the colony to absorb the neutral red indicator, and appear pink to brick-red, which

was apparent in my results. The drop in pH also causes the precipitation of bile salts,

explaining the presence of the hazy precipitate surrounding the organisms. At this point I was

certain it was E. aerogenes because it is the only Gram negative rod, TSA aerobic and TSA

anaerobic positive organism of the options showing lactose fermentation.

My Gram positive organism was S. aureus and my Gram negative organism was E.

aerogenes. All of the purposes stated in the introduction were met. I am now confident of my

ability to perform the tests and to know which test to perform in which circumstance. I was

able to identify my organisms, through the process of elimination and form conclusions based

on my test results.
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References

McPherson, Elizabeth Fish. Microscopy and A Survey of Microorganisms. Third Edition.

New York: Pearson Custom Publishing, 2008.