Unknown # 2 Lab Report

Introduction:

The purpose of this lab is to determine the kinds of bacteria present in a given tube, that

are unknown to the student. All of the possible bacteria were discussed in the lab. The unknown

test tube contained at least three bacteria, which could be gram negative and gram positive. In

order to determine the specific types of bacteria present, a series of tests were performed which

included gram staining and various biochemical test.

Methods and Materials:

The unknown test tube of bacteria selected was unknown B48. After selecting the

unknowns, they were stored in a refrigerator. To begin, the unknown was streaked onto several

plates including tryptic soy agar (TSA), mannitol salt agar (MSA), MacConkey (MAC), nutrient

agar NA, eosin-methylene blue (EMB). For streaking plates, an inoculating loop was heated in

an open flame until it was a bright orange color to make it sterile. In order to not kill bacteria’s,

the loop was cooled for 60 seconds before dipping it in the unknown test tube. Throughout the

process of finding the unknown bacteria, any time a test tube was opened it was followed by

flaming the opening of the test tube, and the opening of the test tube was also flamed prior to re-

capping all test tubes. This technique prevented contamination of bacteria from hands and

environment. After the inoculating loop had been cooled it was dipped in the unknown bacteria

test tube and then spread onto each individual plate in a specific manner. The purpose of this

streaking was to isolate the bacterial colonies growing on plate. After the streak plates were

done, they were placed in an incubator for a period of at least 24-hours. After that bacteria grew

on the streaked plates, the plates were viewed. The objective was to find individual colonies of
bacteria growing that were not touching any other colonies. Shown below in the figure 1 are the

isolated colonies on a streaked plate.

Figure 1.

Using a sterile inoculating needle, isolated colonies from each plate was transferred onto a TSA

slant. TSA slants were labelled according to what plate the colony came from. After the bacteria

were inoculated in slants, they were kept in incubators for at least 24 hours for bacteria to grow

on slants. After the bacteria had grown onto the various TSA slants, they were all transferred

onto clean glass slides using sterile inoculating loop. The smear was allowed to dry on glass slide

and then was heat fixed. All the slides were then gram stained and then viewed under a

microscope. The gram stain gave some clue to determine the bacteria weather the bacteria was

gram positive or gram negative, and also allowed us to view the shape of the bacteria. To gram

stain, gram’s crystal violet was used as the primary stain and then after 1 minute was rinsed off

with deionized water. Then grams iodine which is mordent was flooded over the slides and
rinsed with alcohol. After rinsing with alcohol, it was quickly removed and then rinsed with

deionized water. Then lastly slides were flooded with safranin and again rinsed with water. This

resulted in the gram-positive bacteria being purple and the gram-negative being stained with pink

color. After determining the shape of the bacteria under microscope, the next step was to

determine what biochemicals tests needed to be performed. To determine that the following flow

chart (figure 2) was followed which was provided by instructor.

Figure 2

The flow chart was a good source to start and to determine what biochemical tests needed to be

performed. For one of the gram-positive bacteria by looking at the MSA plate, the nitrate and

catalase test were performed. The purpose of nitrate test is to determine if the enzyme nitrate

reductase is present. To perform this test, the bacteria is transferred to nitrate broth using a sterile

inoculating loop. For the best results the bacteria were in incubator for 48-72 hours. After two

days, 7 drops of nitrate solution A and B were added to the nitrate broth. If the solution turns red,

it means it’s positive for the enzyme nitrate reductase. A catalase test was also performed by

placing small amount of bacteria onto NA plate and flooding the plate with H2O2. If the bacteria

produced bubbles when flooded with H2O2, then it is positive for catalase. For my gram-negative

bacteria, by looking at my MAC plate I inoculated bacteria from MAC slant into SIM stab to see
if a black precipitate is formed. Then, I inoculated my bacteria into urea broth to test for the

enzyme urease. If urease was present the broth will turn from a light orange to hot pink. Next, I

inoculated bacteria from EMB and MAC into lactose broth to differentiate between gram (-)

lactose fermenters and non-fermenters. After allowing them to grow for 24-48 hours in lactose

broth it was determined that the bacteria were lactose fermenter or not and further biochemical

tests for gram negative bacteria were performed like IMVIC. Based on the biochemical tests and

due to some wrong techniques performed I was only able to determine two unknown bacteria in

my unknown B48.

Results:

By looking at MSA plate I was aware of what could be the two possible bacteria. So, to

confirm that I performed nitrate and catalase test as mentioned above. which came out to be

positive for both tests and so it was determined that the bacteria was staph aureus and not

micrococcus. Bubbles were formed for the catalase test and nitrate broth turned red after nitrate

solution A and B were added. Figure 3 shown below is positive result for nitrate test and figure 4

shows positive results for catalase test.

 Figure 3 Figure 4

Next, for my second bacteria due to amber color colonies on MAC plate, I tested it for

SIM stab. Which came out to be negative and confirmed that it’s not proteus. Then I performed

urea test which also came out to be negative. Figure 5 shown below is negative test for SIM stab.

Figure 5
 Next, I inoculated bacteria in lactose broth and the bacteria fermented sugar with gas

produced in durham tube. It was a strong lactose fermentation so, I performed citrate test from

IMVIC tests. The citrate test results were negative. So, if citrate is negative and SIM stab is also

negative, I came to a conclusion that the bacteria is E. coli.

Conclusion:

Due to failure in performing some technique and getting wrong results I was able to

determine only two bacteria for my unknown B48, which are Staph aureus and E. coli.