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Nikila Nallabelli

Mrs. Peterson

GBBE

28 March, 2018

Conclusion

Unknown Number: 13

Unknown number 13 is identified as Serratia marcescens. As found on page 3 of my

unknown lab, my partner and I observed that the bacteria in the snap-tube looked foggy, there

were bubbles at the top, and it had a bad odor. The odor helps identify this bacteria specifically

because Serratia marcescens in the reference packet was one of the only bacteria that had an odor

to it. One error that could have taken place during this part of the lab is accidental contamination

from a vertical column of air while smelling the bacteria. This would partly contaminate the rest

of the tests in the lab and we would be unable to correctly classify our bacteria. We then

inoculated nutrient agar with our bacteria and observed the changes the next day. Through

observation of the growth for the plates at 24, 48, and 72 hours on pages 4-7, it was evident that

25 degrees was the optimal temperature for this bacteria because it grew best at this temperature.

According to the reference packet, the optimal temperature for S. marcescens is 20 degrees

which is very close to the optimal temperature tested for our bacteria. This proves that unlike

many other bacteria, our bacteria flourishes at lower temperatures. When observing the nutrient

agar plate we noticed that the color of this bacteria was pinkish red which was a key factor that

helped us identify this bacteria. An error that could have taken place during this part of the

unknown lab is either contamination from a vertical column of air or contamination from an

inoculating loop that was not sterile. This would alter the results concluded from this lab because
it could cause the introduction of outside bacteria or bacteria that is being used by other students

in the classroom. Therefore, through small errors as those listed previously, the whole

identification may have a higher chance of being incorrect. After the optimal temperature test,

we tested the bacteria through a gram-staining procedure in order to determine the structure of

the cell wall of the bacteria. We observed, after optimal temperature observation on pages 4-7,

that the gram stain on page 5 was pink, indicating that it was gram negative, and had tiny rod

shaped bacteria that were about 1x.3 micrometers in size, indicating that it was bacillus. Through

comparison to the reference packet, it can be seen that S. marcescens is bacillus and has a rod

shaped structure that is about 1x.5 micrometers in size. It is apparent that the identity determined

from the gram test and the size and shape of the bacteria a almost exactly alike once again

proving that S. marcescens is a valid representation of unknown bacteria #13. A possible error

for this gram staining test is the step with the ethanol dripping. If the ethanol was left on the slide

for too long or too less time, the test results could potentially come out opposite or inconclusive

therefore making the identification of the bacteria harder. This would sway the results of the

gram test, one of the most important tests to carry out correctly in order to correctly identify the

given bacteria. The catalase test on page 4 was one of the key factors that allowed us to identify

our bacteria. Since the reaction was almost immediate, it didn’t take long to narrow down the

possibilities of bacterias that it could be. Since this test also matches the description for that of S.

marcescens, there is a very high chance that our bacteria is S. marcescens. Although the

unknown lab thus far for our bacteria went perfectly, other sources of error for many people are

previously listed. A possible error for this bacteria is that enough time wasn’t given to observe a

reaction occuring. This could be an issue because it would then classify your bacteria as negative

for the catalase test which is incorrect and would lead you to misidentify the bacteria. The next
test was the Starch Agar & Hydrolysis test on page 8. For this test, unknown 3 had a negative

response because the iodine had no impact on how the bacteria looked on the starch agar. This

trait, although not mentioned in the reference packet, is pretty different from what you would see

from certain other bacteria. Although we exhibited no errors for this test, one possible error for

other groups could be accidental contamination through a vertical column of air when applying

either the bacteria or the iodine to the agar. Since the iodine is hard to squeeze out of the bottle

and since the bottle is so big, the angle of the slant at which the lid of the starch plate is raised

would increase thus allowing more opportunity for contamination. This would, once again, lead

to misidentification of the bacteria. The next test was the nutrient broth test on page 8 in which

we identified he broth to be turbid, flocculent, and granular. This matched the description

provided for S. marcescens almost exactly thus proving that S. marcescens can be the

identification of unknown bacteria #13. One source of error for this test is accidentally shaking

the test tube before observing it. This could cause for miscalculations on if the bacteria is aerobic

or anaerobic, etc., or it could cause miscalculations on how the broth looks (clear, turbid, etc.).

The motility hanging drop test took us two tries to confirm. Although it has minimal accuracy,

we observed motility, on page 9, for this unknown which matches exactly with the reference

packet information for S. marcescens. One possible error for this step is using too much broth

when putting a small amount on the cover slide during the test. This caused us as a group to have

so much bacteria that was spread out and hard to find. This could’ve stopped us from observing

the motility in this test which could’ve thrown our identification off but we did the test a second

time in order to confirm our observations. The next test conducted was the nutrient agar slant on

page 9. On the agar slant we observed that there was moderate growth which was filiform, pink

and creamy, and greasy. These characteristics were not listed at all in the reference packet so we
were unable to use this test to identify or specify our bacteria. But, the other tests help us

conclude that S. marcescens is the correct identification of unknown #13. No errors on our part

occurred during this test but as we observed others do their work, we saw that many

contaminated their bacteria by allowing a vertical column of air before firing it in the bunsen

burner. This would contaminate the bacteria making it an invalid source to continue with the lab,

therefore making it important to prohibit any source of contamination. The phenol red dextrose

tested positive for acid production and negative for CO2 gas production. While sucrose tested the

same way in the phenol red test, the lactose had a very small amount of acid production and no

gas production. All of the characteristics for these three tests in phenol red listed on page 12

match exactly with the description of these tests listed in the characteristics of S. marcescens.

Therefore, this proves that unknown #13 should be S. marcescens. The possible errors for this

test would be accidental gas inclusion in the test tube when trying to observe the solution. This

could cause regular air to go into the tube causing a miscalculation for the results of this test and

therefore the misidentification of the unknown bacteria. The gelatin stab on page 11 was another

test that tested for motility. We saw that there was minimal liquefaction and that the type of

composition was filiform. This was exactly what was shown as a result of the gelatin stab test in

the reference packet thus confirming my prediction that unknown #13 is S. marcescens. One

possible error for this test is that the gelatin stab was not allowed to cool for long enough in the

ice bath which would not allow for accurate results and may show liquefaction or motility on

accident since the ice bath didn’t have the time to freeze the changes occurring throughout the

reaction. This would, once again, cause for the misidentification of the unknown bacteria. The

next test was the potato agar & hydrolysis test on page 12 that showed minimal growth for

unknown #13 which couldn’t be compared to the reference packet since there was not mention of
the potato agar in the description of S. marcescens. One source of error for the potato agar is

contamination from vertical column of air. Although our test had no errors, this is a possible

error that could throw people off when they try to identify their bacteria based on the

characteristics of the potato agar in relation to that of the one they are referring to. When looking

for the characteristics of each colony on page 13, we noticed that unknown #13 had round

colonies with radiating margins that were smooth and convex. When comparing this to the

photoshoot, our plate and colonies look exactly like those for S. marcescens. Therefore, through

all of the tests, we concluded that unknown #13 has to be S. marcescens. One source of error for

this test is the misidentification of configuration because certain bacteria have huge colonies

which are very hard to see thus resulting in some error. Although we weren’t faced with this

problem, other people were. This would lead them to a possible but improbable misidentification

of their bacteria. The MacConkey agar on page 15 showed a lot of growth and little fermentation

which matched exactly with the description of the results of the MacConkey agar test for

S. marcescens. This shows, once again, that unknown #13 is closest to S. marcescens. One

possible error for this test is contamination form a vertical column of air. This could lead to

contamination which could lead to altered conclusions because there is not anything to refer to

for these special agar tests. For the E.M.B. agar test on page 15, there was a lot of growth but the

color stayed relatively the same as similarly described for the same agar tested for S.

marcescens, once again proving the similarity between unknown #13 and S. marcescens. As

mentioned before, one possible error for this test is contamination form a vertical column of air

which could lead to contamination thus leading to altered conclusions because there isn’t

anything to refer to for these special agar tests. After conducting all of the tests mentioned, my

partner and I came to the obvious and logical conclusion that Unknown #13 was S. marcescens.
Unknown Number: 32

Unknown number 32 is identified as Bacillus cereus. We began the unknown lab with a

snaptube of the contents labeled “32” and we observed on page 3 that it was clear, there was a

small white solid in it, and that it had a strong odor. Although there is not mention of the initial

characteristics for B. cereus in the reference packet, later tests prove that unknown #32 is B.

cereus. A source of error for this part could be accidental contamination from a vertical column

of air while smelling the snaptube of bacteria. This would contaminate the rest of the tests in the

lab because it is the main source for the initial test and we would be unable to correctly classify

our bacteria at the end of the lab. The next test conducted was to test which temperature would

be the optimal temperature on pages 4-7. We saw that all three plates grew tremendous amounts

of bacteria over a 72 hour period so we went off of unknown A’s optimal temperature, 25

degrees, for the rest of our tests. On the page of the reference packet for B. cereus, it says that the

optimum temperature can be from about 30 to 48 degrees celsius. This matches closely with the

results of unknown #32 thus showing the first connection between the two. Sources of error for

this test can include either contamination from a vertical column of air or contamination from an

unsterile inoculating loop. Although we seemed to have no contamination, other groups may

have. The next test was the gram stain test on page 5 through which we determined that unknown

#32 was quite evidently gram positive streptobacillus. According to the reference packet, B.

cereus is also apparently gram positive streptobacillus. Therefore, one more connection between

unknown #32 and B. cereus is established. One source of error for this lab would be the Ethanol

drip because having ethanol on the slide for too long or too less would cause the results of the

test to be altered. Since gram staining is a major factor to identifying the unknown, messing it up
could cause the misidentification of the unknown. The next test was the catalase test on page 4.

Although our catalase test tested negative and that for B. cereus tested positive we have

identified our source of error. Since we saw the catalase test for unknown A react very fast, we

did not wait long enough to see if unknown #32 would react. If we had waited a couple more

seconds, we would’ve seen a reaction. This is a source of error that many like us have fallen for.

The test following this was the starch agar & hydrolysis test on page 8. In this test, we observed

that there was a positive response to iodine which is not mentioned in the reference packet.

Although it may not be mentioned in the packet, the other tests lead us to conclude that unknown

#32 is B. cereus. One source of error for this test is contamination through a vertical column of

air when applying either the bacteria or the iodine to the agar. Since the iodine is hard to squeeze

out of the bottle and since the bottle is so big, the angle of the slant at which the lid of the starch

plate is raised would increase thus allowing more opportunity for contamination. This would,

once again, lead to misidentification of the bacteria. The nutrient broth test on page 9 showed

that the broth was membranous, flaky, and clear. This directly corresponds with the description

of flakiness in the nutrient broth for B. cereus therefore establishing yet another connection

between it and unknown #32. One source of error for this test is either contamination from an

unsterilized inoculating loop or contamination from a vertical column of air which could lead to

changes in broth characteristics and finally to misidentification of the unknown bacteria. When

completing the motility hanging drop test on page 9, we noticed that the bacteria are motile as

does the bacteria B. cereus. Therefore, unknown #32 could be B. cereus. One source of error for

this test could be using too much broth when putting a small amount on the cover slide during

the test. This caused us as a group to have so much bacteria that was spread out and hard to find.

This could’ve stopped us from observing the motility in this test which could’ve thrown our
identification off but we did the test a second time in order to confirm our observations. The

nutrient agar slant test showed abundant growth that was white, fuzzy, and appears to be effuse.

The slant agar was not tested in the reference packet but if it were, it would probably be similar

due to the fact that B. cereus may be unknown #32. One source of error for this test is a vertical

column of air before running the test tube over the flame which could contaminate the bacteria,

hindering accurate results and an accurate identification of the unknown in the future. The

phenol red dextrose produced an acid but no gas, the phenol red lactose produced no gas or acid,

and the phenol red sucrose produced an acid but no gas, all located on page 12. Through the

phenol red tests it can be clarified that unknown #32 is extremely similar to B. cereus because

they have the same phenol red test results. One source of error for the phenol red test is an

accidental gas inclusion in the test tube when trying to observe the solution. This could cause

regular air to go into the tube causing a miscalculation for the results of this test and therefore the

misidentification of the unknown bacteria. The gelatin stab on page 11 showed a saccate

structure with liquification as confirmed by the reference packet for B. cereus. The similarity

between unknown #32 and B. cereus proves that they are the same bacteria. One source of error

for this test is contamination from the unsterilized inoculating loop which could contaminate the

stab causing unnatural motility of bacteria which could eventually lead to misidentification of the

unknown. The potato agar & hydrolysis test was negative for unknown #32 because there was no

growth at all before so nothing happened after iodine was added. This test was also not

performed for the B. cereus bacteria in the reference packet. Therefore, no conclusions can be

drawn about this test. The source of error for this test would be contamination from a vertical

column of air because adding iodine would require opening up the lid at a higher angle due to the

large dropper that holds the iodine. There would be a higher chance of contamination this way
which could lead to inaccurate results and misidentification of the unknown bacteria. The

colonies for unknown #32 were identified as filiform, flat, and branching which matches the

description of B. cereus. Throughout the past tests and this test it is easier to recognize how

similar unknown #32 is to B. cereus. The source of error for this test would be the

misidentification of configuration because certain bacteria have huge colonies which are very

hard to see thus resulting in some error. The skim milk agar show light growth and positive

hydrolyzation which matches perfectly with the description of B. cereus which makes sense

considering that they may be the same bacteria. The source of error for this test would be a

vertical column of air which would contaminate the specialty agar which would be detrimental

because they are important to recognize the bacteria for the unknown. The spirit blue test was

also positive with extensive growth which also matches the description of B. cereus practically

confirming that the two are the same bacteria. One source of error would be cross contamination

because more than one bacteria are on the same agar. This would lead to mixed results and

possible misidentification of the unknown. Through the examination of each of the tests, it can

be confidently stated that unknown #32 is Bacillus cereus.

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