Micro Lab Report final - lab PDF

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MICROBIOLOGY LAB REPORT IDENTIFICATION OF AN UNKNOWN BACTERIA INTRODUCTION

In the massive field of microbiology, being able to iden>fy unknown bacterium is very important. When thinking of one of the biggest parts of microbiology, food microbiology, the ability to iden>fy food spoilage contaminants could be life or death. Being able to know whether an item of food is edible could save many lives. Another aspect of microbiology is microbial ecology. In this field, iden>fica>on on unknowns helps characterize biodiversity. There are many different techniques that go into iden>fica>on of unknowns and this systema>c and precise process is not an easy one. Nonetheless, it allows scien>sts to be able to iden>fy different bacteria in all sorts of different aspects, especially in medicine where this skill issued by physicians in order to correctly diagnose their pa>ents. METHOD

(List the various tests you will be performing)

https://www.youtube.com/watch?v=3os3odC_UFU&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh4 l_a&index=20 Carbohydrate (Sugar) Durham Tube Tests https://www.youtube.com/watch?v=Qm0x8SsCR0w Methyl Red https://www.youtube.com/watch?v=U_3qcDhrw3w Vogues Proskauer https://www.youtube.com/watch?v=cWEULCNfS7c&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh4 l_a&index=24 Nitrate Reduction Test https://www.youtube.com/watch?v=LZ_mVPRLayQ&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh 4l_a&index=13 Simmons Citrate Test

https://www.youtube.com/watch?v=m27wzevB9bA&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh 4l_a&index=15 Indole Test (listed in the manual under the section for SIM) https://www.youtube.com/watch?v=u9cX8mZoh88&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh 4l_a&index=41 SIM test continued https://www.youtube.com/watch?v=SZnD2lFaLsc&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh4l_ a&index=21 Phenylalanine Test https://www.youtube.com/watch?v=NW4dFIgBM74&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh 4l_a&index=25 KIA Test (Kleiger Iron Agar test) https://www.youtube.com/watch?v=yAz0mF4hMmA&list=PL2OnaK_Res6SoH3ZuBTTehfGCovF h4l_a&index=26 Litmus Milk Test https://www.youtube.com/watch?v=1TSFObauCNU&list=PL2OnaK_Res6SoH3ZuBTTehfGCovFh 4l_a&index=33 Catalase Test

RESULTS

Test

Observation

Results

Interpretation

Streak

Does your bacteria create a color on the quadrant streak plate? yes, pinkish purple bacteria

Single colonies of the pink bacteria were observed

The purpose of this technique is to confirm that a bacterium has been successfully isolated. You use it to obtain a pure colony.

Gram Stain

Was your bacteria gram positive or gram negative?

Pink colored, rod shaped bacteria

Gram negative bacteria do not have thick peptidoglycan so when treated with safranin it becomes a red pinkish color

Gram negative

Endospore Stain

Does your bacteria have endospores? Only pink cells were observed

Appearance Before Test Appearance After Test Methyl Red

Before MR Test After MR Test Yellow transparent liquid

Non spores forming bacteria

Spore forming cells turn green, but as no green cells were observed its non spore forming bacteria

Negative MR

Negative: yellow/orange color which means less acid is produced.

Brief Description of Test and Interpretation

This test is used to detect the ability of an organism to produce and maintain acid products from glucose fermentation.

Voges– Proskauer

Before VP Test

Nitrate

Before Nitrate Test Yellow transparent liquid

AOer VP Test

After Nitrate Test No zinc added

This test issued to detect acetone in bacterial broth culture

Positive

Color changes to cherry indicate presence of acetonin.

Nitrate has been reduced to nitrile due to the color change from clear yellow to orange red.

Positive

To test if bacteria can reduce nitrates to nitriles

Simmons Citrate

Before (Dark Green)

Tryptone

Before Test Transparent light-yellow liquid

AOer Test

This tests for an organisms ability to use citrate as a sole carbon source and ammonium ions as a nitrogen source. Its used for differentiating gram negative bacteria on the basis of citrate utilization

Positive

Since color changed to blue this indicates bacteria can utilize citrate as a carbon source.

This test is important for identifying enteric bacteria. If indole is produced it will accumulate in the culture during 24 hour incubation.

Negative

If red on top of the tube then indole is positive, since its yellow, indole is negative.

Sulfur reduction (SIM)

Before Test Cloudy light yellow

AOer Test

This test helps identify sulfur reducing agents which are useful in the diagnostic test of identification of enteric bacteria.

KIA (Kliger’s Iron Agar)

Before Test

AOer Test

This test is used for Negative differentiation of enteric gram negative bacilli on the basis of dextrose and lactose fermentation and H2S production.

Orange agar

Negative SIM

Color would be black if participate formed.

Since the color is not red, there is no fermentation. Since color is not dark, there is no H2S production.

Phenylalanine Before Test Cloudy light strawcolored liquid

AOer Test

This test is used to test the ability of an organism to produce enzyme deaminase. This enzyme removes the amino group from PPA.

Negative

Since the color stayed straw color, the organism is negative.

Phenol Red Lactose

AOer Test

This test studies lactose fermenta>on as well as pH balance

Positive

This microorganism is able to ferment lactose due to the color change from red to yellow, but is not able to produce gas due to the lack of bubbles.

Before Test Liquid hot pink

Phenol Red Mannitol

Liquid, transparent, hot pink

This test is used to study mannitol fermentation. Protease peptone and beef extract serve as sources of carbon and nitrogen.

Positive

Since the color changed from red to yellow, this organism is able to ferment mannitol.

Phenol Red Dextrose

Liquid, hot pink

This tests determines wether Positive the microbe can use the sugar glucose for carbon and energy.

Since the color changed from red to yellow, this organism is able to ferment glucose.

Litmus Milk

Lavender opaque

This test is used to differentiate microorganisms based on metabolic reactions in litmus milk which includes clot formation, fermentation, digestion and the formation of gas.

Since the test turned pink, it is positive for the fermentation of lactose

Positive for lactose and acid production.

Catalase test

Upon addition hydrogen peroxide bubbling seen

This test detects the production of catalase enzyme. This enzyme is a common enzyme that is found is all living organisms.

Positive

Since bubbles were produced the test is positive, meaning there is catalase activity present.

Discussion/Conclusion After filling out the chart shown above for columns appearance after and interpretation, which of the following bacteria do you think you have: Planococcus, Escherichia, Micrococcus, or Serratia

After running all these complex tests on the culture you can identify which bacteria is present based on the results. Every test serves a unique purpose and piecing all the results together will help determine the unknown bacteria culture. The first test, streaking, is used in order to isolate the bacteria and identify different colonies. The next test is used to identify whether the bacterium is gram-negative or gram-positive, in this case it was gram positive due to the pinkish color forming when treated with safranin. Once the endospore test was performed it was easy to tell no spore forming bacteria was identified, showcasing that the bacteria are non-endospores. The next test, being the methyl red test, is used to determine is the acid produced or not. Since the tube was yellow-orange, the test result was negative, meaning the bacteria had less acid production. The VogesProskaueer test is used to analyze whether acetonin is present in the culture medium, and indeed it was due to the cherry red color of the broth. Another test is needed in order to determine whether the bacteria is able to convert nitrates into nitriles, this test indicated the bacteria is nitrate reductase. The Simmons Citrate test is used to determine if the bacteria is able to produce carbon using citrate, which came up positive. Another test that is useful in determining an unknown culture is the Tryptone test, which tests if the bacteria can digest tryptophan, the test in this case proved the bacteria is indole negative, meaning it cannot produce indole. The next step in determining un unknown is the SIM test which is useful in narrowing the specification for bacteria. The test result showed the bacteria is not an enteric bacteria. Once this test is done, the next test is the KIA test which tests to see carbohydrate fermentation, which yielded a negative result. The bacteria tested negative for phenylalanine and positive for the fermentation of lactose which could be inferred from the positive phenol red lactose test. The bacteria also tested positive in the Phenol Red Mannitol test which means the bacteria is able to ferment mannitol. The unknown culture uses sugar in order to gain energy, as seen by the dextrose positive test result. Using all these different tests and identifying different characteristics of different bacteria, helped in the final determination of the unknown bacteria which is identified as Serratia....