Unknown Organism

Enterobacter Aerogenes

Jaya AttaluriBIOL 2420-41101

Professor Danita Bradshaw-Ward

November 3, 2019

Eastfield College

Introduction

Present general information about microorganisms. Include nutrition requirements, pH, etc. You should also include general information about metabolism and the purpose of conducting biochemical tests. You should also provide a brief introduction of your organism.

Microorganisms are microscopic organisms that cannot be seen without the aid of a microscopic. Some examples would be bacteria, viruses, or fungus. Different microorganisms have a wide variety of requirements when it comes to their environments.

Each species of microbes have their own cardinal temperature points. Psychorotrophs are microorganisms that are capable of surviving and growing in temperatures that range from 0 degrees C to 25 degrees C. Mesophiles are microorganisms that growth in temperatures between 15 degrees C to temperatures over 45 degrees C. Thermophiles are microorganisms that can survive in very hot climates, with maximum growth temperatures that can exceed over 100 degrees C. pH, a scale that is used to describe the concentration of hydrogen ions that are present in a solution.

The higher the concentration of ions, the more acidic the solution.

Microorganisms also go through metabolism in order to obtain all the nutrients and energy required to grow. Aerobic respiration breaks down nutrients into carbon dioxide, water and up to 36 ATP. This process involves an electron transport chain where oxygen is the final electron acceptor. With anaerobic respiration, the products are carbon dioxide, lactic acid water and only 2 ATP. There are five types of microorganisms when addressing how oxygen levels can affect microbial growth: aerobic organisms, obligate anaerobes, facultative anaerobes, microaerophiles, aerotolerant anaerobes.

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Because of these types of differences between microorganisms, biochemical testing is required to narrow down Materials and Methods

It is important to note that before conducting any of these tests, aseptic technique was used to minimize contaminations from other pathogens that could cause false test results. Aseptic technique in this laboratory consists of flaming the inoculating loop or needle until it glows bright orange to kill any bacteria. Then, flaming the neck of the test tube that is being used to reduce the chances of an airborne pathogens contaminating the culture inside.

Tests Conducted Materials Procedure

Starch Hydrolysis Starch agar

Iodine Day 1:

1. Flame the loop and the neck of the tube containing the bacteria. Using the loop, inoculate the starch agar by making a single line streak down the center of the agar

2. Incubate the plate inverted at 37 degrees for 48 hours

Day 2:

1. After removing the plate from the incubator, flood it with iodine

2. Get rid of excess iodine

3. Observe the plate for a clear zone and record the results.

Carbohydrate Fermentation Phenol Red Lactose, Phenol Red Dextrose, Phenol Red Sucrose Day 1:

1. Flame the loop and the necks of all three broths. Inoculate the Phenol Red Lactose broth, Phenol Red Dextrose broth, Phenol Red Sucrose broth with the assigned bacteria using an inoculating loop

2. Incubate at 37 degrees C for 48 hours.

Day 2:

1. Examine tubes for color and gas production

Triple Sugar Iron TSI Agar Slant Day 1:

1. Flame loop, the tube that contains the organism and the TSI agar slant. Inoculate the TSI agar with the bacteria by stabbing the butt of the slant and streaking the slant with an inoculating needle.

2. Incubate at 37 degrees C for 48 hours.

Day 2:

1. Examine the slant for color changes and record results

Methyl Red and Vogues Proskauer Test MRVP broth

Sterile test tube

Methyl Red Reagent

Barritt’s A reagent

Barritt’s B reagent Day 1:

1. Flame the loop and the necks of all the tubes containing the bacteria and the MRVP broth. Using a loop, inoculate the MRVP broth with the bacteria.

2. Incubate at 37 degrees C for 48 hours

Day 2:

1. Transfer 1 mL of the broth to an empty sterile test tube

2. MR Test- Add 5 drops of Methyl Red reagent to one of the test tubes. Observe any color changes and record results

3. VP test- Using the other test tube, add 5 drops of Barritt’s A reagent and gently swirl. Then, add 5 drops of Barritt’s B reagent but do not swirl or agitate the mixture this time. Let the tube sit at room temperature for 10 minutes. Examine for color changes and record the results.

Citrate Utilization Test Simmons Citrate slant Day 1:

1. Flame the loop and the necks of the tubes containing the bacteria and the Simmons citrate slant. Inoculate the Simmons Citrate slant with a loop by streaking the slant with the inoculum.

2. Incubate at 37 degrees C for 48 hours

Day 2:

1. Examine the slant for color changes and record results

Sulfide, Indole & Motility (SIM) test SIM Agar deep

Kovac’s reagent Day 1:

1. Flame the loop and the necks of the tube containing the organism and the SIM agar deep. Using an inoculating needle, stab the SIM agar deep with the bacteria.

2. Incubate at 37 degrees C for 48 hours.

Day 2:

1. Sulfide Test- Observe for the presence of black color in media and record results.

2. Indole test- add 5 drops of Kovac’s reagent to the top of the SIM agar tube and observe for the presence of a red color and record the results

3. Motility- observe for the presence of growth throughout the media and record results.

Gelatin Hydrolysis Gelatin Deep tube

Day 1:

1. Flame the inoculating needle and the tubes containing the bacteria and the gelatin deep agar. Using an inoculating needle, stab the gelatin deep tube with the bacteria.

2. Incubate at 37 degrees C for 48 hours

Day 2:

1. After removing the gelatin deep tube from the incubator, place it in a refrigerator at 4 degrees C for 30 minutes.

2. After removing the gelatin deep from the refrigerator, determine whether the media is liquid or solid and record the results.

Lipid Hydrolysis Tributyrin agar Day 1:

1. Flame the inoculating loop and the tube containing the organism. Using the loop, inoculate the agar plate by making a single line streak down the center of the agar.

2. Incubate the plate inverted at 37 degrees C for 48 hours

Day 2:

1. After removing the plate from the incubator, observe for a clear zone around the bacterial growth and record the results

Urea Hydrolysis Urea broth Day 1:

1. Flame the loop and the necks of the tubes containing the bacteria and the urea broth. Using a loop, inoculate the urea broth with the bacteria.

2. Incubate the broth at 37 degrees C for 48 hours.

Day 2:

1. Examine the tube for color changes and record results.

Nitrate Reduction trypticase nitrate broth

Nitrate A reagent

Nitrate B reagent Day 1:

1. Aseptic technique to reduce risk of contamination. Using a loop, inoculate the broth with the bacteria

2. Incubate at 37 degrees C for 48 hours.

Day 2:

1. Aseptic technique to reduce risk of contamination. After removing the tube from the incubator, add 5 drops of Nitrate A reagent and gently swirl. Add 5 drops of Nitrate B reagent and gently swirl.

2. Wait 10 minutes then observe for color change.

3. If the color of the broth is not red, then add a small amount of zinc dust and record the results

Oxidase Test TSA plate

Sterile wood applicator stick

Oxidase Dry slide

Day 1:

1. Flame the loop and the tube containing the bacteria. Using a loop, inoculate the agar by making a single line streak down the center of the plate.

2. Incubate the inverted plate at 37 degrees C for 48 hours.

Day 2:

1. Using a sterile wood applicator stick, remove a small amount of bacterial growth from the TSA agar.

2. Smear bacterial growth onto the Oxidase Dry Slide. Color change should occur within 10 – 20 seconds. Any change after 20 seconds is ignored.

3. Observe for color change and record the results.

Catalase Test

TSA plate

3% Hydrogen peroxide Day 1:

1. Flame the loop and the tube containing the bacteria. Using a loop, inoculate the agar by making a single line streak down the center of the plate.

2. Incubate the plate inverted at 37 degrees C for 48 hours.

Day 2:

1. Add a drop of 3% Hydrogen peroxide to a small area of growth on the TSA plate.

2. After examining the plate for bubbles, record the results.

Gram stain Crystal violet

Iodine

Ethanol

safranin

Microscope slides

Forceps

Bibulous paper

Marking pen

Compound microscope 1. Label the slides. Flame the loop and the tube containing the bacteria

2. Prepare smears of the organisms by obtaining a small sample of organisms and smearing the cells in a loopful of water on the slide.

3. Allow it to air dry and heat fix the smear

4. Flood the slide with the primary stain (crystal violet) and allow it to remain on the slide for 1 minute

5. Wash the slide with tap water for approximately five seconds.

6. Flood the slide with the mordant (Gram’s Iodine) and allow it to remain on the slide for one minute.

7. Wash the slide for approximately five seconds with tap water.

8. Flood the slide with the decolorizer (ethanol) until no more crystal violet washes off the slide.

9. Wash the slide for approximately five seconds with tap water.

10. Flood the slide with safranin and allow it to remain for one minute

11. Wash the slide for approximately five seconds.

12. Using the bibulous paper, remove any excess water from the slide by gently blotting it dry

13. Using the microscope, view the stain under the oil immersion lens.

Results

Carbohydrate Fermentation

All three broths have a yellow hue and gas in the Durham tube which indicates that this organism is positive for both acid and gas production. The yellow hue of the broth is an indicator that an acid was produced which means that this organism is able to ferment the sugars in the broths. The gas in the Durham tube indicates that carbon dioxide was a byproduct of the fermentation that took place.

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Triple Sugar Iron

The color of the slant and the butt are both yellow indicating that this organism is able to ferment lactose and glucose and/or sucrose. The reason that there is a large gap in the tube is because a gas was produced as a result of the fermentation of these sugars.

Methyl Red and Vogues Proskauer Test

For the methyl red test, the broth remained yellow, a negative test result, indicating that there was no acid production from the glucose.

For the Vogues Proskauer Test, no red ring appeared on top of the broth. This indicates that this organism does not have the ability to breakdown glucose into acetylmethylcarbinol.

MR Test:

Sulfide, Indole & Motility Test

For the sulfide test, no hydrogen sulfide was produced, a negative test result. This indicates that this organism does not have the ability to reduce the sulfur- containing compounds.

For the Indole test, a red ring formed around the top of the broth, a positive test result. This indicates that this organism contains the enzyme tryptophanase.

For the motility test, the agar was completely clear except for the site of inoculation, a negative test result. This indicates that this organism does not the ability to swim away from the original site of the inoculation.

Sulfide test: Indole test:

Gelatin Hydrolysis

After placing the tube in the refrigerator for thirty minutes, the medium was solid, a negative test result. This indicates that there was no gelatinase production.

Starch Hydrolysis

After pouring iodine into the plate, there was no clear zone near the bacterial growth, a negative test result. This indicates that this organism does not produce amylase and therefore, does not have the ability to hydrolyze starch.

Casein Hydrolysis

There was no clear zone near the bacterial growth, a negative test result. This indicates that this organism does not have the ability to produce enzymes that can break down casein.

Lipid Hydrolysis

The agar around the bacterial growth is completely opaque, a negative test result. This indicates that this organism does not have the ability to produce lipases that hydrolyze tributyrin.

Urea Hydrolysis

There was no color change in the broth even after it was inoculated by the organism. It remained orange, a negative test result. This indicates that this organism does not have the ability to hydrolyze urea.

Nitrate Reduction

The top of the broth has changed color from yellow to cherry red indicating that nitrate is reduced into nitrite, a positive test result. Only the reagents Nitrate A and B were added to produce this reaction. The broth started turning red almost immediately.

Oxidase Test

After smearing the bacterial growth onto the oxidase dry slide, a color change didn’t occur within the indicated time range (10-20 seconds), a negative test result. This indicates that this organism does not produce cytochrome c oxidase.

Catalase test

Bubbles appeared after the addition of hydrogen peroxide, a positive test result. This indicates that this organism produces catalase.

Gram stain

After viewing the organism under the oil immersion lens, it was apparent that this organism was a gram negative bacilli.

Conclusion

Using these results, I was able to narrow it down to two organisms: Enterobacter Aerogenes and Klebsiella Pneumonia.

References

Emerson, D., Agulto, L., & Liu, H. (2008). Identifying and Characterizing Bacteria in an Era of

Genomics and Proteomics. BioScience, 58(10), 925-936.

Gunsalus, I. C., Horecker, B. L., & Wood, W. A. (1955). Pathways of carbohydrate metabolism

in microorganisms. Bacteriological reviews, 19(2), 79–128.

Oliver, T. (2017). Undergraduate Microbiology Laboratory Manual. Bluedoor.

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Unknown Organism. (2019, Dec 08). Retrieved from http://paperap.com/unknown-organism-best-essay/

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