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Gram staining was used to see if the bacteria were gram positive or negative as well as the shape of the bacteria cells (Gram negative, pink bacilli). Endospore and capsule staining had been done to see if there were any endospores or capsules present (No endospore present; Capsule present). KOH testing was done to double check on whether the unknown was gram negative or positive (Stringy, gram negative). MacConkey was to again check if the sample was a gram negative or positive as well as to if there was any lactose fermentation (No growth, hard to grow).
The Anaerobic and Aerobic tests were to see which how the bacteria was affected by the different environments it had been placed in (Growth; Growth). Oxidase testing had been done to see if the bacteria could use oxygen as a final electron acceptor in the electron transport chain (No color change). Catalase testing was done to see if the microbe has catalase (No bubbles).
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Amylase test was done to see if the microbe could use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth (Negative, no starch as carbon source). Urease test was used to see if any ammonia had been made (No color change, no urea hydrolysis). The Gelatin test was done to see if the microbe could use the protein gelatin as a source of carbon and energy for growth (Liquid on surface, gelatinase enzyme present). Citrate test was to see if the microbe could use citrate as a source of carbon and energy for growth (No growth).
The Methyl Red test had been used to see whether the microbe could perform mixed acids fermentation when supplied glucose (No color change). Voges-Praskauer was done to see whether the microbe produces 2,3-butanediol as a fermentation product from glucose (No color change). SIM tests were done to see if there was any sulfur reduction, indole production, or motility ((S) No sulfur production (I) Tryptophanase enzyme absent (M) Minimal to no movement). KIA testing was used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates (Glucose fermentation only). The Nitrate Reduction test had been done to see if the bacteria could break down nitrate into nitrogen gas (No nitrate reduction, NO3). Kirby Bauer was to see the effectiveness of several antibiotics on the bacteria, or the degree of antibiotic resistance it has (Ampicillin: 10mm, Penicillin: 30mm, Nalidixic Acid: 0mm, Streptomycin: 25mm, Tetracycline: 25mm, Chloramphenicol: 17mm, Kanamycin: 38mm, Tea Tree Oil: 20mm). The molecular identification of unknown bacteria using 16S rRNA sequence had been done to identify and compare bacteria present within a given sample (Not enough sequences because not enough DNA was sequenced).
In the beginning of the semester an unknown bacteria sample had been given to each student in the class. The one that had been given to me and is the focus of study in this report was labeled Imid 7 with a pesticide identified as Imidacloprid. The goal with the unknowns was to narrow down and pinpoint what type of bacteria it is. This had been done by testing for specific characteristic it may have with various experiments to discover the unknown’s characteristics and morphology.
Prepare a clean glass slide on a piece of paper towel and label (E.coli, unknown, and Staph.) where three drops of water will be placed along the slide with an inoculating loop sterilized with the Bunsen burner, make sure to sterilized it between every use. Before transporting a sample of each colony into each of the water drops with a sterilized inoculating loop, make sure it is cool before use. Then let the drops air dry completely before heat fixing it over the Bunsen burner. Then putting the slide over something to catch the liquid waste under it proceed to flooding the slide with crystal violet and letting it sit for about a minute then wash it off with distilled water briefly. Repeat this step but instead of crystal violet use gram iodine. Once that is done tilt the slide at a 45 degree angle over the waste catcher and pour ethyl alcohol down it for about ten seconds and then briefly rinse with distilled water again. Then repeat the step with crystal violet and gram iodine on the slide again but this time with safranin and instead of letting it rest for a minute only do about thirty seconds. Let the slide air dry completely before putting it under the microscope. While it is drying clean the microscope lens with lens paper carefully in case it is dirty. Slowly increase the magnification on the slide until you need to use immersion oil to be able to see the bacteria clearly.
Prepare a clean glass slide on a piece of paper towel and place a drop of water on the slide with an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it between every use. Before transporting a sample of a colony into the water drop with a sterilized inoculating loop, make sure it is cool before use. Then let the drop air dry completely before drawing a rectangle around it with a wax pencil. Flood leifson flagella stain inside the rectangle for about seven to fifteen minutes or when a golden film develops. Then rinse the slide with distilled water before letting the slide air dry again. Once completely dry put under the microscope with immersion oil, flagella will stain red.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, smear some of your unknown onto a slide and cover it with a piece of Kim wipe that was cut to fit. Heat fix it over a hot plate but continuously add malachite green to the Kim wipe the entire time. Once done heating remove the Kim wipe quickly and let the slide dry and cool before putting it under the microscope with immersion oil.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, transfer a colony of your unknown onto a slide with a drop of India ink. Then get another slide and smear the drop and bacteria sample across the slide as evenly as possible and then let air dry. Once completely dry put under the microscope with immersion oil.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, transfer a colony of your unknown onto a slide and add a drop of three percent KOH onto it. Mix the sample well with an inoculating loop.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, streak your unknown into a MacConkey plate and incubate it overnight at 37°C.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, streak your unknown into two plates, each labeled aerobic or anaerobic. Then place the plate labeled anaerobic into the GasPak EZ Anaerob Container System while the aerobic plate should be put into an incubator at 37°C under aerobic conditions as a control.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, smear a sample of your unknown onto a portion of a strip and watch it for any changes in color.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, smear your unknown onto a slide. Then add a drop of three percent hydrogen peroxide and mix it well with a toothpick. If copious bubbles are observed, the microbe is positive for catalase.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, streak your unknown into a plate and incubate it overnight, then add iodine reagent. Clear halos surrounding colonies indicate the ability to digest the starch in the medium.
Get a test tube of urea broth and inoculate it with a sample of your unknown and incubate the test tube at 35°C in ambient air for eighteen to twenty-four hours. If it turns pink then urease enzyme is present.
Get a test tube with gelatin in it and with an inoculating stick sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, transfer a sample of your unknown into it by stabbing it into the gel, make sure not to stab all the way through, then incubate the test tube at 25°C for up to a week. If the gelatin turns liquidy the microbe could use the protein gelatin as a source of carbon and energy for growth.
With an inoculating loop sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, streak your unknown into a citrate plate and incubate it overnight at 37°C.
Inoculate a broth with your unknown and incubate it at 37°C for twenty-four hours. Then add five drops of methyl red into it, mixed acids fermentation results in a red color change.
Inoculate a broth with your unknown and incubate it at 37°C for twenty-four hours. Then add five drops of Barret’s A and shake the test tube gently to mix. Then add five drops of Barret’s B before slanting the test tube to allow maximum oxygen exposure to the reaction mixture and allowed to stand for up to thirty minutes, development of a red color indicates a positive test.
Get a test tube with gelatin in it and with an inoculating stick sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, transfer a sample of your unknown into it by stabbing it into the gel, make sure not to stab all the way through, then incubate the test tube at 35-37°C for twenty-four hours, if there is any grow then that means that the bacteria is mobile. Then add a couple of drops of kovacs into the test tube until there is about a few mm of it on top of the gelatin, if it turns pink then there is tryptophanase enzyme present.
Get a KIA slant test tube and with an inoculating stick sterilized with the Bunsen burner, make sure to sterilize it and cool between every use, transfer a sample of your unknown into it by stabbing it into the gel at the bottom of the slant, make sure not to stab all the way through. Then pull the stick out and when it is no longer in the gel but on top of it rub the stick gently up the gel slant until you reach the top of it. Then incubate the test tube for twenty-four hours.
| Symbol | Interpretation |
|---|---|
| Red/yellow | K/A Glucose fermentation only; Peptone catabolized |
| Yellow/yellow | A/A Glucose and lactose and/or sucrose fermentation |
| Red/red | K/K No fermentation; Peptone catabolized |
| Red/no color change | K/NC No fermentation; Peptone used aerobically |
| Yellow/yellow with bubbles | A/A,G Glucose and lactose and/or sucrose fermentation; Gas produced |
| Red/yellow with bubbles | K/A,G Glucose fermentation only; Gas produced |
| Red/yellow with bubbles and black precipitate | K/A,G, H2S Glucose fermentation only; Gas produced; H2S produced |
| Red/yellow with black precipitate | K/A, H2S Glucose fermentation only; H2S produced |
| Yellow/yellow with black precipitate | A/A, H2S Glucose and lactose and/or sucrose fermentation; H2S produced |
| No change/no change | NC/NC No fermentation |
Get a nitrate broth that contains a Durham tube and inoculate it with your unknown before incubating it at 35±2°C for twenty-four to forty-eight hours. Then examine the tube for gas bubbles (Non-fermenter: Denitrification-production of nitrogen gas. Fermenter or unknown status: Source of gas is unknown; requires addition of reagents.) and then add eight drops from each of the reagents A and B. Mix it well before letting it sit for about ten minutes. If the solution turns red then nitrate reduction to nitrite occurs but if there is no color change then it is an incomplete test and requires the addition of about a pinch of zinc dust. After adding in the zinc let it sit again for ten minutes, if the liquid doesn’t change color then nitrate reduction to nongaseous nitrogenous compounds occurs. If it turns red then there is no nitrate reduction.
Get two Mueller-Hinton agar plates and label on it where you will be adding your antibiotics. Then aseptically dilute your unknown bacterial broth culture to the optimal OD level, (0,0) of 0.2 at 600nm, before spreading 100µL of diluted culture evenly across the entire surface of each of the plates. Allow the culture to soak before placing four antibiotic disks into each of the plates, make sure to spread them evenly from each other. Then add 10 µL of each of the antibiotic stock (Ampicillin: 1µg/µL, Penicillin: 1µg/µL, Nalidixic Acid: 3µg/µL, Streptomycin: 1µg/µL, Tetracycline: 3µg/µL, Chloramphenicol: 3µg/µL, Kanamycin: 3µg/µL, Tea Tree Oil: 3µg/µL) to each of the antibiotic disks, make sure to put them there they are correctly labeled. Then incubate the plates overnight at 37°C.
| Tests | Results |
|---|---|
| Gram Stain | Gram negative, bacilli. |
| Flagella Staining | Not present. |
| Endospore Staining | Not present. |
| Capsule Staining | Present. |
| KOH | Gram negative. |
| MacConkey | No growth, hard to grow. |
| Anaerobic | Both anaerobic and aerobic plates had growth. Though aerobic has double the growth of the anaerobic sample. |
| Oxidase | Negative. |
| Catalase | No bubbles. |
| Amylase | No starch, does not contain the amylase enzyme. |
| Urease | No ammonia is made. |
| Gelatin | Positive |
| Citrate | No growth & No Color change. |
| Methyl Red | Does not perform mixed acids fermentation when supplied glucose. |
| Voges-Praskauer | Microbe does not produce 2,3-butanediol as a fermentation product from glucose. |
| SIM | No black precipitate. Did not turn pink after adding drops of kovacs, so no tryptophanase enzyme. Not that much spreading of growth so the bacteria is not mobile. |
| KIA Slant | (Head/Butt) Red/Orangish, K/A. |
| Nitrate Reduction | No nitrate reduction. |
| NCBI BLAST of 16S rRNA gene sequence | Not enough DNA was isolated. |
| Kirby Bauer (zone of inhibition) in mm | Tetracycline: 25 Ampicillin: 10 Penicillin: 30 Streptomycin: 25 Natidixic Acid: - Chloramphenicol: 17 Kanamycin: 20 Tea Tree Oil: 38 |
I was able to make a successful Gram stain in lab, after a few failed attempts, and was able to determine that
the gram charge was negative and that the cells were rod-shaped (bacilli). The Flagella test had been done in
lab but for the first attempt, the unknown samples were too old to be able to determine whether the bacteria
had flagella accurately, and so we had done that test again when a newer sample had been made. In the endospore,
capsule, and KOH tests, there had been no complications.
In the MacConkey test, there had been no growth, which meant that the bacteria was not gram-negative which
contradicted the KOH test done earlier. This may mean that I will need to double-check on what the unknown is
since a mistake could have been made in one or both of these tests. In the Anaerobe, Oxidase, Catalase, Amylase,
Urease, Gelatin, Citrate, Methyl Red, Voges-Praskauer, SIM, KIA, Nitrate Reduction, and Kirby Bauer tests, there
had been no complications.
I was unable to do the NCBI BLAST of 16S rRNA gene sequence because my sample did not have enough sequences
because not enough DNA was sequenced. Through all of these tests, it was concluded that the unknown, Imid 7,
bacteria is of the enterobacteriaceae family and the shigella genus. Shigellosis is an infectious disease that
causes diarrhea, fever, and stomach cramps. Those infected with this disease are usually inflicted for about
five to seven days and can be prevented by washing your hands with soap and other hygienic methods (Shigella).
Imidacloprid is a neonicotinoid insecticide, used to control termites, some soil insects, sucking insects, and
as a flea product for pets.
Identification and Characterization of Bacterial Strain Imid 7 from Unknown Sample. (2024, Jan 02). Retrieved from https://studymoose.com/document/identification-and-characterization-of-bacterial-strain-imid-7-from-unknown-sample
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