StudySheetMBG353

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Seneca College *

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353

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Chemistry

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Jan 9, 2024

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Name of Media Test For Organism Observation Of Media Results/Conclusion Lactose Broth Lactose 1 Still Red, No Gas formation (-/-), fermentation -ve 2 Red to yellow color, Bubble trapped (+/+), fermentation +ve Glucose Broth Glucose 1 Still Red, no gas formation (-/-) fermentation -ve 2 Red to Yellow Colour Bubble trapped (+/+) fermentation +ve Starch Agar Plate Presence of amylase 1 Clear Zone Around Growth (+,starch hydrolyzed + ve amylase) 2 Blue Colour Formed around growth -,starch not hydrolyzed -ve amylase Urea Slant Presence Of urease 1 Bright Pinkish purple on slant +, urease +ve 2 No colour change still yellow -,urease -ve Tryptophan Broth Test for Indole 1 No change, still work - 2 Red Ring Formed on top + MR Broth Mixed Acid Fermen tation 1 No Red formation, still Clear - 2 Red to Light Red colour + VP Broth Acetyl Methyl Carbinol presence 1 Light Red colour formed + 2 No colour change, still clear - Citrate Citrate 1 Bright blue color +

Slant growth on slant 2 No colour change, Still Dark Blue -

2. The IMVIC pattern for the bacterium E.coli as follows: ++-- In the same manner, write down the IMVIX patterns for organisms #1 and #2 from the results in your table: #1 - -++ #2 ++- - 1. What is the function of Iodine in the starch hydrolysis test? The function of Iodine is to react with starch to form a dark blue-colored complex. If the area is clear, that means the organism broke down the starch that its due to its production of amylase. 2. What is the function of the Durham tube in the carbohydrate broth? The function of the Durham tube is to detect the gas production. Gas will be trapped in Durham tube and a bubble will form. The presence of a bubble indicates fermentation and gas production of fermentation. 3. a) Both the MR and VP tests determine whether a bacteria can use an organic compound as a source of energy. What is this compound? b) Even though the energy source is the same, the MR and VP tests determine separate metabolic. What are the end products of these pathways for each test? For the MR, it will be mixed stable acids (lactic, acetic, formic acid). For the VP, it will be acetyl methyl carbinol. 4. How is indole production detected in the indole test? The presence of indole is present when a red ring is present at the surface of the broth. The presence of Indole when a red ring is formed at the top of the broth. Starch Agar Plate: Testing for Amylase Typtophan Broth: Test for Tryptophan broth transforms to Indole via the addition of kovac reagent MRVP Broth

Mixed Acid Fermentation Metabolizes acids to other acids, from glucose Lactic acid, Formic acid, etc VP Acetyl methyl, carbinol from glucose Metabolizes pyruvic acid to other acids (ie. lactic, formic, and acetic acid), from glucose Lactate, Formic, etc VP Neutral products like Acetyl methyl carbinol and ethyl alcohol from glucose metabolism Interpretation of Results Record your results in the table. Follow the interpretation guidelines below. Carbohydrate Fermentation Positive test for fermentation = acid production (yellow colour). You must also record whether or not gas is produced. Do this by checking the inverted tube for the presence of a “good size” air bubble. Negative test for fermentation = original red/orange colour (no acid has been produced). Indicate your results as acid/gas. Choose between: + / + (acid & gas), + / - (acid but no gas), or - / - (no acid or gas) Starch Hydrolysis After incubation, place several drops of Gram’s iodine onto the bacterial cultures. Try to cover both the culture and the surrounding media – ie. aim for an ‘edge’. Examine the plate for a clear zone surrounding the growth --- a positive result. Negative result? – the media immediately surrounding the bacterial growth appears deep blue.

Urea Hydrolysis Following incubation, observe the tubes for growth and any colour changes. Positive reaction: a deep pink-purple colour. IMViC Tests For the indole test, add about 5 drops of Kovac’s reagent directly to the tubes of tryptone broth. Do not shake the tube. Observe the tube immediately for the formation of a bright red ring at the top of the broth (positive). If the top portion remains yellowish or brownish, this is a negative result. For the MR-VP tests, aseptically transfer 1 ml of each of the cultures to two separate sterile plastic tubes. One plastic tube will be used for the MR test, the other plastic tube will be used for the VP test. Add the following test reagents to the tubes as indicated: MR test: Add 3 drops of the methyl red reagent to one tube. Positive result = red colour (indicates acid production) VP test: Add 15 drops of Barritt’s reagent A, mix well. Then add 5 drops of Barritt’s reagent B, mix well. Let tube sit for 10-15 minutes, shaking often. Positive result = light red colour For the citrate test, observe the tube for growth and colour changes. Positive result = growth + deep blue colour, Negative result = no growth, media remains green

True motility True motility (self-propulsion) may be due to structural appendages called flagella (singular, flagellum). These are thin protein tubes attached to the bacterial cell wall which rotate to provide propulsion for the cell. Not all bacteria have flagella; hence, some types of bacteria are non-motile. Flagella are generally too thin to be seen in live, unstained bacteria even at high magnification. While it is not possible to see flagella using the light microscope, true motility can be detected in live bacteria by observing rapid changes in cell position. True motility will result in the bacteria spinning around (rotational movement) or traveling long distances across the field of view (translational movement), while changing directions from time to time. Brownian motion and streaming All microbes suspended in liquid medium will appear to move quickly back and forth. This type of movement is called Brownian motion and is due to the random vibrations produced when the microbes collide with water molecules. Brownian motion should not be confused with true motility, as even non- motile organisms will display this type of behavior. In this exercise you will examine live bacteria for motility using two techniques: the wet mount and the hanging drop mount. Motility Media Motility media is a semi-solid growth media that is used to determine bacterial motility. It is an example of an indirect method of motility testing. In addition to basic nutrients motility media also contains: • 0.5 % agar – Routine solid media usually contains 1.5 % agar. The lower concentration of agar in the motility media allows the bacteria to migrate away from the line of inoculation. The presence of agar prevents diffusion of the cells, so that any distribution of bacteria throughout the media results only from true motility. triphenyl tetrazolium (TPT) – tetrazolium salts are used as an indicator of bacterial growth. During growth, bacteria are able to take up and reduce the tetrazolium, producing an insoluble, red compound called formazan . In this way, the site of bacterial growth in the tube can be easily identified by the presence of a dark red color.

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