Microlab exercises 20-25
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What results were being observed in fermentation exercise?
- How microbial fermentation can yield acid,, gas, or acid and a gas.
- Also tested for the presence of enzyme, B-galactosidase
What color changes are expected if the fermenting bacteria produce organic acids? What about neutral pH and above neutral?
- Acids will lower pH and change the original color to yellow below pH 6.8.
- Above 7.4: purple-pink
- Between the two: red
What is used to detect gas production in fermentation?
The Durham Tube
How does the Durham Tube work once autoclaved?
- During autoclaving, the air is expelled from Durham tubes, and become filled with the medium.
- If gas is produced, the liuid medium inside the Durham tube will be displaced, entrapping the gas in the form of a bubble.
Which sugar does the enzyme, B-galactosidase, hydrolyze in fermenting bacteria, such as E. Coli? What is the natural substrate, though, for this enzyme and what does it yield?
- Lactose to galactose and glucose.
- Natural substrate: ONPG -> galactose + o-nitrophenol.
Explain what production occured in each tube, if any:
- A. No fermentation
- B. Acid production
- C. Acid and gas production
- D. Alcohol fermentation
Do all microorganisms produce the same end product from pyruvate?
Pyruvate=source from glycolysis. Depending on availability of oxygen. Pyruvate is formed but is used (as intermediary electron acceptors) to produce different fermentation products. (Alcohol, Acid, CO2)
What is the purpose of the phenol red or bromcresol purple in the fermentation tube?
PH indicator that shows gas or acid production by color change
What is the fxn of the Durham tube?
To indicate gas production during fermentation
Wat is the color of phenol red at an acid pH? Above 7.4?
What is the function of B-galactosidase?
Hydrolize substrates of sugars to monosaccharides
What does TSI stand for? What does it detect?
Triple Sugar Iron:used to evaluate the fermentation of sucrose, glucose, and lactose as well as the production of hydrogen sulfide and/or gas.
What is TSI used for in the clinical setting? How?
Most often used in to help identify gram-negative rods, or enteric bacteria, vs. Other gram-negative intestinal bacilli by their ability to catabolize glucose, lactose, and/or sucrose
T or F: When testing with a TSI slant, inoculation of the tube must be done with both a streak and a stab of the butt.
What is the point of using both streak pattern and stabbing of the agar in the TSI slant?
- Results in the formation of two reactions:
- 1. The slant is exposed to atmospheric oxygen, which is aerobic.
- 2. The butt is not exposed so it is anaerobic.
In the TSI results, a yellow butt and red slant is due to the fermentation of glucose, which is indicated by the yellow color. What doesr the red slant mean?
Limited acid formation, because of the limited glucose in the medium.
In the TSI slant, what does it mean when there is excessive acid production, and the slant and butt are yellow.
Fermentation of glucose and lactose
What does it mean if the agar in a TSI slant is split or lifted?
What does a blackening of the agar mean in a TSI slant?
H2S gas production
Distinguish between an acid and an alkaline TSI slant.
- Alkaline: red butt, red slant, no gas, but H2S production (blackenin of agar)
- Acid: yellow slant, yellow butt, gas production, but no H2S production
What is the purpose of the thiosulfate in the TSI agar?
Substrate for hydrogen sulfide production (H2S)
Why is there more lactose and sucrose than glucose in TSI agar? What is reversion?
- So reversion can occur, something to become more acidic.
- Reversion: occurs once there is no sugar left and the MO produces ammonia endproducts which raises the pH and turns the slant back to red (something becomes acidic, runs out of sugar, pH raises)
Two common lipids catabolized by bacteria are __a__ and __b__.
- A. Triglycerides
- B. Phospholipids
Why is it important for bacteria to hydrolize lipids? How is this done?
Phospholipids are functionl components of all cells: Triglycerides are hydrolyzed by enzymes called lipses into glycerol and free fatty Acids (3).
What does rancidity mean?
When lipase-producing bacteria contaminate food products, the bcteria hydrolyze the lipids, especially dairy products, causing the spoilage.
What is considered a lipase-producing bacteria?
A bacteria that produces lipase to hydrolyze lipids for cell components and nutrients.
When a lipolytic bacteria is added to an agar-solidified culture, with lipids, what happens to the pH of the surrounding medium? Why?
It becomes acidic due to the release of fatty acids when the lipids are hydrolyzed by the bacteria.
How do you detect if there is a positive test for lipolysis in a spirit blue agar? (Lipid hydrolysis)
If it turns clear around the lipolytic bacterial colonies due to the acid pH.
What is the function of lipases?
An enzyme that breaks down lipids or fats into smaller molescules of glycerol and fatty acids.
How can you determine whether a bacterium is lipolytic?
If the medium with the inoculated culture becomes acidic surrounding the colony.
What are several pathways that bacteria use to metabolize lipids?
Glycolysis, citric acid cycle, oxidation pathways
What is SIM medium used for?
Used for the differentiation of the enterobcteriacaea. This is based on H2S production, indole production, and motility
How can you Identify H2S production? How can you spot motility?
- Using SIM medium, which contains ferrous ammonium sulfate, it combines with the H2S produced by the bacteria, forming an insoluble, black ferrous sulfide precipitate that cn be seen along the stab line during inoculation.
- If the entire tube is black, this shows motility.
Of what use to bacteria is the ability to produce H2S?
The bacteria that produces hydrogen sulfide uses sulfate rather than oxygen. This is when sulfur is used as the terminal electron acceptor in the electron transport chain. The end result is H2S. This is useful when hydrolyzing proteins into amino acids for nutrients.
What substrates are acted on in SIM medium in order for H2S to be produced?
Thiosulfate and Cysteine
In addition to H2S production and motility, for what other test can SIM medium be used?
How does a black precipitate of FeS indicate the production of H2S?
If H2S is produced, it combines with the ferrous ammonium sulfate to form an insoluble, black ferrous sulfide precipitate.
What are you looking for in the SIM tubes during the IMViC lab?
- Sulfur - a black precipitate
- Indol - a red color using Kovac's reagent
- Motility - tube becomes cloudy
What is the main component of the MR-VP? Using this component,what is produced from the bacteria?
- Acid is produced during fermentation
What does MR-VP stand for?
Methyl Red - Vogues Proskauer
What is the objective of the Methyl Red test? What shows a positive test result?
When enteric bacteria catabolize glucose for energy, the end products may include acids, some very acidic (a pH of 4), others not as much (a pH of 6). Methyl red is a pH indicator, turning red indicating a positive methyl red test, yellow for a negative result.
What is the objective of the Voges-Proskauer Test? What shows a positive test result?
It identifies bacteria that ferment glucose by detecting the presence of acetoin using reagents of 40% KOH and Barrit's reagent. In the presence of acetoin, a cherry-red color develops.
Whatever is the objective of the citrate utilization test? How is this done? How is a positive citrate test shown?
- Determines the ability of bacteria to use citrate as a sole carbon source for their energy needs.
- Simmons citrate agar slants are used for this test, where sodium nitrate is the only carbon source.
- - It also contains both NH4, as a nitrogen source, and a pH indicator
- - CO2 is released when the bacteria oxidizes the citrate, which combines with the sodium and water to form sodium carbonate (an alkaline product)
- - This raises the pH, turning the pH indicator to a blue color, representing a POSITIVE citrate test.
What organic molecule is necessary to detect mixed acid fermentation by bacteria?
Methyl Red, which is pH indicator, which turns red during an acidic medium due to the catabolization of glucose.
Why is it important for the citrate test to have a slant agar?
O2 is necessary for citrate utilization.
When using Kovacs' reagent, what is it detecting and what does it mean when its a negative result?
- The reagent reacts with indole, indicating hydrolyzation of tryptophan (indole is not used during this reaction, and accumulates in the medium)
- A negative result indicates tryptophan was not hydrolized
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