Microlab exercises 26-30
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The casein hydrolysis test is used to detect the presence of ______.
This molecule is a large milk protein incapable of permeating the plasma membrane of bacteria. Also, its presence is the reason milk is white.
What enzyme do bacteria use to breakdown casein for their carbon source?
Secretion of proteolytic enzymes into amino acids
What shows a positive result in the casein hydrolysis test? A negative result?
- Positive (left): A clearing of the cloudy agar is the result of a hydrolytic reaction that yields soluble amino acids.
- Negative (right): There is no protease activity, and the medium surrounding the bacterial colony remains opaque.
T or F: the casein in the sterile skim milk agar makes the agar cloudy
This term means the breakdown of proteins or peptides into amino acids by the action of enzymes.
How can the production of an enzyme, gelatinase, by bacteria have a negative effect on the host?
It can often be correlated with the ability of a bacterium to breakdown tissue collagen and spread throughout the body of a host.
In the gelatin hydrolysis lab, following the incubation of the cultures, they are placed in a refrigerator and ice bath until the bottom resolidifies. If the gelatin has been hydrolyzed, the medium will __a__ after refrigeration. If gelatin has not been hydrolyzed, the medium will __b__ during the time it is in the refrigerator.
- A: remain liquid
- B: resolidify
After taking an incubated gelatin deep tube from the refrigerator and tilt it to the side, it retains its liquid form. What does this mean? How is bacteria involved in this process?
- Boiling tissue collagen changes into gelatin, which is a soluble mixture of polypeptides and amino acids. Bacteria can hydrolyze gelatin by secreting gelatinase, an enzyme, which can be used as nutrients.
- Hydrolyzed gelatin is no longer able to gel and is now a liquid.
What are the forms of gelatin at 35 and 5 degrees celcius?
- 35: gelatin is a liquid
- 5: gelatin is a solid
Why did you refrigerate the gelatin cultures before observing them for liquefaction?
Gelatin after incubation will turn into a liquid form, and only after refrigerating it will it show if it retains its liquid form for a positive gelatin hydrolysis.
Why can't most strict anaerobes tolerate O2?
Some bacteria reduce the O2 into toxic superoxide free radicals (O2-, H2O2, OH-). Most strict anaerobes lack the enzymes, including catalase, to protect itself from these molecules.
How can catalase production be detected and what shows a positive result?
By adding the substrate, H2O2, to a TSA slant culture, if catalase was produced by the bacteria, gas bubbles of O2 will represent a positive catalase test.
This enzyme is characterized with thrombin like acitivity. They clot blood plasma by a mechanism that is similar to normal clotting.
This term refers to the ability of an organism to cause disease (ie, harm the host). This ability represents a genetic component of the pathogen and the overt damage done to the host is a property of the host-pathogen interactions.
Certain bacteria are coagulase positive, forming a fibrin clot around themselves. How is this beneficial to the bacteria?
The fibrin clot avoids attacks by the host's defenses.
What are you looking for in the coagulase test to show a positive result?
Coagulase-positive staphylococci will cause plasma to clot by using coagulase to initiate the clotting cascade.
Why are citrate and EDTA (acting as anticoagulants) usually added?
This is to help prevent false-positive results from the normal clotting factors of the body.
What can cause a culture to be considered coagulase negative?
If they are unclotted after 4 hours.
When is DNase produced by bacteria and how is it harmful to the host cell?
In addition to coagulase production, most pathogenic bacteria produce this enzyme, DNase, which degrades host DNA.
How do you test for DNase production? What shows a positive result?
- A DNase agar, with dissolved methyl green, can only bind to intact DNA (not DNA that has been hydrolyzed by DNase) is inculated with desired bacteria.
- A positive result (right) will show a pink area (halo) around the culture.
What is the role of the enzyme, Cytochrome c oxidase? What is the product of its reaction?
- It uses O2 as an electron acceptor during aerobic respiration.
- It reduces cytochrome c to form water and oxidized cytochrome c
How did we test for a positive cytochrome oxidase? What indicated a positive result? A negative result?
- Using a wooden applicator, a bacterial sample was rubbed on a moistened filter paper with oxidase reagent.
- Positive: The strip became a dark purple in the presence of free O2 and the oxidase
- Negative: no color change or a light pink
Are all bacteria that are oxidase positive are strict aerobes. Why or why not?
- No: all bacteria that are oxidase positive are aerobes, but not strict aerobes.
- Oxidase negative bacteria can be anaerobic, aerobic, or facultative, which means they do not have cytochrome c oxidase.
- - They respire using other oxidases in electron transport
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