Microbiology Lab Final Review

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What is phenol red?

A red, water-soluble dye. An indicator (chemical compound that changes color when exposed to certain conditions) that is sensitive to pH. It will change from red to yellow color when the pH value decreases.

What does it mean when the phenol red sucrose, lactose, or dextrose broth tubes stay red or if they turn yellow?

Phenol red is used to determine the ability of the bacteria to break down specific mono- and disaccharides. Specifically, phenol red test for the enzymes that break down glucose, lactose, and sucrose. When carbohydrates are fermented, acid is produced and the broth will turn yellow in the presence of acid.

What is the basis for the catalase test?

Determining the presence of catalase is the basis for the catalase test.

How is it performed?

The catalase test is preformed as follows: 1) Bring two small clean test tubes to your table. 2) Using aseptic technique, add 0.5ml of Staphylococcus aureus to one tube and 0.5ml of Streptococcus pyogenes to another. 3) Place the pipettes into the beaker of disinfectant at your table. 4) Now with a dropper add 0.5ml of hydrogen peroxide to each tube. 5) Oberve the results. The bacteria that make catalase will cause the solution to bubble.

What types (in terms of oygen requirement) of bacteria are catalase + and catalase-.

Catalase converts hydrogen peroxide to water and oxygen. Aerobes make catalase. However, oxygen is toxic to anaerobic bacteria, so anaerobes do not make the enzyme, catalase. The catalase test is frequently used to differentiate gram positves.

What is the basis for making a spread plate (Why would you want to make one?)

The basis for making a spread plate is to create a solid and uniform growth of bacteria. Spread plates are used to test disinfectants and antibiotics.

What is the "stab and streak" method and for which tests is this method used?

Using the inoculating needle and aseptic technique, stab and streak the agar. "Stab" means to push the needle to the bottom of the tube, and "streak" means to zig-zag over the surface of the agar. This method is used for Triple Sugar Iron (TSI) Agar, Citrate Test, and the Urease Test.

How do we test whether or not a bacterium can cause hemolysis of red blood cells?

To determine whether or not a bacterium can cause hemolysis of red blood cells, we utilize a blood agar. Hemolysis on blood agar is a way to differentiate gram positive bacteria.

Name the three types of hemolysis and their differences

1) Beta-hemolytic bacteria make hemolysin. Hemolysin is an enzyme that can break down red blood cells. When beta-hemolytic bacteria are streak across blood agar, a clear ring is seen around the colonies. 2) Alpha-hemolysis is really not hemolysis at all. These bacteria do not make homolysin, but they do secrete chemicals that result in the discoloration of red blood cells to a green color. 3) In gamma-hemolysis, no change occurs when the bacteria are plated on blood agar because the bacteria do not produce a hemolysin.

Give examples of alpha-, beta-, and gamma-hemolysis.

Alpha-hemolysis (Escherichia coli)
Beta-hemolysis (Streptococcus pyogenes)
Gamma-hemolysis / no hemolysis (Staphylococcus epidermidis)

List each step of the gram stain and what happens to the bacteria (gram+ and gram-) each step.

1) Primary stain (crystal violet) will stain both gram + and gram - bacteria purple.
2) Mordant (iodine) binds with crystal violet. Both gram + and gram - are purple.
3) Decolorizer (alcohol) creates holes in the thin layer of peptidoglycan of gram - bacteria and completely dissolves the LPS layer. Gram + are not affected. Gram + bacteria are purple. Gram - bacteria are colorless.
4) Counter stain (safranin) will stain the decolorized, gram -, bacteria pink. Gram + bacteria remain purple.

What is the basis for doing a triple sugar iron test?

The purpose of this test is to determine whether organisms can ferment glucose, sucrose and/or lactose with or without production of gas. The ability of the organism to produce hydrogen sulphide in an acid environment is also tested.

What two things can the TSI test tell you and what would a positive result look like?

The TSI test indicates the microorganism's ability to ferment sugars and to produce hydrogen sulfide. A positive results would be yellow (3 sugars). Negative would remain red. Black would indicate the presence of hydrogen sulfide.

What is meant by an exoenzyme? What is an example of an exoenzyme?

An exoenzyme is an enzyme that acts outside the cell that secretes it. Amylase is an exoenzyme.

What does the enzyme amylase do?

Amylase is secreted by some bacteria outside of the cell so that it can break down complex sugar (starch) which then is absorbed and used by the cell.

What is the basis for the amylase test and how is it performed?

The amylase test is used to detect the production of the enzyme amylase in bacteria.
The amylase test is performed as follows:
1) Obtain a starch hydrolysis plate and divide it into two sections.
2) Lable one side Staphylococcus epidermidis and the other Bacillus cereus.
3) Using an inoculating loop, zig-zag a line of bacteria across the half moon of the agar.
4) Incubate the plate

What specific reagent (chemical) and media type do you use for this test?

After incubation, the plate is treated with the specific reagen (chemical), iodine. A starch hydrolysis plate is the type of media used for the amylase test.

What does a positive amylase result look like? Negative?

If amylase is present, the starch will be broken down around the bacteria and you will see a clearing around the colonies. If amylase is not present, the iodine will cause the starch to turn a deep brown-black color .

What is the basis for the mannitol salt test and how is it performed?

The mannitol salt test is used to differentiate gram + cocci. This medium prohibits the growth of bacteria that can not tolerate high salt osmolarity, they will ferment the sugar, mannitol. The mannitol salt test is performed as follows:
1) Obtain cultures of Staph aureus and Staph epidermidis
2) Divide the mannitol salt agar plate in half and label.
3) With the inoculating loops aseptically zig-zag across the 1/2 of the agar plate for each bacteria type. Incubate and observe.

What does a positive and negative mannitol salt test result look like?

The phenol red indicator in the mannitol salt agar will turn yellow when the mannitol is fermented. Staphylococcus aureus will result with a positive mannitol salt test (turns agar yellow). A negative mannitol salt test would result with the agar ramaining pink.

What is the basis for the urease test and how is it performed?

The urease test is used to detect the enzyme urease, which breaks down urea into ammonia. Ammonia will raise the pH of the media if it is present. The test is performed as follows:
1) Obtain 2 tubes of urease agar and aseptically inoculate each wit Echerichia coli and Proteus vulgaris.
2) Inoculate using the "stab and streak" method with an inoculating needle.
3) Incubate. Always incubate a control tube that is not inoculated.

What does the enzyme urease do?

Urease, an exoenzyme, breaks down urea to ammonia and carbon dioxide.

What are the positive and negative results of a urease test.

Urease agar turns pink when urease is present due to the alkaline pH caused by the formation of ammonia. A negative result would be yellow.

What is methyl red?

Methyl red is an indicator dye that turns red in acidic solutions (unlike phenol red which will turn yellow).

What is the basis for the Methyl Red Vogeus Proskauer (MRVP). VERY IMPORTANT (many students often are confused as to how to do this)! How

REMEMBER, the MRVP test is actually TWO TESTS: methyl red and Voges Proskauer test. The purpose of the MRVP test is to determine which enteric bacteria produce either acid or neutral products. After aseptically inoculating two MRVP broth cultures with bacteria (then incubate), half of the MRVP broth culture is then used for the methyl red test and the other half for a Voges-Proskauer test.
FIRST LAB: 1) Obtain a culture of E. coli and Enerobacter.
2) Obtain 2 MRVP broth culture tubes and label.
3) Inoculate each tube aseptically with bacteria.
4) Incubate
SECOND LAB: 1) Obtain 4 clean glasses test tubes and label them "E.coli VP" and another "Enterobacter VP." Label the third tube E. coli MR" and the fourth tube "Enterobacter MR."
2) Using the aseptic technique, pipette 1ml of the MRVP broth that was incubated into each tube.

What is the purpose of the Voges Proskauer test?

The Vogeus Proskauer test indicates the presence of neutral products (acetoin).

What is the purpose of the methyl red test?

The methyl red test determines the ability of bacteria to break down glucose which then forms acidic compounds.

What is the basis for the citrate test? How is it performed?

The citrate test is used to differentiate gram negative enteric bacteria. It can detect bacteria that can use citrate (a Krebs cycle intermediate) as their only carbon source. Performed as follows:
1) Obtain a culture of E.coli and Enterobacter aerogenes.
2) Obtain and label two citrate agar (Simmons citrate) slants.
3) Inoculate both of these tubes with the "stab and streak" method.
4) Incubate and observe the tubes.

What does a positive result look like?

If a bacterium can ferment citrate, it will form organic acids and carbon dioxide, causing the agar to turn from green to blue (positive result). A negative result will be green.

What types of bacteria would be more suseptible to the antibiotic penicillin? Why?

Gram positive bacteria like Staph- and Streptococcus would be more suseptible to the antibiotic penicillin because penicillin inhibits the cross-linkages between the NAM-NAG layers of the peptidoglycan cell wall. Results in faulty cell wall and lysis of bacteria: bacteriocidal. Syphilis (gram -) is also suseptible to penicillin.

What is the difference between a selective medium and a differential medium (must be able to define them specifically or else full credit will not be given)?

A selective media only support the growth of specific bacteria. Differential media (like phenol red broth) undergo a color change when specific products are present. Differential media or indicator media distinguish one microorganism type from another growing on the same media.

What is meant by a "zone of inhibition"?

A clear area where bacteria do not grow.

How would you test whether or not a certain chemical inhibited the growth of a bacterium?

Spread plates are used to test whether or not a certain chemical inhibited the growth of a bacterium. They are used to test disinfectants and antibiotics. For example, it was noted that there was a "zone of inhibition" around a contaminant penicillium mold on a bacterial spread plate.

What is a dichotomous key? What is it used for?

The dichotomous key is a series of paired statements worded so that only one of two choices applies to any particular organism. Based on which of the two statements applies, the dichotomous key either directs the user to another pair of statements, or it provides the name of the organism in question. The purpose is to follow "the path" that leads to the identity of the unknown organism.

Would an obligate anaerobe grow on an agar plate in a normal incubator? Why or why not?

No, the use of Petri plates presents problems for the culture of anaerobes because each dish has a loose-fitting lid that allows the entry of air.

How would you draw out how to make a streak plate?

A Petri dish is divided into thirds. The goal of a streak plate is to obtain individual colonies in the third (last) sector by "picking up" bacteria from one section and carrying them into the next. Streak plates are a great way to separate mixed cultures.

How would you draw out how to make a spread plate?

A spread plate is made to create a solid and uniform growth of bacteria. After dipping a cotton swab into the culture, use the swab to fill 3/4 of the plate with very closely spaced zig-zag lines. The part immediately under your wrist will remain empty at first. Rotate the agar 1/4 turn and also turn the swab 1/4 and repeat. Repeat this procedure two times.

What is the purpose of IMViC tests? Name three IMViC tests.

IMViC tests are used to differentiate gram negative enteric bacteria like Enterobacter, Klebsiella, and Escherichia coli. Methyl red, Voges-Proskauer (VP), and the citrate test are three IMViC tests.

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