Lab Quiz 4 Flashcards by Rene Galindo

What is the purpose of a Gelatinase test?

5-17: Gelatinase

Allows us to determine the ability of a microbe to produce gelatinases

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What is the name of the medium?

5-17: Gelatinase

Gelatin Strip

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What is the substrate, enzyme, and product(s)?

5-17: Gelatinase

Substrate: Gelatin
Enzyme: Exoenzyme Gelatinase
Products: AA + Polypeptides

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What is the appearance of a positive test? Negative?

5-17: Gelatinase

Positive: Blue
Negative: No Change

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What were the reagents used? pH indicator?

5-17: Gelatinase

None

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What were the two bacteria that was used?

5-17: Gelatinase

2 type of bacteria:
– Pseudomonas aeruginosa (Pa)
– E. coli (Ec)

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What is the purpose of a motility test?

5-28: Motility Test

To be able to detect bacterial motility which is an important differential characteristic

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What was the medium of this experiment?

5-28: Motility Test

TTC Agar

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What is the substrate, enzyme, and end product(s)?

5-28: Motility Test

Substrate: TTC
Enzyme: Various Reductases
Product: Formazan

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What is the appearance of a positive? Negative?

5-28: Motility Test

Positive: Fuzzy Red Growth
Negative: Solid Non-fuzzy Red Line

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What reagents and pH indicator were used?

5-28: Motility Test

None

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What is the purpose of temperature on microbial growth?

2-9: Effect of Temperature on Microbial Growth

A qualitative procedure to understand the effect of temperature on bacterial growth

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What is the purpose of a nitrate reduction test?

5-8: Nitrate Reduction Test, First Step

This test tells us if bacteria can use nitrate as a final electron acceptor in anaerobic respiration. This is also to test if the gram-negative rods either do not reduce nitrate or reduce it beyond nitrite.

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What is the medium?

5-8: Nitrate Reduction Test, First Step

Nitrate Broth

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What is the substrate, enzyme, and end product(s)?

5-8: Nitrate Reduction Test, First Step

Substrate: Nitrate (NO3)
Enzyme: Endoenzyme nitrate reductase
Product: Nitrite (NO2)

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What is the appearance of a positive? Negative?

5-8: Nitrate Reduction Test, First Step

Positive: Red
Negative: Proceed to step 2

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What reagent and pH indicators were used in step 1?

5-8: Nitrate Reduction Test, First Step

Reagent: Nitrate reagent A + B
pH indicator: None

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What is the substrate, enzyme, and end product(s)?

5-8: Nitrate Reduction Test, Second Step

Substrate: Nitrate (NO3)
Enzyme: Many endoenzyme nitrate reductases
Products: Ammonium (NH4+, no gas) or NO,N2O,N2 gases (denitrification)

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What is the appearance of a positive result? Negative?

5-8: Nitrate Reduction Test, Second Step

Positive: Clear after zinc
Negative: Red after zinc

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What reagent and pH indicator were used?

5-8: Nitrate Reduction Test, Second Step

Reagent: Nitrate reagents A, B, Zinc
pH indicator: None

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What is important about the zinc added in step two?

5-8: Nitrate Reduction Test, Second Step

If the broth turns red it is because nitrate was reduced by zinc. If it does not it means there was no nitrate(NO3) to reduce because it had already been reduced beyond nitrite (NO2)

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What is the purpose of the Kirby-Bauer Test?

7-3: Kirby-Bauer Test

To perform a culture and antibiotic sensitivity test on an organism

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What was the medium of this test? (5)

7-3: Kirby-Bauer Test

Mueller-Hinton Agar standardizes the following
- pH:7.4
- Soft agar
- 4mm depth agar for lateral diffusion
- Temp: 37 C
- Amount of drug

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What were the three levels of susceptibility?

7-3: Kirby-Bauer Test

Sensitive: A normal dose is effective against bacteria
Intermediate: A higher than normal dose of an antibiotic is required
Resistant: No acceptable dose is effective

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What is synergy, broad spectrum, narrow spectrum? ## Footnote 7-3: Kirby-Bauer Test

Synergy: when the effect of two drugs combines to be greater than their sum Broad spectrum: Drugs that are effective against most bacteria typically reserved as final options as it can harm native flora Narrow spectrum: Highly targetted drugs that are effective against a specific mecanism of bacterial growth. Must know what bacteria you are dealing with

What is the purpose of the Membrane Filtration Test? ## Footnote 8-12: Membrane Filtration Test

To test water samples for the presence of coliform bacteria (presumptive)

What was the medium of this experiment? ## Footnote 8-12: Membrane Filtration Test

Endo agar or Nutrient Agar

What is the appearance of a positive? Negative? ## Footnote 8-12: Membrane Filtration Test

Positive: Green Metalic Sheen (rapid lactose fermenter) Dark pink/Red (slow lactose fermenter) Negative: Light Pink/Colorless (no lactose fermentation)

Most nitrate-reducing bacteria contain the enzyme ________________________ and perform a single-step reduction of nitrate. ## Footnote 5-8 Nitrate Reduction

Nitrate Reductase

What is denitrification? ## Footnote 5-8 Nitrate Reduction

The loss of biologically available nitrogen; occurs by release of nitrogenous gas

Write the chemical formulas for nitrate, nitrite, potassium nitrate, ammonia, ammonium ion, molecular nitrogen, and nitric oxide. ## Footnote 5-8 Nitrate Reduction

Nitrate: NO3 Nitrite: NO2 Potassium Nitrate: KNO3 Ammonia: NH3 Ammonium ion: NH4+ Nitrogen: N2 Nitric Oxide: NO

Why is the conversion of nitrate to nitrite an example of reduction? ## Footnote 5-8 Nitrate Reduction

Because you go from NO3 to NO2 which is a gain in electrons for nitrogen as nitrogen's oxidation state goes from +5 to +3 (oxidation state going down is reduction)

Why is the conversion of nitrite to the ammonium ion an example of reduction? ## Footnote 5-8 Nitrate Reduction

Because you go from NO2 to NH4+ and the oxidation state goes from +2 to -3 which is reduction

Why do bacteria need nitrogen (which 2 macromolecules require nitrogen)? (2 answers)* ## Footnote 5-8 Nitrate Reduction

Nucleic acids and Peptides

Why are there Durham tubes in the nitrate broth? ## Footnote 5-8 Nitrate Reduction

Because if denitrification occurs, N2 gas will be produced

Explain thoroughly each of the following results in this experiment. a. After incubation, turbid nitrate broth turns red when test reagents A and B are added. b. After incubation, turbid nitrate broth does not turn red when reagents A and B are added. c. A turbid tube that did not turn red when reagents A and B were added turns red when zinc is added. d. A turbid tube that did not turn red when reagents A and B were added does not turn red when zinc is added. e. There is gas in the Durham tube after incubation with a non-fermenting organism. ## Footnote 5-8 Nitrate Reduction

A. (+) Nitrate is present and bacteria did reduce nitrate B. Zinc must be added to determine if zinc can reduce nitrate (-) or if no nitrite is present (+). Either no reduction or full reduction occured C. (-) Red indicates bacteria did not reduce, the zinc did D. (+) Bacteria were able to reduce nitrate broth and either underwent ammonification or underwent full denitrification E. (+) Full denitrification occured resulting in gas in durham tube

Describe the results that would occur in an uninoculated control tube when reagents A and B are added to it?* Why?* ## Footnote 5-8 Nitrate Reduction

The tube would stay transparent as there is no nitrite to react with

What results should an uninoculated control tube give when ONLY zinc is added to it?* Why?* ## Footnote 5-8 Nitrate Reduction

The broth should remain transparent as the zinc is for reducing the broth but is not an indicator

What results should an uninoculated control tube give when reagents A, B, and zinc are added to it?* Why?* ## Footnote 5-8 Nitrate Reduction

The Tube will turn red because the zinc will reduce nitrate and the indicator will show the presence of nitrite

What do reagents A and B test for? What does the zinc test for? ## Footnote 5-8 Nitrate Reduction

- Reagents A and B test for nitrite presence. - Zinc test if reduction occured and will turn nitrate into nitrite if it has not already occured

Why is the conversion of nitric oxide to molecular nitrogen an example of reduction? ## Footnote 5-8 Nitrate Reduction

Because the electronegativity went from +2 to 0

After incubation, gas in the Durham tube in a tube inoculated with a non-fermenting organism means that _____________________ has occurred. Is this a + or a – test for nitrate reduction? What type of gas has been produced? ## Footnote 5-8 Nitrate Reduction

Denitrification Positive Nitrogen

What is the original source of nitrate in the nitrate broth? Write the formula for this compound. ## Footnote 5-8 Nitrate Reduction

KNO3

The quantitative sensitivity disk method of antibiotic sensitivity testing is called the _________________ method. ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

Kirby-Bauer

In this method, the diameter of the ___________________________ around the disk is measured to the nearest _____________________ (unit). ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

Zone of inhibition Milimeters

The inhibition zone diameter that is produced in this experiment will indicate the ___________________ of a bacterium to each antibiotic. ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

Sensitivity

How do you determine if an organism is susceptible to a particular antibiotic in this test? How do you determine if the organism is resistant? ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

The susceptibility zone is measured and compared to a document that states according to what antibiotic is being used a diameter under X is resistant and over X is susceptible

What will a negative test for antibiotic sensitivity look like? Describe such a result and explain what it means. ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

A negative would be shown as no ZOI or a small ZOI. This means that the antibiotic was unable to stop or kill the bacteria

What will a positive test for antibiotic sensitivity look like? Describe such a result and explain what it means. ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

A large ZOI that is greater than the susceptibility break point ex. Breakpoint <10mm, actual D = 18mm

Why is it important that the sensitivity disks used in this experiment contain a specific, standardized amount of antibiotic?* How is this test standardized (i.e. what is standardized in this test)?(4) ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

- To ensure all results are reliable and safe for humans - The agar is 4mm (thinner than usual) to promote lateral diffusion - pH is kept to 7.2-7.4 to mimic blood pH - Agar is incubated at 37 degrees C to mimic human body temp - Agar is incubated for 18 hours to select for younger bacteria

Why is it important that the results of antibiotic sensitivity test guide treatment of bacterial infections? ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

It allows us to understand how different drugs interact with bacteria and what is an effective medication that could be used to adress a bacterial infection appropriately

Penicillin is an antibiotic produced by the mold ________________________ (genus), which is also responsible for __________________________ (type of food). ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

Penicillium Cheese

What is “intermediate susceptibility” to an antibiotic?* Under what conditions would it be appropriate to use an antibiotic to which a particular organism was intermediately susceptible?* ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

- When the ZOI is above resistant but below susceptibility ex. R<5, I=6-8, S>9 - Appropriate if the person is allergic to other antibiotics or can be used with another drug for enhanced effects

True or false: All bacteria found within the clear area around the disks are dead. Why did you answer true or false? ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

False. There are bacteriostatic antibiotics which are not bacteriocidal. Often times with gram negative infections when they are lysed they release lipid-A which is toxic and can cause shock and even death. Therefore a bacteriostatic drug is better to pause development and allow the patient's body to fight the infection in a safer way

What is a broad-spectrum antibiotic? When would it be better to use a broad-spectrum antibiotic? Why? ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

A broad-spectrum antibiotic is an antibiotic that many bacteria are susceptible both gram positive and gram negative. Broad spectrum antibiotics are generally reserved as a last ditch effort as it also kills natural flora. Otherwise targetted antibiotics are usually more appropriate

What is a narrow spectrum antibiotic? When would it be better to use a narrow spectrum antibiotic? Why? ## Footnote 7-3 Antibiotic Sensitivity Testing (Kirby-Bauer Method)

Narrow spectrum antibiotics are antibiotics that are effective at targetting a specific bacteria. This is effective when you know exactly what the bacterial infection is and will not affect your natural flora

Give 2 reasons why sodium sulfite and basic fuchsin are included in Endo agar. ## Footnote 8-12 Endo Agar

- They inhibit the growth of gram-positive bacteria - They both test for lactose fermentation

Endo agar inhibits the growth of _______________________ bacteria. ## Footnote 8-12 Endo Agar

Gram positive

The fermentable carbohydrate in Endo agar is _____________________. ## Footnote 8-12 Endo Agar

Lactose

What will colonies of lactose fermenters look like growing on Endo agar? ## Footnote 8-12 Endo Agar

Lactose fermentation -> Acids -> If rapid fermenter green metalic sheen, If slow fermenter dark pink/red

What appearance is created in this agar by lactose-fermenting organisms that produce large amounts of acid? ## Footnote 8-12 Endo Agar

- A metalic sheen green on the top of the agar and black appearance from the back of the plate

Name one coliforms (genus and species) that produce large amounts of acids when grown on Endo agar. ## Footnote 8-12 Endo Agar

E. coli

What appearance will be created by lactose non-fermenters growing on Endo agar? ## Footnote 8-12 Endo Agar

Light pink/colorless growth

Bacteria that will grow on Endo agar are Gram _______. ## Footnote 8-12 Endo Agar

Negative

Endo agar is used to detect fecal contamination in water and dairy products. What is the connection between fecal contamination and the bacteria that produce a metallic sheen on this agar?* ## Footnote 8-12 Endo Agar

Water and milk are tested for because E.coli lives in the intestines and their presence signals fecal contamination. The metalic sheen growth on endo agar is a visual indicator that the bacteria was a lactose fermenter and is presumptively a fecal coliform

If an organism that cannot ferment lactose grows on Endo agar, what is it using as a food source?* What kind of pH would this organism be expected to produce in the medium?* What will the colonies of this kind of organism look like?* ## Footnote 8-12 Endo Agar

Peptones, this organism would have a alkaline pH from peptone digestion. This organism would grow in light pink or colorless colonies - **It is important to note, nonfermenter could still be possible salmonella or shigella**

What will be the appearance of a presumptive coliform organism growing on this agar? Why? ## Footnote 8-12 Endo Agar

- Metalic sheen green if the organims is a rapid fermenter of lactose - Dark pink/red if the organism is a slow fermenter of lactorse

If an organism will not grow on Endo agar, that is a presumptive indication that the organism is __________________. ## Footnote 8-12 Endo Agar

Gram Positive