Lab Quiz 4 Flashcards
What is the purpose of a Gelatinase test?
5-17: Gelatinase
Allows us to determine the ability of a microbe to produce gelatinases
What is the name of the medium?
5-17: Gelatinase
Gelatin Strip
What is the substrate, enzyme, and product(s)?
5-17: Gelatinase
Substrate: Gelatin
Enzyme: Exoenzyme Gelatinase
Products: AA + Polypeptides
What is the appearance of a positive test? Negative?
5-17: Gelatinase
Positive: Blue
Negative: No Change
What were the reagents used? pH indicator?
5-17: Gelatinase
None
What were the two bacteria that was used?
5-17: Gelatinase
2 type of bacteria:
– Pseudomonas aeruginosa (Pa)
– E. coli (Ec)
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
What was the medium of this experiment?
5-28: Motility Test
TTC Agar
What is the substrate, enzyme, and end product(s)?
5-28: Motility Test
Substrate: TTC
Enzyme: Various Reductases
Product: Formazan
What is the appearance of a positive? Negative?
5-28: Motility Test
Positive: Fuzzy Red Growth
Negative: Solid Non-fuzzy Red Line
What reagents and pH indicator were used?
5-28: Motility Test
None
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
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.
What is the medium?
5-8: Nitrate Reduction Test, First Step
Nitrate Broth
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)
What is the appearance of a positive? Negative?
5-8: Nitrate Reduction Test, First Step
Positive: Red
Negative: Proceed to step 2
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
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)
What is the appearance of a positive result? Negative?
5-8: Nitrate Reduction Test, Second Step
Positive: Clear after zinc
Negative: Red after zinc
What reagent and pH indicator were used?
5-8: Nitrate Reduction Test, Second Step
Reagent: Nitrate reagents A, B, Zinc
pH indicator: None
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)
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
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
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
What is synergy, broad spectrum, narrow spectrum?
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?
8-12: Membrane Filtration Test
To test water samples for the presence of coliform bacteria (presumptive)
What was the medium of this experiment?
8-12: Membrane Filtration Test
Endo agar or Nutrient Agar
What is the appearance of a positive? Negative?
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.
5-8 Nitrate Reduction
Nitrate Reductase
What is denitrification?
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.
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?
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?
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)*
5-8 Nitrate Reduction
Nucleic acids and Peptides
Why are there Durham tubes in the nitrate broth?
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.
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?*
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?*
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?*
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?
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?
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?
5-8 Nitrate Reduction
Denitrification
Positive
Nitrogen
What is the original source of nitrate in the nitrate broth? Write the formula for this compound.
5-8 Nitrate Reduction
KNO3
The quantitative sensitivity disk method of antibiotic sensitivity testing is called the _________________
method.
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).
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.
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?
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.
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.
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)
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?
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).
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?*
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?
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?
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?
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.
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.
8-12 Endo Agar
Gram positive
The fermentable carbohydrate in Endo agar is _____________________.
8-12 Endo Agar
Lactose
What will colonies of lactose fermenters look like growing on Endo agar?
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?
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.
8-12 Endo Agar
E. coli
What appearance will be created by lactose non-fermenters growing on Endo agar?
8-12 Endo Agar
Light pink/colorless growth
Bacteria that will grow on Endo agar are Gram _______.
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?*
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?*
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?
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 __________________.
8-12 Endo Agar
Gram Positive
