Practical II Flashcards
What does a nitrate reduction test used for?
Identify bacteria that are able to reduce NO3 (nitrate).
What is the first step in a nitrate reduction test?
Reactants A & B are added to nitrate.
The result of the first step in the nitrate reduction test is the solution turns red. What is the interpretation of these results?
Nitrate (NO3) is reduced to nitrite (NO2) which reacts with reagents A & B to form red ppt. This is called a +1.
The result of the first step in nitrate reduction is the solution does not turn color. What is the next step?
Add zinc. If the solution then turns red, this is considered a negative test. The organism did not reduce nitrate (NO3) to nitrite (O2). Zinc did that. Zinc reduced nitrate (NO3) to nitrite (NO2) which reacted with reagents A & B to form nitrite (NO2).
In the nitrate reduction test, After the addition of zinc, what is the interpretation of a solution that again does not change color?
Nitrate (NO3) had been reduced beyond nitrite (NO2). This is a +2 because:
NO3 = negative
NO2 = +1
More reduced forms (N2O, NO, N2 gas) are +2.
What are the 3 possible results in the nitrate reduction test?
Add reagents A & B:
- Turns red = +1 Nitrate was reduced to nitrite
- Does not turn red = add zinc
- Zinc does not turn solution red = +2 (organism had reduced nitrate (NO3) beyond nitrite (NO2)
- Zinc does turn solution red = negative (zinc reduced nitrate (NO3) to nitrite (NO2))
What is the result of the nitrate reduction test if reagents A & B turns the solution red?
+1 - Nitrate was reduced to nitrite
What is the result of the nitrate reduction test if reagents A & B do not turn the solution red
Unknown, must add zinc to check
What is the result of the nitrate reduction test if after the addition of zinc, the solution does not turn red?
+2 - organism had reduced nitrate (NO3) beyond nitrite (NO2)
What is the result of the nitrate reduction test if after the addition of zinc, the solution turns red?
Negative - zinc reduced nitrate (NO3) to nitrite (NO2)
What are the negative, +1 and +2 results in the nitrate reduction test?
Negative = reagents A & B don't change the solution to red but the addition of zinc turns the solution red (because zinc reduced nitrate (NO3) to nitrite (NO2). \+1 = reagents A & B change the solution to red. \+2 = reagents A & B and also zinc don't change the solution color (was reduced beyond nitrite (NO2).
In the nitrate reduction test, which tubes turn red?
Negative = reagents A & B don't change the solution to red but the addition of zinc turns the solution red (because zinc reduced nitrate (NO3) to nitrite (NO2). \+1 = reagents A & B change the solution to red.
In the nitrate reduction test, which tubes are clear?
+2 = reagents A & B and also zinc don’t change the solution color (was reduced beyond nitrite (NO2).
In the nitrate reduction test, what does the addition of zinc do?
It differentiates between a negative and +2 test.
Gram stain procedure
Crystal violet (purple) Iodine Alcohol wash Safranin (pink) Gram + purple Gram - pink
What is the result of a Gram stain?
Gram + = purple
Gram - = pink
What does MacConkey Agar select for?
Selects for Gram -
acid production from lactose fermentation
What does starch hydrolysis test for?
The presence of amylase (enzyme that breaks down starches)
What does the catalase test identify?
Production of the enzyme catalase which converts H2O2 to H2O and O2
What are the results of MacConkey Agar?
Since MacConkey Agars selects for Gram - bacteria:
Positive = colonies and media appear pink/purple
Negative = colonies and media appear white/yellowish
What are the results of starch hydrolysis?
Since starch hydrolysis tests for the presence of amylase:
Positive = iodine creates halo around around colonies
Negative = no halo
What are the results of the catalase test?
Since catalase is an enzyme that converts H2O2 to H2O and O2:
Positive: Bubbles produced after H2O2 is added
Negative: No bubbles after H2O2 is added
What does the MR/VP test indicate?
Which pathway is used for fermentation of glucose
What does the MR test indicate?
Generation of acid from glucose fermentation (pH < 4.4) (mixed fermentation)
What are the results of the MR test?
Positive: Methyl red turns culture red (acid from glucose fermentation)
Negative: Methyl red turns culture yellow/orange (No acid from glucose fermentation)
What does the VP test indicate?
Production of acetoin (pH > 6) (no mixed fermentation)
What are the results of the VP test?
After 25 minutes of adding VP reagents A & B:
Positive: reddish color develops on top of tube
Negative: No reddish color develops
In the MR/VP tests, what is the positive result?
Solution turns red.
What does the oxidase test indicate?
Presence of cytochrome oxidase C (enzyme for electron transport chain)
What are the results of the oxidate test?
Positive: Swabbed colony turns blue after adding drops of reagent.
Negative: Swabbed colony does not turn blue after adding reagent
What does the citrate test indicate?
Citrate as sole carbon source
Ammonium salts as sole nitrogen source
What are the results of the citrate test?
Positive: media turns deep blue
Negative: media remains green
What species does the mannitol salt agar indicate?
Staphylococcus species
How does mannitol salt agar indicate Staphylococcus species?
Acidic end products from mannitol fermentation
What are the results of the mannitol salt agar test?
Positive: media turns yellow
Negative: media remains red
What does the carbohydrate fermentation test indicate?
Acid production and/or gas due to carbohydrate fermentation
What are the four results from the carbohydrate fermentation test?
Acid/gas positive: media turns yellow/bubble in Durham tube
Acid positive: media turns yellow/no bubble
Gas positive: media remains red/bubble in durham tube (alcohol fermentation)
Acid/gas negative: media is red with no bubble
Negative: media remains red/no bubble
What does the hydrogen sulfide test indicate?
Production of hydrogen sulfide through cysteine purification or anaerobic cellular respiration
What are the results of the hydrogen sulfide test?
Positive: black precipitant forms in the SIM media deep
Negative: SIM media remains yellow/brown
What does carbohydrate fermentation in broth with durham tubes indicate?
Production of acid and/or gas due to fermentation of a specific carbohydrate
Carbohydrate fermentation indicates a production of what?
Acid
What is the similarity and the difference between the MacConkey agar and Methyl Red tests?
MacConkey and MR both test for glucose fermentation.
MacConkey is limited to Gram - bacteria.
Of the three tests for carbohydrate fermentation, what specifically do they test for?
- Carbohydrate with Durham Tubes tests for acid due to carbohydrate fermentation (and gas production)
- MacConkey Agar and MR: Acid due to glucose fermentation
What are the four results of the carbohydrate with durham tubes test?
- Acid/gas positive: turns yellow/with bubble
- Acid positive: turn yellow
- Gas positive: remains red/bubble
- Negative: remains red/no bubble
What does the hydrogen sulfide test indicate?
The presence of hydrogen sulfide which can be released via several pathways.
How is the presence of hydrogen sulfide detected using SIM media?
Hydrogen sulfide can be detected using iron salt (ferrous sulfate) mixed in media. Hydrogen sulfide combines with iron salt to produce ferric sulfide which is a black ppt.
What is SIM media?
A medium that contains a sulfur source and iron salt to detect hydrogen sulfide production.
What is bacterial transformation?
When a bacterial cell picks up DNA from its environment.
What is chemically competent?
When a bacterial cell can pick up DNA from the environment because of a chemical treatment.
What is naturally competent?
When a bacterial cell can pick up DNA from the environment without outside influence.
Treating bacterial cells with calcium chloride and heat-shocking them is a method of doing what?
Making bacterial cells chemically competent.
What is an antibiotic?
An antibiotic is a chemotherapeutic agent that kills or inhibits the growth of bacteria.
What is a chemotherapeutic agent?
Any chemical used to fight disease.
Name three disinfectants?
Bleach, soap, hydrogen peroxide (H2O2)
What is a disinfectant?
Chemicals that are harmful to microorganisms that are only used on inanimate objects.
What would be the most effective disinfectant on this disc diffusion assay?
Bleach followed by H2O2. Soap does not display a detectable zone if inhibition. Water is the control.
How do you measure a zone of inhibition?
The diameter of the zone from left to right including the disc.
What is the result of a susceptible bacteria as tested using the Kirby-Bauer test?
If a zone of inhibition is equal to or larger than the susceptible measurement, the organism will be classified as susceptible.
What is an intermediate result of a Kirby-Bauer test?
A results that is between the susceptible and resistant measurements. The antimicrobial agent has a moderate effect on the growth of the cells.
What is a resistant bacteria as measured by the Kirby-Bauer test?
If the zone of inhibition is equal to or smaller than the resistant measurement, the organism will be classified as resistant.
What is a zone of inhibition?
Clear zones surrounding the paper disc in a disc diffusion assay or Kirby-Bauer test. These zones are areas of the plate where bacterial growth is inhibited. Generally, the larger the zone of inhibition, the more effective the substance that is being tested is at preventing bacterial growth.
In a zone of inhibition, is bacteria growth encouraged or inhibited?
Inhibited
The larger the zone of inhibition, is the substance that is being tested is at preventing bacterial growth more effective or less effective?
More effective.
A cell is transformed by the pGLO plasmid. What must occur before it will be resistant to ampicillin?
- Beta-lactamase breaks down ampicillin which allows cells to grow in ampicillin environments.
- Cell transcribes and translates the bla gene from the pGLO plasmid to produce beta-lactamase protein before it will be resistant to ampicillin.
On a pGLO plasmid, what is an ori site?
The location for DNA polymerase to start replication. This allows the plasmid to be replicate.
On a pGLO plasmid, what is a bla gene?
Codes for a protein (beta-lactamase) that provides ampicillin resistance to the bacterial cell, allowing transformed cells and their progeny to survive on a medium containing ampicillin.
On a pGLO plasmid, what is a GFP gene?
Codes for a green fluorescent protein (GFP), which is a protein that fluoresces green when stimulated with ultraviolet light.
On a pGLO plasmid, what is a araC gene?
Regulates the transcription of the GFP gene and is turned on only in the presence of the sugar arabinose. Therefore, transformed cells will possess this plasmid with the GFP gene, but they will only express the green fluorescent protein if arabinose is present in the medium.
What will be the transformation reaction with E. coli cells grown in medium with no pGLO protein:
- What are the expected number of colonies
- Will the cells fluoresce?
- Lawn - All E. coli can grow on this plate as there is no selection from ampicillin.
- No fluorescence - The GFP gene is not present in any cells, so no E. coli should appear green.
What will be the transformation reaction with E. coli cells grown in medium with ampicillin:
- What are the expected number of colonies
- Will the cells fluoresce?
- None
- No fluorescence
The lack of the bla gene found on the GFP plasmid means that none of the E. coli should be able to grow on this plate.
What will be the transformation reaction with E. coli cells grown in medium with ampicillin and arabinose sugar:
- What are the expected number of colonies
- Will the cells fluoresce?
- None
- No fluorescence
The lack of the bla gene found on the GFP plasmid means that none of the E. coli should be able to grow on this plate.
What will be the transformation reaction with E. coli cells with the pGLO DNA grown in medium:
- What are the expected number of colonies
- Will the cells fluoresce?
- Lawn - All E. coli can grow on this plate as there is no selection from ampicillin.
- No fluorescence - The GFP gene is not present in any cells, so no E. coli should appear green.
What will be the transformation reaction with E. coli cells with the pGLO DNA grown in medium and ampicillin:
- What are the expected number of colonies
- Will the cells fluoresce?
- Individual colonies - Only transformed E. coli will be able to grow as expression of the bla gene will make them resistant to ampicillin.
- No fluorescence - There is no fluorescence since arabinose is not present to induce the expression of the GFP gene.
What will be the transformation reaction with E. coli cells with the pGLO DNA grown in medium with ampicillin and arabinose sugar:
- What are the expected number of colonies
- Will the cells fluoresce?
- Individual colonies - Only transformed E. coli will be able to grow as expression of the bla gene will make them resistant to ampicillin.
- Will exhibit fluorescence - Cells will fluoresce since arabinose is present to induce the expression of the GFP gene.
What are the genes in the pGLO plasmid that do the following:
- origin of replication
- Provides ampicillin resistance
- Contains gene for fluorescent protein
- Contains gene that regulate transcription of #3
- Sugar required to fuel #4
- ori site
- bla gene
- GFP gene
- araC gene
- arabinose
What is the enzyme that provides the pGLO plasmid with ampicillin resistance?
Beta-lactamase
Why are gram + bacteria more susceptible to antimicrobial agents?
Their thick peptidoglycan layer absorbs surrounding material, even toxins, making them more susceptible and less and slower to develop resistance.
Why are gram negative bacteria less susceptible to antimicrobial agents?
They have thinner peptidoglycan layer surrounded by several layers of cell membranes which resist toxins. Harder to kill, more likely to develop resistance.
Which type of bacteria is being described here?
Their thick peptidoglycan layer absorbs surrounding material, even toxins, making them more susceptible and less and slower to develop resistance.
Gram +
Which type of bacteria is being described here?
They have thinner peptidoglycan layer surrounded by several layers of cell membranes which resist toxins. Harder to kill, more likely to develop resistance.
Gram negative
What type of agar is used in the Kirby-Bauer test (for bacterial resistance)?
Mueller-Hinton agar
What type of test uses Mueller-Hinton agar?
The Kirby-Bauer test for bacterial resistance
