microbio exam final practical review

A more standardized protocol for the Kirby-Bauer Method that allows for greater reliability in results would be to use : (a) agar plate media poured to a specific depth, and (b) bacterial cultures of specific cell densities (MacFarland standards).

True
False

True

A Staphylococcus aureus strain shows sensitivity to penicillin as demonstrated by the Kirby-Bauer method, One can correctly assume based on this result, that all Staphylococcus aureus strains would be sensitive to penicillin.

True
False

False

Acid-Fast Stain
Interpretation

Acid-fast = pink/red
non-acid-fast = blue

Acid-Fast Stain
Reagents used?

carbol fuchsin (primary)
acid-alcohol
methylene blue (counterstain)

Antibiotic Sensitivity (Kirby-Bauer Test)

Zone of Inhibition: Measured in mm
Larger zone = more sensitive
Compare to standard chart to determine sensitive, intermediate, or resistant

Below is the experimental protocol of Lab 10 to examine the effectiveness of specific germicides. The original culture is a 24 hour old nutrient broth culture of the test bacterium. Other test tubes contain nutrient broth to which various manipulations were carried out; four are controls.
If there is growth in the control #2 tube the experiment results cannot be interpreted. Why does growth in this tube prevent the use of the other results and interpretation of the effectiveness of the agents?

It indicates that the broth being used throughout the experiment was likely contaminated.
It demonstrates that the bacteria being used are resistant to disinfectants.
It demonstrates that the disinfectants were not diluted correctly.
It indicates that the bacteria will grow without incubation.

It indicates that the broth being used throughout the experiment was likely contaminated.

Below is the experimental protocol of Lab 10 to examine the effectiveness of specific germicides. The original culture is a 24 hour old nutrient broth culture of the test bacterium. Other test tubes contain nutrient broth to which various manipulations were carried out; four are controls.
What is the purpose of control tube #4?

To demonstrate the tubes being used were sterile.
To demonstrate that bacteria will grow in the broth.
To demonstrate that the beads being used were sterile.
To demonstrate that bacteria can grow in some disinfectants.

To demonstrate that the beads being used were sterile.

Below is the experimental protocol of Lab 10 to examine the effectiveness of specific germicides. The original culture is a 24 hour old nutrient broth culture of the test bacterium. Other test tubes contain nutrient broth to which various manipulations were carried out; four are controls.
Which control tube is used to compare to test broths 1, 2, and 3 in order to evaluate the effectiveness of the germicide?

Control tube 1
Control tube 2
Control tube 3
Control tube 4

Control tube 3

Blood Agar

• not selective
• differentiates with hemolysis (α, β, γ)

Citrate test

Tests for ability of organism to utilize citrate

• Negative: green
• Positive: blue
• stab and streak inoculation

CNA agar

• selective for gram positive growth
• differentiates with hemolysis
  (A) beta-, (B) alpha-, and (C) gamma-hemolysis

EMB agar

• selective for gram negative growth
• differentiates Lactose fermenters
  Metallic green = E. coli; purple = fermenter

For the standard plate count method, the statistically accurate number of CFUs that should be counted on an agar plate is between
10 and 100.
20 and 200.
30 and 300.
10 and 1000

30 and 300.

Gram Stain
Reagents used?

crystal violet → iodine → alcohol (95% ethanol) → safranin
Alcohol is the decolorizer

Gram Stain
Interpretation

Gram positive = purple
Gram negative = pink

HEA agar

• selective for gram negative growth
• differentiates Lactose/sucrose fermentation and H₂S production
  Orange/yellow = fermenter; Black centers = H₂S

If an organism that is incubated on a Kligler iron agar slant produces a red slant and yellow butt, this indicates that the organism
ferments both glucose and lactose.
ferments lactose but not glucose.
ferments glucose but not lactose.
does not ferment either glucose or lactose.

ferments glucose but not lactose.

In the Kirby-Bauer method, the zone of inhibition

is a measure of the circumference of the circle surrounding the antibiotic disk.
is smallest in diameter for bacteria that are most sensitive to the antibiotic (i.e., least resistant)
is an area where no bacterial growth appears
all the above

is an area where no bacterial growth appears

In the Lab 10 experiment, what was the logic in choosing Staphylococcus aureus and Escherichia coli as the test organisms?

One produces endospores whereas the other does not.
One is Gram-positive and one is Gram-negative.
They are both pathogens.
They are both highly drug resistant.

One is Gram-positive and one is Gram-negative.

info about O-F test

• O-F medium contains glucose & peptone and a pH indicator bromthymol blue
• Two tubes of O-F media are stab-inoculated per test organism; O-F is semi-solid, wire loop must be inserted into agar (¾ depth)
• Following inoculation, one tube is overlayed with oil (anaerobic); the other tube does not have an oil overlay (aerobic tube).
• control is green
• yellow tubes: fermentative
• green for anaerobic tube and yellow for aerobic tube: oxidative
• both tubes green: unable to utilize glucose; oxidizes peptone, turns medium basic (blue).

info about Phenol Red Test

• Will use phenol red broth to test for ability of bacterium to ferment glucose, sucrose, & lactose
• Durham tube in the medium tests for gas production
  Control: organeish red broth
• Fermentation: production of acidic end products; media turns yellow
• Non-fermentative bacteria: no color change (remains red) or turn alkaline(pink color) due to degradation of peptone
  A/G: Fermentation w/acid & gas (yellow with gas)
  A/‒: Fermentation w/acid; no gas (yellow)
  ‒/‒: No fermentation (control color)
  K: Degradation of peptone (hot pink)

KIA agar test

• Differentiates bacteria on the basis of glucose fermentation, lactose fermentation, and sulfur reduction
• stab and streak inoculation
• Glucose-only fermenter (red slant/yellow butt)
• Glucose & Lactose fermenter (yellow slant/yellow butt)
• Non-fermentation of either glucose or lactose (red through out or part of it)
• Gas production (cracks in the medium)
• Sulfur reduction (black though out)

MAC agar

• selective for gram negative growth
• differentiates Lactose fermenters (Pink colonies)

Match the test with the proper definition; answer choices are only used once.
Red color formation is positive for this following reagent addition
Formation of a blue color is positive
indicates a mixed acid fermenter
yellow color formation indicates fermentation

Voges-Proskauer test
Citrate test
Methyl Red test
Phenol Red carbohydrate test

Methyl Red test

checks for mixed acid fermentation
add methyl red
-red color is positive; orange or yellow is negative.

Motility Test

• semisolid medium
• stab inoculation
• positive: red defused growth radiating outward from stab line (organism motile)
• negative: red growth only along stab line (organism not motile)

MRVP broth

a combination medium used for two tests;contains peptone, glucose, & phosphate buffer.

MSA agar

• selective for gram positive growth
• differentiates Mannitol fermenters (yellow color)

Negative Stain
Reagents

Uses nigrosin to stain background; cells appear clear
No heat-fixing - preserves size/shape

Nitrate Reduction Test

test used to determine if bacteria can respire anaerobically; whether an organism reduces nitrate to nitrite

• broth inoculation with durham tube
• Reagents are added to determine; 8 drops of reagent A + 8 drops of reagent B
• Positive: color change to red occurs after addition of reagents; positive for nitrate reduction
• Negative: no color change occurs
• If negative after reagents then add Zinc
• Negative: red color after addition of zinc
• positive: no color change after addition of zinc

Of the germicides test in Lab 10, which is/are generally classified as an antiseptic?
Lysol
Isopropyl alcohol (IPA)
Fabuloso
two of the above
none; A, B, and C are all antiseptics

Isopropyl alcohol (IPA)

Oxidation-Fermentation (O-F) Test

differentiates bacteria onthe basis of fermentative or oxidative metabolism of carbohydrates
stab-inoculation into semi-solid agar
wire loop must be inserted into agar (¾ depth)

oxidation-fermentation (O-F) test differentiates bacteria onthe basis of

fermentative or oxidative metabolism of carbohydrates

PEA agar

selective for gram positive bacteria growth

Phenol red lactose broth was inoculated with a particular bacterial species. Following 24 hours incubation at 37°C, the bacteria grew and the result below was observed. An un-inoculated control is shown for comparison. What is the interpretation of this result? [The broth is turbid with red color}

It is a lactose fermenter
It is non-reactive; it cannot utilize any of the nutrients in the broth.
It can form gas
It can grow but cannot ferment lactose
None of the above are correct
Two of the above are correct

It can grow but cannot ferment lactose

purpose of biochemical tests

to identify the metabolic capabilities of bacteria;often used for the purpose of identifying bacterial isolates.

SIM agar test

Determines 3 bacterial activities - sulfur reduction, indole production, and motility

SIM Indole

• Kovac's reagent added
• Positive: Red layer; tryptophan broken down into indole
• Negative: none colored layer; tryptophan not broken down

SIM motility

• Positive: growth radiating outward from stab line
• Negative: no radiating growth

SIM: sulfur reduction

Positive: black in the medium = sulfur reduction
Negative: no black in medium = sulfur not reduced
stab inoculation

Spore Stain
Interpretation

Spores = green
vegetative cells = pink

Spore Stain
Reagents

malachite green → heat → water → safranin

The cell density of a liquid sample is determined using the standard plate count method with the serial dilution scheme that is shown. What is the original cell density (OCD) of the liquid sample in CFU/mL?

1.02 x 10^7
102
1.02 x 10^8
1.02 x 10^6
1.02 x 10^3

1.02 x 10^8

The function of the tetrazolium salt (TTC) in motility test medium is to
produce more vigorous growth of organisms.
enhance the visibility of the motility results.
select for the growth of motile organisms.
select for the growth of non-motile organisms.

enhance the visibility of the motility results.

The results of the Kirby-Bauer method of assessing antibiotic sensitivity can indicate whether or not the antibiotic has a bactericidal or bacteriostatic effect.

True
False

False

This test specifically determines if a bacterium can respire anaerobically using the nutrients in the growth medium.

urea test
SIM
nitrate reduction test
none of the above

nitrate reduction test

Though the assumption is that a CFU arises from a single bacterial colony, it may not always be the case, depending on the bacterial type.
True
False

True

True or false - only those bacteria that are motile will produce a red color when grown in Motility Test Medium
True
False

False

Upon completion of an oxidation-fermentation test, you observe that your organism has produced an acid result in the aerobic tube and a neutral result in the anaerobic tube. You can reasonably conclude that the organism exhibits _ metabolism of carbohydrates.
fermentative
oxidative
both oxidative and fermentative
no

oxidative

Urea test

Detect presence of urease enzyme

• positive: hot pink broth
• negative: yellow broth

Use the dilution scheme to answer. The concentration of bacteria in the original sample is __ CFU/ml.

8.60 x 10^2
8.60 x 10^7
4.30 x 10^7
4.30 x 10^8
8.60 x 10^6

8.60 x 10^7

Using the Kirby-Bauer Method to determine antibiotic sensitivity, E coli was found to have a zone of inhibition measurement of 10 mm in the presence of ampicillin. Which statement is true concerning this data?

This E coli strain is highly sensitive to ampicillin.
If one had an E coli infection, it's highly likely (based on the data above) that ampicillin would not be a good choice for treatment.
E coli has only intermediate sensitivity to ampicillin.
None of the above are true.

If one had an E coli infection, it's highly likely (based on the data above) that ampicillin would not be a good choice for treatment.

Voges-Proskauer test

checks for acetoin, which is a step before 2,3-butanediol is made.
add VP reagents A and B
Postive VP Test:
-color change to red, 2.3-butanediol fermentation (acetoin produced).
Negative VP Test:
-No color change, no 2,3 butanediol fermentation (acetoin is not produced).

What is the difference between a disinfectant and an antiseptic?

Disinfectants kill bacteria whereas antiseptics just inhibit the growth.
Antiseptics kill bacteria whereas disinfectants just inhibit the growth.
Disinfectants can be used on anything whereas antiseptics are only for instruments.
Antiseptics are used on people whereas disinfectants are used on objects.

Antiseptics are used on people whereas disinfectants are used on objects.

What is the purpose of the inclusion of a Durham tube in differential culture media?
To allow for ease in the visualization of bacterial growth.
To indicate pH changes that occur during growth.
To visualize the production of gas during growth.
To stabilize the oxygen concentration in the media during incubation.

To visualize the production of gas during growth.

When all the possible steps of the nitrate reduction test are complete and the culture tube still fails to produce a color change, this indicates the organism
is not capable of nitrate reduction.
cannot ferment nitrogen.
is capable of nitrate reduction.
cannot grow in nitrate broth.

is capable of nitrate reduction.

Which choice among A-D is true regarding the urea test?
A positive test is indicated by formation of a purple color.
Urea positive bacteria form ammonia and carbon dioxide as the end products of the reaction with urea.
A positive urea test indicates the bacterium possesses the tryptophanse enzyme.
The urea broth tube has a Durham tube to check for gas production.
None of A - D is true; all are false.

Urea positive bacteria form ammonia and carbon dioxide as the end products of the reaction with urea.

Which is applicable to a bacterium producing a positive Voges-Proskauer test?
it means it can break down hydrogen peroxide
a blue color is formed
it means it can ferment lactose
it means it produces acetoin
it means it is a mixed-acid fermenter

it means it produces acetoin

Which of the following among A - D is not applicable to the Kirby-Bauer Method?

It is a laboratory method to assess the sensitivity of bacterial strains to antibiotics.
It utilizes the quadrant streak plate method to assess bacterial growth in the presence of antibiotic.
Several antibiotics can be tested on a single culture plate.
Results of the test can determine whether then bacterial strain is resistant, sensitive, or has intermediate sensitivity.
A - D are all applicable to the Kirby-Bauer Method.

It utilizes the quadrant streak plate method to assess bacterial growth in the presence of antibiotic.

Which of the following choices among A-D is a false statement?

Sulfur reduction: can determine this in SIM, KIA, and Phenylalanine deaminase
SIM can determine if a bacterial type is capable of producing indole.
The appearance of cracks in the "butt" of an inoculated KIA medium indicate the bacteria is motile.
It is proper procedure when inoculating motility test medium to stab it one timewith an inoculum; i.e., in and out along the same path.
Two of the above choices among A-D are false.

Two of the above choices among A-D are false.

Which statement among A-C is false regarding the standard plate count method?
It measures both live and dead cells in the liquid sample.
It utilizes a standard dilution procedure called serial dilution.
It is a viable count method to quantitate cell density of a liquid sample.
None of A-C is false; all are true statements.

It measures both live and dead cells in the liquid sample.

Why do organisms that have the capacity to utilize citrate as the sole carbon source tend to produce an alkaline reaction in Simmons citrate agar?
The conversion of citrate into pyruvate produces carbon dioxide as alkaline waste.
The metabolism of citrate always produces acidic waste products.
The organism will also utilize ammonium phosphate as a nitrogen source, resulting in alkaline waste.
none of the above

The organism will also utilize ammonium phosphate as a nitrogen source, resulting in alkaline waste.