Lab Quiz 7

If you fixed a sample from a week-old culture of staphylococcus aureus on a glass slide and Gram stained it, what would your results look like?

The organisms may appear to be Gram-variable; that is, some cells will appear purple, while others will appear pink.

What is the difference between doing a simple stain versus a differential stain on a bacterial slide?

The simple stain will only tell you morphological shape and size, while the differential stain will allow for determination of size, shape, and type of the cell wall.

A fellow student is unsure why his Gram-negative Escherichia coli is purple. Upon examining the Gram stain kit, you notice that it didn't have alcohol. What caused the bacteria to be purple instead of pink?

Without alcohol during the decolorizing step, the bacterial cell wall was not altered, which prevented the Gram stain's iodine and crystal violet from being rinsed away

You forget to label your slides. At what step, will you first be able to distinguish gram positive cells?

ethanol

In a completed Gram stain, gram-negative bacteria are colorless.

false

If some cells in a pure culture of Staphylococcus epidermidis stain blue, and some cells stain red, the bacteria are __________.

impossible to determine because of a staining error

The purpose of a mordant in the Gram stain is to

prevent the crystal violet from leaving the cells.

In the Gram stain, after staining with crystal violet, all cells will be __________.

purple

You are performing a Gram stain on gram-negative bacteria and you stop after the addition of the mordant. What is the appearance of the bacteria at this point?

purple

After Gram staining, a gram-negative cell will be __________.

red

The counterstain used in the Gram stain is a basic dye.

true

After you add crystal violet and rinse the slide, what should be your next step?

Add Gram's iodine.

What is the best procedure for decolorization?

Add decolorizing agent until run-off is clear.

After Gram's iodine is added and then rinsed, what is the next step in the Gram stain procedure?

Add decolorizing agent.

What is the role of iodine in the Gram stain process?

Iodine is a mordant in the Gram stain technique, which functions to intensify the primary stain.

Based on your observation of the indicated bacterium in this Gram stain, select ALL appropriate statements.

- The bacteria have a staph arrangement. - The indicated bacterium is Gram-positive. - The bacteria pictured are cocci. - Eosin Methylene Blue (EMB) or MacConkey agar would NOT be good choices of media to grow the indicated bacteria.

Based on your observation of the indicated bacterium in this Gram stain, select ALL appropriate statements.

- The indicated bacterium is Gram-negative. - The indicated bacterium is a bacillus. - The indicated bacterium produces endotoxin (also known as lipopolysaccharide, LPS).

Based on your observation of the indicated bacterium in this Gram stain, select ALL appropriate statements.

- The indicated bacterium is Gram-positive - The indicated bacterium has a strepto- formation - Eosin Methylene Blue (EMB) or MacConkey agar would NOT be good choices of media to grow the indciated bacterium - The indicated bacterium is a bacillus

Based on your observation of this Gram stained specimen, select ALL appropriate statements.

- The pictured bacteria are most likely non-acid fast. - The pictured bacteria are diplococci - The pictured bacteria produce lipopolysaccharide (LPS), also known as endotoxin. - It can be ruled out that this specimen is taxonomically found in the Staphylococcus genus.

Based on your observation of the Gram stained bacterial specimen, select ALL appropriate statements.

- The pictured bacteria produce endotoxin (also known as lipopolysaccharide, LPS). - The indicated bacterium is Gram-negative. - The bacteria pictured are cocci.

Gram-negative bacteria

- crystal violet NOT retained during decolorization - stain pink/red by gram stain - one or two layers of peptidoglycan

Gram-positive bacteria

- crystal violet retained during decolorization - many layers of peptidoglycan - stain purple by gram stain

Arrange the steps of the Gram staining procedure in their correct order. Do not overlap any steps. - flood slide with iodine + rinse - flood slide with safranin + rinse - blot slide - flood slide with crystal violet + rinse - view slide under microscope - flood slide with iodine + rinse

- flood slide with crystal violet + rinse - flood slide with iodine + rinse - decolorize + rinse - flood slide with safranin + rinse - blot slide - view slide under microscope

After performing a Gram stain, both purple cocci and red rods are observed. What is the best explanation for this observation?

A mixed-culture was Gram-stained.

A student performs a Gram stain on a bacterial sample that has a mix of gram-negative and gram-positive organisms. The student accidentally forgets the decolorizer step. What would the outcome of the Gram stain be?

All organisms would appear purple (gram-positive).

If you FORGOT to do the decolorizing step, what colors would the Gram-positive and Gram-negative cells be when viewed at the end of the procedure?

All would be purple.

Which of the following describes the correct cellular morphology and Gram reaction for Staphylococcus aureus?

Clusters of gram-positive cocci

Which of the following represents an incorrect association regarding the reagents used during the Gram Staining procedure?

Counterstain = Methylene blue

Which of the following sequences arranges the steps of the Gram stain into the correct order?

Crystal violet ( Iodine ( Alcohol ( Safranin

Which of the following basic stains is used in the Gram staining procedure?

Crystal violet and safranin

Which of the following organisms is gram-negative?

Escherichia coli

Which of the following substances is used as a decolorizing agent in the Gram stain?

Ethyl alcohol

Allowing the primary stain to sit on your slide for more than 3 minutes will have no effect on the results of your Gram stain.

False

In a Gram stain, the wash steps are not required if you need to perform the stain quickly due to time constraints.

False

The Gram stain allows for determination of bacterial species based on their metabolic activities.

False

What is the purpose of using Gram's iodine during the Gram staining procedure?

Gram's iodine is a killing agent, binds to crystal violet, and serves as a mordant

After the Gram stain, microbial cells appear as red rods. What is the best description of these cells?

Gram-negative bacilli

Which of the following statements is INCORRECT regarding the Gram staining procedure? - The primary stain, crystal violet, is able to stain all bacterial cells. - Gram staining separates cells based on the composition of their cell walls. - The decolorizing agent is designed to remove the primary stain from only gram-negative cells. - Gram-negative cells carry a net negative charge in their cell walls; gram-positive cells carry a net positive charge.

Gram-negative cells carry a net negative charge in their cell walls; gram-positive cells carry a net positive charge.

Which of the following describes the correct cellular morphology and Gram reaction for Bacillus cereus?

Gram-positive rods

Why is it recommended that Gram staining be performed on young bacterial cultures (not older than 24 hours)?

Over time, gram-positive cells lose their ability to retain the crystal violet stain after decolorization.

After Gram-staining a sample of Gram-negative bacteria, all the cells appear purple. What is the best explanation for this result?

Not enough alcohol was used.

As bacterial cells age, their peptidoglycan begins to break apart. What would be the effect on decolorization?

Old Gram-positive cells will be decolorized.

If you forget to add the decolorizing agent when performing a Gram stain, what color will the gram-negative cells be?

Purple

After you add crystal violet to the slide, what should be your next step?

Rinse with distilled water.

How does safranin affect Gram-positive cells?

Safranin penetrates the cell wall, but is masked by the darker crystal violet stain.

After Gram-staining a sample of Gram-positive bacteria, all the cells appear red. What is the best explanation for this result?

The alcohol was left on too long.

Why is it important that crystal violet be a contrasting color to safranin?

The contrast allows you to differentiate between gram-negative and gram-positive cells by observing the color of the organism.

What is the most critical step in the Gram staining procedure?

The decolorization step

What happens to the Gram-positive cell wall during decolorization?

The decolorizing agent dehydrates the peptidoglycan.

What happens to the Gram-negative cell wall during decolorization?

The decolorizing agent dissolves the outer membrane.

Which of the following statements about gram-negative cell walls is FALSE? - Their Gram reaction is due to the outer membrane. - They are sensitive to penicillin. - They have an extra outer layer composed of lipoproteins, lipopolysaccharides, and phospholipids. - They are toxic to humans. - They protect the cell in a hypotonic environment.

They are sensitive to penicillin.

In a completed Gram stain, gram-positive bacteria are purple.

True

The Gram stain differentiates between bacteria based on the thickness of their peptidoglycan layer.

True

You performed the Gram stain. You are expecting to find purple Gram-positive cocci and pink Gram-negative bacilli. Instead, you observe purple cocci but don't seem to see any bacilli. How could you explain this?

You forgot the safranin step.

You performed the Gram stain on a smear. You are expecting to find purple Gram-positive bacilli and pink Gram-negative cocci. Instead, you observe pink bacilli and pink cocci. Which of the following is NOT a possible explanation for this unusual result?

You skipped the safranin step.

In the figure, which diagram of a cell wall contains teichoic acids? (positive or negative)

a

A Gram stain is done on __________.

a heat-fixed smear of bacteria

Which step in the Gram stain is the critical step in differentiating gram-positive cells from gram-negative cells?

alcohol-acetone

In the figure, which diagram of a cell wall contains porins?

b

In the figure, which diagram of a cell wall is a gram-negative cell wall? (single or double membrane?)

b

In the figure, which diagram of a cell wall is decolorized by a brief exposure to alcohol? (gram positive or negative)

b

Each of the following statements concerning the gram-positive cell wall is true EXCEPT - it protects the cell in a hypertonic environment. - it is sensitive to lysozyme. - it contains teichoic acids. - it maintains the shape of the cell. - it is sensitive to penicillin

it protects the cell in a hypertonic environment.

While gram-positive cells have a thick layer of __________ in their cell walls, gram-negative cells have a thin layer, surrounded by an additional lipopolysaccharide layer.

peptidoglycan

After performing a Gram stain, gram-negative cells appear __________, while gram-positive cells appear __________.

pink or red; purple

Which of the following reagents of the Gram-stain remains bound by both Gram-positive and Gram-negative cells at the end of a correctly performed stain?

safranin

Which of the following places the steps in the correct sequence? 1-Staining 2-Making a smear 3-Fixing

2-3-1

The Gram stain is an example of a __________ stain, because the process uses two contrasting stains to separate bacteria into groups based on cell wall composition.

differential

Sterilize your loop __________.

before and after transferring bacteria

You are performing a Gram stain on gram-negative bacteria and you stop after the decolorizer step. What is the appearance of the bacteria at this point?

colorless

If you forgot to add the counterstain to the mixed culture sample at the end of the staining procedure, gram-negative cells would appear __________ and gram-positive cells would appear __________.

colorless; purple

You are performing a Gram stain on gram-positive bacteria and you stop after the addition of the counterstain. What is the appearance of the bacteria at this point?

purple

You are performing a Gram stain on gram-positive bacteria and you stop after the addition of the first dye. What is the appearance of the bacteria at this point?

purple