Gram-Staining Short Answer Quiz

Question 1

A differential stain is a _______ procedure involving ________ _________

Answer 1

staining
2 stains

Question 2

What is the function/purpose of a differential stain?

Answer 2

• helps determine if specimen sample is a pure (one type bacteria; same Genus and Species)/mixed culture (more than one type bacteria)
• distinguishes between different organisms (bacteria)
  -> bacteria with different types of cell walls, presence/absence of capsule
• helps determine cell shape, arrangement, size, and identificaiton

Question 3

Name some examples of stains that are differential stains

Answer 3

gram stain
negative/capsule stain
endospore stain

Question 4

Define and include the function of a gram stain

Answer 4

Gram Stain (type of differential stain)
- common technique used to differentiate between two large groups of bacteria based on their different cell wall components/different amount of peptidoglycan
-> ex: Gram Positive bacteria vs Gram Negative bacteria

Question 5

Compare and Constrate Gram Negative and Gram Positive cell wall components

Answer 5

Gram Negative
- less/thin layer peptidoglycan
- have LPS
- have outermembrane
- stains RED

Gram Positive
- lots/thick layer peptidoglycan
- no LPS
- no outermembrane
- stains PURPLE

Question 6

With regards to gram staining, what two populations are we distinguishing between?

HINT: What does Gram Negative and Gram Positive mean?

Answer 6

bacteria with a thick layer of peptidoglycan in their cell wall (gram positive) and bacteria with a thin layer of peptidogycan in their cell wall (gram negative)

Question 7

Gram staining involves a TWO part procedure.
Name and describe these procedures.

Answer 7

Smear: taking a culture sample from a plate (ADD H2O to slide) or brother (DONT add H2O to slide) -> preparing a slide with your specimen sample on it

Stain: series of steps used to stain the colorless sample on your slide so it can be visible (purple or pink/red) under BF microscope

Question 8

What type of stain is Crystal Violet and Safranin considered?

Answer 8

Crystal Violet = primary stain
Safranin = counterstain

Question 9

What is Iodine? What is the function of iodine?

Answer 9

Iodine = mordant (NOT A STAIN!)

helps crystal violet stick to the the cell wall/peptidoglycan of bacteria

Question 10

Name/describe the first step in the gram staining procedure

Include: Reagent used, time, etc

Answer 10

STEP 1:
- Reagent Used: Crystal Violet (primary stain)
- Staining Time: 45 seconds
- Description: Crystal violet is applied and colors ALL cell walls of bacteria on the slide (gram + and gram - bacteria all pick up CV); RINSE AFTER

Question 11

Name/describe the second step in the gram staining procedure

Include: Reagent used, time, etc

Answer 11

STEP 2:
- Reagent Used: Iodine (NOT A STAIN; mordant)
- Staining Time: 60 seconds
- Description: Application of iodine helps the dye, crystal violet, stick to the cell wall of bacteria; RINSE AFTER

Question 12

Name/describe the third step in the gram staining procedure

Include: Reagent used, time, etc

Answer 12

STEP 3:
- Reagent Used: 95% Alcohol (decolorizer)
- Staining Time: 20 seconds
- Description: Run alcohol continously down the slide. This will dissolve LPS on cell wall of gram negative bacteria and will penetrate gram negative cell wall and decolorize the peptidoglycan in the cell wall (bacteria now “colorless”); RINSE

ATP, Gram Negative are colorless, and Gram Positive remain purple

Alcohol ONLY decolorizes gram negative bacteria (if done properly)

Question 13

Name/describe the fourth step in the gram staining procedure

Include: Reagent used, time, etc

Answer 13

STEP 4:
- Reagent Used: Safranin (counterstain)
- Staining Time: 45 seconds
- Description: Application of safranin will color peptidoglycan layer of cell wall of gram negative bacteria, because CV was removed earlier by 95% alcohol (doesn’t color gram positive because you cannot stack dyes!); RINSE

Question 14

What setting on the microscope is used to observe gram stained bacteria?

Answer 14

100x objective lens on BF

Question 15

How are the dyes that are used in gram staining able to bind to the peptidoglycan layers of the cell wall?

Answer 15

Colorless bacterial cell wall are negatively charged, while chromophores (ions) of the the dyes (CV, safranin) are postitivley charged. Opposite charges attract, allowing for the dyes to bind to the peptidoglycan layers in bacterial cell wall

Question 16

In terms of alcohol, why doesn’t gram positive bacteria pick up safranin when it is applied at the final step?

Answer 16

When decolorizing, only gram negative bacteria become colorless and gram positive bacteria remain purple. When safranin is applied, gram positive will NOT pick up the pink/red color because you did not remove the color earlier and you are not able to stack dyes.

Question 17

Describe the role of Alcohol

Include: AKA, function

Answer 17

• AKA “decolorizer”
• Dissolves lipids (LPS portion of Gram Negative cell walls)
• Penetrates Gram-Negative cell walls and decolirize the peptidoglycan in the cell wall

Question 18

Why does alcohol penetrate Gram-Negative cell walls better than Gram-Positive cell walls?

Answer 18

Alcohol dissolves lipids and Gram-Negative bacteria contain LPS in its cell wall, whereas Gram-Positive bacteria do not contain LPS

Question 19

If you over/under decolarize, what false gram rxn/variance will it lead to? Explain why.

Answer 19

Over-decolarize = false gram negative
- Applying alcohol over the required time means that you decolorize everything, including crystal violet from peptidoglycan layers of gram positive bacteria (gram + should not be decolorized bc its doesnt have LPS). This means that peptidoglycan layers of gram positive bacteria are now colorless, so when safranin is applied, it will pick up the red/pink stain, therefore leading to a false gram negative result.

Under-decolarize = false gram positive
- Applying alcohol under the required time means that you did not fully decolorize the crystal violet from peptidoglycan layers of gram negative bacteria. This means crystal violet is still present in peptidoglycan layer of gram negative bacteria, leading to a false gram positive result (looks purple, but should be red/pink).

Question 20

Describe Snapping V

Answer 20

bacillus is in the process of dividing via binary fission, creating a V shape

Question 21

Gram stain ______ can result in:
1. False Gram-Positive Results
2. False Gram-Negative Results

Answer 21

variance (false results)

Question 22

What are the two reasons for variance/false results?

Answer 22

• improper use of decolorizer (over/under time)
• “old bacteria” - cell walls begin to degenerate after ~24 hours