Gram-Staining Short Answer Quiz Flashcards
A differential stain is a _______ procedure involving ________ _________
staining
2 stains
What is the function/purpose of a differential stain?
- 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
Name some examples of stains that are differential stains
gram stain
negative/capsule stain
endospore stain
Define and include the function of a gram stain
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
Compare and Constrate Gram Negative and Gram Positive cell wall components
Gram Negative
- less/thin layer peptidoglycan
- have LPS
- have outermembrane
- stains RED
Gram Positive
- lots/thick layer peptidoglycan
- no LPS
- no outermembrane
- stains PURPLE
With regards to gram staining, what two populations are we distinguishing between?
HINT: What does Gram Negative and Gram Positive mean?
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)
Gram staining involves a TWO part procedure.
Name and describe these procedures.
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
What type of stain is Crystal Violet and Safranin considered?
Crystal Violet = primary stain
Safranin = counterstain
What is Iodine? What is the function of iodine?
Iodine = mordant (NOT A STAIN!)
helps crystal violet stick to the the cell wall/peptidoglycan of bacteria
Name/describe the first step in the gram staining procedure
Include: Reagent used, time, etc
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
Name/describe the second step in the gram staining procedure
Include: Reagent used, time, etc
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
Name/describe the third step in the gram staining procedure
Include: Reagent used, time, etc
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)
Name/describe the fourth step in the gram staining procedure
Include: Reagent used, time, etc
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
What setting on the microscope is used to observe gram stained bacteria?
100x objective lens on BF
How are the dyes that are used in gram staining able to bind to the peptidoglycan layers of the cell wall?
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
In terms of alcohol, why doesn’t gram positive bacteria pick up safranin when it is applied at the final step?
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.
Describe the role of Alcohol
Include: AKA, function
- AKA “decolorizer”
- Dissolves lipids (LPS portion of Gram Negative cell walls)
- Penetrates Gram-Negative cell walls and decolirize the peptidoglycan in the cell wall
Why does alcohol penetrate Gram-Negative cell walls better than Gram-Positive cell walls?
Alcohol dissolves lipids and Gram-Negative bacteria contain LPS in its cell wall, whereas Gram-Positive bacteria do not contain LPS
If you over/under decolarize, what false gram rxn/variance will it lead to? Explain why.
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).
Describe Snapping V
bacillus is in the process of dividing via binary fission, creating a V shape
Gram stain ______ can result in:
1. False Gram-Positive Results
2. False Gram-Negative Results
variance (false results)
What are the two reasons for variance/false results?
- improper use of decolorizer (over/under time)
- “old bacteria” - cell walls begin to degenerate after ~24 hours
