Differential Stains
these stains allow a microbiologist to detect differences between organisms or differences between parts of the same organism
Why are differential stains used more frequently?
they not only allow determination of cell size, morphology, and arrangement (as with a simple stain) but information about other features as well
The _________ is the most commonly used differential stain in bacteriology
Gram Stain
What are other differential stains other than Gram stain used for? ( 2 reasons)
1. used for organisms not distinguishable by the Gram stain
2. used to distinguish important cellular attributes such as acid-fastness, a capsule, spores, or flagella
What are these other differential stains (other than Gram) called?
structural stains
Gram stain
a differential stain in which the decolorization step occurs between the application of two basic stains
primary stain
crystal violet
mordant
enhances crystal violet staining by forming a crystal violet-iodine complex
what acts as a mordant in a gram stain?
iodine
4 steps in Gram staining
1. primary stain
2. mordant
3. decolorization
4. counterstain
______ is the most critical step in the gram staining procedure
decolorization
_______ cells are decolorized by the solution whereas _______ cells are not
gram negative
gram positive
common counterstain
saffranin
why can gram negative cells be counterstained?
because they have been decolorized
Gram positive appear ___ in color and gram negative appear _____ in color
purple
reddish pink
What is it about the walls of gram positive and gram negative that allow them to resist decolorization or not?
it is in the cell wall.
gram negative have a higher lipid content in cell wall and a thinner peptidoglycan layer than the gram positive. the decolorizer extracts the lipid making gram negative wall more porous and incapable of retaining the crystal violet iodine complex, thereby decolorizing it.
in gram positive, the thicker peptidoclycan and cross linking due to teichoic acids trap the crystal violet iodine complex and make it less susceptible to decolorization
What 3 ways does poor technique play into gram variable results:
1. over-decolorize = add too much decolorizer thus giving gram positive a reddish color
2. under-decolorize = adding not enough decolorizer thus giving gram negative a purple color
3. inconsistency in preparing emulsions
What is the sign of a good emulsion?
a good emulsion dries to a faint haze on the slide
Until correct gram technique is mastered, what is recommended?
a control smear of gram positive and gram negative stains
what is a good alternative control?
a smear from the gumline
crystal violet and safrannin are both _______
basic stains
__________ is what makes the gram stain differential
declorization step
When making emulsions, what should you do?
make the emulsions as close to one another as possible. spreading them out on the slide makes it difficult to stain and decolorize them easily.
What happens if a stain solution is not adequately filtered or is too old?
crystal violet crystals appear and block the view of bacteria
interpretation of gram stains can be messed up by these 4 things
1. crystals appear from stain
2. improperly made stain solution can disrupt the field
3. stain precipitate can be mistakenly identified as bacteria if slide is not rinsed thoroughly or stain not allowed to dry on the slide (variability in size is a clue they are not bacteria)
4. age of culture
How does age of the culture affect gram staining?
gram positive walls may lose ability to resist decolorization and give artificial gram negative result
use cultures 24 hours or less.
2 bacteria known for aging fast
bacillus and staphylococcus
Three things that gram stain allows us to do:
Determine cell
1. morphology
2. arrangement
3. size