Stains Flashcards
what do the simple stains use to color organisms?
chromogen ( colored molecule)
List the stains we are responsible for
Simple stains Negative stains gram stain capsule stain acid fast stain Endospore stain
what are the parts of the chromogen in simple stains?
Chromophore
auxochrome
what is the chromophore in simple stains?
portion of chromogen that gives it color
what is the auxochrome in simple stains?
charged portion of the chromogen
how does the chromogen work to stain a cell?
the auxochrome is attracted to the negative charge of the bacteria and the organism becomes stained
simple stains are also considered what?
basic stains
what are some simple stain dye example?
methylene blue
crystal violet
safranin
what are the steps of a simple stain?
1) begin with heat fixed emulsion
2) cover with stain for the specified time
3) rinse with water
4) blot dry
What is the negative stain used for?
to determine the morphology and cellular arrangement in bacteria that are too sensitive to be heat fixed
what kind of charge does the chromogen in negative stain carry?
a negative charge (the chromogen is acidic)
what is a benefit of a negative stain?
produces minimal cell shrinkage
what stains are used in the negative stain?
nigrosin stain
eosin stain
what are the step of the negative stain
1) place a drop of acid stain on the end of the slide
2) add organisms to the drop of stain
3) using another clean slide smear the liquid across the slide
4) do not heat fix! just let dry
what is the purpose of the gram staim?
it is a differential stain between gram + and gram -
what are the properties of a gram - organism
- high lipid content
- low peptidoglycan
- stains red
what are the properties of the gram + stain?
- low lipid concentration
- high amount of peptidoglycan
- stains purple
how does a gram - react to the gram stain
the decolorizer exracts lipid so that the cell is more porous and cannot hold the stain
how do gram + organisms react to the gram stain?
the peptidoglycan holds onto the stain better and resists decolorization
what are the reagents used in the gram stain?
crystal violet (primary stain)
iodine (mordant)
ethanol/acetone (decolorization agent)
safranin (counterstain)
what is a mordant (iodine in gram stain)?
agent that enhances the crystal violet color by forming a crystal-violet iodine complex
what are the steps in a gram stain?
1) begin with heat fixed emulsion
2) cover smear with crystal violet for 1 minute
3) rinse with water
4) cover smear with iodine for 1 minute
5) rinse with water
6) decolorize with 95% ethanol
7) rinse with water
8) counter stain with safranin for 1-2 minutes
9) blot to dry
what is the purpose of a capsule stain?
to detect cells that produce an extracellular capsule
what do capsules contain?
- mucoid polysaccharides (polypeptides)
they usually repel most stains
what does the capsule stain colorize?
background and cell proper
what types of dyes does the capsule stain use?
- acid stains (congo red, nigrosin)
- basic stain
what does the capsule look like at the end of the staining process?
a halo between the background and the cell
what is special about cells that contain a capsule?
the capsule increases virulence and makes them less susceptible to phagocytosis
what are the steps in the capsule stain?
1) add a drop of serum at end of the slide (optional to emulsify the cells)
2) add a drop of congo red at the end of the slide
3) add organisms to drop of congo red
4) using a clean slide, spread the drop across the slide
5) let ari dry
6) flood with maneval stain for 1 minute
7) rinse with water
what is the purpose of the acid fast stain?
a differential stain to test for mycobacterium
what is that lame saying that is pretty helpful that rublee kept wanting us to know for the acid fast stain?
“acid fact : mycobacterium : mycolic acid”
what is mycolic acid?
- a waxy substance that gives acid fast cells a higher affinity for primary stain and resistance to decolorization of acid-alcohol solution
- typically repel aqueous stains
what is unique about mycobacterium that a person would want to test for it?
mycobacterium are often pathogens (TB)
what is the purpose of heat steaming in an acid fast stain?
it melts the wax to help the stain enter
what are the reagents used in acid fast stains?
carbofuchsin (stains acid fast cells) acid alcohol (decoloization) methylene blue (stains acid fast negative cells )
what color are acid fast positive cells?
red
what color are acid fast negative cells?
blue
what are the steps of an acid-fast stain?
1) begin with heat-fixed emulsion
2) cover smear with strip of bibulous paper
3) apply carbolfuchsin stain and steam for 5 minutes (add additional carbolfuchsin when needed)
4) remove paper and rinse with water
5) decolorize with acid alcohol
6) rinse with water
7) counterstain with methylene blue for 5 minutes ( no heat)
8) rinse
9) blot dry
what is the purpose of the endospore stain?
to presence of an endospore
what is an endospore?
it is the dormant form of bacteria that is formed in unfavorable conditions
what are some features of an endospore?
- resistant to heat and chemicals due to tough outer covering made of keratin
- keratin resists staining so you have to steam to get color in spore
what are some spore locations and shapes?
- central (subtilis) and terminal (terminalis)
- spherical and elliptical (oval)
what are the reagents for the endospore stain?
malachite green
water
safranin
what color are the spores in the endospore stain?
green
what are the steps of the endospore stain?
1) begin with heat-fixed emulsion
2) cover smear with bibulous paper
3) apply malachite green
4) steam for 5 minutes adding more stain when needed
5) remove paper and rinse with water
6) counterstain with safranin for 1-2 minutes (no heat)
7) rinse with water
8) blot to dry
endospore producers will have what kind of gram stain?
gram +
when are endospores most likely to be seen?
when incubated overnight at 37 degrees C
what shape will spore producers usually be?
longer bacillis or clostridia
if a stain is gram - what does it say about the acid fast stain?
the cell won’t be acid fast +, it will be acid fast - (blue)
what will mycobaterium look like in a test tube?
chalky and dry
endospore producers will have what kind of acid fast stain?
acid fast + ( red)
