Structural Staining (P) Flashcards
What happens in Schaeffer-Fulton’s method (of endospore staining)?
A primary stain (malachite green) is forced into the spore by steaming the bacterial emulsion
What are the characteristics of malachite green?
1) It is water soluble
2) It has low affinity for cellular material
What is the result from the characteristics of malachite green?
Vegetative cells may be decolorized with water
What is the purpose of safranin?
It is applied to counterstain any cells which have been decolorized
What are the results at the end of staining?
1) Vegetative cells will be pink
2) Endospores will be dark green
What are the reagents used in endospore staining (Shaeffer-Fulton’s method)?
1) Malachite green
2) Water
3) Safranin
What is the primary stain used in endospore staining (Schaeffer-Fulton’s method)?
Malachite green
What is the decolorizing agent used in endospore staining (Schaeffer-Fulton’s method)?
Water
What is the counterstain used in endospore staining (Schaeffer-Fulton’s method)?
Safranin
What is the procedure of endospore staining (Schaeffer-Fulton’s method)?
1) Application of malachite green (primary stain)]
2) Application of heat (mordant)
3) Application of water (decolorizer)
4) Application of safranin (counterstain)
What is the mordant used in endospore staining (Schaeffer-Fulton’s method)?
Heat
What are the characteristics that should be considered in the differentiation of genera and species identification of bacteria?
1) Flagella (which is possessed by most motile bacteria)
2) Shape
3) Number
4) Position
Can bacterial flagella be stained?
Yes
How does flagella staining differ from staining of other bacterial structures?
It differs from staining other bacterial structures because flagella staining usually requires extraordinary care for the slides, stain, and cells
What is the purpose of painstakingly preparing flagellar stains?
These are prepared to coat the surface of the flagella with dye or a metal such as silver
What is the procedure of preparation of smear for flagellar staining (modified Ryu’s method)?
1) Take a slant of flagellated cell structure
2) Add 2 drops of sterile distilled H20
3) Incubate (20 mins)
4) Take a drop of suspension and add on slide kept in slanting position
5) Air dry the smear and no heat fixation treatment is given to the smear
6) Flood the air-dried smears with the staining solution for 1 - 5 mins
7) Wash the staining solution off in tap H20. After the smears have dried, examine them under the OIO of microscope. Cell bodies and flagella stain violet
What is the resulting color of staining flagella?
Violet (cell bodies are also violet)
What is the characteristic of capsules to reagents in simple staining?
Capsules stain very poorly with reagents used in simple staining
Capsule stain can become what (depending on the method)?
A misnomer
Why do capsule stain become a misnomer?
Because the capsule may or may not be stained
What are (-) staining methods?
These contrasts a translucent, darker colored background
What are the results of (-) staining methods?
1) Cells are stained
2) Capsule is unstained
What are used to form the background in (-) staining methods?
1) India ink
2) Nigrosin
3) Congo red
Compare india ink and nigrosin
India ink: difficult to obtain nowadays
Nigrosin: easily acquired
What is required for a (+) capsule stain?
A mordant
What is the purpose of mordant in a (+) capsule stain?
It precipitates the capsule
What are the counterstains that can be used in capsular staining?
1) CV
2) Methylene blue
What happens in counterstaining (in capsular staining)?
The bacterial cell wall takes up the dye
Result for capsules against a dark bg?
Capsules appear colorless
Stained cells are present
What is the procedure of congo red capsule staining?
1) Place a loop-full of Congo red on a slide
2) Mix a small amt of your organism into the drop of Congo red
- > spread the organism or dye suspension well on the slide
3) Let the slide thoroughly air dry
- > do not methanol fix!
4) Fix the dried slide with acid alcohol (for 15 secs)
5) Rinse with distilled H20 and cover the slide with acid fuchsin for 1 - 5 mins
6) Rinse with H20 and allow to air dry
7) Examine the slide under oil immersion
- > cells stain red / pink, and the capsules appear as colorless halos against a dark blue bg
