Lab Exercise 4 - Acid-Fast and Spore Stain

What type of staining is acid-fast staining?

a differential stain

What type of staining is endospore staining?

a structural stain

name of the acid-fast staining method

Ziehl-Neelsen method

trait of acid-fast microorganisms

high content of mycolic acid in their cell walls

mycolic acid is waxy, resists staining; steam forces primary dye to penetrate the cell wall

fastness

the tendency for a microorganism to retain dye when challenged by a decolorizer

reagents used in acid-fast/Ziehl-Neelsen staining

carbol fuchsin with steam

acid alcohol

methylene blue

purpose of carbol fuchsin in acid-fast staining

primary dye

with steam, gets forced into the cell wall of acid-fast cells

purpose of acid alcohol in acid-fast staining

decolorizer

washes carbol fuchsin off of non-acid-fast cells

purpose of methylene blue in acid-fast staining

secondary dye/counterstain

colors non-acid-fast cells

results of acid-fast staining

acid-fast cells appear cerise pink/violet (positive result is pink)

non-acid-fast cells appear blue

genera that test acid-fast positive

Mycobacterium (e.g. M. tuberculosis, M. leprae)

Mycobacterium tuberculosis

the causative agent of tuberculosis

an acid-fast bacterium

Mycobacterium leprae

the causative agent of leprosy (Hansen’s agent)

an acid-fast bacterium

name of the method for spore staining

Schaeffer-Fulton method

spores

dormant forms of bacteria

endospores are within the bacterium

free spores are released from the “parent” bacterium

reagents used in spore staining

malachite green

water

safranin

purpose of malachite green in spore staining

primary dye

forced into spores with steam

(also colors non-spore cells, but then gets washed off; only retained by spores)

purpose of water in spore staining

decolorizer

washes malachite green off of non-spore structures

purpose of safranin in spore staining

secondary dye/counterstain

colors non-spore cells

results of spore staining

no spores: only pink cells

endospores: green ovals/circles inside pink cell bodies

free spores: small green oval bodies outside of pink cell bodies

genera of spore-forming bacteria

Bacillus (e.g. B. anthracis)

Clostridium (e.g. C. botulinum, C. tetani, C. difficile)

relationship between spore formers and Gram-positive/negatives

all spore formers are Gram-positive, but not every Gram-positive is a spore former

(Gram-negatives can not be spore formers)