Stains (Gram Stain, Acid Fast and Endospore Stain)

Session 6 and 8 BIO315L

gram stain

most useful and widely employed differential stain in bacteriology

distinguishes or differentiates bacterial groups based on cell wall structure and divided into two groups: gram negative or gram positive

differential staining

distinguishes between types of bacteria because of physical and chemical differences

gram stain procedure

heat fixation → crystal violet (primary stain) (60 sec) → iodine treatment (mordant) (60 sec) → decolorization (95% ethanol or isoproponal-acetone) (5-10 sec) → counter stain safranin (2 min)

rinse with tap water between each step

gram stain results

gram positive → deep purple

gram negative → pinkish to red

ideal time/age of culture to interpret results

better to Gram stain young, vigorous cultures (less than 24 hours old)

gram variable

some cells in the same culture will be gram positive and some, gram negative

microscope setting to use

examine with bright-field microscopy using 100X oil-immersion

acid-fast stain

differential stain used to identify bacteria in the genus Mycobacterium (some of which are pathogens)

also useful for identifying other organisms which could be pathogenic such as members of the Nocardia genus and parasites in the genus Cryptosporidium and the genus Isospora

acid-fast bacteria

gram-positive bacteria that contain high concentrations of a waxy lipid in their cell walls called mycolic acids

waxy layer surrounding the cells resists penetration of normal stains and dyes and thus prevents the use of simple stains or other commonly used differential stains like Gram stain

two methods of acid fast staining

differ in application of primary stain Carbolfuschin

Ziehl-Neelsen and Kinyoun methods

Carbolfuschin

lipid soluble stain that penetrates the waxy layer more readily than typical aqueous stains

Ziehl-Neelsen method

penetration of mycolic acid layer is achieved through steam-heating of the smear while primary stain is applied; produces phenol as dangerous product and can only be performed inside a biosafety cabinet

Kinyoun method

cold staining method used in the lab; concentration of Carbolfuschin is increased and/or detergent is added to permit the penetration of the stain without heat

amount of time organisms are in contact with stain is also increased

result of acid-fast

negative → blue or brown

positive → red to bright pink color

acid fast stain procedure

carbolfuschin (primary)

time (mordant)

acid-alcohol (decolorizer)

methylene blue (counterstain)

Bacillus and Clostridium

capable of producing an endospore that is resistant to chemical insult, desiccation, high heat, and UV radiation; requires no food since spores are metabolically dormant

endospore

develops within the bacterial cell; spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell

spore

outside the cell

endosporulation

most well-studied process in Bacillus subtilis; transition from a growing cell to a dormant endospore is initiated by starvation and is controlled by a complicated genetic program that takes 8 hours to complete

endospore position

characteristic (used to identify the bacteria); may be central, sub-terminal, or terminal

acid fast microscopy

bright field microscopy using the 100X objective under oil immersion

endospore stain procedure

malachite green (primary stain)

heat (mordant)

water (decolorizer)

safranin (counterstain)

endospore microscopy

bright field microscopy using the 100X objective under oil immersion

endospore stain result

positive → light minty green

negative → red

endospore wet mount

done under phase contrast microscopy; live preparation is done in order to see motility characteristics of specimens