L5: Differential staining (grams staining)

Gram staining

is a fundamental differential stain that is used as in the preliminary identification of bacteria

Hans Christian Gram

danish bacteriologist who developed the gram stain

Crystal Violet, Iodine, Acetone, Safranin

4 components/reagents of gram stain (VIAS)

Crystal Violet

primary stain used for differential staining

Crystal violet

it stains the CELL MEMBRANE of all bacterial species

Iodine

it acts as the MORDANT that creates an INSOLUBLE complex with crystal violet

Acetone/alcohol

acts as decolorizer

Acetone

it washes off the crystal violet from cell walls that do not have thick peptidoglycan layer

thick peptidoglycan layer and teichoic acid

components of gram positive cell wall (PT)

thin peptidoglycan layer and glycocholic saccharide

components of gram negative cell wall (PS)

gram positive

cells that retain the crystal violet complex and its purple color

Crystal violet-iodine complex

complex created when we add crystal violet and iodine

Safranin

secondary stain used for gram negative bacteria that have been previously decolorized

pink

what color does the secondary stain demonstrate

violet

what color does the primary stain demonstrate

Staphylococcus aureus and Escherichia coli

Specimen used in gram staining

1 loopful

amount of liquid culture used in gram staining

2-3x

how many times do we need to pass over the slide in order to heat fix

60 seconds

how long should we flood the smear with crystal violet

60 seconds

how long should we flood the smear with iodine

just until the slide is clear/<1 second

how long should we flood the smear with acetone

60 seconds

how long should we flood the smear with safranin

2% NaOH

the most common decontaminating agent

Oxalic acid

decontaminating agent that prevent pseudomonas

2% NACL-NaOH

decontaminating agent for sputum smear

N-acetyl-l-cysteine-sodium hydroxide

meaning of NACL-NaOH