Staining Techniques

Simple Stain

colors cell

charge sticks to negative charge in cell

methylene blue, crystal violet, safranin

Differential stain

differentiate between different cell types

colors some cells but not others

Gram stain is most common differential stain

Gram stain

crystal violet stains cells reversibly

iodine complexes with crystal violet (irreversibly stains gram + cells in peptidoglycan cell wall)

wash with ethanol (removes crystal violet from gram - cells)

stain with safranin counterstain (turns gram - pink, while gram + remains purple)

Gram +

more thick peptidoglycan layers

only has 1 cell membrane

eg Staphyloccocus aureus, bacillus

Gram -

thinner/fewer peptidoglycan layers

inner and outer cell membrane

famous example E. Coli

Acid Fast Stain

carbolfuchsin stains mycolic acid (in mycobacterium)

counterstain with methylene blue

dont stain gram well dueto waxy cell walls

counter stain/background looks blue

primary stain/cells look pink/red

Differential Stain

giemsa stain for blood film

uses methylene blue, eosin (pink) and azure B

differential stain for blood

RBCs pink due to Eosin

Leukocytes purple due to methylene blue/azure B

Negative stain

stains background and leaves cells unstained

no need to heat fix cells → preserves morphology

nigrosin stain (background dark, cells light purple/white)

no counterstain

Antibody Stain

satins linked to antibodies

targets epitopes on specific bacteria

can contain fluorophores

Flagella Stain

use sterile water on slide then touch w/ loop then colony

dont wiggle loop, will break flagella

add coverslip

add RYU stain and then observe under microscope after 5-10 minutes

Endospore Stain

tells if spore producing

primary stain is malachite green

for gram +

heat fix, saturate with malachite green over steam, rinse, counterstain w/ safranin