Staining (Lab 5 & 6)

why do we stain?

to create contrast

what is fixation

heat of chemical, kills and binds the microorganism to the slide

cell shapes

bacillus, coccus, coccobaccillus, spirillum

cell arrangement

diplo, tetrad, strepto, staphylo

bacillus

rod-shaped

coccus

grain-like

coccobaccilus

short rods

spirllium

coiled

diplo

tetrad

strepto

staphylo

simple stain

uses 1 dye, tells you about the arrangement and morphology

positive gram stain

direct;

what is the cell wall like of a positive gram stain

thick peptidoglycan, small pores

what color is a positive gram stain

purple

negative gram stain

indirect

what color is a negative gram stain

pink

what is the cell wall of a negative gram stain like

thin peptidoglycan, large pores, additional outer envelope

positive/direct stain dyes

crystal violet, safranin, methylene blue, malachite green

negative/indirect stain dyes

acidic; specifically, nigrosin, which colors the slide and leaves the bacteria colorless

gram stain is a ....

differential stain

why is it considered a differential stain

it uses more than one dye

Before gram stain dying process...

get a slide, add a drop of water, take a inoculataing loop and sterilize it, dip in the bacteria plate, add to slide, and let dry

Gram stain; step 1

crystal violet (primary stain), 60 seconds

Gram stain; step 2

wash

Gram stain; step 3

gram idodine (modorant)

what is the point of a modorant

to show if a cell is positive, if skipped you may get a false negative, 60 seconds

Gram stain; step 4

rinse

Gram stain; step 5

alcohol/ethanol (decolorizer), drip it on until it runs clear

what is the point of a decolorizer

to differentiate bacteria by washing the purple dye out of Gram-negative cells while leaving Gram-positive cells purple

Gram stain; step 6

rinse IMMEDIATELY

Gram stain; step 7

Safranin (counter-stain)

what is the point of a counter-stain

to color Gram-negative bacteria pink

Gram stain; step 8

rinse

Gram stain; step 9

blot dry

after crystal violet gram + & - are

purple

after iodine gram + & - are

purple

after alcohol gram + & - are

+ = purple

- = no color

after safranin gram + & - are

+ = purple

- = pink