Lab 5: Smear Preparation and Staining (simple, gram and spore)

what is the first step when staining

attach bacteria to a slide

cell morphology

cocci, bacilli, spiral

arrangement of cells

single, chains, colonies

internal structures

endospores 

bacilli (singular is bacillus)

rod shape: rounded, flat or tapered ends, motile or nonmotile

cocci (singular is coccus)

spherical; occur singly, in chains, tetrads or irregular masses, usually nonmotile

spiral 

helices, usually motile 

goals of smear prep 

adheres cells to the slide, prevents cells from shrinking, kills cells 

what is it important to prepare

thin smears

smear prep from liquid

1 loopful onto slide

smear prep from solid media

1 loopful of water, then loopful of cells

smear prep steps 

1st label slides by drawing circles on the bottom of the slides, then use the aseptic technique by flaming the loop and the tube head, and spread cells thinly on the slide allowing them to dry without heat, finally, once the slide is dry hold the bottom of the slide to opening of the incinerator for 10 seconds 

since cells in simple stains have little contrast, how do we enhance their features?

by dyeing them with stains

what does the simple stain reveal about bacterial cells

cell morphology

simple stain basic dye 

methylene blue, basic fuschin, crystal, and contain cationic chromophores

what is the net charge of bacteria

negative

acidic dye of simple stain 

contain anionic chromophores 

cationic vs anionic chromophores

cationic chromophores are used to stain bacteria because bacteria have negative net charge while anionic chromophores stain background of slides

steps of simple stain

smear prep, add dye to smear and let it sit for one minute, wash stain of with water and blot water drops with bibulous paper

Hans christian gram (1884) 

studied diseased lung tissue and differential stains such as gram positive and gram negative 

gram stains

cell walls contain peptidoglycan

gram positive stains

have thick peptidoglycan that traps the dye

gram negative stains 

have thin peptidoglycan layer and outer membrane 

crystal violet in gram stain

primary stain

gram’s iodine in gram stain

mordant

alcohol in gram stain 

decolorizer

safranin in gram stain

counterstain

what bacterias are used in the gram stain

M.luteus which is positive and E.coli which is negative

gram stain steps 

apply crystal violet (primary stain to look for gram positive bacteria) and then wash with water then apply iodine (not a stain but a mordant that sets the dye), rinse with alcohol (acts as decolorizer for gram negative bacteria) the rinse with water then add safranin as counterstain to stain the gram positive bacteria