Lab 3-7 Gram Staining

what is the purpose of lab 3-7 (Gram staining)?

to differentiate between gram positive and gram negative bacteria

what does it mean to "differentiate"?

to show a difference

what two dyes will be used in this lab?

1st dye: primary dye (crystal violet)

2nd dye: counterstain (safranin)

what do the staining procedures show us? (what cell type and parts?)

cell type: gram + or gram -

parts: endospores mother cell

what is the first dye called?

primary dye

what is the second dye called?

counterstain

what dye are we using as a counterstain?

safranin

what is the clinical significance of gram staining? (2)

- help guide drug treatment

- finding the right antibiotic for gram + or gram -

all prokaryotes have what component?

cell wall

what is a major component of the cell wall?

peptidoglycan

which cell type has a thicker cell wall? (gram +/-)

gram +

list the steps of gram staining

1. heat fixation

2. primary stain

3. add mordant (iodine)

4. decolorization

5. counterstain

6. view slide

what is the purpose of heat fixing?

gently kill and adhere organisms on slide

what is the primary dye called? what is the charge?

crystal violet, positive charge

what are we differentiating in this experiment?

gram - from gram +

what is the purpose of iodine?

binds to crystal violet dye to form a complex to prevent it from being washed out

what is the purpose of the decolorization step?

disrupt outer membrane of gram -, and easily remove CV-I

which organism will stay purple, why?

gram + because of thick outer membrane

what reagent is used to decolorize the cells?

ethanol

describe the chemistry behind the procedure (for each step) for a gram -

1. heat fix slide

2. add CV to organism

3. add iodine so that CV sticks and doesnt get washed off

4. add ethanol (ethanol will disrupt outer membrane

5. add safranin

6. record results

what charges do the primary and counterstain have?

positive charge

describe the chemistry behind the procedure (for each step) for a gram +

1. heat fix slide

2. add crystal violet to organism, organism is stained purple

3. add iodine so that CV will stick to the organism

4. add ethanol

5. add safranin

6. record results

what is the problem with decolorizing the cells for too long? how would it affect the results?

will strip CV-I, gram + will be pink

what is the problem if you do not decolorize long enough? how would it affect the results?

not enough alcohol will end up with a purple gram -

why would the age of your culture also affect your results?

age affects our results because of the build up of dead organisms, can result in a pinkish color since the cell wall is unable to hold on to CV

which staining procedure would you want to have cultures older than 24 hours?

out of all the phases of growth for a bacterial cell, which phase would be best to use a cell for a gram stain?

exponential phase because death is minimal

what would be problematic about accidentally forgetting to stain with iodine? how would this affect the results?

crystal violet dye will wash off when we rinse with water

what do gram - organisms look like at the completion of a gram stain? explain.

pink

would a protozoan organism stain purple or pink?

pink because eukaryotes do not have an outer membrane (no peptidoglycan)