LAB 4: STAINING ii = DIFFERENTIAL STAINING

what type of staining is Gram staining? - RM

differential - RM

what is the main difference between a gram+ and gram- bacterium? - QUEEN

+G=thick cell wall and -G=thin cell wall an outer membrane - QUEEN

what type of staining is acid-fast staining? - JIN

differential - JIN

what’s an acid-fast bacteria? - YUHH

bacteria that has mycolic acid - YUHH

what’s an non-acid fast bacterium? - NAHHH

bacteria doesn’t have mycolic acid - NAHH

what type of staining is endospore staining? - SUGA

differential - SUGA

when do endospore-forming bacteria produce endospores? - JINNN

during harsh conditions - JINNN

1st stains/reagents used in gram staining

crystal violet

2nd stains/reagents used in gram staining

grams iodine

3rd stains/reagents used in gram staining

decolorizer

4th stains/reagents used in gram staining

safranin

simple gram staining step 1

create a smear slide - START

simple gram staining step 2

crystal violet (sit for 1 minute)

simple gram staining step 3 - JISOO

wash - JISOO

simple gram staining step 4

gram’s iodine (sit for 1 minute)

simple gram staining step 5 - JK

wash - JK

simple gram staining step 6

decolorize for 5 sec

gram staining step 7 - HOBI

wash - HOBI

simple gram staining step 8 - HOBI

safranin (sit for 1 minute)

simple gram staining step 9

wash and blot - FINAL

differential staining purpose

distinguish different bacteria

what are the three types of differential staining?

gram, acid-fast, and endospore

gram and acid-fast staining are based on

differences in bacteria’s cell envelope

three layers of bacteria’s cell envelope

membrane, wall, and outer membrane

bacteria’s cell membrane composition

bilayer of phospholipids

bacteria’s cell wall composition

peptidoglycan

bacteria’s outer membrane composition

bilayer of phospholipids and lipopolysaccharides

who developed a staining technique to differentiate the cell envelopes of different bacteria?

hans christian gram

grams staining technique purpose

distinguish based on thickness of cell wall

gram-positive bacteria cell wall

thick cell wall

gram-negative bacteria cell wall

thin cell wall

what are the +G and -G bacteria stain with beginning?

crystal violet (purple)

what are the +G and -G bacteria stain with second?

gram iodine

what does gram iodine do to +G

helps trap crystal violet in +G

why is there less trapping of crystal violet in -G?

its cell wall in thinner

what are the +G and -G bacteria stain with third?

decolorizer of ethanol

who stain is lost during decolorizing? - ROSE

gram negative - ROSE

the ethanol destroys what of the gram negative cell?

outer membrane

who retains the purple color during decolorization?

gram positive

who looses the purple color and becomes colorless during decolorization? - RM

gram negative - RM

what are the +G and -G bacteria stain with finally?

safranin (counterstain)

the safranin (counterstain) stains -G what color?

pink/red

in conclusion , gram positive bacteria are stained

purple

in conclusion , gram negative bacteria are stained

pink/red

gram staining

pink=negative, purple=positive

why can’t gram staining be used on some bacteria? - RIT

they have mycolic acid - RIT

what is mycolic acid?

hydrophobic molecule

what method is used on bacteria with mycolic acid?

ziehl-neelsen method

ziehl-neelsen method purpose

distinguish between those with and without mycolic acid

what are acid-fast cells? - SUGA

bacteria with mycolic acid - SUGA

what non acid-fast cell? - DUDE

bacteria without mycolic acid - DUDE

in acid-fast staining what’s the 1st dye used

carbol fuchsin

when u add carbol fuchsin, acid-fast cells are what color

red

what is the counterstain in acid fast staining

methylene blue

when do bacterial species form a resistant endospore? - FUN

when environment is too harsh for vegetative cell - FUN

what is the vegetative cell in bacteria?

metabolically active bacteria cell

when nutrients return, what happens to the endospore

it germinates

what does it mean when the endospore germinates?

forms new vegetative cell and metabolically active

in endospore staining, endospores are stained with

malachite green

in endospore staining, what color is the endospore?

green

in endospore staining, vegetative cell are stained with - VEGE

safranin - VEGE

in endospore staining, what color is the vegetative cell?

red

endospore staining purpose

to differentiate between vegetative cell and endospore

endospore staining: vegetative cell location

long and red

endospore staining: endospore location

green pea

gram stain step 1

get clean slide

gram stain step 2

add drop of water in center

gram stain step 3

add first bacteria, sterilize, add second then smear (circular motion)

gram stain step 4

air dry

gram stain step 5

heat fix 3 times

gram stain step 6 - JIN

add crystal violet and let sit 1 minute - JIN

gram stain step 7 - LISA

wash at 45 degree angle - LISA

gram stain step 8 - GASSED UP

add grams iodine and let sit for 1 minute - GASSED UP

gram stain step 9 - ROSE

wash at 45 degree angle - ROSE

gram stain step 10 (DONT OVERDO)

decolorize at 45 degree angle for 5-10 seconds until colorless

gram stain step 11 - JISOO

wash at 45 degree angle - JISOO

gram stain step 12 - OG

add counterstain safranin and let sit for 1 minute - OG

gram stain step 13

wash at 45 degree angle

gram stain step 14 (BIBULOUS PAPER) - DRY

blot until dry - DRY

gram stain step 15

view cells under 100x with oil

in diagram form, show the similarities and differences between a cell envelope found in a gram-positive bacterium vs. the cell envelope found in a gram-negative bacterium. - PUS

+G=thick cell wall, -G=thin cell wall & outer membrane - PUS

what bacteria is this?

+G, thick cell wall and NO outer membrane

what bacteria is this?

-G, thin cell wall WITH outer membrane

what is the purpose of the decolorizer in gram staining produce?

to destain the gram negative cells

what would be the color of the cells of a gram stain if you forget to use the ethanol?

purple and blue

acid-fast cells do not stain well using the gram staining method - WHY?

they have hydrophobic mycolic acid - WHY?

under what conditions do bacteria produce an endospore? - DRAKE

harsh conditions - DRAKE

are endospores hard to kill?

yes

what d we do to destroy endospores?

autoclave (high pressure sterilize)