lab quiz 1 (chapter 5) Preparation of smears and simple staining

basic stain:

+ ion

acidic stain:

- ion

how do you make a smear?

-spread small amount of bacterial broth culture on clean slide -allow broth culture to dry -fix the smear

PUTTING THINGS AWAY:

1) blot oil from objective lens with lens paper 2) return microscope to proper location 3) discard/save slides

process:

1) clean slide well with abrasive soap/cleanser -even new slides bc oily film -handle by the edge/end 2) make marker to make 2 dime size circles @ bottom of each slide -so wont wash off 3) label each circle according to bacterial culture 4) sterilize inoculating loop -hold in hottest part of flame (at edge of inner blue area) -electric incinerator until red hot -heat to REDNESS 5) allow loop to COOL -so bacteria picked up isnt killed -hold it without touching/setting down -cooling takes about 30 seconds 6) prepare smears SOLID MEDIA -place 1-2 loopfuls of distilled water @ center of one circle w/sterile inoculating loop -sterilize loop -using the cooled loop, scrape a SMALL amount of culture off a slant (without taking agar) -redo last 2 steps if hear sizzle of boiling water when touch loop to agar (killed bacteria) -emulsify (to milky suspension) the cells in the drop of water -spread suspension to fill a majority of the circle( should look like diluted skim milk) -sterilize loop BROTH CULTURE -flick tube of broth culture lightly with finger to resuspend sedimented bacteria -place 2-3 loopfuls of culture on circle -sterilize loop between each loopful -spread culture in the circle -sterilize loop 7) let smears dry -dont blow on (will spread bacteria into the air) -dont flame or heat wet slide (will distort cells shapes/cause spattering in the air) 8) hold slide with forceps/clothespin 9) fix the smears (DONT FIX UNTIL SMEARS ARE COMPLETELY DRY) a) heat fix: -pass slide quickly through blue flame with smear side up 2-3 times -hold 1 cm above electric incinerator exterior for 20 seconds -place on 60 degree slide warmer for 10 minutes b) cover smear with 95% methanol for 1 minute -tip slide to let the alcohol run off -let slide air dry before staining

staining smears (pictures version):

1) cover smear with methylene blue -leave for 30-60 seconds 2) gently wash off methylene blue with distilled water -squirt water so it runs through the smear 3) blot it dry

2 ways to "fix" a smear:

1) heat-fixation 2) chemically-fixed

when a smear is "fixed", what happens?

1) kill the bacteria 2) coagulated proteins from cells will cause cells to stick to the slide 3) autolysis 4) preserves microbes with minimal shrinkage/distortion when stained

preparing a bacterial smear from LIQUID medium :

1) mark the smear areas with a marking pencil on the underside of a clean slide 2) place 2-3 loopfuls of the liquid culture on the slide with a sterile loop 3) spread the bacterial within the circle 4) allow the smears to air dry @ room temperature 5A) pass the slide through the flame of a burner 2-3 times (HEAT FIX) 5B) cover the smears with 95% methanol for 1 minute, and then let the smears air dry (CHEMICALLY FIX)

preparing a bacterial SMEAR from SOLID medium (pictures/simplified version):

1) mark the smear areas with a marking pencil on the underside of a clean slide 2)place 1-2 loopfuls of water on the slide 3) transfer a very small amount of the culture with a sterile loop. MIX with the water on the slide 4) allow the smears to air dry @ room temp 5A) pass the slide through the flame of a burner 2-3 times (HEAT FIX) 5B) cover the smears with 95% methanol for 1 minute/ let smears air dry (CHEMICALLY FIX)

simple stains can be used to determine cell: 1) 2) 3)

1) morphology (shape) 2) size 3) arrangement

basic stain "stains" how?

1) permeates cell wall 2) adheres by WEAK ionic bonds to bacterial cell 3) bacterial cell is NEGATIVELY charged

if bacteria are growing on solid media (after spreading small amount of bacterial broth culture on clean slide/allow it to dry) what do you do?

1) remove a small amount of the bacterial culture 2) mix with a drop of water on the slide 3) allow bacteria to air dry

synthetic aniline can be:

1) salt 2) acid 3) base

why must a smear be "fixed"? (main purpose)

1) to kill the bacteria 2) allow bacteria to stick to slide

staining smears (long version):

1) use clothespin/forceps/sliderack to hold slide 2) cover smear with methylene blue -leave for 30-60 seconds 3) wash off excess stain with distilled water from wash bottle -let water run through smear 4) gently blot smear with a paper towell or absorbant paper 5) let dry

EXAMINING SMEAR/STAINED BACTERIA:

1) use low, high-dry, and oil immersion objectives 2) put oil directly on smear 3) coverslips are not needed 4) record observations

example of chromophore:

METHYLENE BLUE + methylene blue chloride -> <- Methylene blue + (chromophore) + cl- ion is charged molecule

simple stain that stains the background but leaves the bacteria unstained is called:

Negative stain

how do you heat fix a slide?

Using a slide holder pass the smear in the upper part of the flame two to three times. avoid overheating to avoid turning it into charcoal. USE HEAT touch on back of hand to make sure its not flaming hot, if it is, you probs made charcoal

basic stains adheres by ____ ___ _____ to bacterial cell, that is ____ charged:

Weak ionic bonds negatively charged

if the chromophore is a negative ion, the stain is considered a:

acidic stain

methylene blue + makes a ______ stain:

basic

if the chromophore is a positive ion, the stain is considered a :

basic stain

most bacteria are stained with a basic/acidic stain?

basic stain

synthetic aniline is derived from:

benzene

what is a chromophore?

charged colored ion

acids and bases that make up synthetic aniline are composed of what?

chromophore

aniline is a derivative of:

coal tar

how do you chemically fix a slide? use WHAT? how LONG?

cover smear with 95% methanol for 1 minute

a simple stain that stains the bacteria is called:

direct stain

where can stained bacterial slides be stored?

in a "slide box"

do basic stains "stain" + or - charged bacterial cells?

negatively charged

after removing oil from a slide by blotting it with paper towel, what should you do about any residual oil?

nothing, it doesnt matter

is methylene blue positively or negatively charged?

positively (+)

synthetic aniline dyes are usually:

salts

staining procedures that use only one stain are called:

simple stain

To stain bacteria, a thin film of bacterial cells called a ________, must be placed on a slide.

smear

most stains used in microbiology are:

synthetic aniline dyes

what is autolysis?

the cell breaks -in the case of fixing bacteria 1) bacterial enzymes are denatured 2) enzymes cannot digest cell parts 3) causes cell to break