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44 notecards = 11 pages (4 cards per page)

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Micro lab exam 2

front 1

simple stains

back 1

staining procedure that only uses one stain and all bacteria are stained similarly

used for cell morphology, size, arrangement

front 2

differential stains (gram)

back 2

first step in identifying bacteria

uses two contrasting stains seperated by a declorizing agent

front 3

Acid fast

back 3

used to keep stain in place when washed with acid

front 4

Spore stain

back 4

used to observe endospores, capsules and flagella

front 5

types of staining techniques

back 5

aa

front 6

smears

back 6

speading a small amount of bacteria broth culture or solid inoculum mixed with a drop of distilled water on a clean slide allowing to dry

front 7

heat fixing a sample

back 7

passed over incommator several times or placed over a slide warmer

front 8

chemically fix the sample

back 8

cover smear with 95% methanol for 1 min

front 9

what is the purpose of heating/ fixing the smear?

back 9

to kill the bacteria and preserves with minimal distortion/shrinkage

front 10

simple stains

back 10

bacteria cell surfaces negatively charged and basic dyes are used as stains

use of a single basic dye is called a simple stain

doesnt tell you if its gram positive or negative

front 11

common basic dyes

back 11

1. methylene blue

2. crystal violet

3. safranin

4. malachite green

front 12

simple staining steps

back 12

1. use a clothespin to hold slide

2. cover slide with methylene blue and leave for 30-60 sec

3. wash off excess stain with distilled water

4. blot smear with paper towel and air dry

front 13

which bacteria is a rod?

back 13

bacillus megaterium

front 14

which bacterium is larger

back 14

bacillus megaterium

front 15

what value is a simple stain?

back 15

enhances the contrast between the bacterium and its surrounding material and permits greater clarity of detail

front 16

a

back 16

B

front 17

another method of fixing a smear?

back 17

chemically fixing

front 18

how does alcohol chemically fix the bacteria?

back 18

1. removes water (imp for mounting the cell)

2. denatures proteins (metabolism of the cell is stopped and cell dies)

3.it dissolves and removes lipids (cell membrane of bacteria is harmed by alcohol)

front 19

negative staining

back 19

does not stain bacteria stains background

front 20

dyes used for negative staining

back 20

india ink

nigrosin

front 21

steps in negative staining

back 21

1. add drop of nigrosin on clean slide

2.aseptically add organism

3.place second clean slide on the surface of the first slide and draw it back into the drop

4.when drop flows across the width of spreader slide

5. push spreader slide to other end

6. air dry

front 22

which cell is a rod?

back 22

bacillus subtilis

front 23

why is the size more accurate in a negative stain than in a direct stain?

back 23

No heat fixing or chemicals are used so there is no distortion of the cell

front 24

what microscopic technique gives the field similar appearance to that seen in a negative stain

back 24

dark field microscope

front 25

could any dye be used in place of nigrosin for negative staining?

back 25

no only a cell that does not penetrate the cell, india ink works

front 26

why can carbolfuchsin be used as a direct stain and as a negative stain?

back 26

ph is basic

front 27

Acidic congo red does not conatin suspended particiles like nigrosin but can give the appearance of a negative stain. what is the basis for this stain?

back 27

b/c it it acidic ( negative charge ) stain cannot bind to negatively charged bacteria

front 28

Gram staining steps

back 28

1. apply primary stain (crystal violet) 30 sec bacteria stained purple

(gram plus and minus are blue)

2. apply mordant (gram iodine) 10 sec (gram plus and minus blue)

3. apply decolorizing agent (ethanol)10-20 sec (gram plus blue gram minus colorless)

4. apply secondary stain or counter stain (safranin) 30sec the dye stains decolorized bacteria red (gram plus blue glam minus red

front 29

Colors

Crystal violet

Safranin

back 29

crystal violet-purple

safranin-red

front 30

use of mordant

back 30

grams iodine binds to the crystal violet preventing the compound from being clear thru the cell wall of the gram positive bacteria

front 31

gram positive cell wall

back 31

thick layer of peptidoglycan

front 32

gram negative cell wall

back 32

thin peptidoglycan layer covered by a lipopolysaccharide layer

front 33

no data

back 33

gram negative bacteria

front 34

no data

back 34

gram positive bacteria

front 35

no data

back 35

mycobacteria

front 36

no data

back 36

fungi

front 37

acid fast staining

back 37

differential stain

front 38

principal behind this staining

back 38

carbolfuschin binds to mycolic acids on the outside of the acid-fast bacteria which is not washed away with the acid-alcohol mixture that decolorizes all other bacteria

front 39

dyes used for acid fast staining

back 39

carbofuschin and methylen blue

front 40

acid fast steps

back 40

1. prepare fixed smear of culture

2. cover the smear with carbolfuchsin for 5 min

3. wash with distilled water

4. wash with decolorizer (acid alcohol) for 1 min

5. wash water

6. counterstain with methylene blue for 1 min

7. examine slide under microscope

front 41

Endospores

back 41

"resting bodies"

do not metabolize resistant to heat chemicals and harsh environments,

are formed when nutrients are not available

generally impermeable to stains so heat must be applied

front 42

capsule

back 42

increase virulence(disease causing ability)

decrease being phagocytiezed

front 43

Flagella

back 43

motility

location and presence are helpful in identifying bacteria

front 44

Steps in staining endospores

back 44

1. make smear let air dry, and heat fix

2. tear small piece of paper towel and place over smear

3.cover smear and paper with malachite green and steam the slife for 5 mins (keep wet)

4.remove paper and throw away, wash smear with h20

5.counterstain with safranin for 30 sec

6.wash with h20 30 sec

7.examine under microscope