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Exercise 3-7 Micro Lab: Gram Stain

front 1

Differential Stains

back 1

these stains allow a microbiologist to detect differences between organisms or differences between parts of the same organism

front 2

Why are differential stains used more frequently?

back 2

they not only allow determination of cell size, morphology, and arrangement (as with a simple stain) but information about other features as well

front 3

The _________ is the most commonly used differential stain in bacteriology

back 3

Gram Stain

front 4

What are other differential stains other than Gram stain used for? ( 2 reasons)

back 4

1. used for organisms not distinguishable by the Gram stain

2. used to distinguish important cellular attributes such as acid-fastness, a capsule, spores, or flagella

front 5

What are these other differential stains (other than Gram) called?

back 5

structural stains

front 6

Gram stain

back 6

a differential stain in which the decolorization step occurs between the application of two basic stains

front 7

primary stain

back 7

crystal violet

front 8

mordant

back 8

enhances crystal violet staining by forming a crystal violet-iodine complex

front 9

what acts as a mordant in a gram stain?

back 9

iodine

front 10

4 steps in Gram staining

back 10

1. primary stain
2. mordant
3. decolorization
4. counterstain

front 11

______ is the most critical step in the gram staining procedure

back 11

decolorization

front 12

_______ cells are decolorized by the solution whereas _______ cells are not

back 12

gram negative
gram positive

front 13

common counterstain

back 13

saffranin

front 14

why can gram negative cells be counterstained?

back 14

because they have been decolorized

front 15

Gram positive appear ___ in color and gram negative appear _____ in color

back 15

purple
reddish pink

front 16

What is it about the walls of gram positive and gram negative that allow them to resist decolorization or not?

back 16

it is in the cell wall.

gram negative have a higher lipid content in cell wall and a thinner peptidoglycan layer than the gram positive. the decolorizer extracts the lipid making gram negative wall more porous and incapable of retaining the crystal violet iodine complex, thereby decolorizing it.

in gram positive, the thicker peptidoclycan and cross linking due to teichoic acids trap the crystal violet iodine complex and make it less susceptible to decolorization

front 17

What 3 ways does poor technique play into gram variable results:

back 17

1. over-decolorize = add too much decolorizer thus giving gram positive a reddish color

2. under-decolorize = adding not enough decolorizer thus giving gram negative a purple color

3. inconsistency in preparing emulsions

front 18

What is the sign of a good emulsion?

back 18

a good emulsion dries to a faint haze on the slide

front 19

Until correct gram technique is mastered, what is recommended?

back 19

a control smear of gram positive and gram negative stains

front 20

what is a good alternative control?

back 20

a smear from the gumline

front 21

crystal violet and safrannin are both _______

back 21

basic stains

front 22

__________ is what makes the gram stain differential

back 22

declorization step

front 23

When making emulsions, what should you do?

back 23

make the emulsions as close to one another as possible. spreading them out on the slide makes it difficult to stain and decolorize them easily.

front 24

What happens if a stain solution is not adequately filtered or is too old?

back 24

crystal violet crystals appear and block the view of bacteria

front 25

interpretation of gram stains can be messed up by these 4 things

back 25

1. crystals appear from stain
2. improperly made stain solution can disrupt the field
3. stain precipitate can be mistakenly identified as bacteria if slide is not rinsed thoroughly or stain not allowed to dry on the slide (variability in size is a clue they are not bacteria)
4. age of culture

front 26

How does age of the culture affect gram staining?

back 26

gram positive walls may lose ability to resist decolorization and give artificial gram negative result

use cultures 24 hours or less.

front 27

2 bacteria known for aging fast

back 27

bacillus and staphylococcus

front 28

Three things that gram stain allows us to do:

back 28

Determine cell

1. morphology
2. arrangement
3. size