preparation + staining

Question 1

why do we stain bacteria

Answer 1

bacteria are colorless, transparent, and difficult to see

Question 2

what is staining

Answer 2

coloring the microorganism with a dye for visualization

Question 3

what can we see via staining

Answer 3

certain structures within the organism or the organism itself

Question 4

what must we do to a microbe before staining it (1 word)

Answer 4

we must fix it

Question 5

what is fixing

Answer 5

attaching the microbe to the slide, killing it, and preserving internal and external structures in their natural state

Question 6

how is fixing done

Answer 6

a smear is made, the slide is air dried, and it is fixed by passing it through a flame several times

Question 7

define smear

Answer 7

a thin film of bacteria on the surface of the slide

Question 8

what are two ways we can make a smear

Answer 8

plate or broth

Question 9

describe a plate smear

Answer 9

a drop of water is put onto the slide first, and then an inoculating loop is used to get the cells, then the cells are spread into the water

Question 10

describe a broth smear

Answer 10

put inoculating loop into a broth, and then smear it onto the slide

Question 11

describe the orientation of the slide when fixing it through a flame

Answer 11

smear side up

Question 12

what two methods can be used for fixing microorganisms

Answer 12

heat fixation and chemical fixation

Question 13

describe heat fixation

Answer 13

preparing the slide then passing it through a flame

Question 14

describe chemical fixation

Answer 14

a fixative is added to the smear

Question 15

in chemical fixation, give examples of fixative that can be added to the smear

Answer 15

ethanol, methanol, acetic acid, formaldehyde

Question 16

which method of fixation (heat or chemical) is better for preserving the internal morphology of cells

Answer 16

chemical

Question 17

describe the features of the dyes used to stain organisms

Answer 17

they’re ionic, and they have chromophore groups attached

Question 18

what do chromophore groups do

Answer 18

give the dye its color

Question 19

what two classes can ionizable dyes be divided into

Answer 19

basic and acidic dyes

Question 20

what type of charged groups do basic dyes have

Answer 20

positive

Question 21

what types of molecules do basic dyes bind do? give examples

Answer 21

negatively charged molecules

nucleic acids, many proteins

Question 22

which type of dye (basic or acidic) is most used in microbiology? why?

Answer 22

basic; because they’re attracted to negative charges and bacterial cell surfaces are negatively charged

Question 23

give examples of basic dyes

Answer 23

methylene blue, malachite green, crystal violet, safranin

Question 24

what type of charged groups do acidic dyes have

Answer 24

negative

Question 25

what types of molecules do acidic dyes bind to? give examples

Answer 25

positively charged things some proteins

Question 26

describe how acidic dyes interact with bacteria

Answer 26

they repel bacteria due to having the same charge

Question 27

give examples of acidic dyes

Answer 27

eosin, acid fuchsin, congo red

Question 28

what are the two types of staining procedures

Answer 28

simple staining and differential staining

Question 29

what is simple staining

Answer 29

microbes are stained with a single agent (1 color)

Question 30

describe the steps of simple staining

Answer 30

prepare smear, air dry, fix the bacteria, apply the dye, rinse off excess, blot dry, examine

Question 31

what are the uses of simple staining

Answer 31

used to highlight the entire microbe, allows one to get info about the size, shape, and arrangement of cells

Question 32

what is differential staining

Answer 32

used to divide bacteria into separate groups based on their staining properties

Question 33

what are two types of differential staining

Answer 33

gram stain and acid fast stain

Question 34

what is a gram stain

Answer 34

divides bacteria into two groups based on their ability to take up and retain a dye

Question 35

what two groups are made in a gram stain

Answer 35

gram positive and gram negative

Question 36

what type of smear is used in a gram stain; heat or chemical

Answer 36

heat fixed

Question 37

describe the steps of a gram stain

Answer 37

apply crystal violet dye, wash, then apply iodine = everything purple rinse, use ethanol for decolorization (gram negative will be colorless) wash, apply safranin (counterstain), turns colorless into pink

Question 38

what dyes are used in a gram stain

Answer 38

crystal violet and safranin

Question 39

what are the results of the gram stain

Answer 39

gram negative = red gram positive = blue

Question 40

what is an acid stain used for

Answer 40

used to stain a small group of organisms that do not readily take up stains + differentiates between 2 groups of bacteria

Question 41

what two groups of bacteria are defined in an acid fast stain

Answer 41

acid fast vs non acid fast

Question 42

define acid fast organism

Answer 42

organisms that do not readily take up stains due to high content of lipid in their cell walls

Question 43

what type of smear is used in an acid fast stain: chemical or heat fixed

Answer 43

heat fixed

Question 44

describe the steps of an acid fast stain

Answer 44

add primary stain (carbofuchsin) while slide is over steaming water cool + rinse, add decolorizer then acid alcohol wash, then apply counterstain (methylene blue)

Question 45

in an acid fast stain, why do we apply the primary stain while the slide is over steaming water

Answer 45

the heat is used to push the dye through the cell wall since acid fast organisms dont readily take up dye

Question 46

what two dyes are used in an acid fast stain

Answer 46

carbofuchsin red and methylene blue

Question 47

what are the results of the acid fast stain

Answer 47

acid fast = red non acid fast = blue

Question 48

define endospore

Answer 48

a resistant structure that allows some bacteria to withstand unfavourable conditions

Question 49

what is the name for the procedure to stain an endospore

Answer 49

schaeffer-fulton procedure

Question 50

describe how we stain an endospore

Answer 50

using a heat fixed smear, add malachite green in the presence of steam (this will penetrate the endospore) cool + rinse, add safranin as the counterstain, wash

Question 51

what are the visual results of the endospore stain

Answer 51

endospore=green cytoplasm=red

Question 52

what does the endospore stain allow us to see

Answer 52

presence of an endospore + location of the endospore

Question 53

what are flagella used for

Answer 53

locomotion of bacterial cells

Question 54

what do we do for a flagella stain

Answer 54

add carbolfuchsin

Question 55

why would we want to do a flagella stain

Answer 55

aids in identifying bacteria based on presence and location of the flagella

Question 56

what is a negative stain

Answer 56

its when we stain the background instead of the organism

Question 57

what color will bacteria be after a negative stain

Answer 57

colorless; only the background is stained

Question 58

what dye is used in a negative stain

Answer 58

an acidic dye, ie eosin

Question 59

what is a negative stain used for

Answer 59

revealing the presence of a capsule

Question 60

what is a capsule

Answer 60

a substance that may surround some bacterial cells

Question 61

what is the charge on a capsule

Answer 61

negative