Lab Final Review - Week #2

Question 1

What is a stain (chemically)?

Answer 1

an organic compound made up of a benzene ring plus a chromophore and an auxochrome group

Question 2

The ability of a stain to bind to macromolecular componenets like proteins or nucleic acids depends on?

Answer 2

The electrical charge found on the chromogen portion and the electrical charge of the organisms being stained

Question 3

Acidic stains

Answer 3

Anionic; negative chromogen -> binds to positively charge components like proteins

Question 4

Basic stains

Answer 4

Cationic; positive chromogen -> binds to negatively charged components like nucleic acids

Question 5

What is a chromogen?

Answer 5

It is the colored compound in a stain

Question 6

What time of stain is usually used for bacterial staining? Why?

Answer 6

Basic stains, because of the negative charge on bacterial surfaces

Question 7

Simple staining

Answer 7

Use of a single stain, which produces a distinctive contrast between the organism and the background. It is useful in the visualization of the morphilogical shape an arrangement of an organism

Question 8

Differential staining

Answer 8

Use of two contrasting stain; at least 4 chemical reagents are needed to accomplish this. It is useful in separating microorganisms into groups & visualization of structures

Question 9

What are the 4 primary rules in preparing bacterial smears for staining?

Answer 9

1. clean slides
2. label slides
3. smear bacteria
4. heat fixation

Question 10

How would you prepare a bacterial smear using a broth culture?

Answer 10

Use one or two loopfuls and spread to about the size of a dime

Question 11

How would you prepare a bacterial smear using cultures from a solid medium?

Answer 11

dilute the culture by placing one or two loopfuls of water on the slide in which the cell swill be emulsified. Spread to the size of a nickel

Question 12

Why is heat fixation of the prepared slide so important?

Answer 12

So that the bacterial smear does not wash away during the staining procedure. Heat fixating coagulates the bacteria and fixes them to the glass surface

Question 13

How could a smear that is too thick yield a false result?

Answer 13

Because of the thickness it could prevent stain to be washed away that otherwise would be

Question 14

What are the 3 most commonly used basic stains?

Answer 14

1. methylene blue 2. crystal violet 3. carbol fuchsin

Question 15

How are simple stains clinically relevant?

Answer 15

Although they are not differential or diagnostic, simple stains are a quick procedure that allow for determining whether the sample has the presence of a foreign bacterial pathogen

Question 16

What type of stain is used for negative staining?

Answer 16

Acidic stain ( such as India ink or nigrosin)

Question 17

What are the 2 practical applications of negative staining?

Answer 17

1. because heat fixation is not used, the cells maintain their natural size and shape
2. it is possible to observe bacteria that are difficult to stain

Question 18

What is the principle application of negative staining?

Answer 18

To determine if an organism possesses a capsule (it can also show spore formation)

Question 19

What is a capsule?

Answer 19

A gelatinous outer layer that makes the microorganism more virulent

Question 20

The technique of negative staining if often used to identify what important infectious agent?

Answer 20

Cryptococcus neoformans

Question 21

What is the most important differential stain used in bacteriology?

Answer 21

The Gram stain

Question 22

Gram stain allows for the division of bacterial cells into what two groups?

Answer 22

Gram-positive and gram-negative, this reaction and differentiation is based on the chemical composition of their cell walls.

Question 23

What type of cell wall do gram-positive cells have?

Answer 23

They have a thick peptidoglycan layer

Question 24

What type of cell wall do gram-negative cells have?

Answer 24

They have a very thin peptidoglycan layer and are surrounded by outer lipid layers

Question 25

What is peptidoglycan composed of?

Answer 25

It is a polysaccharide composed of two chemical subunits: N-acetylglucosamine and N-acetylmuramic acid. Peptidoglycan chains are cross linked by means of the enzyme transpeptidase

Question 26

What are the 4 reagent steps of a gram stain?

Answer 26

1. primary stain 2. mordant 3. decolorizing agents. 4. counterstain

Question 27

What is the primary stain? What does it do?

Answer 27

Crystal Violet, it stains all cells purple

Question 28

What is the mordant? What does it do?

Answer 28

Gram's Iodine, it increases the cell's affinity for the stain by binding to the stain forming an insoluble complex. Now, all cells appear purple-black

Question 29

What is the decolorizing agent? What does it do?

Answer 29

Ethyl Alcohol 95%, it serves a dual function: 1. protein dehydrating agent 2. lipid solvent. Because of this it ultimately decolarizes gram negative cells

Question 30

What is the counterstain? What does it do?

Answer 30

Safranin, it stains pink those cell walls which have previously been decolorized

Question 31

What is the most critical phase of the gram stain procedure and why?

Answer 31

The decolorizing step because over-decolorization will result in loss of the primary stain even in the gram positive stain, causing them to stain and appear as negative. And under-decolorization will not remove all stain from the gram negative organisms causing them to appear gram postive

Question 32

Why is it important to rinse the slide with water between each step in the gram staining procedure?

Answer 32

Because it removes excess reagent, thereby preparing the slide for the subsequent reagent

Question 33

Why is best to gram stain cultures that are less than 24 hours old?

Answer 33

Because as cultures age they lose their ability to retain the primary stain, causing them to appear gram-variable.

Question 34

Why is gram staining clinically important? 2 reasons

Answer 34

1. it is much more rapid than culturing a specimen | 2. it allows for the correct type of antibiotic to be used

Question 35

How much diluent and how much bacteria ought to be used when making a smear from an agar plate?

Answer 35

2 loopfuls of water and a small amount (tip of loop or needle) of bacteria

Question 36

What cell component is bound by negative stain?

Answer 36

none

Question 37

What color would a gram negative bacteria appear if stained with methylene blue?

Answer 37

purple

Question 38

What color would a gram negative bacteria appear if stained by nigrosin?

Answer 38

it would not be colored at all

Question 39

What color would a gram negative bacteria appear after gram-staining?

Answer 39

pink