Lab Quiz #4:

Question 1

This type of stain allows to detect differences between organisms or differences between parts of the same organism

Answer 1

Differential Stains

Question 2

What information does a differential stain allow you to see?

Answer 2

Cell size, morphology, and arrangement

Question 3

What color do Gram-positive cells appear?

Answer 3

Purple

Question 4

What color do Gram-negative cells appear?

Answer 4

Reddish-pink

Question 4

What are the steps to a Gram Stain?

Answer 4

Primary stain is crystal violet
Iodine is added as a mordant to enhance crystal violet staining by forming a crystal violet-iodine complex.
Decolorization follows and is the most critical step in the procedure.

Gram-negative cells are decolorized by the solution (of variable composition-generally alcohol or acetone) whereas Gram-positive cells are not.

Gram-negative cells can thus be colorized by the Counterstain Safranin.

Gram-positive cells appear purple.

Gram-negative cells appear reddish-pink

Question 5

What is the most critical step in the Gram stain procedure and why?

Answer 5

Decolorization because Gram-negative cells are decolorized by the solution, whereas Gram-positive cells are not.

Question 6

Why are Gram-negative cells more susceptible to decolorizing?

Answer 6

The Gram-negative cell walls have a higher lipid content and a thinner peptidoglycan layer which means the alcohol in the decolorizer extracts the lipid, making the Gram-negative layer more porous and incapable of retaining the crystal violet-iodine complex

Question 6

What are the quick steps of Gram-stains?

Answer 6

1. Crystal Violet (primary stain) (1 minute)
2. Iodine (mordant to enhance crystal violet) (1 minute)
3. Alcohol (decolorizer)
4. Safranin (counterstain) (1 minute)

Question 7

What causes the most inconsistency around Gram-staining?

Answer 7

Over-decolorizing by leaving the alcohol on too long and get reddish Gram-positive cells and under-colorizing and producing purple Gram-negative cells

Question 8

What steps makes a Gram-stain differential than just a simple stain?

Answer 8

The decolorization step

Question 9

What are solutions consisting of a solvent (usually water or ethanol) and a colored molecule, the chromogen?

Answer 9

A stain

Question 9

What is the portion of the chromogen that give it its color?

Answer 9

Chromophore

Question 10

What is the charged portion of a chromogen and allows it to acta as a dye through ionic or covalent bonds between the chromogen and the cell?

Answer 10

Auxochrome

Question 11

What is attracted to the negative charges on the surface of most bacterial cells?

Answer 11

Basic stains
where the auxochromes becomes positively charged as result of picking up a hydrogen ion or losing a hydroxide ion

Question 12

What are some common basic stains?

Answer 12

Methylene blue, crystal violet, and safranin

Question 13

Basic stains are applied to bacterial smears that have been what?

Answer 13

Heat fixed

Question 14

What kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible?

Answer 14

Heat-fixing

Question 15

What are the steps for bacterial smear preparation?

Answer 15

1. add bacteria in broth culture with loop onto slide
2. allow smear to air dry
3. pass the smear over fire to heat-fix
4. allow slide to cool then begin staining protocol
5. Basic staining can be crystal violet (30 to 60 seconds
   Safranin (stain for up to 1 minute)
   Methylene blue (stain for 30 to 60 seconds)

Question 16

What is the cytological basis for the acid-fast differential stain?

Answer 16

The presence of mycolic acids in the cell walls of acid-fast organisms

Question 17

What is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution?

Answer 17

Mycolic acid

Question 18

What are some acid-fast staining procedures?

Answer 18

Ziehl-Neelsen (ZN) and the Kinyoun (K) method

Question 19

Which method uses heat as part of the staining process?

Answer 19

Ziehl-Neelsen (ZN)

Question 20

Which method uses a cold stain as part of the staining process?

Answer 20

Kinyoun (K) method

Question 20

The waxy wall of acid-fast cells repels typical aqueous stains which results in what?

Answer 20

Most acid-fast positive organisms are only weakly Gram-positive

Question 21

In the ZN method what is used as the primary stain because it is lipid-soluble and penetrates the waxy cell wall?

Answer 21

The phenolic compound carbolfuchsin

Question 22

What is the process to further enhance the staining by carbolfuchsin?

Answer 22

Steam-heating the preparation to melt the wax and allow the stain to move into the cell

Question 23

What is used to decolorize non-acid fast cells?

Answer 23

Acid alcohol

Question 24

What is the acid-staining process using the ZN method?

Answer 24

1. Carbolfuchsin (primary stain) cells are reddish/purple
2. Steam-heating to melt the wax and allow the stain to move into the cell
3. Acid alcohol (decolorization) removes stains from acid-fast negative cells
4. Methylene blue (counterstain)

Acid-fast cells are reddish/purple and non-acid fast cells are blue

Question 25

What color are acid-fast cells?

Answer 25

Reddish/purple

Question 26

What color are non-acid fast cells?

Answer 26

Blue

Question 27

The Kinyoun method uses what type of carbolfuchsin and why?

Answer 27

A slightly more lipid-soluble and concentrated carbolfuchsin because these properties allow the stain to penetrate the acid=fast walls without the use of heat but make this method slightly less sensitive than the ZN method.

Question 28

What is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol?

Answer 28

Acid-fast stain