Ch. 40: Staining Specimens

Question 1

What are the two most commonly used stains?

Answer 1

Gram stain and Ziehl-Neelsen (acid-fast) stain

Question 2

What are examples of simple stains?

Answer 2

Crystal violet and methylene blue

Question 3

What are simple stains usually used to stain?

Answer 3

Yeasts

Question 4

What is lactophenol cotton blue stain used for?

Answer 4

To confirm the identity of fungal organisms

Question 5

List the four components of gram staining.

Answer 5

1. Primary stain
2. Mordant
3. Deocolorizer
4. Counterstain

Question 6

What is a mordant stain?

Answer 6

A substance that fixes a dye to a structure (Fixes the primary stain to bacterial cell wall)

Question 7

What is primarily used as a primary stain for gram staining?

Answer 7

Crystal violet

Question 8

What is primarily used as a mordant stain for gram staining?

Answer 8

Gram’s iodine solution

Question 9

What is primarily used as a decolorizer for gram staining?

Answer 9

95% ethanol or acetone

Question 10

What is primarily used as a counterstain for gram staining?

Answer 10

Basic fuchsin or safranin

Question 11

When wanting to look at a slide of bacteria after culturing, is it best for the colonies to be young or old?

Answer 11

Young

Older colonies may not yield proper results, and the stained bacteria often become excessively decolorized

Question 12

With each step of gram staining, how long should the stains be left on the slide?

Answer 12

Crystal violet, iodine, and counterstain: 30 seconds
Decolorizer: 10 seconds

Question 13

Is it possible to over or under decolorize a sample?

Answer 13

Yes

Question 14

What is the purpose of heat fixing before gram staining?

Answer 14

Prevents the sample from washing off, helps to preserve cell morphology, kills the bacteria and renders them permeable to stain

Question 15

What are bacteria called when they retain the crystal violet-iodine complex and stain a purple color?

Answer 15

Gram-positive organisms

Question 16

What are bacteria called when they lose the crystal violet or purple color and stain red with safranin?

Answer 16

Gram-negative organisms

Question 17

To ensure proper staining quality, it is recommended to have continually growing organisms in the lab. What are these organisms called?

Answer 17

“Control” organisms; using known gram + and - organisms should be stained at least once a week and with each new batch of stain

Question 18

What is a gram-variable reaction when staining?

Answer 18

When an organism stains gram-positive and gram-negative

Question 19

What may be the cause of gram-variable reactions?

Answer 19

• Excessive decolorization
• An overly thick sample smear
• Excessive heat fixation
• Old culture
• Poor-quality stain

Question 20

What is used to check gram-variable reactions?

Answer 20

A potassium hydroxide (KOH) test

Question 21

What is the proper procedure for a KOH test?

Answer 21

1. A loopful of 3% KOH solution is placed on a slide
2. A generous quantity of growth is removed from the culture and transferred to the drop of KOH solution
3. Stir the liquid on the slide with a loop for 30 seconds and lift the loop slowly from the slide
4. Evaluate and record results

Question 22

When performing a KOH test, if the organism is gram-negative what will be the result?

Answer 22

When lifting the loop from the slide, the organisms develop a mucoid appearance and produce a sticky strand

Question 23

When performing a KOH test, if the organism is gram-positive what will be the result?

Answer 23

The mixture stays homogenous and does not form a strand when the loop is lifted from the slide

Question 24

What % of KOH solution is used for the evaluation of fungal samples?

Answer 24

10-20%

Question 25

What is DMSO?

Answer 25

Dimethyl Sulfoxide

Can be used on a KOH test to speed up the activity of the KOH solution

Question 26

Ziehl-Neelsen stain is used to stain what specific organisms?

Answer 26

Mycobacterium and Nocardia species

Question 27

What are the three components of an acid-fast stain?

Answer 27

1. Primary stain - DMSO & carbol fuchsin
2. Acid-alcohol decolorizer
3. Counterstain - Methylene blue

Question 28

How is an acid-fast stain performed?

Answer 28

Air dry and heat fix slide, put primary stain on slide, heat the slide until the stain steams, cool for 5 minutes and then rinsed, use decolorizer for 1-2 minutes until red color is gone, and rinse slide again. Use counterstain and then rinse the slide a final time