Ch. 40: Staining Specimens Flashcards
What are the two most commonly used stains?
Gram stain and Ziehl-Neelsen (acid-fast) stain
What are examples of simple stains?
Crystal violet and methylene blue
What are simple stains usually used to stain?
Yeasts
What is lactophenol cotton blue stain used for?
To confirm the identity of fungal organisms
List the four components of gram staining.
- Primary stain
- Mordant
- Deocolorizer
- Counterstain
What is a mordant stain?
A substance that fixes a dye to a structure (Fixes the primary stain to bacterial cell wall)
What is primarily used as a primary stain for gram staining?
Crystal violet
What is primarily used as a mordant stain for gram staining?
Gram’s iodine solution
What is primarily used as a decolorizer for gram staining?
95% ethanol or acetone
What is primarily used as a counterstain for gram staining?
Basic fuchsin or safranin
When wanting to look at a slide of bacteria after culturing, is it best for the colonies to be young or old?
Young
Older colonies may not yield proper results, and the stained bacteria often become excessively decolorized
With each step of gram staining, how long should the stains be left on the slide?
Crystal violet, iodine, and counterstain: 30 seconds
Decolorizer: 10 seconds
Is it possible to over or under decolorize a sample?
Yes
What is the purpose of heat fixing before gram staining?
Prevents the sample from washing off, helps to preserve cell morphology, kills the bacteria and renders them permeable to stain
What are bacteria called when they retain the crystal violet-iodine complex and stain a purple color?
Gram-positive organisms
What are bacteria called when they lose the crystal violet or purple color and stain red with safranin?
Gram-negative organisms
To ensure proper staining quality, it is recommended to have continually growing organisms in the lab. What are these organisms called?
“Control” organisms; using known gram + and - organisms should be stained at least once a week and with each new batch of stain
What is a gram-variable reaction when staining?
When an organism stains gram-positive and gram-negative
What may be the cause of gram-variable reactions?
- Excessive decolorization
- An overly thick sample smear
- Excessive heat fixation
- Old culture
- Poor-quality stain
What is used to check gram-variable reactions?
A potassium hydroxide (KOH) test
What is the proper procedure for a KOH test?
- A loopful of 3% KOH solution is placed on a slide
- A generous quantity of growth is removed from the culture and transferred to the drop of KOH solution
- Stir the liquid on the slide with a loop for 30 seconds and lift the loop slowly from the slide
- Evaluate and record results
When performing a KOH test, if the organism is gram-negative what will be the result?
When lifting the loop from the slide, the organisms develop a mucoid appearance and produce a sticky strand
When performing a KOH test, if the organism is gram-positive what will be the result?
The mixture stays homogenous and does not form a strand when the loop is lifted from the slide
What % of KOH solution is used for the evaluation of fungal samples?
10-20%
