Lab Final Review - Week #2 Flashcards
What is a stain (chemically)?
an organic compound made up of a benzene ring plus a chromophore and an auxochrome group
The ability of a stain to bind to macromolecular componenets like proteins or nucleic acids depends on?
The electrical charge found on the chromogen portion and the electrical charge of the organisms being stained
Acidic stains
Anionic; negative chromogen -> binds to positively charge components like proteins
Basic stains
Cationic; positive chromogen -> binds to negatively charged components like nucleic acids
What is a chromogen?
It is the colored compound in a stain
What time of stain is usually used for bacterial staining? Why?
Basic stains, because of the negative charge on bacterial surfaces
Simple staining
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
Differential staining
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
What are the 4 primary rules in preparing bacterial smears for staining?
- clean slides
- label slides
- smear bacteria
- heat fixation
How would you prepare a bacterial smear using a broth culture?
Use one or two loopfuls and spread to about the size of a dime
How would you prepare a bacterial smear using cultures from a solid medium?
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
Why is heat fixation of the prepared slide so important?
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
How could a smear that is too thick yield a false result?
Because of the thickness it could prevent stain to be washed away that otherwise would be
What are the 3 most commonly used basic stains?
- methylene blue 2. crystal violet 3. carbol fuchsin
How are simple stains clinically relevant?
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
What type of stain is used for negative staining?
Acidic stain ( such as India ink or nigrosin)
What are the 2 practical applications of negative staining?
- because heat fixation is not used, the cells maintain their natural size and shape
- it is possible to observe bacteria that are difficult to stain
What is the principle application of negative staining?
To determine if an organism possesses a capsule (it can also show spore formation)
What is a capsule?
A gelatinous outer layer that makes the microorganism more virulent
The technique of negative staining if often used to identify what important infectious agent?
Cryptococcus neoformans
What is the most important differential stain used in bacteriology?
The Gram stain
Gram stain allows for the division of bacterial cells into what two groups?
Gram-positive and gram-negative, this reaction and differentiation is based on the chemical composition of their cell walls.
What type of cell wall do gram-positive cells have?
They have a thick peptidoglycan layer
What type of cell wall do gram-negative cells have?
They have a very thin peptidoglycan layer and are surrounded by outer lipid layers
What is peptidoglycan composed of?
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
What are the 4 reagent steps of a gram stain?
- primary stain
- mordant
- decolorizing agents.
- counterstain
What is the primary stain? What does it do?
Crystal Violet, it stains all cells purple
What is the mordant? What does it do?
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
What is the decolorizing agent? What does it do?
Ethyl Alcohol 95%, it serves a dual function: 1. protein dehydrating agent 2. lipid solvent. Because of this it ultimately decolarizes gram negative cells
What is the counterstain? What does it do?
Safranin, it stains pink those cell walls which have previously been decolorized
What is the most critical phase of the gram stain procedure and why?
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
Why is it important to rinse the slide with water between each step in the gram staining procedure?
Because it removes excess reagent, thereby preparing the slide for the subsequent reagent
Why is best to gram stain cultures that are less than 24 hours old?
Because as cultures age they lose their ability to retain the primary stain, causing them to appear gram-variable.
Why is gram staining clinically important? 2 reasons
- it is much more rapid than culturing a specimen
2. it allows for the correct type of antibiotic to be used
How much diluent and how much bacteria ought to be used when making a smear from an agar plate?
2 loopfuls of water and a small amount (tip of loop or needle) of bacteria
What cell component is bound by negative stain?
none
What color would a gram negative bacteria appear if stained with methylene blue?
purple
What color would a gram negative bacteria appear if stained by nigrosin?
it would not be colored at all
What color would a gram negative bacteria appear after gram-staining?
pink
