Ch 2.4 Staining Flashcards
What is a simple stain (positive stain)?
One dye is used to highlight structures in a specimen. All the organisms in the sample will seem the same color.
What is a negative stain?
A negative stain creates an outline of an organism on a colorful background. This type of stain is absorbed by the background and not by the cells in the specimen.
What is a capsule stain?
Capsule staining is a negative staining technique used to differentiate between bacteria and yeasts that do have a capsule and those that do not. The first step is to add a couple drops of either nigrosin or India ink to the specimen to stain the background. The cell and its capsule remain clear. Counterstaining can then be used to stain the cell while leaving the capsule a clear color. Capsules on bacteria will appear either as a halo around the cell or clear.
What is a gram stain?
Gram staining is used to tell apart bacteria with different types of cell walls. This differential staining procedure involves four steps. The first step is a crystal violet primary stain is added to a specimen smear. This stains the cells a purple or blue color. Next, a mordant, iodine, is used to make the dye less soluble so it can clump and stay on the peptidoglycan cell walls. After that, alcohol (usually ethanol) is added to decolorize and wash away the stain. Gram-positive cells usually stay purple while gram-negative cells become colorless. Lastly, safranin, a counterstain is added. The counterstain stains the decolorized gram-negative cells pink. The purple cells are gram-positive cells while the red/pink cells are gram-negative cells.
What are acid fast stains?
Acid-fast staining is a differential staining technique that helps distinguish two different types of gram-positive cells. It aids in the differentiation of gram-positive cells that do not have waxy mycolic cells in their cell walls and those that do. There are 2 methods for this type of staining, the Ziehl-Neelsen technique, and the Kinyoun technique. What contrasts the two methods is the use of heat during the primary staining process. Both techniques use carbolfuchsin as a primary stain. However, Zeihl-Neelsen uses heat to immerse the carbolfuchsin into the acid-fast cells. When the primary stain is used, waxy, acid-fast cells keep the carbolfuchsin even after a decolorizing agent is used. Next, a secondary counterstain called methylene blue is used that makes non-acid fast cells blue. Cells with waxy mycolic cells in their cell walls will stain red or pink while those who do not have those cells will stain blue.
What is an endospore stain?
Endospore staining is another differential staining technique that is used to differentiate endospores from the rest of the cell. This staining technique uses 2 stains. Malachite green (the primary stain) is added to the specimen. Heat is used to help push the stain into the endospore. Decolorization is then done by washing the cell with water. In this step, the endospore continues to retain the green stain. After that, the counterstain safranin is added to stain the cells pink. If endospores are present, they will appear green either in pink vegetative cells or separate from the pink cells. If there are no endospores present, there will only be pink vegetative cells. This type of staining technique is important for identifying Bacillus and Clostridium.
What is a flagellar stain?
Flagellar staining is used to see whether a bacterium has flagella. Finding the number and location of flagella is important when trying to classify and identify a bacterium. This staining technique begins by first using a mordant (tannic acid or potassium alum) to coat the flagella. Later, pararosaniline or basic fuchsin is used to stain the specimen. If flagella are present, they should be visible.
What needs to happen before cells can be stained?
It needs to be prepared either by wet mount or fixation. Wet mount is when a specimen is put on a slide in a drop of liquid with a coverslip placed on top. Fixation is done either by chemically treating a specimen or heat fixing. When a specimen is chemically treated, chemical agents are used to kill microorganisms to preserve their structure. To heat-fix, a slide is spread with a thin layer of a specimen and a heat source is used to momentarily heat up the slide.
