Staining Flashcards
The technique of using pigments or dyes (stain) to better visualize or enhance the details of a cell when viewed under a microscope.
STAINING
WHY DO STAINING?
Better contrast between the cells and the background
Confirm the presence (absence) of some cell structures
Differentiate viable from non-viable cells
- Positively charged
- Simple staining
- Cells are stained
Basic Dyes
examples of basic dyes
Crystal Violet, Safranin, Malachite green, Methylene blue
- negatively charged
- Negative staining
- background is stained
Acidic Dyes
examples of acidic dyes
Nigrosin, Rose bengal, Picric acid
Preparatory Steps
A small amount of culture spread thinly on a glass slide typically used for microscopic examination
Heat-Fixing kills the cells, preserves the specimen and allows the smear to firmly adhere to the slide
Hans Christian Gram
Based on the thickness of the peptidoglycan layer of the bacterial cell wall
GRAM STAINING
GRAM STAINING stapes and time
crystal violent - 1 minute
wash
iodine or mordant 1 minute
wash
alcohol wash - 10 seconds
wash
safranin or fuchsin - 1 minute
wash
traps the Crystal Violet-Iodine complex and is not easily washed off by the decolorizer
Thicker peptidoglycan
cannot retain CV-I complex and is easily washed off.
Thinner peptidoglycan
utilize acidic dyes e.g. nigrosin and india ink
negative charges of the dye and the cell repel each other
● Results in clear cells with dark background
NEGATIVE STAINING
CAPSULE STAINING steps
- Mix a loopful of Bacillus subtilis grown in nutrient agar with 20% sucrose with a small drop of India ink on a clean glass slide.
- The ink suspension of bacteria is spread over the slide by dragging a spreader slide. Allow air drying of the suspension at room temperature.
- Gently heat dry the slide to fix the organisms to the slide.
- Stain the smear with crystal violet for one minute.
- Wash off the crystal violet stain.
- Blot dry with clean tissue paper and examine under oil immersion objective.
Combine simple staining and negative staining
The capsule is not stained, the field and cells are stained instead
CAPSULE STAINING
are non-ionic, acid or basic dyes will not adhere to it
Capsules
