sTAINING, MOUNTING AND PREP Flashcards
Why is it important to stain cells before viewing them?
Staining provides contrast between different structures in the sample to make them visible so they can be identified. contrast increases as different components within a cell take up stains to different degrees.
What is differential staining?
When multiple stains are used and each stains binds to a specific cell structure - staining each structure differently so structures can be easily identified.
Acetic Orcein
binds to DNA
bind chromosomes a dark red
Eosin
cytoplasm
dark red or p!nk
Iodine
starch
blue black
violet under microscope
iodine in potassium iodide solution
cellulose
yellow
Haematoxylin
RNA/DNA
purple/blue
Methylene blue
all purpose stain
stains DNA blue
What is a wet mount used to observe
living organisms and aquatic samples
explain process of a wet mount
- use a pipette to add a drop of liquid -water or immersion oil…if stain is used it is important to ensure it is not toxic to live organism
- use tweezers t carefully place the specimen
- drop coverslip at a 45 degree angle to avoid air bubbles which obstruct image.
what is a dry mount used to observe?
hair, pollen, parts of insects, sectioning of muscle tissue and plants
explain the process of a dry mount
- solid specimens are sectioned (cut into thin slices so light can pass them)
- use tweezers to place specimen in the middle of the slide
- add coverslip on top
Explain the process of a squash slide
- prepare a wet mount
- use lens tissue to gently press down on cover slip - careful not to break it. depending on material- damage to coverslip can be avoided by squashing sample between two microscopes slides
what would the squash slide technique be used for
soft samples e.g root tip squashes to look at cell division
explain the process of a blood smear
edge ion a slide is used to smear the sample and crate a thin, even coating on another slide
place cover slip ontop
example of where blood smear where be used for
e.g blood to view blood
why do images of cells tend to have low contrast
cells do not absorb a lot of light
resolution is limited by the wavelength of light and diffraction of light as it passes sample
cytosol of cell and other structures are often transparent
What is firstly done to prepare a sample for staining?
sample is first placed on slide and allowed to air dry
then this is heat-fixed by passing it though a flame. Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to breakIf you heat the slide before it is completely dry, then you end up “boiling apart” the cells. The bacterial frost is air-dried before staining to fix the bacteria on the slide so that it does not wash away with a subsequent staining procedure specimen will adhere to the microscope slide and then take up stains.
What are examples of positive dyes and how do they act?
direct,cationic,basic or positive dyes e.g methylene blue, basic fuchsin and crystal violet directly bind to and satin the negatively charged surface of bacterial cells.
What are examples of negative dyes and how do they act?
e.g nigrosin, congo red are negatively charged and are repelled by negatively charged cytosol. These dyes stay outside of cells, leaving the cell unstained, which stand out against gained background. NEGATIve StaIn tEcNiQuE
