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Learners should be able to demonstrate and apply their knowledge and understanding of:
HSW4 Carry out experimental and investigative activities, including appropriate risk management, in a range of contexts.
HSW5 Analyse and interpret data to provide evidence, recognising correlations and causal relationships.
Why is staining required in microscopy?
Why is staining required in microscopy?
Because cells do not absorb a lot of light, they have low contrast against their background. This means they are hard to distinguish on the slide from the background.
The cytosol (aqueous interior) of the cell is transparent and other parts of the cell are also transparent.
Stains increase the contrast of the cells. Different parts of the cells take up different stains to different degrees. The take up of stains differently by different organelles means you can distinguish these organelles within the cell.
How are cells stained?
How are cells stained?
Samples are placed on a slide and air dried.
The slide is passed through a flame to heat-fix the sample. This adheres the sample to the slide and enable the sample to take up stains.
What stains are used?
What stains are used?
Crystal violet - a positively charged dye that binds to the negatively charged cytosol of a cell.
(A) Germinal tube and blastospore of Candida albicans (crystal violet stain)
(B) Pseudohypha chlamydospore of C. albicans (crystal violet stain).
https://www.sciencedirect.com/topics/neuroscience/crystal-violet
Methylene blue - Another positively charged dye that binds to the negatively charged cytosol of a cell.
Human Cheek Cells stained with Methylene Blue under 400x magnification.
From Wikimedia Commons
Nigrosin - A negatively charged dye that is repelled by the negatively charged cytosol. The dye stays outside of the cells and the cells stay unstained.
Negative staining permits visualization of the usually transparent and unstainable capsule of many organisms, most importantly Cryptococcus neoformans.
https://universe84a.com/collection/nigrosin-preparation-3/
Congo red - A negatively charged dye that is repelled by the negatively charged cytosol. The dye stays outside of the cells and the cells stay unstained. It can also stain cell walls in plants and fungi.
Differential staining
Differential staining
Staining to differentiate between organelles in a cell.
Using Wrights stain to distinguish between leucocytes and erythrocytes in a peripheral blood smear. It is a mixture of eosin (red) and methylene blue dyes.
Wright's stain, with red blood cells taking up eosin Y, azure B (a type of methylene blue) giving nuclei a purple color, and methylene blue coloring the cytoplasm of this plasmablast (an immature plasma cell).
You can see here that Wrights stain here has allowed investigators to distinguish between the nucleoli, the rest of the nucleus and the blue cytoplasm.
Wrights stain can also be used to distinguish between different types of white blood cells.
Differential staining to distinquish between microbial organisms.
Species of bacteria can be divded into two groups. Gram positive and Gram negative.
Gram positive bacteria are surrounded by a single thick peptidoglycan cell wall. Gram negative bacteria have a much thinner peptidoglycan cell wall, but in addition they have an outer membrane containing lipopolysaccharides surrounding the cell.
Gram positive bacteria are bacteria are susceptible to penicillan. Gram negative bacteria are not susceptible to penicilla. Gram staining is used by microbiologists to distinguish between these two groups.
Gram Staining Protocol
Gram Staining Protocol
Flood crystal violet solution over fixed smear.
After 30 – 60 seconds, pour off the crystal violet solution and rinse with gentle running water.
Flood the Gram’s Iodine solution over the smear
Leave the iodine solution for 30 – 60 seconds and pour off the excess iodine and rinse with gentle running water
Shake off the excess water over the smear
Decolorize the smear by passing the decolorizing solution till the solution runs down in clear form. Alternatively, add a few drops of decolorizing solution and shake gently and rinse with distilled water after 5 seconds.
Rinse with distilled water to wash off decolorizer.
Shake off the excess water over the smear
Pour counter stain safranin over the smear
Leave for 30 – 60 seconds and wash with gentle running water
Air dry or blow-dry the smear.
Past paper questions
Past paper questions
Q1
Q1 Answer
Q2
Q2 answer
D
Q3
Q3 answer
C
Q4
Q4 Answer
Q5
Q5 Answer
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