microscopy Flashcards
sample preparation
• Dry mount - Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning. The specimen is placed on the centre of the slide and a cover slip is placed over the sample.
For example hair, pollen, dust and insect parts can be viewed whole in this way, and muscle tissue or plants can be sectioned and viewed in this way.
-wet mount-• Wet mount - Specimens are suspended in a liquid such as water or an immersion oil. A cover slip is placed on from an angle, as shown. For example, aquatic samples and other living organisms can be viewed this way.
• Squash slides - A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip. Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides. Using squash slides is a good technique for soft samples. Care needs to be taken that the cover slip is not broken when being pressed. For example, root tip squashes are used to look at cell division.
• Smear slides - The edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A cover slip is then placed over the sample. An example of a smear slide is a sample of blood. This is a good way to view the cells in the blood.
staining
Crystal violet or methylene blue are positively charged dyes, which are attracted to negatively charged materials in cytoplasm leading to staining of cell components.
Dyes such as nigrosin or Congo red are negatively charged and are repelled by the negatively charged cytosol. These dyes stay outside cells, leaving the cells unstained, which then stand out against the stained background. This is a negative stain technique.
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Differential staining can distinguish between two types of organisms that would otherwise be hard to identify. It can also differentiate between different organelles of a single organism within a tissue sample.
• Gram stain technique is used to separate bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria (Figure 5). Crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye. The slide is then washed with alcohol. The Gram-positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope. Gram-negative bacteria have thinner cell walls and therefore lose the stain. They are then stained with safranin dye, which is called a counterstain. These bacteria will then appear red. Gram-positive bacteria are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls. Gram-negative bacteria have much thinner cell walls that are not susceptible to penicillin.
• Acid-fast technique is used to differentiate species of Mycobacterium from other bacteria. A lipid solvent is used to carry carbolfuchsin dye into the cells being studied. The cells are then washed with a dilute acid-alcohol solution. Mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red. Other bacteria lose the stain and are exposed to a methylene blue stain, which is blue.
You will often look at slides that have been pre-prepared.
A number of stages are involved in the production of these slides:
• Fixing - chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible.
• Sectioning - specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly with a knife called a microtome.
• Staining - specimens are often treated with multiple stains to show different structures.
• Mounting - the specimens are then secured to a microscope slide and a cover slip placed on top.
textbook
Dry mount
• Your specimen needs to let light through it for you to be able to see it clearly under the microscope. So if you’ve got quite a thick specimen, you’ll need to take a thin slice to use on your slide.
Use tweezers to pick up your specimen and put it in the middle of a clean slide.
• Pop a cover slip (a square of thin, transparent plastic or glass) on top.
wet mount-• Start by pipetting a small drop of water onto the slide.
Then use tweezers to place your specimen on top of the water drop.
• To put the cover slip on, stand the slip upright on the slide, next to the water droplet. Then carefully tilt and lower it so it covers the specimen. Try not to get any air bubbles under there — they’ll obstruct your view of the specimen.
• Once the cover slip is in position, you can add a stain. Put a drop of stain next to one edge of the cover slip. Then put a bit of paper towel next to the opposite edge. The stain will get drawn under the slip, across the specimen.
microscope
1) Start by clipping the slide containing the specimen you want to look at onto the stage.
2)
Select the lowest-powered objective lens(the three ones) (i.e. the one that produces the lowest magnification).
3) Use the coarse adjustment knob to bring the stage up to just below the objective lens.
4) Look down the eyepiece (which contains the ocular lens).
Use the coarse adjustment knob to move the stage downwards, away from the objective lens until the image is roughly in focus.
5) Adjust the focus with the fine adjustment knob, until you get a clear image of what’s on the slide.
6) If you need to see the slide with greater magnification, swap to a higher-powered objective lens and refocus.
