2.1- Microscopy

Question 1

How does a light microscope work?

Answer 1

A compound light microscope has two lenses:

• Objective lens - placed near the specimen
• Eyepiece lens – through which the specimen is viewed

The objective lens produces a magnified image which is magnified again by the eyepiece lens.
Illumination is provided by a light under the sample.

Question 2

What are the types of sample preparation?

Answer 2

• Dry mount
• Wet mount
• Squash slides
• Smear slides

Question 3

Explain dry mount and wet mount sample preparation.

Answer 3

Dry mount- solid specimens are viewed whole or cut into very thin slices with a sharp blade (sectioning). A cover slip is placed over the sample.
(Eg. Hair, pollen, muscle tissue)

Wet mount- specimens are suspended in a liquid (water or immersion oil). Cover slip is places from an angle. Used for aquatic samples.

Question 4

Explain squash slides and smear slides sample preparation.

Answer 4

Squash slides- a wet mount is prepared, then a lens tissue is used to gently press down the cover slip. Good for soft samples.

Smear slides- the edge of a slide is used to smear the sample, creating a thin coating on another slide. A cover slip is placed on top. (Cells in blood)

Question 5

Why is staining used?

Answer 5

In light microscopy, the sample is illuminated from below with a white light:

• image has low contrasts as cells don’t absorb much light.
• resolution is limited due to wavelength and diffraction of light.
• cytosol and other cell structures are often transparent.

Stains increase the contrast as different components within a cell take up stains to different levels. It allows different components to become more visible and identifyable.

Question 6

How do you prepare a sample to stain?

Answer 6

1. Place on a slide and air dry.

2. Pass it through a flame to heat fix.

Question 7

What is differential staining?

Answer 7

Can distinguish between two types of organisms that would otherwise be hard to identify.
or
Differentiate between organelles of a single organism:

• gram stain technique.
• acid-fast technique.

Question 8

Explain the gram stain technique.

Answer 8

• used to separate bacteria into Gram-positive and Gram-negative.

1. Apply crystal violet to specimen, then iodine, which fixes dye.
2. Wash the slide with alcohol.

• Gram positive bacteria = retain crystal violet stain and appear blue/purple.
• Gram negative bacteria= lose stain due to thinner cell walls. Counterstained with safranin dye and will now appear red.

Gram positive bacteria are susceptible to the antibiotic pencillin, which inhibits the formation of cell walls.

Question 9

Explain the acid-fast technique.

Answer 9

-Used to differentiate species of mycobacterium from other bacteria.

1. Lipid solvent carries carbolfuchsin dye into sample cells.
2. Cells are washed with a dilute acid-alcohol solution

Mycobacterium- retain carbolfuchsin stain = red
Other bacteria- exposed to methylene blue = blue.

Question 10

What are the stages in the production of pre-prepared slides?

Answer 10

1. Fixing- chemicals are used to preserve specimens in as near natural state as possible.
2. Sectioning- specimens are dehydrated with alcohols and placed in mould with wax/resin. Can be sliced thinly.
3. Staining- treated with multiple stains.
4. Mounting- secured to slide with a cover slip on top.