Chapter 2 Microbiology

Microscopy

Define “refraction”

Refraction is the bending of light, caused by the slowing of its speed as it passes through a material.

What is the refractive index?

It is the measure of how greatly a substance slows the velocity of light.

What is the term for the place where light rays from a light source are concentrated in a microscope?

The focal point

What is the term for the distance between the centre of the lens and focal point?

The focal length

How does focal length relate to lens strength?

The shorter the focal length, the more magnified an object is and thus the stronger the lens.

What are the 8 types of microscopes?

1. Bright-field microscope

2. Dark-field microscope

3. Phase contrast microscope

4. fluorescence microscope

5. Scanning electron microscope

6. Transmission electron microscope

7. Confocal microscope

What does it mean when a microscope is parfocal?

It means the image must stay in focus as the lenses are changed/moved.

How do you calculate the total magnification?

It is the objective lens multiplied by the ocular

What is resolution definition?

The ability of a lens to distinguish between subjects right next to each other. Dependent on the numerical aperture of the lens.

How do you calculate numerical aperture?

nsinx

n = refractive index of the medium

x = ½ the angle of the cone of light entering the object → the larger the x value = the better the resolution.

What is the working distance?

The distance between the objective lens and the cover glass

What is the oil immersion objective?

Oil allows many light rays that could not enter the objective in air alone, to do so. Oil allows light beams to bend towards the objective lens → thus increasing the numerical aperture and resolution.

Describe Bright-field microscopy

It is a method of microscopy that produces a dark image on a bright background, relying on staining or light absorption for contrast.

Describe dark-field microscopy

The specimen is placed under the condenser, so only light reflected and refracted by the specimen forms an image of a brightly illuminated specimen on a dark background.

Commonly used for unstained specimens, visualisation of live bacteria, distinguishing of structures.

Describe phase-contrast microscopy

Deviated light (through specimen) and undeviated light (surroundings) allow small differences in the refractive index of cells to make them more visible. Used for live cell imaging and observation of unstained fungi, bacteria and protozoa.

Describe differential interference contrast

Two beams of light at right angles are generated by prisms. They are combined after passing through the specimen. Detects differences in refractive index and thickness of the specimen → giving rise to a 3D and brightly coloured image of the live, unstained specimen.

Describe fluorescence microscopy

Image is visible due to fluorescent light emitted by specimen, fluorchromes used as a stain. Visually enhances 3D features at small scales.

Describe a transmission electron microscope

Forms an image from radiation passed through the specimen. Gives high resolution visualisation of cellular organelles, viruses, tissue sections and bacterial structures.

Describe a scanning electron microscope

Produces an image from electrons released from atoms on the objects surface.

Describe confocal microscopy

Uses laser beam to illuminate specimen - fuorescent stain (fluorochrome). Produces a much sharper image that is ideal for studying biofilms.