Microbiology 3050 Light Microscopy

Compound light microscopes use …

visible light to illuminate specimens

types of light microscopy include …

bright field, dark field, phase contrast, and fluorescence

Light is refracted or bent when …

passing from one medium to another, done by the refractive index

In the refractive index F =

focal point, f = focal length

The shorter the focal length the …

greater the magnification

What is bright field microscopy?

a standard optical microscopy technique where white light passes directly through a specimen, producing a dark image on a bright, illuminated background

When using a Bright field microscope specimens are visualized because of …

differences in contrast (density) between the specimen and its surroundings

Total magnification =

mag. of ocular lenses x mag. of objective lenses

What is the upper limit of magnification?

1000x

What is the upper limit of resolution?

0.2 micrometers

Microscope resolution (d) is the ability to …

distinguish two objects as distinct and separate when viewed under a microscope

wavelength (lambda) of light means a shorter wavelength =

greater resolution

d =

(0.5 lambda) / (n(sintheta)), none of these change magnification only the resolution

NA or numerical aperture =

the measure of light gathering ability which includes n and sin(theta)

The higher the NA the …

higher the resolution

n stands for the …

refractive index, increasing n increases NA

What can you do to change the refractive index?

Add immersion oil

We want d or resolution to be …

as low as possible, this gives the image better clarity

sin(theta) =

the angle of the cone of light entering the lenses

Fixation preserves …

specimens and fixes them in position, organisms are usually killed and firmly attached to microscope slides

We air dry bacteria on slides before heat fixating them because …

we don’t want to boil the bacteria (let them die peacefully)

Why do we heat fix the bacteria?

This keeps bacteria in place so that we don’t wash them away when staining

Heat fixing is usually used for bacteria and …

archaeons, and chemical fixation is usually used for larger more delicate organisms

What is the first step in microscopy?

Fixation

What is the second step to Microscopy?

Staining

What does staining do?

makes internal and external structures of cells more visible by increasing contrast with background

What two features do stains have?

chromophore groups (a chemical group with conjugated double bonds which give stains their color) and the ability to bind to cells (most often with a charge, a lot of dyes are salts)

Basic stains have what charge?

positive (so they are used to bind to negatively charged structures = bacteria)

Acidic stains have what charge?

negative (they don’t bind to bacteria for this reason but they can be used to stain backgrounds instead of the cells

Simple staining uses …

one single stain, used for visualization of morphological shape and arrangement

What is differential staining?

use of two contrasting stains separated by a decolorizing agent to detect presence or absence of structures

What two differential stains are used for identification?

Gram stain (cell wall) and Acid-fast stain (mycolic acids)

What two differential stains are used for visualization of structures?

Spore stain and Capsule stain

The Gram stain technique was named after …

Christian Gram

Gram staining is the most widely used …

differential stain procedure, divides bacteria into two groups positive (one thick layer of peptidoglycan(PG) in their cell walls) and negative (two layers of membrane and one thin layer of PG)

In the Gram staining procedure crystal violet is applied …

first and it sits for 1 minute(turns it purple), then iodine solution is added for another minute which sticks the crystal violet in place in the thick layer of PG assuming there is one

After iodine sis added…

decolorize with alcohol briefly for about 20 seconds, it is possible to completely wash away the crystal violet and iodine even if its gram positive

What is the last step of the Gram stain?

counterstain with safranin for 1-2 minutes (this will turn anything not still purple pink)

Acid-fast staining is useful for …

members of the genus Mycobacterium because they have high lipid content with mycolic acid in their cell walls

Mycolic acid is very …

waxy and it makes it hard for stains to stick to it, so acid-fast staining is specific for bacteria with it like the ones that cause TB and leprosy

Endospore staining is a heating and …

double staining technique, bacterial endospore is one color and vegetative cell is a different color, we use malachite green stain

Endospores are …

resistant, long lasting structures

Capsule staining uses a …

negative stain - capsules are colorless against a stained background, we often don’t heat fix these because it could destroy the capsule

Flagella staining applies a …

mordant to increase thickness of flagella

Capsule staining gives cells that have capsules little …

halos