Theme 2- Microscopy

Light microscope

Uses visible light to observe specimens

3 types of light microscopy

Bright field LM, Dark filed LM, Phase-contrast Microscopy

Equation for total magnification

Total magnification = objective lens * ocular lens

Resolution

The ability of the lenses to distinguish two points

Shorter wavelength of light

Provide the greater resolution

Refractive index

A measure of the light-bending ability of a medium

Immersion oil

Keeps light from bending in case air bends light and misses the small high-magnification lens

Bright field illumination

Dark objects are visible against bright background

What did the bright field illumination show

Internal structures and can use stains for specimens

Dark field illumination

Light objects are visible against a dark background and are invisible in the ordinary light microscope

Why cannot the Dark field illumination be stained

Distorted by staining

Phase-Contrast Microscopy

accentuates diffraction of the light that passes through a specimen

Diffraction

Scattering of light rays as they “touch” a specimen’s edge

What is the phase-contrast microscopy used to show

Internal structures and we don’t have to fix/kill the microorganism

Electron Microscopy

Uses electrons instead of light. The shorter wavelength of electrons gives greater resolution

What two kinds of electron microscopy

Transmission Electron Microscopy ( TEM), Scanning Electron Microscopy ( SEM)

Transmission Electron Microscopy ( TEM)

Beam of electrons passes through specimen then an electromagnetic lens to a screen/film

What specimens in TEM be stained with

Heavy-metal salts

What are the disadvantages of TEM

• ultra thin studies only

• No 3D images

• Fixed, dehydrated and viewed under a vacuum

Scanning Electron Microscopy

Beam of electrons that scans the surface of a whole specimen and secondary electrons emit from the specimen produce the image

Advantages of a SEM

No sectioning of sample, 3D images

Staining

Coloring the microbe with a dye that emphasizes certain structures

What is the process of staining

1. Thin layer of material spread over a slide ( smear)

2. Air dry

3. Fix by passing through a flame

Basic dye

The chromosphere is a cation. E.g Crystal violet, Methylene blue, malachite green, Saran in

Acidic dye

The chromosphere is an anion. E.g eosin, acid fuschin, nitros in, India ink

Simple stain

Use of a single basic dye, stain the entire microbe to show the cellular shape or basic structures

Mordant

Can be used to hold the stain or coat the specimen to enlarge it. E,g see flagella

What are the two types of differential stains we get And what are they used to distinguish

Gram stain and Acid-fast stain used to distinguish between bacteria

What are the steps of a gram stain

1. Application of crystal violet ( purple dye)

2. Adds iodine as the mordant

3. Alcohol wash ( decolorization)

4. Application of sacra in ( counterstain )

What colour of the decolourizing agent in the gram negative

Colourless

What is the end product colour of the counterstain if it is gram-positive

Purple

What is the end product colour of the counterstain if it is gram-negative

Red

What does the acid-fast stains distinguish

Differentiates bacteria into distinctive groups but only binds to bacteria that has a waxy cell wall. E.g Mycobacterium, Norcadia

Steps on an acid-fast stain

1. Application of primary stain - Carbolfuchsin

2. Application of heat as the mordant Alcohol wash

3. Application of acid alcohol as the decolorizer

4. Application of methylene Blue as the counter stain

What is the end product colour of the counterstain if it is acid-fast

Red

What is the end product colour of the counterstain if it is non-acid-fast

Blue

Negative staining

Staining the background instead of the cell

What structures don’t require fixing

Cell shape, size and capsule

Capsule/ negative stain

Cells, India ink and safranin

Endosperm stain

Malachite green, heat-dye to penetrate, wash, safranin

Flagella stain

Mordant, carbolfuschin