2 Microscopy and Staining 1

MCB 1
MICROSCOPY

Zaccharias Janssen
Robert Hooke
Anton van Leeuwenhoek
Joseph Jackson Lister developed better microscopes
1940 electron microscope

units of measurement
1 micrometer (m) = 10
-6
m
1 nanometer (nm) = 10
-9
m

TYPES OF MICROSCOPES:

1. LIGHT microscopes light waves and mirrors
a. Simple
Short focal length
Only 1 lens
Magnification ~ 300x
b. compound or complex
2 sets of lenses
magnification ~ 1000x

2. ELECTRON microscopes electron beams and magnetic fields; in vacuum
for examining objects smaller than 0.2 m in diameter

Features of a good microscope
adequate magnifying power
provide good contrast
possess high resolving power
serves your purpose

Types of Light microscopes:

1. Bright Field
microscopic field is brightly lit; objects under study are darker
gross morphology

2. Dark Field
background is black; object bright or luminous
for specimens that are
invisible in the ordinary light microscope
cannot be stained by standard methods
distorted by staining

3. UV
make use of shorter wavelength of light (180nm-400nm)
images are made visible by recording on a photographic emulsion or by displaying on
a TV screen
detection of substances (e.g. DNA)
2 Microscopy and Staining 2
MCB 1

4. Fluorescence
modification of UV microscope
makes use of fluorochromes (fluorescent dyes)
detection of immunological reactions antigen-antibody reaction

5. Phase Contrast
detailed examination of internal structure
not necessary to fix or stain cells
principle is based on variations in the refractive index

6. Differential Interference Contrast
principle is based on variations in the refractive indices
advantage: no diffraction halo associated with phase contrast
disadvantage: the three-dimensional appearance may not represent reality

Types of Electron microscopes:

A. Transmission Electron Microscope (TEM)
Examine viruses
Internal ultrastructure in thin sections of cells

B. Scanning Electron Microscope (SEM)
Surface features of cells and viruses
Lacks resolution of TEM but reveals a three- dimensional image

special EM stains: osmic acid, permanganate, lead, uranium, lanthanum

EXAMINATION OF MICROORGANISMS

A. Living or natural state
Advantages:
Observation of unaltered/undistorted characteristics of the organism
Cellular processes can be studied
Motility can be observed
Simple to prepare
Disadvantage:
The refractive index of the cell is almost similar to that of water
Two techniques used:
Wet mount technique
Hanging drop technique
B. Stained Preparations
Advantages:
Provides contrast
Slides can be preserved
Specimens are killed.
Disadvantages:
More complicated and tedious to prepare
More expensive
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MCB 1
3 Basic Steps in Staining Microorganisms:

1. Smear preparation
smear a thin dry film of microorganisms

2. Fixation
Heat fixation: Direct flame
Steam fixation
Chemical fixation: Alcohols

Purpose of fixation:
1. kills the cells
2. makes the cells sticky so they adhere to the slide
3. increases apparent diameter of cells

3. Staining application of biological dyes

Dye (stains) organic compound carrying chromophoric ions

Types of Stains: basic or positively charged dye
acidic or negatively charged dye
neutral

Mechanisms of staining
physical: absorption, adsorption; osmosis; capillary action
chemical: ion-exchange

Staining Procedures

SIMPLE STAINING only one dye

1. Positive/direct cells same color as dye
Methylene blue
Crystal violet

2. Negative/indirect cells colorless or luminous
India ink
nigrosin

DIFFERENTIAL STAINING 2 or more dyes and/or reagents

Gram Staining Gram positive (blue/violet)
Gram Negative (red/pink)
Acid fast Staining diagnosis of tuberculosis


STRUCTURAL STAINING 2 or more dyes and/or reagents
Endospore; Flagella; Capsule; Storage granules