Micro biology Chapter 3; Observing Microrganisms

Light Microscopy

The use of any kind of
microscope that uses visible light to
observe specimens

Compound Light Microscopy:

TThe
modern compound light microscope has a
series of lenses and uses visible light as its
source of illumination.

Illuminator:

Light source

Condenser:

Has lenses that direct the light
rays through the specimen

Objective Lenses

Lenses closest to the
specimen. Further magnifies the image.
• 4X, 10X, 40X, 100X

Oular lens

Type of microscopy ;Darkfield Microscopy

Phase-Contrast Microscopy
• Super Resolution Microscopy
• Two-Photon Microscopy

Type of microscopy ;Fluorescence Microscopy

Immunofluorescence Microscopy
• ELISA

ype of microscopy ;Confocal Microscopy
• Electron Microscopy

Transmission Electron Microscopy (TEM

Type of microscopy ; Dark field microscopy

Used to examine live microrganisims that are either are invisible in the ordinary light microscope that cant be stained by standards methods or are distorted by staining.

Dark field condenser; contains opaque disk to block light that would enter the lense .

Type of microscopy ; Fluorescence microscopy

Take advantage of fluorscence the ability to absorb wave lengths of ultra violet light and giver a lower visible longer wavelength.

Type of microscopy ; Confocal Microscopy

Technique use to reconstruct 3-D images, specimines are stained with fluorchromes so they emit light .

Pinhole aperature- eliminates blurring that occurs with other microscopes

Confocal microscopes are paired with computers to help
construct the 3-D images.
• This allows scientists to obtain an image which can
then be viewed in any orientation

Type of microscopy ; Electron Microscopy

Used to examine structures of extremely small organisms including viruses a beam of electron used in place of light. Use it to examine structures too small USES electromagnetic lenses instead of glass.

Type of microscopy ; Transmission Electron Microscopy

TEM finely focused electron beams that pass through the focus electron beams pass through prepared and the beam os focused on one area.

Preparing Specimens for
Microscopy Staining; Bacterial Smear

thin layer of bacteria placed on
a glass blade and «fixed» by heat or chemicals to
ensure they remain attached to the glass, for
coloring. It can be prepared from a solid medium
or broth.

_{Simple vs Differential Staining; simple}

Simple Stain: an aqueous or alcohol solution
of a single basic dye
• Primary Purpose: to highlight the entire
microorganism so that cellular shapes
and basic structures are visible.
• May use a mordant

Stains

are salts composed of positive and negative ions, one of which is
colored and known as the chromophore.

Basic Dyes:

Cationic (positively charged) dyes

Acidic Dyes: Anionic Dyes

Ex: eosin, acid fuchsin, and nigrosin

Bacteria are

slightly negatively charged at a pH of around 7. Cationic dyes
will be attracted to the cells, while anionic dyes will be repelled. Thus, we use
these two dyes differently.

Negative Staining

Colorless bacteria against dark backgrounds is achieved
using Acidic dyes.
• Used to determine cell shape, size, and capsule presence.

The Gram Stain

1. A heat fixed smear is covered with crystal violet
(basic). The stain colors all cells and is called the
Primary Dye.

2. After 60 seconds, dH2O is used to rinse the slide,
followed by application of iodine (mordant). At
this point, both Gram-Negative and Gram-Positive
bacteria will appear purple.

3. Slide is washed with alcohol (decolorizer), which
removes the crystal violet from the cells of
Gram-negative species.

4. Alcohol is rinsed of with dH2O, and the slide is
then stained with Safranin, the counterstain, for
60 seconds.

5. The slide is washed again, blotted dry, and is now
ready for examination.

Acid fast stain

Acid-Fast stains are differential as they bind
strongly only to bacteria with waxy material in their
cell walls. This stain is specifically used to identify
bacteria in the genus Mycobacterium.

• Mycobacterium tuberculosis 🡪 causative agent
of Tuberculosis
• Mycobacterium leprae 🡪 causative agent of
Leprosy

• Carbolfuchsin (primary stain) and Methylene Blue
(counter stain) are the dyes used in this staining
procedure.
• Carbolfuchsin is more soluble in cell walls with
lipids. Non-acid-fast bacteria lack lipid component.

Special stains

are specifically used to color
parts of a microorganism. This includes:
• Capsules
• Endospores
• Flagella

Negative stains for capsules

Demonstrating the presence of a capsule is a means of
determining the organism’s virulence (the degree to
which a pathogen can cause disease).
• Capsule are difficult to stain because of their
water-soluble capsular materials which can be dislodged
during rigorous washing.
1. Bacteria are placed in a solution containing a
fine colloidal suspension of colored particles
(nigrosin or india ink). This provides a
contrasting background.
2. The cells are then stained with a simple stain.
3. Due to their composition, capsules do not
accept biological dyes, such as safranin, and
thus appear as halos surrounding each cell

Endospre Stain

Endospore: Special dormant structure
which is highly resistant to extreme
environments and is difficult to stain.
• Dyes do not penetrate the
endospore’s walls

Fagella stain

Bacterial flagella are structures of
locomotion too small to be seen with a
light microscope without staining.
• Main dye used is carbolfuchsin