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Microscopy and Staining

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9 views35 pages

Microscopy and Staining

Uploaded by

Alfhathin
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Microscopy

Staining
MICROSCOPY
Most common method used for both the
detection and characterization of
microorganisms
Compound Microscope
Magnification of the Lens
• 1. Ocular (eyepiece) lens – 10x
• 2. Objective lenses
• A. Low power – 10x
• B. High power – 40x
• C. Oil immersion – 100x
Microscope Objective Lenses
Purposes of the Cedar Wood Oil
• Enhances the resolution of the microscope and is used to
fill the space between the objective lens and the glass
slide.
• Prevents light rays from dispersing and changing their
wavelength after passing through the samples.
Microscopic resolution or
Resolving Power
• Refers to the ability of the lenses to separate closely
distant objects.
• Determined by numerical aperture and wavelength of
light.
• Increased by decreasing the wavelength of light coming
from the illuminator.
TYPES OF MICROSCOPE
LIGHT MICROSCOPE
- Visible light passes through the specimen and then through a series of lenses
that reflect light, resulting in the magnification of the organisms that are
present in the specimen.
- To effectively visualize cells through LM, at least 10^5 cells per/millilitre of
specimen are required.
- N.B. – the measuring unit for bacteria is micrometer.
- Ocular micrometer – located within the eyepiece lens and used to determine
the sizes of microorganisms.
Notes to remember
- Refractive index – is a measure of the relative velocity at
which light passes through a material.
- Total magnification is the product of the lens that is used.
BRIGHTFIELD MICROSCOPE

• Most commonly used light microscope in clinical laboratory


• Forms a dark image against a “brighter” background.
• Can distinguish between two dots that are 0.2 um apart.
PHASE-CONTRAST MICROSCOPE

• Permits a detailed examination of internal structure in living organisms.


• It is used to identify medically significant fungi grown in culture.
• The phasic differences are seen through the microscope as degrees of
brightness.
• Staining is not part of the phase-contrast microscope so it can also be used to
examine unstained living cells.
FLUORESCENT MICROSCOPE

• It involves the excitation of fluorochrome using light


• Uses fluorochromes (dyes) to stain or attach to microorganisms.
• Utilized in the observation of chlamydiae, legionellae, mycobacteria, and fungi.
• Some examples of fluorochrome dyes are : acridine-orange, auramine and
rhodamine, calcofluor white and fluorescein isothiocyanate (FITC)
DARK FIELD MICROSCOPE

• Uses dark field condenser that blocks away light that can enter the objective
directly.
• Directs the light to hit the specimen at an oblique angle which makes all other
light that passes through the specimen miss the objective.
• Thus, the background become a dark field in which organisms appear
extremelt bright against a “darker” field.
• Used to detect spirochetes
• Used to examine unstained microorganisms suspended in liquid against a dark
background, and organisms that are invisible under ordinary light.
ELECTRON MICROSCOPE

• Uses electrons instead of light to visualize small objects.


• Uses electromagnetic fields instead of lenses to form an image on a
fluorescent screen like in television screen.
• Useful for studying the morphology of bacteria.
• Has built-in camera to capture images of the cells in black-and-white
transmission of electron micrographs.
• Advantage in using this microscope is that objects smaller than 0.2 um can be
visualized with 100,00x magnification
• Fixative used is glutaraldehyde or osmium tetroxide
ELECTRON MICROSCOPE

• Dehydrating agent used is alcohol or acetone.


• Stains used are lead citrate and uranyl acetate.
Types of Electron Microscope
• 1. Transmission electron microscope (TEM)
• ►Allows visualization of the internal structures of cells.
• ► Has the greatest resolution approximately 1,000x
higher than the light microscope
• ► Used to examine very thin specimens and
microorganisms since it can magnify a million times. With
resolving power of 0.2 nm.
EM
• 2. Scanning electron microscope (SEM)
• ► Scans the surface of the cells or specimens
• ► Specimen is positioned at the bottom of the column
with a resolving power of 200 nm.
Microscopy for Diagnostic
Microbiology
Organism Bright-Field Fluorescence Dark-Field Electron
Group
Bacteria    ‒
Fungi   ‒ ‒
Parasites   ‒ 
Viruses ‒  ‒ 
Staining
Gram stain reagents
Acid Fast Stain reagents
• Used to stain bacteria that have high lipid contents in their
cell walls.
• Stains: Carbol fuchsin, acid-alcohol and methylene blue or
malachite green.

• Principles: Primary stain binds to the mycolic acid in the


cell wall of the acid-fast bacteria, like in mycobacteria, and
is retained after decolorizing with acid alcohol. Acid-fast
bacilli (AFB) retain the primary stain and deep pink or red-
colored, while non-AFB are either blue or green colored.
Modified acid-fast staining method (Modified Kinyoun)

• • Used for the identification of intestinal coccidian


oocysts.
• Ideal for cryptosporidia and cyclospora parasites in
specimens
• • Specimen used is stool and reagents used are the same
with those of acid-fast reagents except for the
concentration of the acid alcohol (1% H2SO4).
Negative Staining
• Utilized to demonstrate the presence of diffuse capsule
surrounding some bacteria.
• Excellent technique for studying bacterial gas vacuoles
and viral morphology.
• Example: India ink or Nigrosin.

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