Microscopy Flashcards by Mark James Mateo

use of a microscope to magnify objects too small
to be visualized with the naked eye so that their
characteristics are readily observable

MICROSCOPY

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APPLICATIONS OF MICROSCOPY

Rapid preliminary organism identification
Rapid final identification of certain organisms
Detection of different organisms present in the same specimen
Detection of organisms not easily cultivated in the laboratory
Evaluation of patient specimens for the presence of cells indicative of inflammation or contamination
Determination of an organism’s clinical significance
Provide pre-culture information
Determine which tests and methods should be used for identification and characterization of
cultivated organisms
Provide a method for investigating unusual or
unexpected laboratory test results

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TYPES OF MICROSCOPY

BRIGHT-FIELD (LIGHT) MICROSCOPY
PHASE-CONTRAST MICROSCOPY
FLUORESCENT MICROSCOPY
DARK-FIELD MICROSCOPY
ELECTRON MICROSCOPY

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visible light is passed through the specimen and then through a series of lenses that bend the light in a manner that results in magnification of the organisms present in
the specimen

PRINCIPLES BRIGHT-FIELD (LIGHT) MICROSCOPY

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PRINCIPLES OF LIGHT MICROSCOPY

A. MAGNIFICATION
B. RESOLUTION
B.1 RESOLVING POWER
C. CONTRAST

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→extent to which detail in the magnified object is maintained

RESOLUTION

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→ability of the lenses to distinguish fine detail and structure

RESOLUTION

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→determined by numerical aperture and wavelength of light

RESOLUTION

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→closest distance between two objects that
when magnified still allows the two objects to be distinguished from each other

RESOLVING POWER

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FACTORS AFFECTING RESOLVING POWER

IMMERSION OIL

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(TRUE OF FALSE)
Shorter the wavelength of light used in the instrument, the greater the resolution

TRUE

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specific optical and viscosity characteristics
designed for use in microscopy

IMMERSION OIL

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→used to fill the space between the objective lens and the glass slide onto which the specimen has been affixed

IMMERSION OIL

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→enhances resolution by preventing light rays from dispersing and changing wavelength after passing through the specimen

IMMERSION OIL

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measure of the light-bending ability of
a medium

Refractive Index

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required for optimal detection and
characterization of bacteria

1000× magnification

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needed to make objects stand out from the background

CONTRAST

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achieved by staining techniques that highlight organisms and
allow them to be differentiated from one another and from
background material and debris

CONTRAST

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Kohler Illumination

designed to provide maximum illumination and resolution when observing images using a microscope

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detailed examination of internal structures in living microorganisms

PHASE-CONTRAST MICROSCOPY

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not necessary to fix or stain the
specimen

PHASE-CONTRAST MICROSCOPY

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based on the wave nature of light ray

PRINCIPLE OF PHASE-CONTRAST MICROSCOPY

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light rays can be in phase (their peaks and valleys
match) or out of phase

PRINCIPLE OF PHASE-CONTRAST MICROSCOPY

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wave peak of light rays from one source coincides with the wave peak of
light rays from another source

Reinforcement (relative brightness)

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wave peak from one light source coincides with the wave trough from another light source

Interference (relative darkness)

Set of light rays

a. direct from light source b. reflected or diffracted from a particular structure in the specimen

scattering of light rays as they touch a specimen's edge

Diffraction

Two sets of light rays are brought together, form an image of the specimen on the ocular lens, containing areas that are relatively light and through shades of _______

gray, to black

PRINCIPLE OF FLUORESCENT MICROSCOPY

Fluors or Fluorochromes

→raised to a higher energy level after absorbing ultraviolet (excitation) light

Fluors or Fluorochromes

→return to their normal, lower energy state, they release excess energy in the form of visible (fluorescent) light

Fluors or Fluorochromes

(TRUE OR FALSE) Fluorescing objects appear brightly against a light background

(False) Fluorescing objects appear brightly against a DARK background

FILTERS IN FLUORESCENT MICROSCOPY

Excitation filter Barrier filter

passes light of the desired wavelength to excite the fluorochrome

Excitation filter

prevents the excitation wavelengths from damaging the eyes of the observer

Barrier filter

FLUORESCENT DYES

Acridine Orange Auramine Fluorescein Isothiocyanate (FITC) Calcofluor White

requires BLUE excitation light

Acridine Orange Auramine Fluorescein Isothiocyanate (FITC)

(WAVELENGTH) ✓Acridine Orange, Auramine, Fluorescein Isothiocyanate (FITC) Exciter filter: Barrier filter:

450-490 wavelength 515 wavelength

requires a VIOLET excitation light

Calcofluor White

(WAVELENGTH) Calcofluor White Exciter filter: Barrier filter:

355-425 wavelength 460 wavelength

→ direct chemical interaction between the fluorescent dye and a component of the bacterial cell

FLUOROCHROMING

Advantage of FLUOROCHROMING

enhances contrast and amplifies the observer’s ability to detect stained cells tenfold greater than light microscopy

Examples of FLUOROCHROMING

✓Acridine orange stain ✓Auramine-rhodamine stain ✓Calcofluor white stain

binds to nucleic acid

ACRIDINE ORANGE

→used to confirm the presence of bacteria in blood cultures

ACRIDINE ORANGE

→stains all nucleic acids---nonspecific

ACRIDINE ORANGE

→bright orange fluorescence

ACRIDINE ORANGE

→does not discriminate between gram-negative and gram- positive bacteria

ACRIDINE ORANGE

→used for detection of cell wall–deficient bacteria grown in culture

ACRIDINE ORANGE

→have affinity to waxy mycolic acids in the cell walls of mycobacteria

AURAMINE-RHODAMINE

→non-specifically bind to nearly all mycobacteria

AURAMINE-RHODAMINE

→appear bright yellow or orange against a greenish background

AURAMINE-RHODAMINE

→used to enhance detection of mycobacteria directly in patient specimens

AURAMINE-RHODAMINE

→initial characterization of cells grown in culture

AURAMINE-RHODAMINE

→bind in the cell walls of fungi

CALCOFLUOR WHITE

→directly detect fungi in clinical material

CALCOFLUOR WHITE

→observe subtle characteristics of fungi grown in culture

CALCOFLUOR WHITE

→visualize some parasites such as microsporidia

CALCOFLUOR WHITE

→antibodies are conjugated to a fluorescent dye

IMMUNOFLUORESCENCE

→dye-antibody conjugate detect, or “tag,” specific microbial agents

IMMUNOFLUORESCENCE

→microorganisms become readily detectable by fluorescent microscopy

IMMUNOFLUORESCENCE

→combines the amplified contrast provided by fluorescence with the specificity of antibody- antigen binding →Legionella spp., Bordetella pertussis, and Chlamydia trachomatis

IMMUNOFLUORESCENCE

Example: FITC→ intense, APPLE GREEN fluorescence →most commonly used

IMMUNOFLUORESCENCE

→involves the alternation of microscopic technique rather than the use of dyes or stains to achieve contrast

DARK-FIELD MICROSCOPY

→condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle

DARK-FIELD MICROSCOPY

→only light that hits objects will be deflected upward into the objective lens for visualization

DARK-FIELD MICROSCOPY

→other light that passes through the specimen will miss the objective, making the background a dark field

DARK-FIELD MICROSCOPY

→ used to detect SPIROCHETES

DARK-FIELD MICROSCOPY

Appear extremely bright against a black field

DARK-FIELD MICROSCOPY

uses electrons instead of light to visualize small object

ELECTRON MICROSCOPY

electrons are focused by electromagnetic fields and form an image on a fluorescent screen

ELECTRON MICROSCOPY

magnifications in excess of 100,000×

ELECTRON MICROSCOPY

ELECTRON MICROSCOPY Two General Types

1. TRANSMISSION ELECTRON MICROSCOPE (TEM) 2. SCANNING ELECTRON MICROSCOPE (SEM)

passes the electron beam through objects and allows visualization of internal structures

TRANSMISSION ELECTRON MICROSCOPE (TEM)

uses electron beams to scan the surface of objects and provides three-dimensional views of surface structures

SCANNING ELECTRON MICROSCOPE (SEM)