Microscopy Flashcards by Cza S.

Microscopic diagnosis for bacteria

Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: +/- [spirochetes (borrelia, leptospira, treponema)]
Electron M: -

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Microscopic Diagnosis for Fungi

Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: -
Electron M: +/-

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Microscopic diagnosis for Parasites

Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: -
Electron M: +/-

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Microscopic diagnosis for viruses

Bright-field M: -
Fluorescence M: +/-
Phase-contrast M: -
Dark-field M: -
Electron M: +/-

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Used for stained and unstained samples

BRIGHT FIELD MICROSCOPE

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Most common methods employed under bright field microscope

o Gram Stain
o Acid Fast Stain
o Potassium Hydroxide

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

Kohler Illumination

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Scanning power

Objective lens magnification:
Ocular lens magnification:
Total Magnification:

Objective lens magnification: 4x
Ocular lens magnification: 10x
Total magnification: 40x

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Low power objective lens

Objective lens magnification:
Ocular lens magnification:
Total magnification:

O bjective lens magnification: 10x
Ocular lens magnification: 10x
Total magnification: 100x

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High-dry power objective lens

Objective lens magnification:
Ocular lens magnification:
Total magnification:

Objective lens magnification: 40x
Ocular lens magnification: 10x
Total magnification: 400x

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Oil immwersion power objective lens

Objective lens magnification:
Ocular lens magnification:
Total magnification:

Objective lens magnification: 100x
Ocular lens magnification: 10x
Total magnification: 1000x

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Observing microbial pathogens

Tools: eyes
Magnification: 0
Application:

Gross examination

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Observing microbial pathogens

Tools: Magnifying glass
Magnification: 5
Application:

Gross examination

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Observing microbial pathogens

Tools: dissecting microscope
Magnification: 2.5-30
Application:

Gross detailed examination & manipulation

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Observing microbial pathogens

Tools: bright-field microscope
Magnification: 10-2000
Application:

Cells stained

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Observing microbial pathogens

Tools: dark-field microscope
Magnification: 10-400
Application:

Cells not readily stained for bright-field microscopy

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Observing microbial pathogens

Tools: phase-contrast microscope
Magnification: 10-400
Application:

Living or unstained cells

Observing microbial pathogens

Tools: fluorescence microscope
Magnification: 10-400
Application:

Preparations using fluorochrome stains, which can directly stain cells or be connected to antibodies that attach to cells

Observing microbial pathogens

Tools: transmission electron microscope
Magnification: 150-10Million
Application:

Determine ultra structure of cell organelles

Observing microbial pathogens

Tools: scanning electron microscope
Magnification: 20-10,000
Application:

Determine surface shapes and structures

• Motility of Spirochetes
• Also used for fluorescent stains – Substitute
• Higher resolving power than bright field

DARK FIELD MICROSCOPE

• Inclusion bodies seen on virus and Chlamydia, living cells/natural state
• Micro lymphocytotoxicity test for Human leukocyte antigen (inverted PCM)

PHASE-CONTRAST MICROSCOPE

• Requires fluorescent stain
• UVL – provided by mercury are lamp
• Substitute: Dark-field microscopy

FLUORESCENT MICROSCOPE

Stain cell wall of fungi (Chitin)

Calcofluor White

Stain high affinity with nucleic acid

Acridine Orange

Truant’s method; High affinity with mycolic acid (Mycobacterium spp.)

Auramine Rhodamine

For immunofluorescent techniques; 3 bacteria (Bordetella, Chlamydia, Legionella)

Fluorescein isothiocyanate (FITC)

• Viral morphology • Resolution: 0.5 nm • Fixatives: Glutaraldehyde and Osmium tetroxide • Dehydrating agents: Alcohol • Stain: Heavy metals (Lead citrate, uranyl acetate)

ELECTRON MICROSCOPE

• Transmission EM – _________ structure • Scanning EM – ____________ structure

• Transmission EM – Internal structure • Scanning EM – External structure

This is useful to obtain high resolution images and for three-dimensional reconstruction of biological models

Confocal Microscopy

This measures surface features by moving a sharp probe over the object’s surface.

Scanning Probe Microscopy

two types of scanning probe microscope

Scanning tunneling microscope Atomic force microscope

Light microscope source

Visible light

Electron microscope source

Expensive

Light microscope vs electron microcope Medium of transmission

Air - High vacuum

Light microscope vs electron microcope Nature of Lens

Glass - Electromagnet

Light microscope vs electron microcope Focusing mechanism

Lens position is adjusted mechanically Current to the magnetic lens

Light M. vs. Electron M. Source of Contrast

Differential light absorption Scattering of electrons

Light M. vs. Electron M. Specimen mount

Glass slide Metal grid

Best resolution Light M. Electron M.

0.2um 0.5nm

Highest practical magnification Light M. Electron M.

1000 – 1500 Over 100,000

Affordability Light M. Electron M.

Cheaper Expensive