Microscopy Flashcards by Rebecca Treacy

List the SI units of length and their abbreviations.

Unit of measure is m
10-3 = 1mm
10-6 = 1 um which is micro metre
10-9 = 1nm which is nano metre

1000nm = 1um
0.001 = 1nm nanometer
Micrometer is larger than a nanometre

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Visible white light consist of light radiation between?

400 - 700nm. Also called white light. Light we see visibly.

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Define reflection.

Light hitting a specimen can be reflected and we can observe the reflected light. The immediate re-emission of light after it hits a mirrored surface E.g mirror

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Define transmission.

Light entering a special environment and which comes out the other side is transmitted. E.g. Light coming through a leaf when you hold it up the to the sun

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Define absorption.

Light entering a specimen is absorbed and does not leave. E.g. Light absorbed by sunglasses

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Define refraction.

Light is bent at an angle by a specimen so it is transmitted at a different angle relative to it entering the specimen. E.g. A pencil in a cup of water and then looks broken at waters edge

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Define luminescence.

The emission of light by a specimen. E.g. Chemical reaction in a glow stick

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Define phosphorescence.

Similar to florescence except light emission by the specimen continues after illumination (excitation) has stopped. E.g. Light still glowing after switch is turned off

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Define fluorescence.

Light, usually of shorter wavelengths, is absorbed by a specimen and the molecules then emit different wavelengths of light which are longer. E.g. White tshirt under uv light

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Going from top to bottom, list the parts of the microscope.

Ocular lens -
Body tube - transmits image from objective lens to the ocular lens
Arms
Objective lens - primary lens that magnify specimen
Stage
Condenser. - focuses light through specimen
Diaphragm - controls the amount of light entering the specimen
Illumination - light source
Course focus - inner knob
Base
Fine focus - outer knob

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How does the compound microscope work?

The image from the objective lens is magnified again by the ocular lens.
The lenses change the light path and make the original object appear upside brown and back to front

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What is total magnification and how is it calculated?

Multiply- objective lenses by the ocular lens magnification. The sum of the magnification of the specimen

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What is resolution?

(Resolving power) is the ability of the lenses to distinguish 2 points that are a specified distance apart
General principle is - shorter wavelength of light used in the instrument, the greater the resolution.

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How is resolving power/resolution calculated?

Wavelength of light/numerical aperture (NA) of the lens

You see smaller objects if the RP is lower

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What is 200nm in micrometers?

Move 3 decimal places to left.

0.2 micrometers

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Through what lens does light pass in a compound microscope?

Objective lens and ocular lens

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What does it mean when a microscope has a resolution of 0.2nm?

It can resolve objects that are 0.2nm apart

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What is the function of the oil in oil immersion microscopy?

Reduces the light refractive as it is the same refractive index as glass.
Oil has the same effect as increasing the objective lens diameter - improves the resolving power of the lens.

Describe transmission electron microscopy

Ultra thin section of specimens
Light passes through the specimen, then an electromagnetic lens, to a screen film.
Specimens may be stained with heavy metal salts, so that an image is seen
Beams of electrons are used instead of light
Electron beam wavelength 0.005nm

Describe scanning electron microscopy

Specimen is dried and coated with heavy metal ions
Beam of electrons scans SURFACE of specimen
Heavy metal ions refract electrons
Requires a vacuum so the subject is dead

Why do electron microscopes have greater resolution than than light microscopes?

They usually use electro magnetic radiation of smaller wavelengths.
Smaller wavelengths = better resolution

How does TEM differ from SEM?

TEM - looks at an object through a specimen

SEM - surface structures, light refracted off the specimen via heavy metal ions

What is staining?

Colouring the microbe with a dye that emphasises certain structures. Can allow minute structures to be detected

What is a smear?

A thin film of a solution of microbes on a slide. Usually fixed to attach the microbes to the slide and to kill the microbes

What are the 2 types of dyes?

Acidic dyes and basic dyes

What are the 2 types of staining?

Positive staining and negative staining

Describe acidic dyes.

Anionic, with negative charges on the chromophore. Anions bind Acidic dyes not attracted to most bacteria as they have a slight neg charge

Describe basic dyes.

Cationic, with positive charges on the chromophore, cations bind. Bind to neg charges on the specimen.

Describe positive staining.

Surfaces of microbes are negatively charged and attract basic dyes

Describe negative staining.

Microbe repels dye, the dye stains the background

What is a simple dye?

Single dye such as methlyene blue, saffranin, crystal violet. Highlights the entire microorganism

What are differential dyes and how do they work?

Two or more dyes. Stain different organisms, different structures of one organism. Gram stain and acid-fast stain Acid fast important for 2 important pathogens - mycobacterium leprae and mycobacterium tuberculosis Also used for pathogenic strain of nocardia

What are special stains?

Stains for particular structures including endospores, capsules and flagella

What are the 4 gram stain results?

Gram positive - stained blue with crystal violet Gram negative - stained red with saffranin Gram non-reactive - stained poorly with gram stain and counter stain Gram variable - can have blue and red cells in population

List the steps in preparing a gram stain.

1. Crystal violet used, primary stain. Then washed 2. Iodine wash, then washed 3. Wash with alcohol, decolourizing agent. Which may remove purple cells 4. alcohol rinsed then red dye (saffranin) used. Counter stain

How does the acid-fast stain work and what is it composed of?

The stain uses carbolfuchsin red. Cells are washed with a hot acid. Counter stain with methylene blue Those retaining red stain of carbolfuchsin are acid fast. Stains lipid components of cell wall

Describe endospore staining?

Primary stain - malachite green, usually with heat. Decolourizing cells - water Counter stain - saffranin

Describe negative staining for capsules.

Stains surface around the cells Usually do not accept most dyes thus appear as a halo surrounding each stained bacterial cell wall Pos cell wall Neg surrounding area

Describe flagella staining?

Mordant on flagella | Carbolfuchsin simple stain

Which stain would be used to identify microbes in the genera mycobacterium and nocardia?

Acid fast

How do unstained endospores appear?

Gram stains, remain unstained against the gram

How do stained endospores appear?

Malachite green

What is penicillins affect on bacterial cells?

Gram positive bacteria tend to be killed easily by it, gram negative are usually resistant because the penicillin cannot penetrate the lipopolysaccharide wall, which is good because gram neg is toxic to human.