Microbiology Chapter 3 - Microscopy & Metric Units

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Chapter 3 of Tortora's 10th Edition Microbiology "Observing Microorganisms Through a Microscope" For Microbiology 208 at Maple Woods with Dr. Scott Quinton

What is Resolving Power?

Ability to see fine details of a specimen, or the maximum distance between 2 points on a specimen at microscope can detect.

What are the major parts of a Microscope?

Base, Fine Focusing Knob, Coarse focusing knob, Illuminator, Diaphragm, Condenser, Stage, Objective Lenses, Arm, Body, Ocular Lens.

What is the Coarse Focusing Knob?

Moves the stage of the Microscope up and down to focus the image.

What is a Illuminator?

The light source for the Microscope.

What is the Diaphragm?

Controls the amount of light entering the condenser.

What is the Condenser?

Focuses light through the specimen.

What is the Stage?

Holds the microscope slide in position.

What are the Objective Lenses?

Primary lenses that magnify the specimen.

What is the body?

Transmits the image from the objective lens to the ocular lens using prisms.

What is the Ocular Lens?

Re-magnifies the image formed by the objective lens.

What is the magnification of the Scanning Lens?

5X.

What is the magnification of the Lower Power lens?

10x.

What is the magnification of the High and Dry lens?

40X.

What is the magnification of the Oil Immersion lens?

100X.

What is the magnification of the Ocular Lens?

10X.

What is total magnification?

Objective Lens x Ocular Lens.

What is the path of light in a microscope?

Light source, illuminator, condenser, stage, ocular lens.

What is Reflection?

Light that is lost.

What is Transmission?

Light that passes through.

1 km = _____ meters

1000 meters

What is the standard unit of measurement for length?

meters

1 dm = ____ meters

0.1 meters

1 cm = _____ meters

.01 meters

1 mm = ______ meters

.001 meters or 10x-3

1 micrometer = _____ meters

10x-6 meters
(0.000 001)

1 nanometer (nm) = ______ meters

10x-9 meters
(0.000 000 01)

1 picometer (pm) = _____ meters

10x-12 meters
(0.000 000 000 001)

If a microbe measures 10 micrometers, how long is it in nanometers?

1000 nanometers

1. Ocular lens
2. Coarse focusing knob
3. Fine focusing knob
4. Base
5. Objective Lenses
6. Stage
7. Condenser
8. Light Source / Illuminator

Label the parts of a compound microscope

Compound microscope

A microscope that uses a series of lenses and uses visible light in order to examine small specimens

Refractive Index

a measure of the light-bending ability of a medium, often changed by stains

immersion oil

material used to preserve the direction of light rays at the highest magnification, this is placed between the glass slide and the objective lens

Darkfield microscope

a microscope used to examine live microorganisms that are either invisible under ordinary light microscopes, cannot be stained by standard methods, or are distorted.

This microscope uses a darkfield condenser containing an opaque disk to block out light entering the objective lens indirectly.

Phase-contrast microscope

a microscope that allows for the detailed examination of internal structures of living organisms, without the need to stain or fix the specimen

As wavelength increases, resolution ______ (increases or decreases)

decreases

Fluorescence microscopy

a microscope technique that makes use of the ability of some substances to absorb UV light and give off visible light, producing a 2-D image of a specimen

Confocal microscopy

a technique in light microscopy used to reconstruct 3-D images in successive slices. Specimen are stained with flourochromes so they will emit light. Often used when studying cancer cells.

Electron microscope

the general type of microscope used for objects smaller than about 0.2 micrometers.

Electron microscope

in this type of microscope, a beam of electrons is used to project an image of the specimen versus visible light rays.

Transmission Electron Microscope (TEM)

a type of electron microscope which produces images of the internal structures in 2-D slices of a specimen

Scanning Electron Microscope (SEM)

a type of electron microscope which produces a 3-D image of the external structure of a specimen

Simple Stains

a stain technique used to color a sample so that it emphasizes certain structures

Differential stains

a stain technique used to distinguish between types of microbes based on their reactions with a given stains

Gram Stains

this differential stain technique uses Crystal Violet as its primary stain, Iodine as its mordant, Alcohol as its decolorant, and Safranin as its secondary stain (counterstain).

What are the steps to conducting a Gram stain?

1. Crystal Violet (primary stain)
2. Iodine (mordant)
3. Alcohol (decolorant)
4. Safranin (secondary stain)

What is the purpose of iodine in Gram stain?

acts as a mordant in Gram stains, adhering the violet to the cell walls

What is the purpose of alcohol in Gram stains?

acts as a decolorant in Gram stains, removing the violet from all but the gram positive cell walls

What is the purpose of the Safranin in Gram stains?

acts as a secondary stain so that gram negative cells can be see under microscope against a clear background

What is the consequence of skipping the primary stain (crystal violet) in the Gram stain sequence?

positive cells will not be discriminated from gram negative ones; all cells will appear pink

What is the consequence of skipping the mordant (iodine) in the Gram stain sequence?

it is likely that the crystal violet will not adhere to the peptidoglycan cell walls of gram positive cells, therefore all cells will appear pink

What is the consequence of skipping the decolorant (alcohol) in the Gram stain sequence?

the violet would remain across the slide, all cells would appear purple

What is the consequence of skipping the secondary stain (safranin) in the Gram stain sequence?

the gram negative cells would appear colorless against a clear background, they will not be seen

What is the consequence of too much of the primary stain (crystal violet) in the Gram stain sequence?

??

What is the consequence of too much of the mordant (iodine) in the Gram stain sequence?

??

in a Gram staining procedure, this is the chemical which adheres the primary stain to the cell walls of the microbes

mordant

in a Gram staining procedure, these cells appear purple

Gram positive

in a Gram staining procedure, these cells appear pink

Gram negative

Acid-fast stain

a differential stain technique that identifies bacterial that have a waxy material in their cell walls;

uses Carbolfuchsin as its primary stain, Acid-alcohol as its decolorant, Methylene blue as its secondary stain

Endospore stain

a differential stain technique that identifies spores;

uses Malachite green as its primary stain, Heat as its mordant, Safranin as its secondary stain

Explain what happens chemically to bacteria's cell walls when iodine is applied in the Gram stain procedure

crystals form inside the cytoplasm of both types of cells

Explain what happens chemically when alcohol is applied in the Gram stain procedure

alcohol dehydrates the peptidoglycan of Gram-positive cells, making it more impermeable to the crystal violet-iodine, trapping the primary stain (crystals) within the membrane

alcohol dissolves the outer membrane of Gram-negative cells and leaves holes in the thin peptidoglycan, allowing the crystal violet-iodine to easily escape across the membrane

Explain what happens chemically to acid-fact bacteria's cell walls the during acid-fast procedure

Carbolfuchsin stain penetrates the cell walls, binding to the cytoplasm of acid-fast bacteria. This resists removal by acid-alcohol.

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