Laboratory Techniques and Methods

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86 Terms

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Microscopy

use of light and electrons to magnify objects

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Magnification

the apparent increase in size of an object

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Thickness of the lens

Curvature of the lens

Speed of light

the image enlarged depends of the following:

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Resolution or Resolving Power

ability to distinguish objects as close as 0.2 micrometer

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0.1 mm (100um)

human eye can see objects as small as about

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shorter wavelength

greater numerical aperture (-the ability to gather light)

Better resolution are due to

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Contrast

different intensity between two objects or its background

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Par focal

ability of the microscope to stay in focus even you shift from different objectives

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Working Distance

distance between the lens and the specimen

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Total Magnification

the number of times the object is enlarge from its original size

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Ocular lens

Remagnifies the image formed by the objective lens

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Body

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

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Objective lenses

primary lenses that magnify the specimen

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Stage

holds the microscope slide in position

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Condenser

focuses light through the specimen

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Diaphgram

controls the amount of light entering the condenser

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Illuminator

light source for the microscope

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Coarse focusing knob

move the stage up and down to focus the image

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Fine focusing knob

Moves stage slowly for precise, final focusing

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Bright-Field

Dark-Field

Phase-Contrast

Differential Interference Contrast (Nomarski)

Fluorescent

Confocal

Different type of light microscopes

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Nomarski

Differential Interfence contrast mirscope is also called

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Light microscopes

Useful magnification 1x to 200x; resolution to 200 nm

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Light microscopes

Use visible light; shorter; blue wavelengths provide better resolution

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Bright-field

Colored or clear specimens against bright background

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Dark-field

Bright specimen against dark background

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Phase-constrast

Specimen has light and dark areas

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Differential interference contrast (Nomarski)

Image appears three-dimensional

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Fluorescent

brightly colored fluorescent structures against dark background

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Confocal

Single plane of structures or cells that have been specifically stained with fluorescent dyes

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Bright-field

simple to use; relatively inexpensive, stained specimens often reuqired

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Dark-field

use a special filter in the condenser that prevents light from directly passing through a specimen; only light scattered by the specimen is visible

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Phase-contrast

use a special condenser that splits a polarized light into two beams

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Phase-contrast

Use a special condenser that splits a polarized light beam into two beams, one of which passes through the specimen, and one of which bypasses the specimen; the beams are then rejoined before entering the oculars; contrast in the image results from the interactions of the two beams

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Differential interference contrast (nomarski)

Use two separate beams instead of a split beam; false color and a three-dimensional effect result from interactions of light beams and lenses; no staining required

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Fluorescent

An ultraviolet light source causes fluorescent natural chemicals or dyes to emit visible light

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Confocal

Use a laser to fluoresce only one plane of the specimen at a time

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Bright-field

Observation of killed stained specimens and naturally colored live ones

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Bright-field

also used to count microorganisms

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Dark-field

Observation of living; colorless; unstained organisms

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Phase-contrast & Differential interference contrast (nomarski)

Observation of internal structures of living microbes

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Fluorescent

Localization of specific chemicals or structures; used as an accurate and quick diagnostic tool for detection of pathogens

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Confocal

Detailed observation of structures of cells within communities

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Transmission

Scanning

Type of Electron Microscope

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Electron microscope

Typical magnification 1000x to 100,000×: resolution to 0.001 nm

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Electron microscope

Use electrons traveling as waves with short wavelengths; require specimens to be in a vacuum, so cannot be used to examine living microbes

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Transmission

Monotone, two-dimensional, highly magnified images; may be color-enhanced

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Scanning

Monotone, three-dimensional, sufrace images, may be color-enhanced

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Transmission

produce a two-dimensional image of ultrastructure of cells

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Scanning

Produce three-dimensional view of the surface of microbes and cellular structures

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Transmission

Observation of internal ultrastructural detail of cells and observation of viruses and small bacteria

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Scanning

Observation of the surface details of structures

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Probe microscopes

Magnification greater than 100,000,000x with resolving power greater than that of electron microscopes

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Scanning tunneling and Atomic force

Individual molecules and atoms visible

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Scanning tunneling

Measures the flow of electrical current between the tip of a probe and the specimen to produce an image of the surface at atomic level

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Atomic force

Measure the deflection of a laser beam aimed at the tip of a probe that travels across the surface of the specimen

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Transmission

Observation of internal ultrastructural detail of cells and observation of viruses and small bacteria

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Scanning

Observation of the surface details of structures

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Scanning Tunneling

Observation of the sruface of objectsm provide extremely fine detail, high magnificatino and great resolution

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Atomic force

Observation of living specimens at the molecular atomic levels

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Staining

artificially coloring the organism with the use of different dyes and reagents

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Simple stain

uses a signle dye

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Crystal violet

Methylene blue

exampes under simple stain

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Simple stain

reveals size, morphology, and arrangement of cells

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Crystal violet

uniform purple stain

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Methylene blue

uniform blue stain

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Differential stains

use two or more dyes to differentiate between cells or structures

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Gram stain

Ziehl-Neelsen acid-fast stain

Schaeffer-Fulton endospore stain

examples under differential stains

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Gram stain

Gram-positive cells are purple; gram-negative cells are pink

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Ziel-Neelsen acid-fast stain

Pink to red acid-fast cells and blue nonacid-fast cells

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Schaeffer-Fulton endospore stain

Green endospores and pink to red vegetative cells

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Gram stain

Differentiates between Gram-positive and Gram-negative bacteria, which is typically the first step in their identification

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Ziehl-Neelsen acid-fast stain

Distinguishes the genera Mycobacterium and Nocardia from other bacteria

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Schaeffer-Fulton endospore stain

Highlights the presence of endospores produced by species in the genera Bacillus and Clostridium

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Negative stain for capsules

Flaggeral Stain

exmaples under special stains

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Negative stain for capsules

Background is dark, cells unstained or stained with simple stain

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Flagellar stain

Bacterial flagella become visible

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Negative stain for capsules

Reveals bacterial capsules

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Flagellar stain

Allows determination of number and location of bacterial flagella

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MgRNA

GRAM (+) contains _______ which when combined to Crystal violet and gram's iodine will form an insoluble compound

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decolorized

-GRAM (-) does not contain MgRNA, thus easily __

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less; more

GRAM (+) are ____ permeable to the decolorizer

- GRAM (-) are ___permeable to the decolorizer

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lower isoelectric pH

GRAM (+) have ______, thus they are more acidic

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higher isoelectric pH

GRAM (-) have _______, thus, it does not easily combine with the basic dye (CV)

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Reasons why Gram (+) becomes Gram (-)

1. Removal of MgRNAby reacting the MQS with bile salt solution- this will precipitate the MgRNA

2. Aging, dying, and autolyzing MQS

3. Using acidic Iodine or Lugol's Iodine 4. Technical error (e.g. over decolorization)

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Neisseria

Veillonella

Moraxella (Branhamella)

1. ALL cocci are Gram(+) EXCEPT

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Mycobacteria

Corynebacteria

Bacillus

Clostridium

Lactobacillus

Listeria monocytogenes

Erysipelotrix insidios

ALL bacilli are Gram(-) EXCEPT