light refraction - Light bends as it moves from glass to air - oil bridges the gap between the specimen slide and lens and reduces refraction

Chapter 3 Flashcards by Carson Gafford

Most important tool for studying microorganisms

Microscope

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Use visible light to observe objects

Magnify images approximately 1,000x

Microscope

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can magnify images in excess of 100,000x

Electron microscope,

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can view individual atoms

Atomic-‐force microscope

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Light passes through specimen, then through series of magnifying lenses

Light Microscopy

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Most common and easiest to use

bright-field microscope

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Magnification
Resolution
Contrast

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the ability of a microscope to determine two closely related objects as separate objects

resolution

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maximum resolving power of most bright field microscopes

2 um
- This is sufIicient to see most bacterial structures
- Too low a resolution to see viruses

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Microscope has two magnifying lenses

compound microscope

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two lenses on compound microscope

oscular and objective lens

MagniIication is equal to the factor of the ocular x the objective
10x X 100x = 1,000

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Uses light condenser to concentrate light on sample

BrightIield Microscope

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deIined by the minimum distance between two objects where those objects appear as separate objects

Resolving power

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depends on the quality of lenses and wavelength of illuminating light

Resolution

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The first to observe bacteria, achieved a reolution of about 1.4 um

AvL

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Resolution is enhanced with lenses of higher magniIication (100x) by the use of

immersion oil

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The oil reduces

light refraction

Light bends as it moves from glass to air
oil bridges the gap between the specimen slide and lens and reduces refraction

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immersion oil has nearly same refractive index as

glass

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Reﬂects the number of visible shades in a specimen

contrast

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higher contrast for microscope is achieved through

specimen staining

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Ampliﬁes diﬀerences between refractive indexes of cells and surrounding medium

Phase-Contrast

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Uses set of rings and diaphragms to achieve resolu:on

Phase-‐Contrast

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reflects number of visible shades, this is great except it kills some of the cells

Phase-Contrast

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Causes specimen to appear three dimensional

Interference Scope

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Most frequently used interference scope is

Nomarski diﬀeren:al interference contrast

Used to observe organisms that are naturally ﬂuorescent or are ﬂagged with ﬂuorescent dye

Fluorescence Microscope

absorbs ultraviolet light and emits visible light

Fluorescent molecule | -Image ﬂuoresces on dark background

Computer used to re-construct three dimensional image of thicker structures

Confocal Scanning Laser Microscope

Provides detailed sectonal views of internal structures of an intact organism

Confocal Scanning Laser Microscope

Laser sends beam through sectons of organism and then Computer constructs 3-‐D image from sec:ons

Confocal Scanning Laser Microscope

Reverse image | -Specimen appears bright on a dark background

Dark-‐Field Microscope

“Like a photographic negative”

Dark-‐Field Microscope

Achieves image through a modiﬁed condenser

Dark-‐Field Microscope

- Used to observe ﬁne detail - Directs beam of electrons at specimen - Electrons pass through or sca7er at surface

Transmission Electron Microscope (TEM)

Shows dark and light areas, darker areas more dense

Transmission Electron Microscope (TEM)

specimen preparation for TEM

- thin sectioning - freeze fracturing or freeze etching - can often introduce artifacts

- Used to observe surface detail | - Beam of electrons scan surface of specimen

Scanning Electron Microscope (SEM)

Specimen coated with metal, usually gold

Scanning Electron Microscope (SEM)

- Electrons are released and reﬂected into viewing chamber | - Some atomic microscopes capable of seeing single atoms

Scanning Electron Microscope (SEM)

set out to examine the details of rabies virus

Eran Perlson- set up a system to grow asymmetric nerve cells in observation chamber and use live cell imaging to track hoe rabies virus particles are transported along the axons.

Most common strategy to change contrast and observe organisms

Dyes and Staining

using stains are made of

organic salts

carry (+) or (-‐) charge on the molecule | Divided into basic or acidic based on charge

dyes

carry positive charge and bond to cell structures that carry negative charge

basic dyes | -commonly stain the cells

carry negative charge and are repelled by cell structures that carry negative charge

Acidic dyes | -commonly stain the background

- uses one stain - Allows for increased contrast between cell and background - All cells stained the same color - No differentiation between cell types

Simple stain | ex/ hematopoietic cell smear

-Used to distinguish one cell type from another

DiﬀerenAal Stains

Two most common bacterial diﬀerenAal stains

gram stain | acid fast stain

- Most widely used procedure for staining bacteria | - Dr. Hans Christian Gram

gram stain

Stained purple/blue

gram positive

stained red or pink

gram negative

primary stain in gram stain

crystal violet

mordant in gram stain

grams iodine | -fixes primary dye in cell

decolorizer in gram stain

usually alcohol | -removes primary dye from gram negative cell

counter or secondary stain in gram stain

safranin | -recolors cells that lose stain through decolorization

Used to stain organisms that resist conventional staining

Acid-‐fast Stain

Acid fast staining is particularly useful in staining members of genus..

Mycobacterium

High lipid concentraAon in cell wall prevents uptake of dye, ‘waxy’ mycolic acid

Acid-‐fast Stain

- Use heat to facilitate staining | - Once stained diﬃcult to decolorize

Acid-‐fast Stain

Presumptive identiﬁcation in diagnosis of clinical specimens

Acid-‐fast Stain

primary dye acid fast

- carbol fuchsin | - colors acid fast bacteria red

decolorizer acid fast

- generally acid alcohol | - removes stains from non acid fast bacteria

counter stain acid fast

- methylene blue | - colors non acid fast bacteria blue

- Example of negative stain | - Allows capsule to stand out around organism

Capsule stain

can appear as clear region in Gram or simple stains

Endospore stain

uses gentle heat to facilitate staining

Endospore stain

Staining increases diameter of ﬂagella | Makes visible with light microscope resolution

Flagella stain

arrangement of prokaryotic cells depends on

plan of division

architecture can specify division planes in Staphylococcus aureus.

Peptidoglycan

- static in location - Extrapolymeric substance (EPS) - dynamic community structure helps recruitment of nutrients and survival in harsh conditions

biofilm

rigidity of bacterial cell wall is due to..

peptidoglycan- ONLY FOUND IN BACTERIA