Chapter 2

Leeuwenhoek's Microscope

single lens, magnify 300X, was able to see bacteria

Light Microscopes

up to 1000X, first micrscope invented, resolution limit is 0.2 um

1 micrometer

10-6 meter

1 mililiter

10^-3 meter

1 nanometer

10^-9 meter

refractive index

measure of how much a substance slows down velocity of light

Focal point

focus light rays at a specific place

focal length

distance between center of lens and focal point

short focal length

more magnification

Bright-field, dark-feild, phase-contrast, fluorescence, confocal

types of light microscopes

compound microscopes

have two sets of lenses, invented by Robert Hooke

Bright-Feild Microscope

used to examine both stained and unstained, produces dark image on brighter background

Total magnification

ocular lense * objective lenses

Resolution

ability of a lens to distinguish objects from each other, rather than single larger object, clarity

shorter wavelength

greater resultion

magnification, resolution, contrasts

3 key features of a microscope

Working distance

distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus

Higher power

shorter distance

Oil Immersion Objective

to keep light from refracting, without this most light is lost

Dark-field, phase-contrast, DIC

types of microscopes used for LIVING UNSTAINED microorganisms

Dark-field Microscope

produces light image on dark background, uses opaque disk to create a silouette

Phase-contrast microscope

produces an image of a darker microbe against a lighter background, brights together TWO sets of light rays, excellent way to view INTERNAL structures

DIC

created image by detecting differences in refractive indices and thickness of different parts, used to observe cell organelles

flourescence microscopy

uses UV light, localization of specific proteins in cells

Confocal micrscopy

laser scanning creates 3D image of specimens, UV light

Electron microscopy

electrons replace the light, allows for microbial morphology to be studied in detail

TEM

for internal structures, virus

SEM

creates surface level 3D

Straining specimens

kills them, increases visibility, accentuates specific morphological features, preserves speciment for future research

Fixiation (step 1)

microorganisms are killed and attached

Heat fixation

preserves overal morphology but destroys subceluar structures

chemical fixation

protects fine celluar substructures and morphology

Basic dyes

have positive charges attract negative bacteria, stain bacteria

acid dyes

negative charge, repels bacteria, strains background

simply staining

single stain is used, used to determine size, shape, and arragnment of bacteria

Differential staining

divides organisms into groups based on properties

Gram stain

divides bacteria into two groups base don differences in cell wall structure

gram-postive

purple, thick peptidoglycan cell walls

gram-negative

pink, have thin peptidoglycan cell walls and layer of lipopolysaccharides

crystal violet

primary stain, all purple

iodine and water rinse

cells remain purple

alcohol

differntial step, gram-negative become colorless

safranin

counterstain, gram-negative become pink

acid-fast straining

useful for staining members of genus mycobacterium

microbacteria

has cell wall with lipids that prevents dyes from binding to cells

Ziehl-Neelson method

staining used concentrated phenol and carbol fuchsin to drive the stain into the cell

capsule straining

used to visualize polysaccharide capsules surrounding bacteria

negative stain

capsules may be colorless against stained background

flagella staining

used to provide information about the presence and distribution patter of flagella (are usually too fine to see)