Chapter 3

Chapter 3

Measurements 

Micrometer (um) 1um = 10^-3 mm = 10^6m one millionth

Nanometer (nm)  1nm= 10^-6 mm = 10^-9 m 1nm= 0.001um

Humans can see 0.1 mm

Bacteria: 0.1-5 um 1x10^-6m

Viruses = 10 - 1000 nm

Microbes = measured in micrometers

 Microscopes

Prokaryotic = Light + electron

Muscle Protein = electron

Light Microscope 

• 1 lens

• Like a magnifying glass

Types:

• Compound light microscopy

• Darkfield

• Phase- contrast

• Differential interference contrast

• Fluorescence 

• Confocal

Compound Light Microscope

• Total magnification= objective lens= ocular lens

• Image from an objective lens is magnified again by an ocular lens

• Resolution: Ability of the lens  to distinguish 2 points and distinguish fine detail and structure

  • Ex: resolution = 0.4nm, can distinguish between 2 points 0.4 nm apart

• Limit of resolution = 0.2 um

• Limit of magnification = ~ 1500 x

Brightfield microscope

• dark objects visible against a bright background

• Light -> condenser tense -> specimen -> objective lens-> ocular lens -> eye

Electron Microscope

• Low wavelength of e-s = high resolution

• Viruses, internal cell structure

• Use electromagnetic lenses

• Black + white images but can add color

Transmission electron microscopy

• Resolution: 0.2 nm

• Magnification: 10,000 - 10,000,000 x

• Internal structure 

Scanning electron microscopy

• Resolution: 0.5 nm

• Magnification: 1,000 - 500,000 x

• Scans surface, 3D

Staining ( Prep for light microscopy)

Staining: Coloring w/ due that emphasizes structures

Smear: Thin film of material with microbes spread over a slide

Fixing: with heat ( pass slide through flame) or Chemical ( methanol/ethanol for 1 minute)

• Kills microbes

• Increase permeability to dyes

• Preserves structure with minimal distortion

Simple Staining 

• One stain/due

• Determines the presence and morphology of bacteria

• Stool and discharge

• Cell shape and structure all bacteria

• Basic and acidic dyes

  • Methylene blue

  • Carbolfuchsin

  • Crystal violet

  • Safranin

Differential Staining 

• Distinguishes bacteria 

• 2 + dyes

• Components of the cell will determine the reaction with dyes

• Gram stain and acid-fast stain 

  • Gram Stain

  • Detects bacteria 

  • 1st step in identifying bacteria 

  • Best on young, growing bacteria 

• Gram Positive 

  • Thick peptidoglycan wells

  • Purple 

• Gram Negative

  • Thin Peptidoglycan walls

  • pink/red

  • Liposacchariddes and phospholipids in the outer membrane

1. Application or crystal violet Both stained purple

2. The application of iodine  (mordant) fix the color

3. Alcohol wash out crystal violet

4. Apply safranin 

   1. Pink- gram-negative

   2. Purple- gram-positive

Acid-Fast Stain

• Binds only to bacteria that have a waxy material in their cell walls, not decolorized by acid-alcohol

• Identifies: mycobacterium and Nocardia

M. tuberculosis

M.leprae

Special Stain

• Distinguishes parts of microbes

• Capsule stain

• Endospore stain

• Flagella stain 

Capsules (non-ionic) no net charge

• Gelatinous structure outer layer

• Made of polysaccharides, glycoprotein, or polypeptide

• Protects bacteria and gives pathogenicity 

• Highly hydrated polymers do not accept most dyes

Negative staining 

• Capsules are non-ionic

• Will not stain w/ a + or - charged stain

• The presence or absence of capsules

• Utilize negative staining technique for outline of capsule

• Negative stains are repelled by negatively charged bacterial cells, preventing cells from taking stain

• Primary Stain: Crystal Violet

• Secondary Stain: Congo Red

  • 1. Congo Red/ India Ink contrasts the background with the capsule

  • 2. Cells stained

  • The capsule appears like a halo of white

• Capsules have toxins

Endospores

• Resistant, dormant structures in cells that can’t ordinarily be stained ( until in harsh environments)

• Occurs in gram-positive bacteria

Schaeffer- Fulton Endospore Stain

• Primary Stain: Malachite Green with heat

• Decolorize cells: H2O

• Counterstain: Safranin

• Spores appear green within red/pink cells

Flagella 

• Too slender

• Carbolfuchsin (mordant)

  • Thickens appearance

• Light microscope

• Number and arrangement of flagella