© 2004 Wadsworth – Thomson Learning Chapter 3 Methods of Studying Microorganisms

© 2004 Wadsworth – Thomson Learning

Chapter 3Chapter 3Methods of Studying Methods of Studying

MicroorganismsMicroorganisms


Properties of Light• Electromagnetic waves

– Visible light• 400-700 nm

– Longer wavelengths• Infrared rays, microwaves,

radio waves

– Shorter wavelengths• Ultraviolet rays, x-rays,

gamma rays

Figure 3.1


Properties of Light• Reflection

– Light hits an opaque object

• Rays bounce off the object

• Transmission– Rays pass through the

object– Must be clear

transparent• Glass

• Water

• Absorption– Some light does not pass

through• Certain wavelengths

can be absorbed

• Different colors result

Figure 3.2


Properties of Light• Diffraction

– Light rays bend when they pass near an opaque object

• Refraction– Bending of light

• Object of different density

• Slows down• Bending of rays

– Refractive index• Determines the speed

of light through a medium

Figure 3.3

Figure 3.4


Microscopy• Magnification

– Enlargement of image– Convex lens

• Refracts light of image

• Contrast– Absorption varies light

intensity– Specimen absorbs light

• Resolution– Distinguish between two

points– Resolving power

• Closest and yet distinguish

• Size of lens

• Wavelength of light

• Refractive index

Figure 3.5


Microscopy• Compound microscope

– Light source– Condenser

• Direct light through object

– Stage mount• Holds the specimen

– Objective lenses• Various magnifying powers

– Ocular lens• Additional magnification

• Total magnification– Objective lens X ocular

lensFigure 3.6


Wet Mount• Simple sample

preparation for microscopic viewing

• Observe living microorganisms

• Usually not stained

• May use a vital stain

Figure 3.8


Stains• Increase contrast• Require fixation of sample

– Heat fixation• Coagulates proteins and causes to stick to slide

– Chemical fixation

• Types of dyes– Acidic: safranin, acid fuchsin, crystal violet, methylene blue– Basic: eosin, basic fuchsin, congo red

• Simple stains– Make cells visible with one dye

• Differential stains– Distinguish between types of microorganisms


The Gram Stain• Primary stain

– Crystal violet

• Mordant– Iodine-sets the stain

• Decolorization– Alcohol or acetone

• Counterstain– safranin

Figure 3.9


Other Light Microscopes• Phase contrast microscopy

– Rings in objective and condenser• Increase contrast of certain parts of specimen

– Cellular movement and internal structure

• Darkfield microscopy– Light is scattered off of object

• Only light entering objectives is from specimen

– Viewing surface structures

• Nomarsky microscopy– Prisms in objective and condenser– Living organisms in animal tissues

• Fluorescence microscopy– Fluorescent material illuminated by UV light


Scanning microscopes

• Concentrating on small field of view

• Confocal microscopy– Same object viewed simultaneously from

opposite sides• Illuminating microscope

– Focused on very small area

• Receiving microscope with photodetector– Connected to computer– Computer generates image

– Three-dimensional• Multiple scans and different depths


Electron microscopes• Electrons instead of light rays• Much greater magnification• Transmission electron

microscope (TEM)– Electrons pass through specimen– Captured on photographic film– Ultra-thin specimen

• Scanning electron microscope (SEM)– Electrons hit specimen and cause

secondary electrons to eject from it– Captures the surface only

Figure 3.15b

Figure 3.18a


Sample preparation for EM• Freeze fracturing • Shadow casting

Figure3.16 Figure 3.17


Viewing atoms and molecules

• Scanned-proximity probe microscopes– Scanning tunneling microscope

• View surfaces that conduct electricity– Metals– Semi-conducting materials

– Atomic force microscope• Biologically important molecules• Attractive and repulsive forces• Diamond probe detects forces• Laser beam detects bending of beam• Resolution of 10 pm: 1/100th of nm


Culturing microorganisms

• Transfer of microorganism– Sterilize transfer loop– Dip loop into broth culture– Streak onto solid medium

Figure 3.21


Sterilization• Eliminating all microorganisms• Culture media must be sterilized• Heat sterilization

– Moist heat• Autoclave• 121oC for 20 minutes

– Dry heat• 170oC for 90 minutes

• Filtration– Membrane filters

• Chemicals

Figure 3.20


Pure culture• Streak plate method

– Streak inoculum onto one portion of the plate

– Sterilize the loop– Streak through the

first inoculum and spread into second section

– Repeat several times– Incubate– Observe isolated

coloniesFigure 3.21


Pure culture

• Pour plate method– Make serial

dilutions of bacterial suspension

– Mix diluted sample with melted agar

– Pour into plate– Incubate– Observe for isolated

coloniesFigure 3.22


Culture media• Defined media

– Produced from pure chemicals

• Complex media– Extracts of natural sources

• Beef, blood, milk, protein, yeast, soybeans• Precise composition not known

• Selective media– Contents select for specific

microorganism

• Differential media– Identification of microorganisms


Growth conditions

• Temperature– Incubators– Water baths

• pH– Growth medium at optimal pH– Buffers maintain pH over period of growth


Growth conditions• Oxygen

– Strict aerobes• Require oxygen

– Strict anaerobes• Oxygen is toxic

– Facultative anaerobes• Use oxygen when available• Can grow without oxygen

– Aerotolerant anaerobes• Can’t use oxygen but not toxic

– Microaerophilic• Need low concentrations of oxygen


Oxygen culturing conditions• Culturing

– Anaerobic chambers• All oxygen is

replaced with other gas

– Shaking machines• Increase oxygen in

the media

– Candle jars• Not anaerobic but

reduces available oxygen

Figure 3.25


Preserving cultures

• Cold storage– Short-term: refrigeration slows growth

• Must continually transfer

– Long-term: freezing• Add substance to reduce freeze-killing

– Glycerol, skim milk, dimethyl sulfoxide (DMSO)

– Lyophilization• Long term—freeze drying• Frozen and dried under vacuum

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© 2004 Wadsworth – Thomson Learning Chapter 3 Methods of Studying Microorganisms