1. © 2004 Wadsworth – Thomson Learning Chapter 3 Methods of Studying Microorganisms

2. © 2004 Wadsworth – Thomson Learning 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

3. © 2004 Wadsworth – Thomson Learning 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

4. © 2004 Wadsworth – Thomson Learning 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

5. © 2004 Wadsworth – Thomson Learning 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

6. © 2004 Wadsworth – Thomson Learning 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 lens Figure 3.6

7. © 2004 Wadsworth – Thomson Learning Wet Mount Simple sample preparation for microscopic viewing Observe living microorganisms Usually not stained May use a vital stain Figure 3.8

8. © 2004 Wadsworth – Thomson Learning 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

9. © 2004 Wadsworth – Thomson Learning The Gram Stain Primary stain –Crystal violet Mordant –Iodine-sets the stain Decolorization –Alcohol or acetone Counterstain –safranin Figure 3.9

10. © 2004 Wadsworth – Thomson Learning 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

11. © 2004 Wadsworth – Thomson Learning 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

12. © 2004 Wadsworth – Thomson Learning 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

13. © 2004 Wadsworth – Thomson Learning Sample preparation for EM Freeze fracturingShadow casting Figure3.16 Figure 3.17

14. © 2004 Wadsworth – Thomson Learning 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: 1/100 th of nm

15. © 2004 Wadsworth – Thomson Learning Culturing microorganisms Transfer of microorganism –Sterilize transfer loop –Dip loop into broth culture –Streak onto solid medium Figure 3.21

16. © 2004 Wadsworth – Thomson Learning Sterilization Eliminating all microorganisms Culture media must be sterilized Heat sterilization –Moist heat Autoclave 121 o C for 20 minutes –Dry heat 170 o C for 90 minutes Filtration –Membrane filters Chemicals Figure 3.20

17. © 2004 Wadsworth – Thomson Learning 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 colonies Figure 3.21

18. © 2004 Wadsworth – Thomson Learning Pure culture Pour plate method –Make serial dilutions of bacterial suspension –Mix diluted sample with melted agar –Pour into plate –Incubate –Observe for isolated colonies Figure 3.22

19. © 2004 Wadsworth – Thomson Learning 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

20. © 2004 Wadsworth – Thomson Learning Growth conditions Temperature –Incubators –Water baths pH –Growth medium at optimal pH –Buffers maintain pH over period of growth

21. © 2004 Wadsworth – Thomson Learning 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

22. © 2004 Wadsworth – Thomson Learning 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

23. © 2004 Wadsworth – Thomson Learning 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