1. Cellular and Colonial Morphology Aseptic Technique, and Streak Plate
Labs 1 and 2

2. Applications of Light Microscopy
Observe less detailed features of intact cell than advanced microscopy (i.e. electron and laser)
Shape, presence of flagella, diagnostic stain, arrangement of cells, some large internal features
<1000X magnification
Brightfield
Stained cells or cells with color/contrast
External features
Phase contrast
Live transparent (unstained) cells
Some internal features
Dark-field
Live transparent cells
Greater resolution; more features; internal and external

3. Path of Light: Brightfield
Illuminator
Filter for shorter λ; blue light
Condenser
Focuses light into specimen
Diaphragm (iris)
Controls amount of light to specimen
Specimen on slide
Diffracts light as it passes throughimage produced
Objective lenses at nosepiece
Magnifies image 10, 40, or 100 times
Image inverted
Head with prism
Direct light path into ocular lens
Ocular lens magnifies image 10X
Image path sent to eye

5. Magnification Versus Resolution
Magnification = increase in apparent size
Objective and ocular lenses
Resolution = clarity
Ability to see 2 nearby objects as distinct objects
Resolving power depends on numerical aperture of lens
Ability to gather light; increase aperture and increase resolution of lens
Increase magnification must increase aperture; aperture limit
Highest resolution possible is 0.2μm with 100X lens
Steps you can take to increase resolution
GET MORE LIGHT TO LENS!
Oil immersion with 100X lens
Same refractive index as glass; prevents loss of light to air
Small wavelength light (blue light)
Adjust diaphragm and condenser as you increase magnification
Course focus and fine focus to focus image
Clean lenses and slide

6. Microscopy Field of vision Parfocal Contrast versus resolution.
Smaller at higher powers; must CENTER object or you will lose it at higher power
Mechanical stage adjuster!!!
Parfocal
Focus under low power then move to high power immediately; don’t move focus in between; then fine focus after you get to high power.
Contrast versus resolution.
you need light to increase resolution, but too much light can decrease contrast; must find a happy medium.
What happens if I go from low power to high power and the cells disappear?
Field of vision narrowed—center specimen
Not enough light—open diaphragm
Slightly out of focus—use fine focus
Resolution not optimal—clean lenses or slide, use oil if using 100X lens, open diaphragm,
The lens is not clicked all the way in place—make sure it clicks

7. Cellular Morphology Prokaryotes Eukaryotes
Rods (strep or single arrangement)
Cocci (staph, strep or single arrangement)
Spirillum
No nuclei present.
Must have cells under oil immersion to see any detail.
Eukaryotes
Cells larger than prokaryotes
Can’t always see nucleus
Fungi
Molds versus single celled fungi
Yeast
Candida albicans; vaginal yeast infection and thrush
Saccharomyces cerevisiae; bakers yeast; brewing yeast

10. Cellular Morphology Continued
More Eukaryotes
Fungi
Molds
Filamentous due to hyphae; multicellular
Penicillium
Yes it makes penicillin; grows on bread
Aspergillus,
Some produce aflotoxins (pathogenic)
Rhizopus
Classic bread mold and house mold
Yeast
Saccharomyces (bread and beer yeast) or Candida albicans (yeast infections)
Algae
Have pigments associated with them; single cell
Dinoflagellate called Peridinium
Protozoa
Single cell
Trypanosoma gambiensae
Causes Sleeping Sickness; nervous disorders; central Africa; tsetse flies
Helminth
Multi cell
Schistosoma mansonni
Causes schistosomiasis

11. Microbes in the Environment
Where do microbes live and where do they not live?
Sterile media versus culture.
Autoclave
To inoculate is to purposely grow an organism by putting it in some media, and to contaminate to to accidentally grow an unwanted organism.
Broth versus solid media
Agar
Slant versus plate media
Incubation

12. Growth Growth in brothcloudy or turbid
Pellicle, sediment, flocculant
Growth on platescolonial morphology
Colony and colonial morphology
Margin, elevation, color and colony shape
Page 52 for terms to use
Observe with dissecting scope
Dissecting scopes used to observe 3D structure of larger objects while microscopes used to see 2D surface of smaller objects.

13. Describing Cultures Terms for broth growth
Terms for colonial morphology

14. Aseptic Technique Types of media Sterile Asepsis Inoculate
Deep
Slant
Broth
Sterile
Asepsis
Inoculate
Demo the technique
Describing your cultures:
Motile versus non-motile
Slant descriptions
Broth descriptions (pellicle, flocculant, sediment, turbid)
Can you tell if a culture is contaminated? How?

15. Streak Plate Streak for isolated colonies Pure culture
All cells came from same parental cell
Genetic information is identical for most part
Isolated colony
Streak plate used to get isolated colonies  pure culture

16. Streak Plate