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Cellular and Colonial Morphology Aseptic Technique, and Streak Plate

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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

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