Presentation on theme: "Cellular and Colonial Morphology Aseptic Technique, and Streak Plate"— Presentation transcript:
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 magnificationBrightfieldStained cells or cells with color/contrastExternal featuresPhase contrastLive transparent (unstained) cellsSome internal featuresDark-fieldLive transparent cellsGreater resolution; more features; internal and external
3 Path of Light: Brightfield IlluminatorFilter for shorter λ; blue lightCondenserFocuses light into specimenDiaphragm (iris)Controls amount of light to specimenSpecimen on slideDiffracts light as it passes throughimage producedObjective lenses at nosepieceMagnifies image 10, 40, or 100 timesImage invertedHead with prismDirect light path into ocular lensOcular lens magnifies image 10XImage path sent to eye
5 Magnification Versus Resolution Magnification = increase in apparent sizeObjective and ocular lensesResolution = clarityAbility to see 2 nearby objects as distinct objectsResolving power depends on numerical aperture of lensAbility to gather light; increase aperture and increase resolution of lensIncrease magnification must increase aperture; aperture limitHighest resolution possible is 0.2μm with 100X lensSteps you can take to increase resolutionGET MORE LIGHT TO LENS!Oil immersion with 100X lensSame refractive index as glass; prevents loss of light to airSmall wavelength light (blue light)Adjust diaphragm and condenser as you increase magnificationCourse focus and fine focus to focus imageClean 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 powerMechanical stage adjuster!!!ParfocalFocus 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 specimenNot enough light—open diaphragmSlightly out of focus—use fine focusResolution 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)SpirillumNo nuclei present.Must have cells under oil immersion to see any detail.EukaryotesCells larger than prokaryotesCan’t always see nucleusFungiMolds versus single celled fungiYeastCandida albicans; vaginal yeast infection and thrushSaccharomyces cerevisiae; bakers yeast; brewing yeast
10 Cellular Morphology Continued More EukaryotesFungiMoldsFilamentous due to hyphae; multicellularPenicilliumYes it makes penicillin; grows on breadAspergillus,Some produce aflotoxins (pathogenic)RhizopusClassic bread mold and house moldYeastSaccharomyces (bread and beer yeast) or Candida albicans (yeast infections)AlgaeHave pigments associated with them; single cellDinoflagellate called PeridiniumProtozoaSingle cellTrypanosoma gambiensaeCauses Sleeping Sickness; nervous disorders; central Africa; tsetse fliesHelminthMulti cellSchistosoma mansonniCauses schistosomiasis
11 Microbes in the Environment Where do microbes live and where do they not live?Sterile media versus culture.AutoclaveTo 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 mediaAgarSlant versus plate mediaIncubation
12 Growth Growth in brothcloudy or turbid Pellicle, sediment, flocculantGrowth on platescolonial morphologyColony and colonial morphologyMargin, elevation, color and colony shapePage 52 for terms to useObserve with dissecting scopeDissecting 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 DeepSlantBrothSterileAsepsisInoculateDemo the techniqueDescribing your cultures:Motile versus non-motileSlant descriptionsBroth 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 cellGenetic information is identical for most partIsolated colonyStreak plate used to get isolated colonies pure culture