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Chapter 3 – Observing microorganisms through a microscope Units of Measurement • Microbes are really small. Really, really small • Requires small units of measurement • ______________________ allows for really small to really large measurements • Units are based on factors of 10 o 1 m = 10 decimeter (dm) = 100 centimeter (cm) = 1,000,000 micrometer (um) • US system does not convert easily o What’s smaller than an inch? o How big is 0.0023 inches? 0.0000045 inches? • Metric system measures length _________________ o Abbreviated as ______ o ___________ indicate the “_________” of the meter o A micrometer (μm) is 1 ____________ of a meter, 0.000001 m, ______ m o A nanometer (nm) is 1 _____________ of a meter, 0.000000001 m, ______ m • To convert units: 0.0024 cm into μm 0.0024 x 0.01 or 10-2 = 24 μm 0.000001 or 10-6 Microscopy: The instruments • Total magnification – The _______________ of a microscopic specimen o How many times is the ____________________ than the ________________? o 100X total magnification = the image in the microscope is _____________________ than the specimen • Resolution (or resolving power) – ability of the microscope lens to _____________________ o How _______________ do two points have to be in order to see ________________? o Resolving power of 4 μm = two points have to be at least 4 μm apart in order to see them as two points. Two points less than 4 μm apart will look like ______________. Light microscopy - Any kind of microscope that uses visible light to observe specimens • Compound light microscopy o Uses ___________________________, visible _____________ to view specimens o Can view small specimens, some fine details o Standard total magnifications range from ____________ up to ____________ o Best resolving power about _______________ • Depending on conditions, a number of light microscopy techniques exist o Brightfield microscopy Uses visible light as illumination Specimen appears __________ against ___________ background (brightfield) Observe _____________ (colored) microbes Can’t resolve _______________ specimens (viruses) ______________, easy to use o Darkfield microscopy Uses special filter that ____________________ Only light that ______________ is seen Specimen appears ____________ against __________ background (darkfield) Used when microbes _____________________ properly o Phase-contrast microscopy Uses special filter that _________________ rays that reflect off the specimen Produces ____________________ Can be used on _____________________ o Differential interference contrast (DIC) microscopy Similar to phase-contrast Uses _____________________ of light separated by prisms • Specimen appears ____________ without stain______________ Images appear __________ o Fluorescence microscopy Fluorescence – ability of objects to __________ short wavelengths of light and ______________ (or __________) longer wavelengths of light Uses ____________ for illumination Some objects fluoresce naturally Some can be stained with __________________________(fluorescent dyes) Only ___________________ can be seen against ___________ background Can be used to see selective specimens within sample • Auramine O sticks only to M. tuberculosis • Can look for bacterium in patient’s tissue sample Imunofluorescence uses _________________________________ • Antibodies bind to ______________________ • Can _____________________ microbes in ________________ o Confocal microscopy Similar to fluorescent microscopy – stain specimens with ________________ Uses ________________ to scan one “slice” of the specimen at a time All “slices” can be put together by computer • Creates ____________, high _______________, _______ images Images can be ________________, viewed from any angle Scanning acoustic microscopy – • Uses __________________ to visualize a specimen • Sound hits specimen, reflected sound waves used to construct image • Resolution about __________ • Used to examine ___________________ attached to another surface o i.e., cancer cells, artery plaques, tooth decay Electron microcopy • Uses beam of ________________ instead of light o 100,000 times smaller than light o Can resolve much smaller objects; viruses, internal cell structures • Images are always __________ and __________ o Can be _______________ colored • Two types of electron microscopes o Transmission electron microscopy Beam of electrons pass through prepared, ___________________ of specimen Total magnification: ______________ to _______________ Resolution: ___________ 2D image Electrons have ___________________ power • Only ___________ section can be viewed (100 nm) Specimens are ___________________ and _____________ • Can _______________ some specimens o Scanning electron microscopy Bounces electrons of specimen, collects reflected electrons to create image Creates ___________ images, useful for ____________ of cells and viruses Total magnification: ____________ to _____________ Resolution: ____________________ Preparations of specimens for light microscopy • Most microbes appear _________________ in microscope • Most common way to prepare specimens is by _________________ (coloring) with a dye • Dyes are generally ________ molecules (combination of one positive and one negative ions) o One ion is colored; called the chromophore • Two general types of dyes o __________ dyes – chromophore is the ____________ ion o __________ dyes – chromophore is the ____________ ion • Most bacteria are ______________ charged o Thus, _____________ dyes work best (opposites attract) • _____________________ involves staining the background o Specimen remains _________________ • Three types of staining techniques o Simple stains Highlights entire ____________ Visualize _______________, ___________ structures Mordant – chemicals that _______________ stain o Differential stains Dyes reacts ___________________ with ________________ bacteria Can be used to ___________________________ them Gram stain • Most common differential staining technique • Classifies bacteria into 2 large groups; gram-_________, gram-______ • Stains structural differences in _________________ Acid-fast stain • Dye binds to _____________________ in cell wall • Used to identify pathogenic members of ________________________ o Special Stains Used to color, isolate ______________________ of microbes • Capsules – _____________________________ of microbe • Endospores – resistant, _____________________ formed within a cell • Flagella – structures that allow a cell to _________________
