Micro Bio – Microbiology Exam Test – Flashcards
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Introduction to observing and staining microorganisms
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oToo small to see àmicroscopes oUnits §Micrometer (um) – 10-6 §Nanometer (nm) – 10-9 |
Principles to observing and staining microorganisms
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o Microscopy – use of visible light or electrons to magnify o Magnification – apparent increase in size o Visible light – electromagnetic radiation visible § Electromagnetic radiation – energy without mass, given from decomposing atoms o Light microscopes – curved glass lenses to magnify images formed by visible light § Light slows down in dense glass § Leading edge slows down before trailing à bends rays § Lens curves to focal point à further away image gets magnified § Quality of lens depends on ability to go through focal point § Resolution – ability to resolve two objects · Minimum distance 2 objects bust be apart to distinguish · 0.61 times wavelength of light divided by numerical aperature · Numerical aperature – constant written on barrel of lens. Light collecting efficiency § Contrast – difference in intensity between two objects or between object and background · Important determinant of resolution · Can’t resolve if no contrast § Microorganisms colorless and have little contrast · Staining · Phased light |
· Types of light microscopy |
o Types § Bright-field § Dark-field § Phase § Fluorescent § Confocal o Use different types of light or condensers |
Bright-Field Microscopy
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o Magnified images in field of bright white light § Cheapest § Most common § Condenser focuses white light on specimen or slide o Simple § Single lens or lens set to magnify § Up to 300x § First type – not used much today o Compound § Two sets of lenses in combo · Objective · Ocular § Most common light microscope today § Magnification = objective X ocular · Maximum resolving power 0.2 um · Max magnification 2000x § High mag objective lenses (100x) called immersion oil · Immersion oil placed between lens and specimen · Due to small opening of high mag lenses and low refractive index of air · Rays à glass à bend at sharp angle through air à miss small opening of high mag · With oil: Rays à glass à oil (same refractive index as glass 1.52) à more rays into oil immersion objective lens |
Dark-Field Microscopy |
o Special condenser and dark field stop passes light rays at sharp angle to specimen o Only those refracted by specimen enter objective o Specimens visible without staining as bright objects in dark field o Some objective and ocular lenses as bright-field o Useful for small, thin, or motile microbes |
· Phase Microscopy
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o Special condenser sends light waves in same phase § Specimens slow waves and get them out of phase § Phase lenses convert light with altered phase into contrast § Produces sharp image of cells and structures without staining o Two types § Phase contrast – contrast differences § Differential interference – produces 3D appearance o Useful for cellular detail and motility in wet mounts of living microbes |
Fluorescent Microscopy
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o Uses special filters and dyes § Microbes stained with fluorescent dye § Illuminated with light with wavelength absorbed by dye (excitation wavelength) § Dye emits absorbed light at longer wavelength (emission wavelength) § Filters in oculars filter out excitation wavelength and detect emitted light o Microbes appear as bright colored objects against dark background o Very sensitive, moreso than other methods o Figure 4-20 shows brightfield picture of auramine O acid-fast stained sputum smear showing no M. tuberculosis cells, while fluorescent picture shows M. tuberculosis cells
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Immunofluorescence |
o Fluorescent dye coupled to antibodies for a specific microbial species o Antibodies bind and fluoresce showing presence of microbe o Combine sensitivity of fluorescent microscopy with specificity of antibodies allowing rapid (within 1 hour) diagnosis if antibodies available § Critical for rapidly fatal diseases like bubonic plague o Very few diagnoses from light microscopy § Vaginitis and meningitis are exceptions o Vaginitis § Common in women § Caused by 3 microorganisms · Candida albicans (yeast) · Trichomonas vaginalis (protozoan) · Gardnerella vaginalis (bacterium) § C. albicans and T. vaginalis seen by light microscopy § If absent, must be G. vaginalis o Meningitis § Uncommon § Life-threatening § Caused by 3 microbes that gram stain differently · Neisseria meningitidis – Gram-negative coccus · Streptococcus pneumoniae – Gram-positive coccus · Haemophilus influenzae – Gram-negative rod |
Confocal Microscopy
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o Laser light, flueorescent dye, and computer o Produces 1um thick plane images o Assembled into 3 D image |
Electron Microscopy |
o Beam of electrons instead of visible light rays § Shorter wavelength (0.01 to 0.001 nm versus 400nm) § Much greater resolving power and magnification than light · 2.5 nm and 100,000x versus 200n and 2,000x o Obtains detailed images of small things § Viruses § Large molecules § External cellular structures o Two types, both work in vacuum § Scanning · Resolving power of 20nm · Magnification 1000x-10000x · 3 D image of object surface like bacteria and viruses § Transmission · Resolving power of 2.5nm · Magnification 10,000-100,000x · Views of external and internal cellular structures, viruses, large molecules · Must be embedded in plastic and sliced thinly · Or could be freeze etched |
Probe Microscopy
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o Tiny pointed tungsten probes o Magnification up to 100,000,000x o Types § Scanning tunneling · Probe passed back and forth above specimen · Electron flow measured · Measures down to .01nm · Specimens must be conductive § Atomic force · Probe passed back and forth over specimen · Deflections in laser beam aimed at probe detected · Measures down to .01nm · Can be used on living cells · Don’t use vacuum or electron beam · No need for conductivity |
Preparing Microbes for Light Microscopy
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o Must be mounted on slide § Make wet mount if living or § Smear if they are going to be stained o Wet Mounts § Liquid suspension on glass slide under cover slip § Used for living specimens § Can determine if microbes motile · Brownian motion (random tumbling) must be distinguished from true motility (swimming in straight line vigorously) § Best done with phase contrast microscope o Microbial Smears § Thin film of microorganisms on glass slide § Done before staining § Bacteria stained with basic dyes (colored positive ions) because surface is normally negatively charged § Smear air dried and bacteria fixed to prevent from washing away · Wire inoculating loop sterilized in flame, then cooled · Samples microbes and transferred into water drop on slide and emulsified · Spread thin with loop · Air dried · Fixed with heat or chemical treatment o Passing slide 3 times quickly over bunsen burner o Chemical fixing can be done with 95% alcohol o Staining |
Staining
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§ Several types · Simple stains · Negative stains · Differential stains · Special stains · Electron microscope stains
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Simple stains |
· Aqueous or alcohol solution with single basic dye · Dye placed on slide for 30 – 60 secs and washed away with water · Slide dries · Observed · Examples o Crystal violet o Safranin o Methylene blue |
Negative Stains
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· Acidic (like nigrosin) dyes or black particles (like india ink) · Stains background and not microbe · Microbes are light silhouette against dark blackground · Determines if microbe contains capsule (important virulence factors) |
Differential stains |
· Stains that react differently depending on different groups bacterial · Quick distinguishing of bacterial groups · Important stains o Gram stain o Acid-fast stain o Endospore stain |
Gram stain |
o Most important and commonly used stain o Distinguishes § Gram-positive – bluish-purple § Gram-negative – red o Carried out as shown in Figure 4-43 § Smear § Heat fix § Cover smears with crystal violet for 30 secs, rinse · GP – purple · GN - purple § Gram iodine for 30 secs, rinse · GP – purple · GN - purple § Gram decolorizer for 15 secs, rinse · GP – purple · GN - colorless § Gram safranin for 30 secs, rinse · GP – purple · GN – bright red o Iodine insoluble complex with crystal violet § Retained by gram-positive cell walls o Mordants – substances that enhance staining § Iodine mordant for crystal violet and gram-positive cells |
Acid-Fast Stain
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o Detects Mycobacterium (and some Nocardia species) o Mycobacterium and Nocardia are acid fast à stain red o Rest are non acid fast à stain blue o Carried out as shown in Figure 4-45 § Smear § Heat fix § Carbolfuchsin stain, heat over steam 5 min, rinse · AF - Red · NAF - Red § Alcohol for 15 sec, rinse · AF - Red · NAF - Colorless § Methylene blue for 30 secs, rinse · AF - Red · NAF - Blue o Mycobacterium don’t stain well because cell walls contain >50% dry weight of waxy lipids called mycolates o Tuburculosis § Mycobacterium is genus responsible for etiologic agents of tuberculosis and leprosy · 1/3 of population (2 billion) have been infected with TB · 15 million in US (5%) have been infected in US § Highly infectious · Expelled in respiratory droplets · Treat and quarantine until people not infectious · Acid fast rapid way to identify TB in sputum because it is specific for Mycobacterium |
Schaeffer-Fulton Endospore Stain
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§ Detects endospore forming bacteria § Endospores à stain green § Vegetative cells à stain red § Endospores impermeable to most chemicals and don’t stain well § Carried out · Smear · Heat fix · Malachite green, steam 5 min o Endospores – green o Vegetative - green · Rinse o Endospores – green o Vegetative - colorless · Safranin stain for 30 sec, rinse o Endospores – green o Vegetative - red § Endospores take up stain cause they are heated |
Special Stains
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o Stains to visualize bacterial components § Flagella § Capsules § Chromatin |
Electron Microscope Stains |
o Electron absorbing heavy metals § Lead § Osmium § Tungsten o Negative stains – background or o Postive stains – molecules or structures in microbe |
