Introduction to observing and staining microorganisms

oToo small to see àmicroscopes

oUnits

§Micrometer (um) – 10^-6

§Nanometer (nm) – 10^-9

  Principles to observing and staining microorganisms

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

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

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

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

 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

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

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

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

§  Several types

·         Simple stains

·         Negative stains

·         Differential stains

·         Special stains

·         Electron microscope stains

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

·         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

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

§  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

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