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Laboratory Tools in MicrobiologyLaboratory Tools in Microbiology
Sofronio Agustin
Professor
Sofronio Agustin
Professor
LECTURES IN
MICROBIOLOGY
LECTURES IN
MICROBIOLOGY
LESSON 3LESSON 3
Topics CoveredTopics Covered
Microscopy
Staining Techniques
Methods of Culturing Microbes
Units of MeasurementUnits of Measurement
1 µm = 10-6 m = 10-3 mm
1 nm = 10-9 m = 10-6 mm
1000 nm = 1 µm
0.001 µm = 1 nm
Simple MicroscopeSimple Microscope
A simple microscope has only one lens
Compound MicroscopeCompound Microscope
A compound microscope has two sets of lenses and is typically used in teaching and research laboratories.
Parts of a student laboratory microscope
Optics : MagnificationOptics : Magnification
A specimen is magnified as light passes through the objective and ocular lenses.
Total Magnification = objective lens X ocular lens(magnifications)
The pathway of light and two stages of magnification of a compound microscope.
Optics: ResolutionOptics: Resolution
Resolution is the ability of the
lenses to distinguish two
points.
A microscope with a
resolving power of 0.2 um
can distinguish between two
points > 0.2 um.
Resolution distinguishes magnified objects clearly.
Optics: ResolutionOptics: Resolution
Resolution can be increased
by using immersion oil and
shorter wavelengths of light.
Optics: RefractionOptics: Refraction
Refractive index is the light-bending ability of a medium.
The light may bend in air so much that it misses the small high-magnification lens.
Immersion oil is used to keep light from bending.
Brightfield MicroscopyBrightfield Microscopy
Dark objects are visible against light background. Used to observe stained or unstained specimens. Most commonly used in laboratories.
Darkfield MicroscopyDarkfield Microscopy
Light objects are visible against a dark background.
Light reflected off the specimen enters the objective lens.
Used to screen for syphilis agent, Treponema pallidum.
Phase-contrast MicroscopyPhase-contrast Microscopy
Accentuates diffraction of light that passes through the specimen.
Used to observe internal cellular detail of live specimens.
Differential Interference Contrast (DIC) Microscopy
Differential Interference Contrast (DIC) Microscopy
Accentuates diffraction of light that passes through the specimen.
Uses two beams of light to produce
more pronounced contrast.
Fluorescence MicroscopyFluorescence Microscopy
Cells are stained with fluorescent dyes called fluorochromes.
Uses UV light as energy source.
Fluorochromes absorb UV light and
emit visible light
Fluorescent staining of fresh sample of cheek scrapings
Confocal MicroscopyConfocal Microscopy
Uses fluorochromes and laser light.
The laser illuminates each
plane in a specimen to produce
a 3-D image.
Electron MicroscopyElectron Microscopy
Very high magnification of up to 100,000 X.
Transmission electron microscope (TEM)
View internal structures of cells
Scanning electron microscope (SEM)
Three-dimensional images
Transmission Electron MicroscopyTransmission Electron Microscopy
Transmission Electron Micrograph of (a) a virus and (b) a protozoan.
Scanning Electron MicroscopyScanning Electron Microscopy
False-color Scanning Electron Micrograph of Paramecium sp.
Summary of Microscope TypesSummary of Microscope Types
Optical and Electron Microscopy:A Comparison
Optical and Electron Microscopy:A Comparison
TEM and SEM ComparedTEM and SEM Compared
Scanning Electron Microscope Transmission Electron Microscope
Scanning Probe MicroscopyScanning Probe Microscopy
Scanning tunneling microscopy uses a metal probe to scan a specimen.
Resolution 1/100 of
an atom.
Scanning Probe MicroscopyScanning Probe Microscopy
Atomic force
microscopy uses a
metal and diamond
probe inserted into
the specimen.
Produces 3-D
images.
Preparation of Specimens for Light MicroscopyPreparation of Specimens for Light Microscopy
A thin film of a solution of microbes on a slide is a smear.
A smear is usually fixed to attach the microbes to the slide and to kill the microbes.
StainsStains
Positive stains
Dye binds to the specimen
Negative stains
Dye does not bind to the specimen, but rather
around the specimen.
Positive and Negative StainsPositive and Negative Stains
Positive stains are basic dyes (cationic) that bind to negatively charged cells.
Negative stains are acidic dyes (anionic) that bind the background.
Positive StainsPositive Stains
Simple- One dye
Differential- Two-different colored
dyes
Ex. Gram stain Special
- Emphasize certain cell partsEx. Capsule stain
Bacterial Stain TypesBacterial Stain Types
Simple StainSimple Stain
Use of a single basic dye is called a simple stain.
A mordant may be used to hold the stain or coat
the specimen to enlarge it.
Differential Stain: Gram StainDifferential Stain: Gram Stain
The Gram stain classifies bacteria into gram-
positive and gram-negative.
Gram-positive bacteria tend to be killed by
penicillin and detergents.
Gram-negative bacteria are more resistant to
antibiotics.
Differential Stain: Gram StainDifferential Stain: Gram Stain
Color of
Gram + cells
Color of
Gram – cells
Primary stain:
Crystal violet
Purple Purple
Mordant:
Iodine
Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin
Purple Red
Differential Stain: Gram StainDifferential Stain: Gram Stain
Differential Stain: Acid Fast StainDifferential Stain: Acid Fast Stain
Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.
Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.
Culture of MicrobesCulture of Microbes
Five basic techniques
Media
Microbial growth
Five Basic TechniquesFive Basic Techniques
1. Inoculate
2. Incubate
3. Isolation
4. Inspection
5. Identification
Summary of Laboratory TechniquesSummary of Laboratory Techniques
Isolation TechniqueIsolation Technique
A single visible colony
represents a pure
culture or single type of
bacterium isolated from
a mixed culture.
Isolation MethodsIsolation Methods
Culture MediaCulture Media
Classified according to three properties Physical state
Chemical composition
Functional types
Culture Media: Physical StateCulture Media: Physical State
Liquid media Semi-solid media Solid media
Liquid MediaLiquid Media
Liquid media are water-
based solutions that are
generally termed broths,
milks and infusions.
Semi-solid MediaSemi-solid Media
Semi-solid media contain a
low percentage (<1%) of agar,
which can be used for motility
testing.
Solid MediaSolid Media
Solid media contain a high percent (1-5%) of agar, which enables the formation of discrete colonies.
Culture Media: Chemical CompositionCulture Media: Chemical Composition
Synthetic or chemically-defined media Nonsynthetic or complex media
Synthetic MediaSynthetic Media
Synthetic media contain
pure organic and
inorganic compounds
that are chemically
defined (i.e. known
molecular formula).
Complex MediaComplex Media
Complex or enriched
media contain
ingredients that are not
chemically defined or
pure (i.e. animal
extracts).
Culture Media: Functional TypesCulture Media: Functional Types
Enriched media
Selective media
Differential media
Enriched MediaEnriched Media
Enriched media are
used to grow
fastidious bacteria.
Differential and Selective MediaDifferential and Selective Media
Selective media enables
one type of bacteria to
grow
While differential media
allows bacteria to show
different reactions (i.e.
colony color).
Differential MediaDifferential Media
Selective MediaSelective Media
Mannitol Salt Agar
MacConkey Agar
Miscellaneous MediaMiscellaneous Media
Examples of
miscellaneous media
are reducing,
fermentation and
transportation media.
Microbial GrowthMicrobial Growth
• Incubation Varied temperatures, atmospheric states
• Inspection Mixed culture Pure culture
• Identification Microscopic appearance
• Maintenance and disposal Stock cultures sterilization