Transcript MICROBIOLOGY

LECTURES IN
MICROBIOLOGY
Laboratory Tools in Microbiology
LESSON 3
Sofronio Agustin
Professor
Topics Covered
 Microscopy
 Staining Techniques
 Methods of Culturing Microbes
Units 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 Microscope
 A simple microscope
has only one lens
Compound 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 : 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: 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: Resolution
 Resolution can be increased
by using immersion oil and
shorter wavelengths of light.
Optics: Refraction
 Refractive index is the lightbending 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 Microscopy
 Dark objects are visible against light background.
 Used to observe stained or unstained specimens.
 Most commonly used in laboratories.
Darkfield 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 Microscopy
 Accentuates diffraction of light that passes through the
specimen.
 Used to observe internal cellular detail of live specimens.
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 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 Microscopy
 Uses fluorochromes and laser
light.
 The laser illuminates each
plane in a specimen to produce
a 3-D image.
Electron 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 Microscopy
Transmission Electron Micrograph of (a) a virus and (b) a protozoan.
Scanning Electron Microscopy
False-color Scanning Electron Micrograph of Paramecium sp.
Summary of Microscope Types
Optical and Electron Microscopy:
A Comparison
TEM and SEM Compared
Transmission Electron Microscope
Scanning Electron Microscope
Scanning Probe Microscopy
Scanning tunneling
microscopy uses a
metal probe to scan a
specimen.
Resolution 1/100 of
an atom.
Scanning Probe Microscopy
Atomic force
microscopy uses a
metal and diamond
probe inserted into
the specimen.
Produces 3-D
images.
Preparation 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.
Stains
 Positive stains
Dye binds to the specimen
 Negative stains
Dye does not bind to the specimen, but rather
around the specimen.
Positive 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 Stains
 Simple


- One dye
Differential
- Two-different colored
dyes
Ex. Gram stain
Special
- Emphasize certain cell
parts
Ex. Capsule stain
Bacterial Stain Types
Simple 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 Stain
 The Gram stain classifies bacteria into grampositive 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 Stain
Color of
Gram + cells
Purple
Color of
Gram – cells
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing agent:
Alcohol-acetone
Purple
Colorless
Counterstain:
Safranin
Purple
Red
Primary stain:
Crystal violet
Differential Stain: Gram Stain
Differential 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 acidalcohol, and are usually counterstained (with a different color basic
stain) to see them.
Culture of Microbes
 Five basic techniques
 Media
 Microbial growth
Five Basic Techniques
1. Inoculate
2. Incubate
3. Isolation
4. Inspection
5. Identification
Summary of Laboratory Techniques
Isolation Technique
 A single visible colony
represents a pure
culture or single type of
bacterium isolated from
a mixed culture.
Isolation Methods
Culture Media
 Classified according to three properties
 Physical state
 Chemical composition
 Functional types
Culture Media: Physical State
 Liquid media
 Semi-solid media
 Solid media
Liquid Media
 Liquid media are waterbased solutions that are
generally termed broths,
milks and infusions.
Semi-solid Media
 Semi-solid media contain a
low percentage (<1%) of agar,
which can be used for motility
testing.
Solid Media
 Solid media contain a high percent (1-5%) of agar,
which enables the formation of discrete colonies.
Culture Media: Chemical Composition
 Synthetic or chemically-defined media
 Nonsynthetic or complex media
Synthetic Media
 Synthetic media contain
pure organic and
inorganic compounds
that are chemically
defined (i.e. known
molecular formula).
Complex Media
 Complex or enriched
media contain
ingredients that are not
chemically defined or
pure (i.e. animal
extracts).
Culture Media: Functional Types
 Enriched media
 Selective media
 Differential media
Enriched Media
 Enriched media are
used to grow
fastidious bacteria.
Differential 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 Media
Selective Media
Mannitol Salt Agar
MacConkey Agar
Miscellaneous Media
Examples of
miscellaneous media
are reducing,
fermentation and
transportation media.
Microbial Growth
• Incubation
 Varied temperatures, atmospheric states
• Inspection
 Mixed culture
 Pure culture
• Identification
 Microscopic appearance
• Maintenance and disposal
 Stock cultures
 sterilization