Tools off the Laboratory:
The Methods for Studying
Microorganisms
Chapter 3
The 5 I’s of culturing microbes
1. Inoculation – introduction of a sample
into a container of media
2. Incubation – under conditions that allow
g
growth
3. Isolation –separating one species from
another
4. Inspection
5. Identification
Isolation
• If an individual bacterial cell is separated
from other cells & has space on a nutrient
surface, it will grow into a mound of cellsa colony
• A colony consists of one species
1
Isolation technique
Media – providing nutrients in
the laboratory
• Most commonly used:
– nutrient broth – liquid medium containing beef
extract & peptone
– nutrient agar – solid media containing beef extract,
peptone
pep
o e & aga
agar
• agar is a complex polysaccharide isolated from
red algae
– solid at room temp, liquefies at boiling (100oC), does
not resolidify until it cools to 42oC
– provides framework to hold moisture & nutrients
– not digestible for most microbes
Types of media
• synthetic – contains pure organic & inorganic
compounds in an exact chemical formula
• complex or nonsynthetic – contains at least
one ingredient that is not chemically definable
• generall purpose mediadi grows a broad
b d range
of microbes, usually nonsynthetic
• enriched media- contains complex organic
substances such as blood, serum, hemoglobin
or special growth factors required by fastidious
microbes
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Enriched media
• selective media- contains one or more
agents that inhibit growth of some
microbes and encourage growth of the
desired microbes
• differential media – allows growth of
several types of microbes and displays
visible differences among desired and
undesired microbes
selective & differential media
3
Selective media
Differential media
Miscellaneous media
• reducing medium – contains a
substance that absorbs oxygen or slows
penetration of oxygen into medium; used
f growing
for
i anaerobic
bi b
bacteria
t i
• carbohydrate fermentation mediumcontains sugars that can be fermented,
converted to acids, and a pH indicator to
show the reaction; basis for identifying
bacteria and fungi
4
Carbohydrate fermentation
media
• magnification – ability to enlarge
objects
• resolving power – ability to show detail
compound light microscope
5
Pathway of light
Effect of wavelength on
resolution
Oil immersion lens
6
Effect of magnification
Types of light microscopes
• Bright-field – most widely used, specimen
is darker than surrounding field
• Dark-field – brightly illuminated
p
surrounded by
y dark field
specimens
• Phase-contrast – transforms subtle
changes in light waves passing through
the specimen into differences in light
intensity, best for observing intracellular
structures
3 views of a cell
7
Fluorescence Microscope
• Modified compound microscope with an
ultraviolet radiation source and a filter that
protects the viewer’s eye
• Uses dyes that emit visible light when
bombarded with shorter uv rays.
• Useful in diagnosing infections
Electron microscopy
• Forms an image with a beam of electrons that
can be made to travel in wavelike patterns when
accelerated to high speeds.
• Electron waves are 100,000X shorter than the
waves of visible light
light.
• Electrons have tremendous power to resolve
minute structures because resolving power is a
function of wavelength.
• Magnification between 5,000X and 1,000,000X
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2 types of electron microscopes
• Transmission electron microscopes
(TEM) – transmits electrons through the
specimen; darker areas represent thicker,
denser parts and lighter areas indicate
more transparent, less dense parts
• Scanning electron microscopes (SEM)–
provides detailed three-dimensional view.
SEM bombards surface of a whole, metalcoated specimen with electrons while
scanning back and forth over it.
Transmission Electron Micrograph
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Scanning Electron Micrograph
Specimen preparation
• wet mounts & hanging drop mounts –
allow examination of characteristics of live
cells: motility, shape, & arrangement
• fixed mounts are made by drying &
heating a film of specimen. This smear is
stained using dyes to permit visualization
of cells or cell parts.
Staining
• cationic dyes - basic, with positive charges
on the chromophore
• anionic dyes - acidic, with negative
g on the chromophore
p
charges
• surfaces of microbes are negatively
charged and attract basic dyes – positive
staining.
• negative staining – microbe repels dye &
it stains the background
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Staining
• simple stains –one dye is used
• differential stains – use a primary stain
and a counterstain to distinguish cell types
or parts.
parts examples: Gram stain
stain, acid
acid-fast
fast
stain and endospore stain
• special stains: capsule and flagellar
stains
Types of stains
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