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PowerPoint to accompany
Foundations
in
Microbiology
Fifth Edition
Talaro
Chapter
3
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Tools of 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
growth
3. Isolation –separating one species from
another
4. Inspection
5. Identification
3
Isolation
• If an individual bacterial cell is separated
from other cells & has space on a nutrient
surface, it will grow into a mound of cells- a
colony
• A colony consists of one species
4
Isolation technique
5
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 & 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
6
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
• general purpose media- grows a broad range of
microbes, usually nonsynthetic
• enriched media- contains complex organic
substances such as blood, serum, hemoglobin or
special growth factors required by fastidious
microbes
7
Enriched media
8
• 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
9
selective & differential media
10
Selective media
11
Differential media
12
Miscellaneous media
• reducing medium – contains a substance
that absorbs oxygen or slows penetration of
oxygen into medium; used for growing
anaerobic bacteria
• 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
13
Carbohydrate fermentation media
14
• magnification – ability to enlarge objects
• resolving power – ability to show detail
15
compound light microscope
16
Pathway of light
17
Effect of wavelength on resolution
18
Oil immersion lens
19
Effect of magnification
20
Types of light microscopes
• Bright-field – most widely used, specimen
is darker than surrounding field
• Dark-field – brightly illuminated
specimens surrounded by dark field
• Phase-contrast – transforms subtle changes
in light waves passing through the specimen
into differences in light intensity, best for
observing intracellular structures
21
3 views of a cell
22
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
23
24
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.
• Electrons have tremendous power to resolve
minute structures because resolving power is a
function of wavelength.
• Magnification between 5,000X and 1,000,000X
25
26
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, metal-coated
specimen with electrons while scanning back
and forth over it.
27
Transmission Electron Micrograph
28
Scanning Electron Micrograph
29
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.
30
Staining
• cationic dyes - basic, with positive charges
on the chromophore
• anionic dyes - acidic, with negative charges
on the chromophore
• surfaces of microbes are negatively charged
and attract basic dyes – positive staining.
• negative staining – microbe repels dye & it
stains the background
31
Staining
• simple stains –one dye is used
• differential stains – use a primary stain and
a counterstain to distinguish cell types or
parts. examples: Gram stain, acid-fast stain
and endospore stain
• special stains: capsule and flagellar stains
32
Types of stains
33