chapter03 lecture
TRANSCRIPT
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Foundations in
MicrobiologySixth Edition
Chapter 3
Tools of the Laboratory:
The Methods for Studying
Microorganisms
Lecture PowerPoint to accompany
Talaro
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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The 5 Is of Culturing Microbes
1. Inoculation introduction of a sample intoa container of media to produce a culture
of observable growth
1. Isolation separating one species fromanother
2. Incubation under conditions that allow
growth3. Inspection
4. Identification
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Isolation
If an individual bacterial cell is separatedfrom other cells and has space on a nutrient
surface, it will grow into a mound of cells -
a colony. A colony consists of one species.
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Insert figure 3.2Isolation technique
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Isolation techniques include:
streak plate technique
pour plate technique
spread plate technique
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Insert figure 3.3
Isolation methods
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Media: Providing Nutrients in the
LaboratoryMedia can be classified according to three
properties:
1. Physical state liquid, semisolid and solid2. Chemical composition synthetic
(chemically defined) and nonsynthetic(complex)
3. Functional type general purpose,enriched, selective, differential, anaerobic,transport, assay, enumeration
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Physical States of Media
Liquid broth; does not solidify
Semisolid clot-like consistency; contains
solidifying agent (agar or gelatin)Solid firm surface for colony formation
contains solidifying agent
liquefiable and nonliquefiable
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Most commonly used solidifying agent is agar
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 and
nutrients
not digestible for most microbes
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Media: Providing Nutrients in the
Laboratory Most commonly used media:
nutrient broth liquid medium containing beef extractand peptone
nutrient agar solid media containing beef extract,peptone and agar
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Media: Providing Nutrients in the
Laboratory Synthetic contains pure organic and inorganic
compounds in an exact chemical formula
Complex or nonsynthetic contains at least oneingredient that is not chemically definable
General purpose media- grows a broad range ofmicrobes, usually nonsynthetic
Enriched media- contains complex organicsubstances such as blood, serum, hemoglobin orspecial growth factors required by fastidiousmicrobes
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Selective media- contains one or more
agents that inhibit growth of some microbes
and encourage growth of the desired
microbes
Differential media allows growth ofseveral types of microbes and displays
visible differences among desired and
undesired microbes
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Miscellaneous Media
Reducing medium contains a substancethat absorbs oxygen or slows penetration ofoxygen into medium; used for growing
anaerobic bacteria Carbohydrate fermentation medium
contains sugars that can be fermented,converted to acids, and a pH indicator to
show the reaction; basis for identifyingbacteria and fungi
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Insert figure 3.10Differential media
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Incubation, Inspection, and Identification
Incubation temperature-controlled chamber at
appropriate temperature and atmosphere
microbe multiplies and produces macroscopically
observable growth
Inspection observation; macroscopic and microscopic
pure culture grows only single known species of
microorganisms
mixed cultures hold two or more identified species or
microorganisms
contaminated culture once pure or mixed culture that
has unwanted microbes growing
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Incubation, Inspection, and Identification
Identification macroscopic and microscopic
appearance, biochemical tests, genetic
characteristics, immunological testing
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Disposal of Cultures
Potentially hazardous cultures and specimens
are usually disposed of in two ways:
steam sterilization incineration
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The Microscope
Key characteristics of a reliable microscope are:
Magnification ability to enlarge objects
Resolving power ability to show detail
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Magnification in most microscopes results
from interaction between visible light wavesand curvature of the lens.
angle of light passing through convex surface
of glass changes refraction
Depending on the size and curvature of the
lens, the image appears enlarged.
extent of enlargement - magnification
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Insert figure 3.14
Student microscope
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Principles of Light Microscopy
Magnification occurs in two phases
The objective lens forms the magnified real image.
The real image is projected to the ocular where it is
magnified again to form the virtual image.
Total magnification of the final image is a
product of the separate magnifying powers of
the two lenses.power of objective X power of ocular = total magnification
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Insert figure 3.15
Pathway of light
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Resolution
Resolution defines the capacity to distinguish orseparate two adjacent objects resolvingpower
function of wavelength of light that forms the
image along with characteristics of objectives Visible light wavelength is 400 nm 750 nm.
Numerical aperture of lens ranges from 0.1 to 1.25.
Oil immersion lens requires the use of oil to preventrefractive loss of light.
Shorter wavelength and larger numerical aperture willprovide better resolution.
Oil immersion objectives resolution is 0.2 m.
Magnification between 40X and 2000X
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Insert figure 3.16Wavelength on resolution
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Insert figure 3.17Oil immersion lens
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Types of Light Microscopes
Bright-field most widely used; specimen isdarker than surrounding field; live andpreserved stained specimens
Dark-field brightly illuminated specimenssurrounded by dark field; live and unstainedspecimens
Phase-contrast transforms subtle changes inlight waves passing through the specimen intodifferences in light intensity, best for observingintracellular structures
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Fluorescence Microscope
Modified compound microscope with an
ultraviolet radiation source and a filter that
protects the viewers eye
Uses dyes that emit visible light when
bombarded with shorter UV rays - fluorescence
Useful in diagnosing infections
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Insert figure 3.21Fluorescent staining
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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 100,000 times shorter thanthe waves of visible light.
Electrons have tremendous power to resolve
minute structures because resolving power is afunction of wavelength.
Magnification between 5,000X and 1,000,000X
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2 Types of Electron Microscopes
Transmission electron microscopes (TEM) transmit electrons through the specimen. Darkerareas represent thicker, denser parts and lighter
areas indicate more transparent, less denseparts.
Scanning electron microscopes (SEM)provide detailed three-dimensional view. SEM
bombards surface of a whole, metal-coatedspecimen with electrons while scanning backand forth over it.
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Insert figure 3.24bTransmission micrograph
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Insert figure 3.25Scanning micrographs
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Specimen Preparation for Optical
Microscopes Wet mounts and hanging drop mounts
allow examination of characteristics of live
cells: motility, shape, and arrangement Fixed mounts are made by drying and
heating a film of specimen. This smear is
stained using dyes to permit visualization of
cells or cell parts.
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Staining
Dyes create contrast by imparting a color tocells or cell parts.
Basic dyes - cationic, with positive charges on the
chromophore Acidic dyes - anionic, with negative charges on
the chromophore
Positive staining surfaces of microbes are
negatively charged and attract basic dyes Negative staining microbe repels dye, the dye
stains the background
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Staining
Simple stains one dye is used; revealsshape, size, and arrangement
Differential stains use a primary stain
and a counterstain to distinguish cell typesor parts (examples: gram stain, acid-faststain and endospore stain)
Special stains reveal certain cell parts notrevealed by conventional methods: capsuleand flagellar stains
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Insert figure 3.27Types of stains