Chapter03 Lecture

8/7/2019 Chapter03 Lecture

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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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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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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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3

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

Chapter03 Lecture

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