chapter 3 lecture outline - learning.hccs.edu

Chapter 3

Lecture Outline

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Identification of Microorganism

One goal of these procedures is to attach a name or identity to the microbe, using information gathered from inspection and investigation. Identification is accomplished through the use of keys, charts, and computer programs that analyze the data and arrive at a final conclusion.


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Inoculation of Microorganism

The sample is placed into a container of medium that will support its growth. The medium may be solid or liquid, and held in tubes, plates, flasks, and even eggs. The delivery tool is usually a loop, needle, swab, or syringe.


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Information Gathering of Microorganism

Additional tests for microbial function and characteristics may include inoculations into specialized media that determine biochemical traits, immunological testing, and genetic typing. Such tests will provide specific information unique to a certain microbe.


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Specimen Collection of Microorganism

Microbiologists begin by sampling the object of their interest. It could be nearly any thing or place on each. Very common sources are body fluids, foods, water, soil, plants, and animals, but even places like icebergs, volcanoes, and rocks can be sampled.


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Incubation of Microorganism

Inoculated media are placed in a controlled environment (incubator) to promote growth. During the hours or days of this process, a culture develops as the visible growth of the microbes in the container of medium.


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Inspection of Microorganism

Cultures are observed for the macroscopic appearance of growth characteristics. Cultures are examined under the microscope for basic details such as cell type and shape. This may be enhanced through staining and use of special microscopes.


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Isolation of Microorganism

Some inoculation techniques can separate microbes to create isolated colonies that each contain a single type of microbe. This is invaluable for identifying the exact species of microbes in the sample, and it paves the way for making pure cultures.


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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

• Magnification in most microscopes results from an interaction between visible light waves and the curvature of a lens.

– The extent of enlargement is the magnification.

© Kathy Park Talaro


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Parts of the Microscope

(a) Courtesy of Nikon Instruments Inc., Melville, New York, USA, www.nikoninstruments.com


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Magnification in Two Phases (1 of 2)

• The objective lens forms the magnified real image

• The real image is projected to the ocularwhere it is magnified again to form the virtual image


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Magnification in Two Phases (2 of 2)


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Total Magnification (1 of 2)

• Total magnification of the final image is a product of the separate magnifying powers of the two lenses

• objective power × ocular power = total magnification


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Total Magnification (2 of 2)


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Resolution

• The capacity to distinguish or separate two adjacent objects and depends on

– The wavelength of light that forms the image along with characteristics of the objectives


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Quantifying Resolution

( )lens objective of aperture Numerical 2

nm in light of Wavelength RPPower Resolving

=

• Visible light wavelength is 400 nm–750 nm

• Numerical aperture of lens ranges from 0.1 to 1.25

• Shorter wavelength and larger numerical aperture will provide better resolution

• Oil immersion objectives resolution is 0.2 μm

• Magnification between 40X and 2000X


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The Purpose of Oil


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Magnification & Resolution


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Concept Check: (1)

In order to get the best resolution possible with a light microscope, you would want:

A. Long wavelengths of light

B. Short wavelengths of light

C. The wavelength doesn’t affect resolution


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Concept Check: (2)

In order to get the best resolution possible with a light microscope, you would want:

A. Long wavelengths of light

B. Short wavelengths of light

C. The wavelength doesn’t affect resolution

Answer: B


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Variations on the Optical Microscope (1 of 3)

• Bright-field – most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens

© Prof. Dr. Heribert Cypionka, www.microbiological-garden.net


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• Dark-field – brightly illuminated specimens surrounded by dark field; used for live and unstained specimens



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• Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures



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Fluorescence Microscope

• Modified microscope with an ultraviolet radiation source and filter.

• Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence

• Useful in diagnosing infections



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Scanning Confocal Microscope

• Uses a laser beam of light to scan the specimen.

• Integrates images to allow focus on multiple depths or planes.

© Anne Fleury


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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 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


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Comparing Microscopes:

TABLE 3.3 Comparison of Light Microscopes and Electron Microscopes

Characteristic Light or Optical Electron (Transmission)

Useful magnification 2,000×* 1,000,000× or more

Maximum resolution 200nm (0.2µm) 0.5nm

Image produced by Light rays Electron beam

Image focused by Glass objective lensElectromagnetic objective

lenses

Image viewed through Glass ocular lens Fluorescent screen

Specimen placed on Glass slide Copper mesh

Specimen may be alive Yes Usually not

Specimen requires special stains or treatment

Depends on technique Yes

Shows natural color Yes No

*This maximum requires a 20x ocular.


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2 Types of Electron Microscopes (1 of 2)

• Transmission electron microscopes (TEM) -transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.

Source: Erskine. L. Palmer, Ph.D.; M. L. Martin/Frederick Murphy/CDC


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2 Types of Electron Microscopes (2 of 2)

• Scanning electron microscopes (SEM) –provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

Source: National Institute of Allergy and Infectious Diseases (NIAD)/CDC


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Specimen Preparation for Optical Microscopes

• Wet mounts and hanging drop mounts –allow examination of characteristics of live cells: size, 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 (1 of 3)

• Dyes are used to create contrast by imparting color

• Basic dyes – cationic, positively charged chromophore

• Positive staining – surfaces of microbes are negatively charged and attract basic dyes


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Staining (2 of 3)

• Acidic dyes – anionic, negatively charged chromophore

• Negative staining – microbe repels dye, the dye stains the background

Source: Larry Stauffer, Oregon State Public Health Laboratory/CDC


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Staining (3 of 3)

• Simple stains – one dye is used; reveals shape, size, and arrangement

• Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)

• Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains


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Staining Examples (1 of 8)

The figure of “Staining Examples.”

a) Simple and Negative Stains

Use one dye to observe cells

Methylene blue stain of Corynebacterium (1,000x)(a1): A © Harold J. Benson


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The figure of “Staining Examples” continues on this slide.

Negative stain of spirilla bacteria made with nigrosin(500×)(a2): Courtesy of Tuatara: Journal of the Biological Society of Victoria University of Wellington. J. E. Sheridan, Jan Steel and M. N. Loper. http://nzetc.victoria.ac.nz/tm/scholarly/Bio18Tuat03-fig-Bio18Tuat03_104a. html


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b) Differential Stains

Use two dyes to distinguish between cell types

Gram stain

Purple cells are gram-positive.

Red cells are gram-negative (1,000x).(b1 left): Source: Bobby Strong/CDC; (b1 right): Source: CDC


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Acid-fast stain

Red cells are acid-fast.

Blue cells are non-acid-fast (750x).(b2): © Richard J. Green/Science Source


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Spore stain, showing spores (green) and vegetables cells (red) (1,000x)(b3): © Steven R. Spilatro, Department of Biology, Marietta College, Marietta, OH


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c) Structural stains

Special stains used to enhance cell details

Capsule stain of rod-shaped bacterium (1,000×)(c1): ©Steven P. Lynch


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Flagellar stain of Proteus vulgaris. Note the fine fringe of flagella (1,500x) (c2): © David Fankhauser


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Fluorescent stains of bacterial chromosome (green) and cell membrane (red) (1,500x)(c3): From I.A. Berlatzky, A. Rouvinski, S. Ben-Yehuda, “Spatial organization of a replicating bacterial chromosome, “PNAS, 105(37):14138-14140, Fig. 4A. © 2008 by The National Academy of Sciences of the USA


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The 6 I’s of Culturing Microbes

• Inoculation – introduction of a sample into a container of media to produce a culture of observable growth

• Isolation – separating one species from another

• Incubation – under conditions that allow growth

• Inspection

• Information gathering

• Identification


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Isolation

• If an individual bacterial cell is separated from 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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Isolation Techniques (1 of 3)

• Streak plate technique

Note: This method works best if the spreading tool (usually an inoculating loop) is resterilized (flamed) after each of steps 1-3.

(a) Steps in a Streak Plate; this one is a four-part or quadrant streak.(a) and (b): © Kathy Park Talaro


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• Pour plate technique

(c) Steps in a Loop Dilution; also called a pour plate or serial dilution(c) and (d): © Kathy Park Talaro


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Inspection (1 of 2)

• If a single species is growing in the container, you have a pure culture but if there are multiple species than you have a mixed culture.

• Check for contaminants (unknown or unwanted microbes) in the culture.

© Kathy Park Talaro


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Ways to Identify a Microbe:

• Cell and colony morphology or staining characteristics

• DNA sequence

• Biochemical tests to determine an organism’s chemical and metabolic characteristics

• Immunological tests

© John Watney/Science Source


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Media: Providing Nutrients in the Laboratory

Media can be classified according to three properties:

• Physical state – liquid, semisolid, and solid

• Chemical composition – synthetic(chemically defined) and complex

• Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration


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Physical States of Media (1 of 2)

• Liquid – broth; does not solidify

• Semisolid – contains solidifying agent

• Solid – firm surface for colony formation

– Contains solidifying agent

– Liquefiable and nonliquefiable


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Agar (1 of 2)

• The most commonly used solidifying agent

• Solid at room temperature, liquefies at boiling (100°C), does not re-solidify until it cools to 42°C

• Provides framework to hold moisture and nutrients

• Not digestible for most microbes


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Agar (2 of 2)


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Most Commonly Used Media

• Nutrient broth – liquid medium containing beef extract and peptone

• Nutrient agar – solid media containing beef extract, peptone, and agar


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Chemical Content of Media (1 of 2)

• Synthetic – contains pure organic and 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


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Selective & Differential 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 those microbes

© McGraw-Hill Education/Lisa Burgess, photographer


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Concept Check: (3)

CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patient’s sample is inoculated and based on the color of the colonies you can identify the pathogen. CHROMagaris best described as:

A. Enriched

B. Selective

C. Differential

D. Complex

© K. Talaro


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Concept Check: (4)

CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patient’s sample is inoculated and based on the color of the colonies you can identify the pathogen. CHROMagaris best described as:

A. Enriched

B. Selective

C. Differential

D. Complex

Answer: C


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Miscellaneous Media

• Carbohydrate fermentation medium – contains sugars that can be fermented, converted to acids, and a pH indicator to show this reaction

© Harold J. Benson

© McGraw-Hill Education. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted

without the prior written consent of McGraw-Hill Education.

3-653-65

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chapter 3 lecture outline - learning.hccs.edu

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