bio 411 master presentation dr. rowe sh 222 x2521, [email protected]

Bio 411 Master Presentation

Dr. Rowe

SH 222

X2521, [email protected]

Overview of Microbiology

• The study of organisms and agents to small to be seen clearly by the unaided eye

• Organisms involved are prions,viroids, virusoids, viruses, bacteria, fungi,protists (protozoa,and algae etc)

Figure 1.1

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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Type of Microbial Cells

• prokaryotic cells lack a true membrane-delimited nucleus – This is not absolute

• eukaryotic cells have a membrane-enclosed nucleus, are more complex morphologically, and are usually larger than prokaryotic cells


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Classification Schemes

• three domain system, based on a comparison of ribosomal RNA, divides microorganisms into – Bacteria (true bacteria),

– Archaea

– Eukarya (eukaryotes)

Domain Bacteria

• Usually single-celled

• Majority have cell wall with peptidoglycan

• Most lack a membrane-bound nucleus

• Ubiquitous and some live in extreme environments

• Cyanobacteria produce amounts of significant oxygen


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Domain Archaea

• distinguished from Bacteria by unique rRNA sequences

• lack peptidoglycan in cell walls

• have unique membrane lipids

• some have unusual metabolic characteristics

• many live in extreme environments


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Domain Eukarya - Eukaryotic

• protists – generally larger than Bacteria and Archaea– algae – photosynthetic – protozoa – may be motile, “hunters,

grazers”– slime molds – two life cycle stages

– water molds – devastating disease in plants

• fungi – yeast - unicellular– mold - multicellular


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Figure 1.2 Universal Phylogenetic Tree

Origins of Life

• microbial fossils– Swartkoppie chert – granular silica– 3.5 billion years old

• fossil record sparse

• indirect evidence and scientific method are used to study origins of life

Figure 1.5

Last Universal Common Ancestor (LUCA)

• the root or origin of modern life is on bacterial branch but nature still controversial

• Archaea and Eukarya evolved independently of Bacteria

• Archaea and Eukarya diverged from common ancestry

Endosymbiotic Hypothesis

• origin of mitochondria, chloroplasts, and hydrogenosomes from endosymbiont

• mitochondria and chloroplasts– SSU rRNA show bacterial lineage– genome sequences closely related to

Richettsia and Prochloron, respectively

• hydrogenosomes– anaerobic endosymbiont

Historical Perspectives

• Spontaneous Generation. Living organisms can develop from non living matter.

• Until the 17th century this was the primary hypothesis to explain how life arises.


• Francesco Redi (1626-1697) Experimented with decaying meats and showed maggots wouldn’t develop in the meat spontaneously if covered in gauze but flies laid eggs on top of gauze which developed into maggots thus temporarily disproving spontaneous generation.

Antony van Leeuwenhoek (1623-1723) Dutch. First

to observe and describe microoganisms under a microscope.


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Discovery of Microorganisms

• Antony van Leeuwenhoek (1632-1723)– first person to

observe and describe microorganisms accurately

Figure 1.11 (a)


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Figure 1.11 (b) and (c)


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The Conflict over Spontaneous Generation

• spontaneous generation– living organisms can develop from

nonliving or decomposing matter

• Francesco Redi (1626-1697)– discredited spontaneous

generation

– showed that maggots on decaying meat came from fly eggs


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But Could Spontaneous Generation Be True for Microorganisms?

• John Needham (1713-1781)– his experiment:

mutton broth in flasks boiled sealed

– results: broth became cloudy and contained microorganisms

• Lazzaro Spallanzani (1729-1799)– his experiment:

broth in flasks sealed boiled

– results: no growth of microorganisms


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Louis Pasteur (1822-1895)• his experiments

– placed nutrient solution in flasks– created flasks with long, curved necks– boiled the solutions– left flasks exposed to air

• results: no growth of microorganisms

Pasteur disproves spontaneous generation by the famous swan necked flask experiment

Figure 1.13


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Final Blow to Theory of Spontaneous Generation

• John Tyndall (1820-1893)– demonstrated that dust carries

microorganisms– showed that if dust was absent, nutrient

broths remained sterile, even if directly exposed to air

– also provided evidence for the existence of exceptionally heat-resistant forms of bacteria

• Ferdinand Cohn (1828-1898)– heat resistant bacteria could produce

endospores


• Joseph Lister -1867 – Developed the concept of antiseptic surgery. – Instruments are heat sterilized and phenol is used

afterward to eliminate infection.

• Pasteur correlated process of wine making and beer making with yeast by- products

• Winogradsky and Biejerinck showed importance of bacteria in biogeochemical cycling (Carbon, sulfur, nitrogen)

Microbiology in the 20th Century

• Robert Koch (1843-1910) Demonstrated the role of microbes in causing disease during his study of anthrax.

• Koch established the relationship between Bacillus anthracis and anthrax by isolating the organisms from infected animals and injecting them into mice.


• Koch’s Postulates

1. Microbe must be present in every case of the disease but not in healthy animal

2.Suspected microbe must be isolated and grown in pure culture

3.Same disease must result when pure culture inoculated into healthy host

4. Same microbe isolated from infected host

Germ Theory

• Idea that microbes caused disease was not well accepted until the late 1800’s

• Pasteur was attributed to be the father of germ theory although Koch and Lister played major roles

IMMUNOLOGY

Pasteur studied chicken cholera and found that if cultures were incubated for very long periods between transfers they would become

ATTENUATED=NO LONGER ABLE TO CAUSE DISEASE.

When the attenuated form was injected into healthy chickens they became resistant to infection

Pasteur developed other means to attenuate a bacteria via heat treatment or chemical treatment.

Vaccine Production

• The idea of injecting attenuated strains to protect healthy animals or humans from microbial infection was termed vaccination

• Pasteur developed the first vaccine against anthrax and the viral disease rabies.

Vaccination

• The acute contagious disease diphtheria caused by a toxin produced by the bacteria Corynebacterium diphtheriae was the first such disease identified

• Von Behring and Kitasato responsible for identifying and creating a vaccine and antitioxin by injection of healthy animals with inaactivated toxin.


• Diseases such as diphtheria,yellow fever, malaria, were shown to be infections of microorganisms

• Penicillin - 1st antibiotic– discovered by Flemming in 1945

• Waksman’s discovered streptomycin in 1952

Story of Louis Pasteur

1. Struggle of science and pseudoscience2. Pasteurization process for wine3. Story of establishment of germ theory4. Story of scientific development of first

human/animal vaccines, rabies, anthrax5. Story of virus disease and bacterial disease6. Story of antiseptic surgery7. Story of modern culture technique and

microscopy and disease


Microorganisms were used as experimental models for developing the area of genetics and molecular biology

• 1962 - Watson and Crick discovered the genetic material DNA

• 1965 - Jacob and Monod revealed how transcription of DNA is regulated

Important Techniques in Microbiology

• Aseptic (sterile) techniques

• Culture media

• Solid Agar media for pure culture technique

• Microscopy-light microscopy, scanning and transmission electron microscopy


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Microscopy

• microorganisms range in size from the smallest, viruses which are measured in nanometers (nm), to the largest, which are about 200 micrometers (μm).


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Lenses

• focus light rays at a specific place called the focal point

• distance between center of lens and focal point is the focal length

• strength of lens related to focal length– short focal length more

magnification


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The Light Microscope

• many varieties– bright-field microscope– dark-field microscope– phase-contrast microscope– fluorescence microscope– confocal microscope

• are compound microscopes– image formed by action of 2

lenses

Light Microscopy

Microscope’s light path


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Microscope Resolution

• ability of a lens to separate or distinguish small objects that are close together

• wavelength of light used is major factor in resolutionshorter wavelength greater

resolution


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•working distance— distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus

Table 2.2

Bright Field Microscope

• Dark image against light background

• Generally must stain bacteria to increase

• visibility

• Magnification is from 50X-1000X

Preparation and Staining

• Fixation-Process whereby the internal and external structures of the cell and the microorganism itself are fixed in place

• Two types for bacteriology-heat fix and chemical fix.

Simple Stains

Basic Dyes are positively charged and bind to negatively charged macromolecules like nucleic acids. Bacterial surfaces are negatively charged so it basic stains are commonly used.

• Crystal violet• Malachite green• safranin

Simple Stains

Acid Dyes are negatively charged and thus bind to positively charged macromolecules such as calcium rich substances.

• Acid fuchsin

• eosin


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Figure 2.17

Differential staining

• Gram stain

• Acid-fast-Use harsh treatment to allow penetration of dye. Heat, basic fuchsin and phenol. A rinse with acid alchohol. Acid fast cells retain the red fuchsin because of the presence of positively charged molecules. Used to stain mycobacterium


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Acid-Fast Staining

• particularly useful for staining members of the genus Mycobacterium

e.g., Mycobacterium tuberculosis – causes tuberculosis

e.g., Mycobacterium leprae – causes leprosy

– high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics


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Acid-Fast Staining

• particularly useful for staining members of the genus Mycobacterium

e.g., Mycobacterium tuberculosis – causes tuberculosis

e.g., Mycobacterium leprae – causes leprosy

– high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics


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Staining Specific Structures• endospore staining

– heated, double staining technique– bacterial endospore is one color and

vegetative cell is a different color

• capsule stain used to visualize capsules surrounding bacteria– negative stain - capsules may be colorless

against a stained background

• flagella staining– mordant applied to increase thickness of

flagella


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Figure 2.19


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The Dark-Field Microscope• image is formed by light reflected or

refracted by specimen• produces a bright image of the object

against a dark background• used to observe living, unstained

preparations– has been used to observe internal

structures in eukaryotic microorganisms

– has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis


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The Fluorescence Microscope• developed by O. Shimomuram, M.

Chalfie, and R. Tsien• exposes specimen to ultraviolet, violet,

or blue light• specimens usually stained with

fluorochromes• shows a bright image of the object

resulting from the fluorescent light emitted by the specimen

• has applications in medical microbiology and microbial ecology studies


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Figure 2.12


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Table 2.3


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Fluorescence Microscopy

• essential tool in microbiology– fluorochrome-labeled probes, such

as antibodies, or fluorochromes tag specific cell constituents for identification of unknown pathogens

– localization of specific proteins in cells


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Figure 2.13Live dead stain


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Figure 2.7

Transmission Electron Microscopy


Figure 2.22 p 30


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Figure 2.22



• Uses magnetic lenses

• Uses electron beam in place of light

• used for virus and cross sections of cells

• specimen must be stained: osmium tetroxide or phosphotungstate are common stains.

THIOBACILLUS KABOBISFREEZE FRACTURE TECHNIQUE


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Figure 2.24

Scanning Electron MicroscopyScanning Electron Microscopy

• specimen scanned with primary electron beam and sample emits a shower of secondary electrons which can be detected

• Visualizes surfaces or surface structures

Scanning Electron MicroscopyScanning Electron Microscopy

Figure 2.26 p 32

a)Staphylococcus aureusb)Cristospira(spirochete)


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Figure 2.27

Figure2.19 p 28

Parameters used in the phenotypic identification of microbes

• Cell shape

• Cell size

• Colony morphology

• Staining behavior

• Physiological and biochemical characteristics

Cell Shape

• Cocci (spherical)

• Rod

• spirochete

Figure 5.10 p 110

Bacterial Nomenclature

• Bacterial species is defined differently for bacteria than for higher organism where it is based on interbreeding

• Species for bacteria is a collection of strains that share many stable properties and differ significantly form other groups of strains

• A strain is a population of organisms that descends from a single organism or pure culture. Strains only differ slightly from one another

Identification of Bacteria

• Variety of tests are used

• Categorization based on Bergey’s Manual of Determinative Bacteriology

bio 411 master presentation dr. rowe sh 222 x2521, [email protected]

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