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EXAM 1 - Flashcards

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Class:MCB 2610 - Fundamentals of Microbiology
Subject:Biology: Molecular/Cell
University:University of Connecticut
Term:Spring 2013
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What is Microbiology Study of microbes

The study of really small life.

Not all micro organisms are small.
What are microbes?
forms of life too small to be seen with the naked eye (bacteria, fungi, algae, protists).
What does the field of microbiology tell us?
The field examines how microbes interact with humans, with food, and how they can be used BY humans (among other aspects).
2 types of Microbial cell Prokaryotic and Eukaryotic
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Prokaryotic cells

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

Ex: Chloroplasts


SIMPLER and NO MEMBRANE BOUND ORGANELLES.

Eukaryotic cells

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

What is the largest known organism on earth?what is it's width? Oregon Mushroom fungus. 2 miles wide

Fungus Argon mushroom...body grows underground and comes up with mushrooms....called the thallus. it is very old if it is all one organism
Why was the three domain system developed?
developed due to advances in electron microscopy, biochemistry and physiology, and ability to determine nucleic acid and protein sequences
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Carl Woese
Carl Woese et al (1990 paper) based on a comparison of ribosomal RNA, divides microorganisms into domains:
Bacteria (true bacteria), (prokaryotes)
Archaea-Archea are prokaryotes with no mitochondria-simpler cells
Eukarya (eukaryotes)

Carl woese looked at the research that had been done on DNA sequences of the 3 DOMAINS of microorganisms.

Five Kingdom System Robert Whittaker

Aristotle classification Two groups-Plants and animals
Domain Bacteria

Usually single-celled, some are NOT.

  • Majority have cell walls with peptidoglycan: specific to bacteria
  • Most lack a membrane-bound nucleus
Ubiquitous (found everywhere) & some live in extreme environments
  • Some cause disease but most are beneficial
  • Many recycle elements (e.g., Cyanobacteria produce amounts of significant oxygen)
Responsible for most of life on earth because of extensive use of O2 they use & produce

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Domain Archaea

  • distinguished from Bacteria by unique rRNA sequences
  • lack peptidoglycan in cell walls and have unique membrane lipids
  • some have unusual metabolic characteristics (e.g., methanogens which produce methane gas)
  • many live in extreme environments (autoclave)
  • Their role in causing diseases is unclear
  • Archeal GUM DISEASE=only known archael disease

Domain Eukarya

generally larger than Bacteria and Archaea

  • Plants and Animals
  • algae – photosynthetic
  • protozoa – may be motile, hunters, grazers
  • slime molds – two life cycle stages (mold-like, protozoan-like)
  • water molds – protists that grow in moist environments - some cause devastating diseases in plants
  • fungi – (e.g. yeasts – unicellular, molds – multicellular)
  • Primarily decomposers, some cause disease

Viruses Acellular Infectious Agents (not considered part of 3 domain system of classification)
viruses smallest of all microbes
Very simple – some consist of only proteins and nucleic acids
requires host cell to replicate, no metabolic energy needed
cause range of diseases, some cancers
viroids and virusoidsinfectious agents composed of RNA (viroids=pieces of RNA)
prions – infectious proteins
Why are viruses NOT considered to be alive?

  • They dont replicate outside of a host cell.
  • They (usually) have little to no biochemical activity outside of a host cell.
  • They are inert & nonreactive outside of a host cell.
  • Microbiology still studies viruses, though, since they are too small to be seen with the naked eye.

Dont require metabolic activities, dont require energy to carry out processes

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Describe these four virus: Poliovirus, bacteriophage, Ebola Virus, Tobacco mosaic virus
Define Life

Metabolism-take in nutrients, take energy and build new molecules

Growth

Reproduction-divide and multiply

Genetic variation/evolution-pass traits through generations

Response/adaptation to external environment….(thermoregulation, osmoregulation)

Homeostasis (maintaining internal organization & order, usually by expending energy), internal pH, solute concentration

Endospores form from a detrimental event...Antibiotic resistance=circle zone around cell making it sensitive to antibiotic.

Why are viruses not included in the 3 domain system of classification?

Need host cell to live,  dont meet all classifications to life. Theyre genetically removed from the 3 domains.

Because the way domain systems are devised, they look at subunit ribosomal DNA. Viruses don't have RNA...because Viruses don't have ribosomes.
  
What are macromolecules (major building blocks) needed for life? (4) Polypeptides: Amino Acids

Nucleic acids: Deoxyribonucleotides and ribonucleotides

Lipids: Diverse structures

Polysaccharides: sugars
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Polypeptides FXN
Amino Acids
Enzymes catalyze the vast majority of biochemical reactions in the cell. Other proteins are structural components of cells

55% of dry weight of cell
Nucleic Acids FXN
Deoxyribonucleotides and ribonucleotides
    Deoxy: Informational: DNA provides instructions for assembly & reproduction of cell  =  2 - 5% of dry weight of cell
    Ribo: Many FXNS, most of which are involved in production of polypeptides. Some serve structural or catalytic FXNS
    =  15 - 20% of dry weight of cell
    >Deoxy, ribo=critical role as storehouses of genetic info.
    >Comparing of DNA sequences allow us to break life into 3 domains--by looking at ribosomal frequency
    LIPIDS FXN
     Diverse structures
    Structural: make up cellular membranes that form physical boundary between the inside of cell and surroundings and membranes of internal organelles

    = 10% of dry weight of cell
    Polysaccharides FXN
    sugars

    Structural (such as cellulose and Chitin) and energy storage (such as glycogen and starch)

    = 6 - 7% of dry weight of cell
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    Most important FXN of polypeptides the function of enzymes as catalysts of chemical RXNS
    Polypeptide: RNA polymerase Location: Cytoplasm of bacteria and archaeons, nucleus of eukarya

    FXN: fxns as an enzyme that produces RNA molecules from DNA template.
    Polypeptide: Glycogen phosphorylase Located in CYTOPLASM

    FXN: Conversion of glycogen into glucose monomers
    Polypeptide: K+ channel Located in PLASMA MEMBRANE

    FXN: Passive transport of K+ across the membrane, from an area of high concentration to an area of low concentration.
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    Polypeptide: Na+/K+ ATPase Located in the PLASMA MEMBRANE

    FXN: Active transport of Na+ and K+ across the membrane, from areas of low concentration to areas of high concentration.
    Polypeptide: Flagellin Located in BACTERIAL FLAGELLUM

    FXN: monomers polymerize to form flagellum which aids in bacterial motility.
    Polypeptide: FtsZ Location: Associated with plasma membrane of bacteria

    FXN: Key component of cell division machinery.
    Enzyme

    Substrate

    Active Site
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    what forms a cell's plasma membrane?

    Polysaccharides and polypeptides can be embedded in a lipid bilayer, forming a cells plasma membrane. This separates the external environment from the interior of the cell.

    Polypeptide/polysaccharide membrane
    What can studying the genetics of microbes teach us about the evolution of life on Earth?
    The very early environment on Earth was drastically different than it is today.
    There was little oxygen in the atmosphere, and the surface of the planet was a soup of chemicals in liquid form.
    This early atmosphere and environment led to the initial synthesis of the first forms of macromolecules (and their use in primitive single-celled life).
    MICROBIAL GENETICS
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    How do we age earth and how old is earth predicted to be?

    Through radioisotope analysis Earth is thought to be between 4.5 to 4.6 billion years old.


    microbial fossils: Swartkoppie chert – granular silica – fossilized mats of microbes3.5 billion years old
    fossil record sparse; indirect evidence and scientific method are used to study origins of life--visually not changed from then to now
    Stromatolite layers of cyanobacteria and minerals    
    STROMATOLITE
    How did the first microbial life arise?
    In the 1950s, a grad student named Stanley Miller formed organic molecules from a primordial soup.
    --Saw clear liquid turn cloud then red--seeing amino acids
    --Wanted to see what earth was going to look like in the beginning.
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    Stanley Millers Set up
    Tools Used for studying Microorganisms
    microscopes
    culture techniques
    molecular genetics
    genomics
    Discovery of Microorganisms Robert Hooke
    Robert Hooke (1635-1703) first to discover eukaryotic tree cells
    Looking at a thin slice of cork with a crude microscope
    Life consisted of little boxes – cells
    •Developed Cell theory – all living things are composed of cells
    Discovery of Microorganisms Leeuwenhoek

    Antony van Leeuwenhoek (1632-1723)

    first person to observe and describe microorganisms accurately with a microscope that could magnify 
    50 – 300 times
    Called microbes animalcules
    Studied his teeth scrapings and diarrhea
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    Leeuwenhoek's microscope
    Leeuwenhoek describing GIARDIA

    I weighed about 160 pounds…and I ordinarily a-morning have a well-formed stool, but now and then hitherto I have had a looseness, at intervals of 2, 3 or 4 weeks, when I went to stool some 2, 3 or 4 times a day… My excrement being so thin, I was at diverse times persuaded to examine it…I will only say that I have generally seen, in my excrement, many irregular particles of sundry size.  All particles aforementioned lay in a clean and transparent medium wherein I have sometimes also seen animalcules a-moving very prettily.  Their bodies were somewhat longer than broad, and their belly, which was flattened was furnished with sundry little paws, wherewith they made such a stir in the medium and among the globules.

    Archaezoa
    No mitochondria
    Multiple flagella
    Giardia lamblia
    Trichomonas vaginalis (no cyst stage)
    Giardia=proteosome 
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    spontaneous generation theory and who discredited the theory?

    Spontaneous generation: living organisms can develop from nonliving or decomposing matter, thought maggots came from nothing.

    The Conflict over Spontaneous Generation

    Francesco Redi (1626-1697) discredited spontaneous generation

    –showed that maggots on decaying meat came from fly eggs

    –His experiment – 3 containers with meat, one covered with paper, one covered with gauze (NO maggots), one open (got maggots)

    Biogenesis=one living thing must produce another living thing.

    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
    Louis Pasteur (1822-1895) disproved spontaneous generation?
    Settled the matter of spontaneous generation in 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
    Biogenesis – Life comes from life
    Louis Pasteur's experiment: a simple yet elegant experiment to disprove spontaneous generation theory in the late 1800's

    Realized the experiment was getting contaminated by air.

    Pasteur filtered air through a cotton plug and then place the cotton in broth. He found the broth became cloudy...Why? 
    ---Micro organisms in cotton that came from air contaminated the broth.
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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
    The Role of Microorganisms in Disease - Germ Theory
    was not immediately obvious

    infectious disease believed to be due to supernatural forces (miasmas – bad vapors)

    establishing connection depended on development of techniques for studying microbes
    Evidence for the Relationship between Microorganisms and Disease
    Agostini Bassi (1773-1856)showed that a disease of silkworms was caused by a fungus *critical finding

    M. J. Berkeley (ca. 1845)demonstrated that the great Potato Blight of Ireland was caused by a water mold

    Heinrich de Bary (1853)showed that smut and rust fungi caused cereal crop diseases
    Evidence for the Relationship between Microorganisms and Disease-Louis Pasteur
    Louis Pasteur (1822-1895)
    demonstrated that microorganisms carried out fermentations
    developed pasteurization – low temperature heating to destroy unwanted microbes in wine and milk – used to reduce spoilage bacteria and kill harmful bacteria – heated to 145F(63C) for 30 min. then cooled, now 161F (72C) for 15 sec
    showed that pébrine disease of silkworms=caused by protozoan
    Pasteurization: heating process to kill organisms and bring back down to cool temperature and inoculate it.
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    Evidence for the Relationship between Microorganisms and Disease-Semmelweis & Lister
    I. Semmelweis (1840s) high incidence of purpural fever in obstetrical patients could be reduced by washing hands (autopsy source)
    J. Lister (1827-1912) provided indirect evidence that microorganisms were causal agents of disease
    developed a system of surgery designed to prevent microorganisms from entering wounds as well as methods for treating instruments and surgical dressings using phenol
    his patients had fewer postoperative infections...1st to use PHENOL
    John Snow – 1st Epidemiologist 1854 Cholea outbreak
    Soho neighborhood of London   500 died in 10 days  
     Mapped affected areas    --Caused by microorganism Briocholera--contaminated the water.
    Centered on Broad Street well – sewer pipe was leaking into well
    Convinced officials to remove pump handle
    Interestingly people who worked at a local brewery were unaffected.
    Final proof that microbes cause disease…

    Robert Koch (1843-1910)

    Established relationship between Bacillus anthracis & anthrax
    Used criteria developed by teacher Jacob Henle (1809-1895)
    these criteria now known as Kochs postulates--four determining factors to see if an organism is a causative agent.
    still used today to establish the link between a particular microorganism and a particular disease
    What did Koch discover in terms of the causes of Anthrax and how did this help him
    Robert Koch determined Bacillus anthracis and Mycobacterium tuberculosis were the causes of anthrax and tuberculosis (respectively).
    His work with anthrax helped sheep herders and cattle ranchers avoid costly animal losses.
    The basic rules Koch established made it possible for others to determine which microbes caused which diseases. They are still in use to this day.  HIS LAB DISCOVERED PETRI DISHES
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    Koch’s Postulates (guidelines)
    the microorganism must be present in every case of the disease but absent from healthy individuals
    the suspected microorganism must be isolated and grown in a pure culture
    the same disease must result when the isolated microorganism is inoculated into a healthy host
    the same microorganism must be isolated again from the diseased host
    Limitations of Koch’s Postulates (guidelines)
    some organisms cannot be grown in pure culture (e.g. Mycobacterium leprae)
    using humans in completing the postulates is unethical (e.g. Ebola hemorrhagic fever)
    molecular and genetic evidence may be used instead 
    What theory explains when/how eukaryotes appeared
    Endosymbiotic theory: Primitive prokaryotic microbes ingested other microbes, starting a symbiotic relationship, forming the first basic eukaryotes.
    Ingested microbes that could use oxygen for a respiratory process to produce chemical energy became mitochondria.
    Ingested microbes that could fix carbon dioxide into organic molecules using light energy became chloroplasts.
    Endosymbiotic Hypothesis

    • Mitochondria, hydrogenosomes, and chloroplasts are all thought to have evolved from bacterial cells that invaded or were ingested by early ancestors of eukaryotic cells
    • Mitochondria and chloroplasts are very similar to extant bacteria and cyanobacteria, respectively
    • Both mitochondria and chloroplasts contain bacterial DNA and ribosomes
    • Both are similar based on SSU rRNA

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    The Development of Techniques for Studying Microbial Pathogens
    Kochs work led to discovery or development of:
    Agar (Fanny Hess): adds to medium in order to solidify polysaccharides.
    Petri dish (Richard Petri)
    nutrient broth and nutrient agar
    methods for isolating microorganisms
    Aseptic technique
    Advantages of using Agar over gelatin as a solidifying agent
    Not broken down by most organisms

    Doesnt melt until it reaches 100C

    Doesnt solidify until it reaches 50C

    Produces a clear product
    Some organisms can degrade gelatin 
    methods for studying viruses (Chamberland)

    Charles Chamberland (1851-1908): developed porcelain bacterial filters used by Ivanoski and Beijerinck to study tobacco mosaic disease

    • determined that extracts from diseased plants had infectious agents present which were smaller than bacteria and passed through the filters
    • infectious agents were eventually shown to be viruses
    • Called filterable particles”: b/c of their nature of going through that porcelain filter.

    methods for studying viruses (Pasteur and Roux)
    Pasteur and Roux
    discovered that incubation of cultures for long intervals between transfers caused pathogens to lose their ability to cause disease – could be used to develop vaccines
    Pasteur and his coworkers
    developed vaccines for chicken cholera, anthrax, and rabies
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    Magic Bullet
    Paul Ehrlich (1910) – German Physician
    1st chemotherapy, synthetic drug salvarsan, arsenic derivative, offered salvation from syphilis
    Famous Microbial Disease
    Some microbial diseases have had a profound impact on humanity—e.g., plague=black death killed 1/3 population of the world.
    HOW THE PLAGUE SPREAD
    In 20th century=dramatic drop in U.S. deaths from infectious diseases. How is this possible?
    Where has this reduction in deaths come from?
    Prevention of infection through
    Use of antiseptics (Joseph Lister)
    Sanitation improvements (sewage treatment)
    Food/water safety (pasteurization)
    Personal hygiene improvements
    Vaccination
    Treatment of infections (antibiotics!)
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    Smallpox Virus
    Koch’s Postulates applied to tuberculosis
    Immunological Studies
    once established, led to study of host defenses - immunology
    Edward Jenner (ca. 1798)
    used a vaccination procedure to protect individuals from small pox
    Milkmaid with cowpox was immune
    Tested on 8 year old volunteer
    This preceded work establishing role of microorganisms in disease.
    Small pox caused by virus, chinese would take scabs and put them in people's noses and they would get some type of immunity.
    The Birth of Modern Chemotherapy
    1928: Alexander Fleming discovered the first antibiotic.
    He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus.
    1940s: Penicillin was tested clinically and mass produced.

    The colony of penicillia and nothing grew around fungal penecillin...showing an inhibition complex within penecillin.
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    What were the earliest molecules?
    original molecule must have fulfilled structural, catalytic, and hereditary functions (these activities are now performed by proteins, DNA, RNA)--if you cut yourself, you can seal a cut.
    ribozymes – Discovered by Thomas Cech in 1981
    RNA molecules that form peptide bonds
    perform cellular work and replication
    earliest cells may have been RNA surrounded by liposomes 
    Ribozymes

    Some RNA molecules have the ability to catalyze reactions (these are known as ribozymes, a combination of ribonucleic acid and enzymes)


    This means RNA could serve the dual purpose of genetic information storage AND catalyzing reactions!

    How Rybozymes worked
    But what about separating interior from exterior?
    A single lipid layer known as a micelle may have been an early form of plasma membrane. (can form spontaneously)
    This could have formed a crude way of separating interior contents from the external environment.

    Micelles=LIPOSOMES that form spontaneously covered RNA molecules.
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    Micelle
    So then, the basic idea of how microbial life arose on Earth is:
    Early conditions formed RNA and micelles.

    These came together into a primitive cell using RNA for storing genetic info and coding.

    Primitive cells eventually changed from using RNA to DNA instead for storing their genetic information.
    So then, the basic idea of how microbial life arose on Earth is:



    Endosymbiotic
    Hypothesis    
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    Microbial SPECIES

    eukaryotic species are animals or plants that naturally interbreed
    Bacteria & Archaea DONT reproduce sexually & are referred to as strains

    • Strain consists of descendents of single, pure microbial culture – share stable properties
    • may be biovars, serovars, morphovars, pathovars

    binomial nomenclature – developed by Carolus Linnaeus (1750)

    • genus and species epithet
    • (e.g., Escherichia coli – italics or underline)
    • Can abbreviate after first use (e.g. E. coli)

    What accounts for differences in the rate a disease spreads?
    Humanitys interaction with infectious diseases is always changing.
    Sometimes cultural/economic differences can mean differences in disease rates (lack of malaria in the United States versus Africa).

    Microbes live in parasites and they cause disease....some microbes can develop resistance to antibiotics.
    How old are microbial fossils?

    • Multicellular fossils dating to about 0.5 billion ybp (years before present) have been found—meaning microbes dominated the planet for approximately 3.5 billion years!
    • Some microbial fossil records do exist, largely in fossilized mats discovered in Australia.-Stromatolites

    Fossilized Cyanobacteria
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    Fossilized Stromatolites
    -Live in australia
    Of all the microbes we can grow in the lab, its estimated there are many more that cant be grown.
    Early microfossils similar to modern filamentous cyanobacteria
    About 3.5 billion years old
        

    Endosporeè2nd photo….go into dormant stage when conditions aren’t right

    Circle around the dot in green, means that that organism is sensitive to the antibiotic


    If it is clear, it is affected....they can modify to survive.

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    Are Microorganisms good or bad? Depends on their environment w/o them, we could not exist.

    Ex: food born pathogens....very few are pathogens and 99% are beneficial to the environment, they turn back oxygen we need.
    Proteins can be Enzymatic and structural
    Most important FXN of polypeptides Catalysts of chemical rxns.
    Ignaz Semmelweis story Doctor had two clinics...one doctors the the other physicians, the physicians would do an autopsy and not wash hands before delivering babies. there were more deaths at physician offices than midwives.
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    Why are pure cultures critical to koch's postulates To know which organism causes the disease.
    Elemental makeup of important cell molecules Proteins:        CHONS
    Lipids:           CHOP
    Carbohydrates:    CHO
    Nucleic Acids:     CHONP
    Microscopy

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

    Lenses and the Bending of Light

           light is refracted (bent) when passing from one medium to another

           refractive index      a measure of how greatly a substance slows the velocity of light

           direction and magnitude of bending is determined by the refractive indices of the two media forming the interface (i.e., glass and air)

           Glass has a higher refractive index than air

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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

    How lenses bend light

    (F = focal point, f = focal length)

    The strength of the lens is related to the focal length

    short focal length Þmore magnification

                Refracted- bent as comes away and focused on focal point- where specimen should be if shorter little f, the higher magnification

     

    Types of Light Microscopes (5)

           Many varieties

          bright-field microscope

          dark-field microscope

          phase-contrast microscope

          fluorescence microscope

          confocal microscope

           Modern microscopes are all compound microscopes

          image formed by action of ³2 lenses

    The Bright-Field Microscope

           routinely used in a microbiology lab

           produces dark image against brighter background–used for both stained & unstained specimens (staining improves contrast)

           has several objective lenses

    -- parfocal microscopes remain in focus when objectives are changed

           total magnification product of the magnifications of the ocular lenses and the objective lenses

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    Microscope Resolution

    Resolution (or resolving power) - ability of a lens to separate or distinguish small objects that are close together

           The wavelength of light used is a major factor in resolution

    shorter wavelength ==> greater resolution

    (blue light 450 – 500 nm can not resolve structures smaller than 0.2 um)- Shorter wavelength

     

    Microscope terminology

           Numerical aperature – ability of the lens to gather light – depends on the property of the lens and on the refractive index of the medium

           Refractive index – how much a substance bends a light ray (staining a specimen changes its refractive index allowing for better contrast)

           The refractive index of air is 1.00 if we increase this by using immersion oil, we can increase the numerical aperature- lenses to gather light

     

    Numerical Aperature

    Smaller working distances give better resolution – can better separate close objects because the light spreads out more

    Shorter focal length

    Working distance slide to lens

    Right has higher magnification

    Theta- angle light have to go through, theta small doesn’t separate angle far,

    Right two points separate farther away- better resolving power 

    Using Immersion oil to increase refractive index

    If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so. This results in an increase in resolution and numerical aperture.

    -Higher objective- put oil- going to help light goes through goes air to glass, glass air then glass, oil will funnel light rays into lens then going out into air 

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    Working distance versus resolving power

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

    -Resolving power – ability to distinguish close objects as separate

    What would be magnification 10 ocular lens and 40x be? 400
    The Dark-Field Microscope

            image is formed by light reflected or refracted by specimen and 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

        been used to identify bacteria (Treponema pallidum)=causative agent of syphilis

           organisms that are alive- motile

    Uses a hollow cone of light so that only light that has been reflected or refracted by 

    specimen enters lens- solid disk, allows specimen 2b bright & background 2b dark

    What is the most important property of the lens?

    Resolution*** resolving makes it clearer*******

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    What does it mean when a microscope has a resolution of .2 um?

    Objects need to be at least .2 um apart to see as two separate object 

    The Phase-Contrast Microscope

    -Converts slight differences in refractive index & cell density into easily detected variations in light intensity.  Uses hollow cone of light

    -Hollow cone of light passes through specimen & is retarded (out of phase). Light then passes through phase plate which advances it & brings light back into phase.

    -Produces image where cells are dark & background is light


    -Excellent way to observe unstained, living cells: studying microbial motility    &   detecting bacterial structures such as endospores & inclusion bodies that have refractive indices different from that of water 

    Why is it sometimes important to view stained organism?
    The Differential Interference Contrast Microscope (DIC)

     Similar to phase-contrast - creates image by detecting differences in refractive indices and thickness of different parts of specimen

    Uses two beams of polarized light to create a

    3-dimensional image of the specimen: Excellent way to observe living cells:

           live, unstained cells appear brightly colored & three-dimensional

           cell walls, endospores, granules, vacuoles, nuclei =clearly visible

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    Why don’t most light microscopes use 30x ocular lenses for greater magnification?

     -because resolution- need to also increase resolution 

    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 (fluorescent dyes)

            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

            Uv light- light hits object and it florescent- change in wave length as comes back to eye

    Epifluorescence Microscopy

    Longer wave length comes back to your eye 

    Immuno-Fluorescence

            essential tool in microbiology

           fluorochrome-labeled probes, like antibodies, or fluorochromes tag specific cell constituents for identification of unknown pathogens

           localization of specific proteins in cells-Put tags on organisms & use as diagnostic tool

           Isolate antibodies & attach fluorochrome to it. Only bind to antigen. If have specimen w/ no tepenima(spelling) on it then it won’t bind.

     

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    What are the advantages of brightfield, darkfield, phase contrast DIC and flourescence microscopy How similar? How different?
    Cells stained with fluorescent dyes

    Living cells (green) dead cells (red), Streptococcus pyogenes (antibody staining)

    Other uses for fluorescence microscopy

            Photosynthetic organisms naturally fluoresce when excited with specific wavelengths.

            Can also be used to localize specific proteins within cells

           One method is to fuse the gene of the protein of interest to the jellyfish (Aequorea) protein, green fluorescent protein (GFP)

    Green Fluorescent Protein fused with Mbl cytoskeletal protein of Bacillus subtilis

    Rod- has protein scaffolding that forms a helix, holds cell into rod shape. Take gene gfp infused with pseudo-skeleton protein and is expressed with a tag on it



    Do all specimen examined by fluorescence microscopy need to be stained with fluorochrome? No some are naturally fluorescent

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    Confocal Microscopy

            confocal scanning laser microscopy (CLSM) creates sharp, composite   3-D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface

            Specimen is usually fluorescently stained

            numerous applications including study of biofilms

            Want to get rid of biofilm? Flush with cl. See if killed anything stain it with fluorescent stain, dead cells red. Cells underneath still living

            Confocal Microscope uses laser light, an aperature eliminates stray light

    Preparation and Staining of Specimens

            increases visibility of specimen

            accentuates specific morphological features

            preserves specimens

    Fixation

            preserves internal and external structures and fixes them in position

            organisms usually killed and firmly attached to microscope slide

            heat fixation – routinely used with bacteria & archaea

           preserves overall morphology but not internal structures, inactivates enzymes & can destroy some proteins

            chemical fixation – used w/ larger, more delicate organisms

           protects fine cellular substructure & morphology

           Inactivates, makes insoluble & immobilizes, proteins & lipids (e.g., ethanol, acetic acid, formaldehyde & glutaraldehyde).

    Dyes and Simple Staining: Dyes

            make internal and external structures of cell more visible by increasing contrast with background

            have two common feature

    chromophore groups

    1.         chemical groups with conjugated double bonds
    2.            give dye its color

    ability to bind cells

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    ionizable dyes have charged groups

            basic dyes have positive charges that bind to negatively charged molecules (e.g., nucleic acids, many proteins, and cell surfaces) – methylene blue, basic fuchsin, crystal violet, safranin, malachite green

            acid dyes have negative charges that bind to positively charged cell structures but are repelled by the cell surface (produces negative staining, nigrosin, good for seeing capsules) – eosin, rose bengal acid fuchsin

    • simple stains

           a single stain is used (easy and quick)- one stain

           can determine size, shape, and arrangement of bacteria


    Crystal violet or methylene blue

    Differential Staining

            divides microorganisms into groups based on their staining properties

           e.g., Gram stain

           e.g., acid-fast stain

            differential stain also used to detect presence or absence of structures

           endospores, flagella, capsules

    -Gram stain most common  

    Gram Staining

           most widely used differential staining procedure

           Developed by Christian Gram, 1884

           divides bacteria (but not archaea) into two groups, Gram positive and Gram negative, based on differences in cell wall structure

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    Gram Stain Steps

    1)  Make a smear of a bacterial suspension on a glass slide

    2) Heat fix by passing through a flame

    3)  Stain with primary stain – Crystal Violet, let stand for 1 minute, rinse with water – (all cells stain)

    4)  Treat with iodine for 1 minute (a mordant which binds with the CV to make a large crystal inside the cell), rinse with water

    5)  Decolorize with alcohol to remove the stain if it is a Gram neg. organism.  Stain can’t be removed in a Gram pos. organism due to thick cell wall peptidoglycan          6)  Counter stain with Safranin

    Colors of gram positive and negative

    Gram positive cells are purple, Gram negative cells are pink

    What step do you think is critical of Gram stain?

    Alcohol decolorizer- if too short not going to remove crystal violate 

    What step can be left out?

    Leave out safranin step so can see that gram postiive  cells purples and gram negative clear 

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    Acid-Fast Staining

           particularly useful for staining members of the genus Mycobacterium (which don’t stain well with the Gram stain)

    e.g., Mycobacterium tuberculosis – causes tuberculosis

    e.g., Mycobacterium leprae – causes leprosy

          high lipid content in cell walls (mycolic acid) prevents dyes from readily binding

    - Uses high heat and phenol to drive basic fuchsin into the cells

     

    Differential Staining of Specific Structures

    Endospore staining – exceptionally resistant to staining (e.g. Bacillus sp. & Clostridium sp.)------Heated, double staining technique-----Bacterial endospore =1 color & vegetative cell=different color

    Capsule stain- visualize capsules surrounding bacteria (India ink or nigrosin)

    --Negative stain - capsules may be colorless against a stained background

    Flagella staining – very thin & can only be seen with an electron microscope

    --Uses a mordant and a stain.  The mordant is used to increase thickness of flagella

                     --Acid fast positive- red 

    Electron Microscopy

            The best light microscope has a resolving limit of 0.2um (max. mag of 1500X)

            In the electron microscope electrons replace visible light as the illuminating beam (resolution of 0.5 nm, max mag of 100,000X)- Electrons replace visible light resolution of 0.5 nanometers

            wavelength of electron beam is much shorter than visible light, resulting in much higher resolution

            allows for study of microbial morphology in great detail

    Limits of Resolution

    Different kinds of ranges, pox virus, poliovirus 

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    Light vs Electron Microscopy
    Rhodospirillum rubrum

    -refer to slides

    Left- light microscope can see the shape

    Structure- left use electron microscope

    The Transmission Electron Microscope (TEM)

           Uses an electromagnet to focus an electron beam

           electrons scatter when they pass through thin sections of a specimen

           transmitted electrons are under vacuum which reduces scatter and are used to produce clear image

           denser regions in specimen, scatter more electrons and appear darker

    -view on a screen

    -Can’t be anything that’s alive 

    Comparison of Light Microscope and TEM

    Comparison of Light

    Microscope: Lamp, uses glass lenses to focus light rays

    TEM: Electrons - Image going to be on a grid

    Specimen Preparation for TEM

           analogous to procedures used for light microscopy

           for transmission electron microscopy, specimens must be cut very thin

           specimens are chemically fixed and stained with electron dense materials, such as heavy metals, that differentially scatter electrons

           -Dehydrate them and heavy metal stain

           Sometimes get artifacts

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    Other TEM Preparation Methods • negative stain

          heavy metals do not penetrate the specimen but render dark background

          used for study of viruses, bacterial gas vacuoles

    Other TEM Preparation Methods shadowing

          coating specimen with a thin film of a heavy metal only on one side

          useful for viral morphology, flagella, DNA

    Other TEM Preparation Methods freeze-etching

          freeze specimen then fracture along lines of greatest weakness (e.g., membranes)

          allows for 3-D observation of shapes of intracellular structures- can look at certain structures

          reduces artifacts

    Disadvantages of TEM

           Electrons can only penetrate very thin specimens

           Usually gives only 2D image

           Specimens must be viewed under high vacuum

           Specimens are dead

           Because of harsh treatment (dehydration, etc) specimens are shrunken and distorted (artifacts)

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    Electron microscopes have better resolution. why?- Shorter wave length 
    The Scanning Electron Microscope

           uses electrons reflected from the surface of a specimen to create detailed image

           produces a realistic 3-dimensional image of specimen’s surface features

           can determine actual in situ location of microorganisms in ecological niches (e.g. human skin, gut, etc) – samples must be dehydrated and coated with a thin film of metal

     

    Electron Cryotomography

            since 1990s, rapid freezing technique has provided way to preserve native state of structures examined in vacuum

            images are recorded from many different directions to create 3-D structures 

            provides extremely high resolution of       cytoskeletal elements, magnetosomes, inclusion bodies, flagellar motors, viral structures

    -If freeze quickly will preserve structures better, and don’t allow artifacts to form can get an image more true to image of what would actually find

    Scanning Probe Microscopy •scanning tunneling microscope

           scanning tunneling microscope (1980)

          magnification 100 million times, can view atoms on surface of a solid

          steady current (tunneling current) maintained between microscope probe and specimen

          up and down movement of probe as it maintains constant current is detected and used to create image of surface of specimen

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    Two different kinds of scanning probe microscopy

           scanning tunneling microscope (1980)



           atomic force microscope

    Scanning Probe Microscopy atomic force microscope

          sharp probe moves over surface of specimen at constant distance

          up and down movement of probe as it maintains constant distance is detected and used to create image

          Can be used to study surfaces that do not conduct electricity well

          Tip measures fluctuations using the computer 

    Atomic Force Microscopy Aquaporin membrane protein

    -Protein in bacterial cell membranes 

    What type of microscope has the lowest magnification?

    - light microscope

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    What type of microscope has highest? standing probe microscope
    Nutritional requirements of microorganisms:• What do microorganisms generally need to grow?

           Macronutrients - All cells need access to large amounts of carbon, nitrogen, phosphorus, sulfur, and oxygen to build macromolecules.


           Micronutrients - are also required by microbes:

           Includes several metal ions (Na+, Mg2+, Mn2+, etc.)

           Often required for protein structure/activity, biosynthesis of ATP by electron transport-related processes

    Micronutrients in depth

    micronutrients (trace elements)……Mn, Zn, Co, Mo, Ni, and Cu=required in trace amounts often supplied in water or in media components

    ubiquitous in nature- found everywhere èserve as part of enzymes and cofactors- without enzymes wouldn’t function normally

    Aid in catalysis of reactions and maintenance of protein structure

    Unique substances

           some organisms also require unique substances such as silicic acid used to construct the silica walls of diatoms

           No matter what an organism’s nutrient requirement they require a balanced mix and if one nutrient is limited or in short supply the organism will have limited growth

     

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    Elements of Life are important in enzyme function and electron transport.
    All organisms require Carbon, Hydrogen, Oxygen, and Electrons

           carbon is backbone of all organic components present in cell

           hydrogen and oxygen are also found in organic molecules

           electrons play a role in energy production used for cellular work and reduction of CO2 to form organic molecules- electrons have metabolic activity and transfer electrons 

    • Acquisition of nutrients

           Autotrophs assimilate carbon from inorganic sources.  

           Heterotrophs assimilate carbon in preexisting organic form.

    • Energy from molecules

           breaking down Chemicals or molecules for energy- chemotrophs

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    • Use light as energy-plants

           cynobacteria-mechanism to take energy from light phototroph

    • Organic molecules organotrophs    
    • Inorganic molecules

           lithotrophs- rock eaters

           Carbon from organic materials 

    • Organism that is a pathogen to body

           chemoorganoheterotroph- get nutrients from using organic molecules inside body 

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    • Acquisition of energy (WHERE)

          Phototrophs capture light energy to produce ATP.

          Chemotrophs capture energy from oxidation of reduced organic or inorganic compounds.

    • Electron sources
    Microorganisms May Change Nutritional Type

           some organisms have great metabolic flexibility and alter their metabolism in response to environmental changes (e.g., when oxygen is limiting they can switch their metabolism to use light as a source of energy)- can feed broad source of nutrients

           This provides distinct advantages to organisms that experience frequent changes in environmental conditions

    Microorganisms also have an absolute Requirement for Nitrogen, Phosphorus, and Sulfur

           These elements are needed for synthesis of important molecules (e.g., amino acids, nucleic acids)

    -Amino acids in proteins, proteins composed of 20 amino acids, dna, rna, need to find a way to get nitrogen 

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    Nitrogen

           nitrogen is needed for amino acids, purines, pyrimidines, some carbohydrates, lipids, enzyme cofactors etc.

           supplied in numerous ways such as metabolism of amino acids, nitrates, ammonia, and some fix N2 from atmosphere

    -Extracts ex yeast, digest organisms 

    Phosphorus and Sulfur

    Phosphorus=needed for nucleic acids, phospholipids, nucleotides, some cofactors, some Proteins---usually supplied as inorganic phosphate

    ·       most organisms use inorganic phosphorus which=directly incorporated into cells

    Sulfur=needed for amino acids cysteine & methionine, some carbohydrates, biotin & thiamine------usually supplied as sulfate via assimilatory sulfate reduction

            most organisms use sulfate and reduce it by assimilatory sulfate reduction

            Need to know exact source of nitrogen...in an extract you dont know what's in there.

    Growth Factors

          have to know exactly what they prefer
     
    Some organisms can synthesize all organic molecules from a single carbon source and inorganic salts, but some require growth factors to support growth

           Growth factors are organic compounds that cannot be synthesized by an organism but are essential for growth

           must be supplied by the environment if the cell is to survive and reproduce 

    There are 3 Major Classes of Growth Factors

    amino acids needed for protein synthesis If you mutate an AA, it cant produce vitamin C...we get vitamin C from food b/c cant make it.

    purines and pyrimidines needed for nucleic acid synthesis

    vitaminsSmall organic molecules that function as enzyme cofactors, needed in very small amounts

    Other less common growth factors include heme (cytochromes, Haemophilus influenzae) & cholesterol (required by some mycoplasmas) need heme by adding blood on blood agents.Wild type ecoli doesnt need GF, makes everything itself, just needs food

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    • Why is it so difficult to grow some organisms in a lab culture?

    Don’t always know what they need to grow, nutritional requirements, pH temp…

    Factors affecting microbial growth: Nutrient concentration

           Nutrient concentration

          Growth rate will depend on the amounts of nutrients in the environment.

          One key nutrient, available in the lowest amount, will dictate how much growth can occur over time (i.e., it will be a limiting factor).

    Low nutrients concentration=grow slower. if you limit N, no matter what they will be limited.

    Factors affecting microbial growth:Slope- rate of growth,

           Effects of oxygen on microbial growth


    Slope=RATE OF GROWTH

    • Effects of oxygen on microbial growth

            Aerobes grow in the presence of oxygen.

           Obligate aerobes REQUIRE oxygen.- if too much then it is toxic

           Microaerophiles grow best when there is less oxygen than normal.

            Anaerobic growth occurs without oxygen.

            Aerotolerant anaerobes aren’t harmed by oxygen but don’t use it, either.

            Obligate anaerobes cannot grow when oxygen is present.

            Facultative anaerobes CAN use oxygen but can also grow in absence of oxygen.

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    Types of Aerobes and Anaerobes based on oxygen requirements.

           Obligate- absolute need for oxygen grow top of test tube

           Faculative- grows better in the presence of oxygen

           Tolerant- not required oxygen but doesn’t use oxygen

           Strict- only gown with no oxygen, O2 poisoness

            micro- toxic to too much o2 

    Factors affecting microbial growth:

           Effects of oxygen on microbial growth

          Often determined by what defenses are available against oxygen’s negative effects in the cell- Oxygen terminal electron receptor when we add in, produces free radicals 

    Basis of Different Oxygen Sensitivities

           oxygen easily reduced to toxic reactive oxygen species (ROS)

          superoxide radical

          hydrogen peroxide

          hydroxyl radical

           aerobes produce protective enzymes

          superoxide dismutase (SOD)

          catalase

          peroxidase...helps fight against accumulation of free radicals

    Oxygen toxicity and how it reacts in the body

    Reactive Oxygen Species

            O2 + e-   à      O2- (superoxide radical)

            O2- + e- + 2H+  à           H2O2 (hydrogen peroxide)

            H2O2 + e- + H+  à       H2O + OH (hydroxyl radical)

    Enzymes that protect against toxic  O2 products

            2O2- + 2H+  superoxide dismutase  O2 + H2O2

            2H2O2 catalase 2H2O + O2

            H2O2 + NADH + H+ peroxidase 2H2O + NAD+

    Put hydrogen peroxide on cut, bubbles b/c catalase in open wound to detoxify

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    Strict Anaerobic Microbes

           all strict anaerobic microorganisms lack or have very low quantities of

          superoxide dismutase

          catalase

           these microbes cannot tolerate O2

           anaerobes must be grown without O2

          work station with incubator

          gaspak anaerobic system

    Growth of strict anaerobes in culture

           Growth in an anaerobic culture medium containing reducing agents (thioglycollate or cysteine)

           Work in an anaerobic chamber

           GasPak jar

           Plastic bag or pouch system

    Packet generates H and CO2, Strip- knows the concentration of gas, will turn color when all O2 is used. An Anaerobic Workstation

    The GasPak Anaerobic System


    Gas generator envelope removes O2

    THese elements are needed for synthesis of important molecules like amino acids and nucleic acids N P S 
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    Factors affecting microbial growth: pH-• Effects of pH on microbial growth

          pH affects macromolecule structures and transmembrane electrochemical gradients.

          Each microbe will have an optimal pH range for growth.- there are not going to be the same organisms living in all these conditions

           Acidophiles = pH < 5.5

           Neutrophiles = pH 5.5 to 8.5- Blood pHis 7.4

           Alkalophiles = pH > 8.5

     

    Factors affecting microbial growth: Solutes and Water Activity

    changes in osmotic concentrations in the environment may affect microbial cells- osmotic concentrations deals with solutes

            hypotonic solution (lower osmotic concentration outside (the cell))

           water enters the cell

           cell swells may burst- if cell wall isn’t strong enough

            hypertonic (higher osmotic concentration outside)

      water leaves the cell

      membrane shrinks from the cell wall (plasmolysis) may occur- possible cell death

           do this to preserve products 

    Extremely Adapted Microbes

            halophiles- love salt

           grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M

            extreme halophiles

           require salt concentrations of 2M and 6.2M- 6.2M where salt precipitates out mostly in archea organisms

           extremely high concentrations of potassium

           cell wall, proteins, and plasma membrane require high salt to maintain stability and activity (e.g., Halobacterium in Dead Sea)

    Effects of NaCl on Microbial Growth

           halophiles

          grow optimally at >0.2 M

           extreme halophiles

          require >2 M

    optimum point- high point of curve on the graph, it is the best condition for growth

     

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    Solutes and Water Activity

    water activity (aw)

           amount of water available to organisms

           reduced by interaction with solute molecules (osmotic effect)

           higher [solute] Þ lower aw

           reduced by adsorption to surfaces (matric effect)......Aw of distilled water is 1.0......Aw of milk is 0.97...........Aw of dried fruits is 0.5

           Osmotolerant microbes can grow over wide ranges of water activity (e.g., Staphylococcus aureus 3M NaCl)

    Osmotolerant microbes can grow over wide ranges of water activity (e.g., Staphylococcus aureus 3M NaCl)

          Ex. Mold on peoples walls , water availability low because using the vapor in the air,

          Organisms have different capabilities of extracting water from the environment, and have different water requirements 

    Why is it difficult for organisms to grow at low aw?

    They need to put energy into getting water which causes them to have a slower growth rate, It  takes energy to find the water and bring into the cell, they don’t have energy for cell division or motility 

    Factors affecting microbial growth:• Effects of osmotic pressure and water availability on microbial growth

            Different solute concentrations can result in influx of water into or efflux from the cell.

           This can cause stress to the cell, causing it to either swell or shrink.

            Water must also be available for biochemical reactions (measured in terms of water availability or aw).

           Interactions with solutes can decrease aw values.

           Pure water aw = 1.0; seawater aw = 0.98; honey aw = 0.6

           Most bacteria require an aw > 0.9 (revisited in Chapter 16).

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    Factors affecting microbial growth: Temperature

            Effects of temperature on microbial growth

           Temperature can also affect macromolecular structure, membrane fluidity, and enzyme function.

           Different microbes have different optimal temperature growth ranges.

    Picture- Pink- organisms growing on the snow, psychrophiles- like low temperatures

    Temperature

            microbes cannot regulate their internal temperature

            enzymes have optimal temperature at which they function optimally

            high temperatures may inhibit enzyme functioning and be lethal

            Enzymes provide microbes with metabolic activities. They function at temperature that works for their enzymes

     

    Effect of Temperature on Growth rate

            organisms exhibit distinct cardinal growth temperatures

           minimal

          maximal

          Optimal

    Membranes melt or solidify....Proteins denature

    There is an Optimum value where the cell will grow best at

    Sharp drop off because enzymes work well until point where they degrade . At a sharp temperature H bonds are broken

    Ex. Making egg in pan

    Temperature Ranges for Microbial Growth

           psychrophiles – 0o C to 20o C-Love low temps

           psychrotrophs – 0o C to 35o C- organisms that will spoil food, refrigerator temperature

           mesophiles – 20o C to 45o C--Pathogens

           thermophiles – 55o C to 85o C--Like High

           hyperthermophiles – 85o C to 113o C--really high up to 121oC.

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    Adaptations of Thermophile

            protein structure stabilized by a variety of means

           e.g., more H bonds- for adaptation....       e.g., more proline....       e.g., chaperones- protective proteins

            histone-like proteins stabilize DNA

            membrane stabilized by variety of means

           e.g., more saturated, more branched & higher molecular weight lipids- ex. Crisco is solid at room temp. & when raise temp. it melts, organisms have different mechanisms for different environments   

             e.g., ether linkages (archaeal membranes)

    How can we use our knowledge of organism growth requirements to grow them in the lab?

            To study organisms we need to be able to grow, transport, and store microorganisms in the laboratory

            culture media is solid or liquid preparation

            Culture media must contain all nutrients required by organism for growth

         Specific requirements of medium depends on organism being cultured

            Culture media can be classified based on:

           chemical constituents from which they are made, physical nature, function 

    • How can different types of microorganisms be grown in the laboratory?

          Media for microbial growth

          Microbes can be grown in the lab on both solid (agar plates) and liquid media (broths).

    • How can different types of microorganisms be grown in the laboratory?

           Media for microbial growth

          Microbes can be grown in the lab on both solid (agar plates) and liquid media (broths).- Anything solid going to have a solidifying agent, nutrient agar has a solidifying agent compared to a nutrient broth 

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    • How can different types of microorganisms be grown in the laboratory?

            Media for microbial growth

         Can be either complex (unknown chemical composition) or defined/synthetic (precisely defined chemical composition)

           Defined- know everything in medium & amount, make media by weighing out exact ingredients

           Complex medium- don’t know extract components might've extract

           Peptones- don’t know requirements of organisms, so give them everything

    Complex Media • 3 common examples

          Nutrient broth

          Tryptic soy broth

          MacConkey Agar

    Some Media Components

           peptones      protein hydrolysates prepared by partial digestion of various protein sources


           extracts      aqueous extracts, usually of beef or yeast


           agar      sulfated polysaccharide used to solidify liquid media; most microorganisms cannot degrade it – usually extracted from red algae

    Functional Types of Media

    Supportive

    Enriched

    Selective

    Differential 

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    Functional Types of Media Supported vs enriched

           supportive or general purpose media

          support the growth of many microorganisms

          e.g., tryptic soy agar

           enriched media

          general purpose media supplemented by blood or other special nutrients

          e.g., blood agar- sheep blood, need extra nutrients

     

    Enriched Media

    Enzymes that can breakdown blood cells, strep throat take a swap and put on streak blood agar and look for colony growth

    Chocolate agar- blood is autoclaved- heated up blood cells and cells broken down which some organisms prefer 

    Selective Media

           favor the growth of some microorganisms and inhibit growth of others

           e.g., MacConkey agar

          selects for gram-negative bacteria

    ex. Salmonella on a piece of chicken, looking for certain growth 

    Differential Media

           distinguish between different groups of microorganisms based on their biological characteristics

           e.g., blood agar

          hemolytic versus nonhemolytic bacteria

           e.g., MacConkey agar

          lactose fermenters versus nonfermenters

           Differential- organisms look different on the plate, add dye, when prevents lactose it changes dye to red 

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    MacConkey Agar

           Differential and selective – contains lactose, dyes, and bile salts – selects for gram negative bacteria and for organisms that produce acid from lactose fermentation

           E. coli is red (ferments lactose to acid)

    Why are peptones, yeast extract and beef extract added to growth media?   Because don’t know what they want. Don’t know the growth requirements for that organism.

    Good source of food, trying to mimic environment, dont know what the growth requirements are.

     

    Growing microorganisms in the laboratory:

            Obtaining a pure culture (population of cells arising from a single cell)

            One of the benefits of a solid medium is that cells are held in place on the surface and can be isolated.

            This can lead to separating a mixture of cells into a pure population.

            There are three basic methods for separating cells on a plate.

            Streak plate method

            Spread plate method

            Pour plate method

    The Streak Plate

           involves technique of spreading a mixture of cells on an agar surface so that individual cells are well separated from each other

          involves use of bacteriological loop

           each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)

    • Obtaining a pure culture: Streak plate method

            Going to take an unknown sample and streak one area then heat the inoculate, streak to next area and heat the inoculate and streak to the next section- trying to get an isolated colony.

            Kosh postulate- pure culture

            Obtaining a pure culture: Spread/pour plate methods- Not as good as t streak but can use for counting

           Series of dilutions, usually high concentration

           Pour plate- subsample into molten agar- in a water bath, mix it gently it will then solidify 

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    • Unculturable bacteria: What if it won’t grow?

          Cultivation independent methods

           DNA from unculturable bacteria can be amplified and sequenced by PCR.- identify sequence on DNA and look at organisms

           Sequences can be used to produce fluorescent probes that will bind to complementary DNA (fluorescent in situ hybridization or FISH).

           See Toolbox 6.2 for more details!

    • Unculturable bacteria: Metagenomics

          DNA is isolated from an environmental sample and sequenced.

          The genetic content of microorganisms in an area can be examined and compared to those from a different area… EVEN IF THEY CAN’T BE GROWN.

          Metagenomic information must still be confirmed in cultured organisms, however—but they may help us to better formulate growth media recipes.

    • Unculturable bacteria: Microbial consortia

          Some microbes may just be too accustomed to growing with their friends and neighbors to be isolated.

    Microbial Growth on Solid Surfaces

           colony characteristics that develop when microorganisms are grown on agar surfaces aid in identification

           microbial growth in biofilms is similar

           differences in growth rate from edges to center is due to

          oxygen, nutrients, and toxic products

          cells may be dead in some areas

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    Bacterial Colony Morphology

    Put organism on plate, get colonies, they look different, middle part of colony have organisms spreading away, growing organisms will be different. The kinds of conditions inside the colony look similar to biofilm.

     

    Is a colony formed as a result of streaking a plate always derived from a single bacterium?

          No, sometimes with grow in clumps or chains 

    Select the kingdoms established by Aristotle over 2300 years ago. Animalia and Plantae

    Select the kingdoms in Haeckel’s classification system.


    Plantae, Protista and Animalia

    Whittaker’s five kingdom system includes: Aristotle’s two kingdoms
    Haeckel’s three kingdoms

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    The endosymbiotic theory explains: the origin of eukaryotes

    Eukaryotes originated over _____ ago by endosymbiosis.
    two billion

    Modern day examples that prove endosymbiosis exist. TRUE
    PCR takes small amounts of DNA and produces large amounts of _____. DNA
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    After the third cycle of PCR, ______ copies of DNA are produced. two
    The ingredients needed for PCR include: DNA and Primers
    All of the following are domains except: Protista
    During evolution, genes move from organism to another. True
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    Endosymbiotic theory: prokaryotes living w/in cells eventually became organelles in prokaryotic cells. False
    What roles do ions play in cells? Ions such as K+, Na+ and Cl- play an important role in controlling osmotic balance within the cell.

    Mg2+ and Mn2+ are critical for the function of DNA polymerases, and Mg2+ is also important for other types of protein-nucleic acid interaction.

    Iron is the central factor that enables enzymes such as cytochromes to carry and shuttle electrons.

    Other less abundant ions are needed as cofactors for enzymes that are present in low levels in the cells (and hence are called micronutrients since only very small amounts of such ions are needed for growth). Ions that are micronutrients include Zn2+, Co2+, Mo+, Cu2+, and Mn2+.
    Select the correct statement. Autotrophs can assimilate carbon from inorganic sources such as carbon dioxide (CO2) whereas heterotrophs must obtain usable carbon from pre-existing organic molecules such as amino acids, lipids, sugars, nucleic acids or smaller organic molecules like pyruvate or acetate.

    From what sources can organisms acquire electrons? All organisms require electrons to use as reducing agents for biochemical oxidation and reduction reactions. 

    Organotrophs acquire electrons by breaking down organic molecules, such as glucose. 

    Lithotrophs remove electrons from inorganic reduced molecules, such as ferrous iron (Fe2+), elemental sulfur, hydrogen gas (H2), hydrogen sulfide (H2S), ammonium (NH4+), and nitrite (NO2-). 
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    How might understanding the natural growth requirements of a microorganism help a researcher?

    It is essential for its cultivation. 

    It is important to understand the natural environments in which the organism can thrive (or fail to thrive). 

    We can make predictions based on what the organism can and cannot grow on, as to what metabolic pathways are present in the cell.
    How do prototrophic and auxotrophic organisms differ? Prototrophs can synthesize all necessary cellular constituents from a single organic carbon source and inorganic precursors. Auxotrophs, in contrast, require that at least one organic precursor molecule be obtained from the environment. As a result, auxotrophs will only grow in niches where the appropriate organic molecules can be found.

    What are the advantages and disadvantages of growing in an oxygen-rich environment? Oxygen, which is an avid acceptor of electrons, enables organisms to extract more energy from compounds via oxidative phosphorylation than can be extracted using any other terminal electron acceptor. 

    Why does growing in a salty environment pose a challenge? Salty environments pose a challenge to cellular life both because of the osmotic stress it places on the cell (in a salty environment we would expect much the water in the cell to escape to the outside).

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    Which of the following cellular components or processes are affected by temperature changes? Temperature affects the rate of chemical reactions by affecting the thermal energy of molecules, making them more or less likely to react if the temperature goes up or down, respectively.

    Temperature affects the folding and conformation of proteins, which can have dramatic effects on whether a particular enzyme can adopt the conformation necessary to carry out its function.

    The fluidity of the membrane bilayer also is strongly affected by temperature. Membranes with a large percentage of unsaturated lipids, which pack loosely, will remain more fluid at low temperatures than membranes rich in saturated lipids, which pack together more closely.
    Correct Complex media are derived in part from components where the specific concentrations of each nutrient are unknown. 

    Examples of complex media include media formulated from crude yeast or animal (beef or brain) extracts. 

    In defined media, the chemical identity and concentration of all components are known.

    Incorrect In defined media, yeast, animal, and plant tissue is present.

    Select the incorrect statement regarding selective and differential media. Differential media like MacConkey, allows the cultivation of both Salmonella and E. coli – but E. coli, which can ferment fructose, will form orange colonies on the agar due to the drop in pH via acid production whereasSalmonella will produce red colonies. 

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    Correct A selective medium is designed such that only one type or a few types of microorganism will grow on it while most other microorganisms will not. 



    Correct An example of a selective medium is one that contains antibiotics that kill off most bacteria but allow the resistant bacteria to grow. This includes the cultivation of Neisseria on Thayer Martin media, which contains a combination of antibiotics that kill most fungi, Gram-positive and Gram-negative bacteria but to which Neisseria is resistant.

    Correct Differential media allows the growth of more than one type of organism, but will permit the identification of specific species based on visual phenotypes such as a change in the color of the medium. MacConkey medium is actually both a selective and a differential medium because it contains bile salts that prevent the growth of many types of bacteria including Staphylococcus aureus.
    Pure cultures can be obtained by: streaking the mixture of bacteria on solid media in a petri dish. If properly performed the sequential streaking of an initial mixture of bacteria onto different sections of the petri dish will result in single colonies of bacteria that are spatially separated from one another. Each colony arises from a single cell that underwent several rounds of cell division.

    spreading several dilutions of bacteria containing liquid onto plates of solid nutrient media one is able to isolate single colonies that grow.

    diluting the bacteria into molten nutrient agar at a temperature that should not kill the bacteria and rapidly pouring a plate from this mixture. The pour plate method is best for isolating individual, non-overlapping colonies but is the most laborious and can kill some of the bacteria if the temperature is not well controlled or if the bacteria is particularly heat sensitive.
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    Unculturable microorganisms can be characterized by determining the types of bacteria present in a sample by: isolating DNA from all cells and amplifying and sequencing the 16S ribosomal DNA.

    True The most common method is to count the number of colony forming units (CFU) present in a sample. This is typically done by making several serial dilutions of the culture to be tested (for example a 1/10 dilution, a 1/100 dilution, a 1/1000 dilution) and spreading a defined volume of each dilution on its own nutrient agar plate. At some dilution the number of colonies that appear on the plate will be of a sufficient number to be counted accurately (less diluted samples will give too many colonies to count accurately and more dilute samples either give no colonies or too few colonies to be meaningful). Each colony on the plate started as a single cell, therefore, we can make a simple calculation to get back to the concentration of cells (CFU per mL) in the original sample based on the number that we count on the plate. Variations of this method include using filtration to capture microbes present in very dilute samples. Placing the filter on solid media allows colonies to grow and be counted.
    The advantages of measuring turbidity include _____.

    getting an instant estimation of the number of cells present in a sample

    rapid performance of the method (counting colonies can take days or weeks depending on how rapidly the bacteria grow)

    sampling a culture many times with a spectrophotometer without having to otherwise perturb the culture for analysis (e.g., making dilutions and plating on solid media)
    Select the incorrect statement regarding the parts of a typical bacterial growth curve. The cells in the culture eventually consume enough nutrients in the culture that the concentration of one or more of the nutrients will be too high to support the maximal growth rate. Also, waste products that may be detrimental to growth (acidic or basic compounds, for example) can build to toxic levels. At this point the growth of the culture slows until it enters “stationary phase”. This phase in the growth curve results in no new biomass or new cells being formed (i.e. bacterial growth has stopped).

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    Select the correct statement regarding the parts of a typical bacterial growth curve. Bacteria that have been taken from spent media and have ceased to grow do not resume growth immediately upon inoculation into fresh media. Instead the culture will have a “lag phase” where the cells adjust their metabolism and protein/lipids to adapt to rapid growth.

    Select the correct statement regarding the parts of a typical bacterial growth curve. After exiting lag phase, the cells gradually enter “log phase” or “exponential phase” where growth of the culture reaches the maximum value for that particular condition. Under this condition the rate of growth is exponential because once cell produces two new cells with each cell division.

    Select the correct statement regarding the parts of a typical bacterial growth curve. After an extended period in stationary phase a large number of cells in the culture lose viability and will not resume growth when transferred to fresh media (the “death phase”). The loss of cellular viability is likely due to an inability to repair damaged membranes and macromolecules.

    How can we determine the growth rate of a microorganism? b) Growth of a culture of microorganism occurring in the log phase can be determined by knowing the starting population of cells in the culture (N0, measured at time “0”) and the population of cells at a later time (Nt, measured at time “t”). 

    TRUE
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    What is a continuous culture? Continuous culture systems (e.g. chemostats) are open systems in which nutrients are fed into a culture vessel at a constant rate. 

    The volume of the culture is kept constant by draining the excess medium (including cells and waste) at the same rate as fresh media is introduced. 

    Continuous culture systems allow the density of the culture to be controlled by the concentration of nutrients in the media while the growth rate of the culture is controlled by the rate at which fresh media is introduced into the system (the dilution rate). 

    Unlike batch cultures, which undergo lag, log, stationary, and death phases, continual culture systems can keep the bacterial culture growing in the log phase for indefinite period of time.
    What are the advantages and disadvantages of removing microbes by filtration? Removing microbes from media or other fluids by filtration is fast.

    Filtration does not involve heating or chemically treating the sample, which in some cases is critical if components in the media are sensitive to heat or it is undesirable to add chemicals.

    Filters have the disadvantage of being expensive.

    Filters are easily clogged by particles in the solution. Viscous liquids display very poor flow rates through most filters.
    How is temperature used to eliminate microbes or control their growth? Some microbes, however, produce spores that are resistant to temperatures.

    Most common pathogens can be removed from milk or juice by brief heating to temperatures below boiling (a process called pasteurization). This process is widely used because it kills most pathogens while preserving the properties of the liquid, however many bacteria are not destroyed during this process and pasteurized milk or juice will spoil after prolonged storage.

    Refrigeration is used to impede the growth of microbes and can preserve specimens, food or media for extended periods whereas, freezing stops the growth of almost all microbes for indefinite periods and is an excellent way to preserve food or specimens for long term storage.
    How do oxidizing agents affect microbes? Oxidizing agents including peroxides, bleach, iodine, chlorine and ozone all work by stripping electrons away from proteins, nucleic acids, lipids and cell walls. 

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    The primary difference between organotrophs and lithotrophs is organotrophs assimilate electrons from organic sources while lithotrophs assimilate electrons from inorganic sources. 

    An organism that assimilates its carbon from organic molecules, its electrons from inorganic sources, and its energy from either organic or inorganic molecules is best characterized as a chemolithoheterotroph.

    An organotroph is an organism that
    Although prototrophic organisms can grow in nutrient poor habitats, growth is normally slow because
    synthesis of materials necessary for metabolism takes time under nutrient poor conditions.

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    How do microbes maintain proper intracellular osmotic pressure? Some microbes have rigid cell walls that enable them to withstand a wide range of external osmotic pressures. 

    Increasing temperature impacts bacterial cell function by increasing fluidity but decreasing selectivity of the plasma membrane.

    If the goal is to estimate the number of bacteria in a broth sample, the most appropriate separation technique would be the pour plate. 

    On a streak plate preparation of a pure culture, colonies were found along the streak like as well as below the surface of the agar. What is a reasonable conclusion for this observation? The agar plate was contaminated before inoculation by the streaker.
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    The agar plate was contaminated before inoculation by the streaker. cells don’t have to be cultured first.

    A serial dilution of an E. coli culture was performed, and 1 ml of various dilutions was plated onto nutrient agar. The resulting data are shown in the table below. What is the estimated concentration of cells/ml in the original broth culture?
    2.9 x 109 cells/ml. 

    When determining the abundance of bacterial cells, why exclude plates with more than 300 or fewer than 10 colonies? From a probability and statistics perspective, anything less than 10 cells is considered too small a sample size.

    To achieve stability in a continuous culture system, the dilution rate must be less than the maximal growth rate so that cell numbers can be replenished before being washed out with excess media.

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    Autotrophs assimilate carbon from inorganic sources like CO2. TRUE
    Organotrophs acquire electrons from organic molecules. True
    Auxotrophic organisms have the enzyme systems necessary to synthesize all the nutrients their metabolism requires. FALSE
    Water activity (aw) is defined as ratio of the vapor pressure of a liquid relative to the vapor pressure of water under standard conditions. Water activity (aw) is defined as ratio of the vapor pressure of a liquid relative to the vapor pressure of water under standard conditions.
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    The minimum requirements for any medium to support the growth of bacteria are a carbon source, an energy source, and a source of electrons.
    False
    In most environmental samples, far more cells can be cultivated as colonies than are evident by direct microscopic observation, a phenomenon known as the “great plate count anomaly”. False
    It is statistically valid to include plates with fewer than 10 colonies provided there are many of them in the sample. True
    In a typical bacterial growth curve, both the growth phase and the death phase are exponential. True
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    Do all specimen examined by fluorescence microscopy need to be stained with fluorochrome? No some are naturally fluorescent
    Pasteur filtered air thru cotton plug & placed cotton in broth. He found broth became cloudy.Y? Micro organisms in cotton that came from air contaminated the broth.
    Which of the 5 I’s listed below is in the most logical order?

    C. Inoculation, Incubation, Isolation, Inspection, Identification 

    In the enzymatic reaction depicted below (A + B􏰀C + D), what is the nature of the reaction?

    D. Exothermic, -􏰁G, Keq > 1, Keq = [C][D]/[A][B] 

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    Which category of light gives the best images for a bright field microscope?

    B. 380nm – 500nm

    The energy required to bring the substrates of a reaction together in the correct way to reach the transition state is called

    B. activation energy 

    Enzymes function as catalysts by

    A. bringing the substrates together at the active site, in effect concentrating them.
    B. bringing the substrates together at the active site correctly oriented for the reaction 

    Competitive inhibition can be overcome by adding excess

    B. substrate

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    The ____________________secretory pathway(s) are capable of injecting macromolecules into another cell.

    E. Type III & Type IV 

    These microbes use CO2 as their carbon source.
    These microbes oxidize inorganic chemical compounds as their source of energy and they use CO2 as their source of carbon.

    B. chemoautotrophs 

    Which microbe does not correctly match its energy source?

    C. photoheterotroph – Fe2+ 

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    Specific amino acids that target proteins to particular locations in a eucaryotic cell are called:

    C. signal peptide sequences 

    Type of proteins that bind to unfolded proteins and can maintain the bound proteins in this unfolded state:

    C. chaperones 

    Microtubules are composed of these proteins.

    A. tubulin 

    Eucaryotic cells move by what mechanism(s)?

    A. amoeboidmovement
    B. flagellar movement

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    Which of the following organelles closely resembles a procaryotic cell?

    C. Mitochondrion 

    Function of a procaryotic cell envelope:

    B. protection against osmotic lysis

    Chemical component(s) of Gram positive cell walls:

    A. peptidoglycan


    C. teichoic acids 

    Unique to the outer layer of a Gram negative bacterium:

    A. lipopolysaccharide 

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    Special proteins that allow compounds to diffuse passively through pores in the membranes of Gram negative bacteria:

    A. porins

    Molecular processes that are coupled in procaryotic cells:

    B. transcription and translation

    Which of the following is incorrectly matched?

    C. flourescence microscopy-exploits the natural fluorescence that cells exhibit

    A modern microscope that removes light that is out of focus, thereby improving the sharpness of the image.

    C. confocal microscopy 

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    A minimal medium used to grow bacteria typically contains what ingredient(s)?

    A. sulfur
    B. nitrogen
    C. phosphorous 

    A complex medium to grow bacteria may contain the following ingredient(s):

    A. organic compounds such as amino acids, vitamins, nucleotides
    B. growth factors
    C. peptone

    D. yeast extract 

    Which of the following pairs is not correctly matched?

    A. van Leeuwenhoek – microscope=correct
    B. Pasteur – fermentation=correct
    C. Jenner – aseptic surgery=incorrect

    D. Koch – established relationship between microbes and disease=correct

    In terms of Pasteur's experiments with the swan-neck flask, which of the following statements are true?

    A. All microbes were killed before beginning.
    B. Food was present in the flask.

    C. There was air involved.
    D. All contamination was removed.


    ALL 

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    Which of the following is probably true about all of the experiments that proved spontaneous generation?

    A. microbes were already present

    Why do biologists consider procaryotes important?

    A. they constitute most of the biomass on earth
    B. they perform essential transformations of materials on which all other life depends C. they are the most numerous organisms on earth
    D. they are biochemically far more diverse than eucaryotes

    The cytoplasm of cells contain the majority of what type of molecules?

    C. proteins 

    Fossilized evidence of microbes dates to how many billion years old?

    C. 3.5 

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    ATTENUATION An attenuated vaccine is a vaccine created by reducing the virulence of a pathogen, but still keeping it viable (or "live").[1] Attenuation takes an infectious agent and alters it so that it becomes harmless or less virulent. These vaccines contrast to those produced by "killing" the virus (inactivated vaccine).
    Selected Advances in Microbiology: Scientists in historical order, Earliest to latest

    Anton Von Leeuvenhock = uses microscope to see microorganisms

    Louis Pasteur = disproves idea of spontaneous generation

    Joseph Lister = practices infection control

    Robert Koch = Identifies bacillis anthracis as cause of anthrax

    Alexander Flemming = Discovers Penicillin

    Salk and sabin = Develops poliovirus vaccines

    Lynn Margulies = Proposes endosymbiotic theory

    Kary Mullis = Invents PCR

    Carl Woese = Proposes 3 domain classification of living organisms

    Craig Venter = Publishes first complete bacterial genome sequence.

    Growing microorganisms in a lab 2 ways Solid agar in petri dish, spread bacteria on surface of agar.... and incubate. Colonies are on Surface of agar. SPREAD METHOD


    Molten agar with bacteria, pour into petri dish....and incubate. Colonies are on surface of agar, and IN agar media.
    NOTE: There is a sharp drop because enzymes are being denatured at those temperatures.


    GROWTH RANGES
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    organisms exhibit distinct cardinal growth temperatures
    minimal
    maximal
    Optimal

    Membranes melt or solidify

    Proteins denature

    Enzymes denature with heat.

    ARCHAEA ENJOY WHAT TEMPS HOT HOT HOT
    Effects of NaCl on Microbial Growth
    halophiles
    grow optimally at >0.2 M
    extreme halophiles
    require >2 M
    Nutrient concentration
    Growth rate will depend on the amounts of nutrients in the environment.
    One key nutrient, available in the lowest amount, will dictate how much growth can occur over time (i.e., it will be a limiting factor).
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    Obligate aerobe; requires Oxygen; grows on top
    Facultative anaerobe; w/ or w/o Oxygen...will grow better at top but also throughout
    Aerotolerant anaerobe: O2 is limited; Grows wherever
    Strict anaerobe: O2 is poison; No way of protecting cells...so therefore oxygen will kill cells.
    Microaerophile: requirement for oxygen but too high is poisonous
    Grows at low levels.
    Immuno fluorescence Fluorochromes attach to specific antibodies...antibodies attach to designated specific bacteria....bacterial cell with bound antibodies combined with fluorochromes begins to glow.

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    List View: Terms & Definitions

      Hide All 323 Print
     
    Front
    Back
     What is MicrobiologyStudy of microbes

    The study of really small life.

    Not all micro organisms are small.
     What are microbes?
    forms of life too small to be seen with the naked eye (bacteria, fungi, algae, protists).
     What does the field of microbiology tell us?
    The field examines how microbes interact with humans, with food, and how they can be used BY humans (among other aspects).
     2 types of Microbial cellProkaryotic and Eukaryotic
     Prokaryotic cells

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

    Ex: Chloroplasts


    SIMPLER and NO MEMBRANE BOUND ORGANELLES.

     Eukaryotic cells

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

     What is the largest known organism on earth?what is it's width?Oregon Mushroom fungus. 2 miles wide

    Fungus Argon mushroom...body grows underground and comes up with mushrooms....called the thallus. it is very old if it is all one organism
     Why was the three domain system developed?
    developed due to advances in electron microscopy, biochemistry and physiology, and ability to determine nucleic acid and protein sequences
     Carl Woese
    Carl Woese et al (1990 paper) based on a comparison of ribosomal RNA, divides microorganisms into domains:
    Bacteria (true bacteria), (prokaryotes)
    Archaea-Archea are prokaryotes with no mitochondria-simpler cells
    Eukarya (eukaryotes)

    Carl woese looked at the research that had been done on DNA sequences of the 3 DOMAINS of microorganisms.

     Five Kingdom SystemRobert Whittaker

     Aristotle classificationTwo groups-Plants and animals
     Domain Bacteria

    Usually single-celled, some are NOT.

    • Majority have cell walls with peptidoglycan: specific to bacteria
    • Most lack a membrane-bound nucleus
    Ubiquitous (found everywhere) & some live in extreme environments
    • Some cause disease but most are beneficial
    • Many recycle elements (e.g., Cyanobacteria produce amounts of significant oxygen)
    Responsible for most of life on earth because of extensive use of O2 they use & produce

     Domain Archaea

    • distinguished from Bacteria by unique rRNA sequences
    • lack peptidoglycan in cell walls and have unique membrane lipids
    • some have unusual metabolic characteristics (e.g., methanogens which produce methane gas)
    • many live in extreme environments (autoclave)
    • Their role in causing diseases is unclear
    • Archeal GUM DISEASE=only known archael disease

     Domain Eukarya

    generally larger than Bacteria and Archaea

    • Plants and Animals
    • algae – photosynthetic
    • protozoa – may be motile, hunters, grazers
    • slime molds – two life cycle stages (mold-like, protozoan-like)
    • water molds – protists that grow in moist environments - some cause devastating diseases in plants
    • fungi – (e.g. yeasts – unicellular, molds – multicellular)
    • Primarily decomposers, some cause disease

     VirusesAcellular Infectious Agents (not considered part of 3 domain system of classification)
    viruses smallest of all microbes
    Very simple – some consist of only proteins and nucleic acids
    requires host cell to replicate, no metabolic energy needed
    cause range of diseases, some cancers
    viroids and virusoidsinfectious agents composed of RNA (viroids=pieces of RNA)
    prions – infectious proteins
     Why are viruses NOT considered to be alive?

    • They dont replicate outside of a host cell.
    • They (usually) have little to no biochemical activity outside of a host cell.
    • They are inert & nonreactive outside of a host cell.
    • Microbiology still studies viruses, though, since they are too small to be seen with the naked eye.

    Dont require metabolic activities, dont require energy to carry out processes

      Describe these four virus: Poliovirus, bacteriophage, Ebola Virus, Tobacco mosaic virus
     Define Life

    Metabolism-take in nutrients, take energy and build new molecules

    Growth

    Reproduction-divide and multiply

    Genetic variation/evolution-pass traits through generations

    Response/adaptation to external environment….(thermoregulation, osmoregulation)

    Homeostasis (maintaining internal organization & order, usually by expending energy), internal pH, solute concentration

    Endospores form from a detrimental event...Antibiotic resistance=circle zone around cell making it sensitive to antibiotic.

     Why are viruses not included in the 3 domain system of classification?

    Need host cell to live,  dont meet all classifications to life. Theyre genetically removed from the 3 domains.

    Because the way domain systems are devised, they look at subunit ribosomal DNA. Viruses don't have RNA...because Viruses don't have ribosomes.
      
     What are macromolecules (major building blocks) needed for life? (4)Polypeptides: Amino Acids

    Nucleic acids: Deoxyribonucleotides and ribonucleotides

    Lipids: Diverse structures

    Polysaccharides: sugars
     Polypeptides FXN
    Amino Acids
    Enzymes catalyze the vast majority of biochemical reactions in the cell. Other proteins are structural components of cells

    55% of dry weight of cell
     Nucleic Acids FXN
    Deoxyribonucleotides and ribonucleotides
      Deoxy: Informational: DNA provides instructions for assembly & reproduction of cell  =  2 - 5% of dry weight of cell
      Ribo: Many FXNS, most of which are involved in production of polypeptides. Some serve structural or catalytic FXNS
      =  15 - 20% of dry weight of cell
      >Deoxy, ribo=critical role as storehouses of genetic info.
      >Comparing of DNA sequences allow us to break life into 3 domains--by looking at ribosomal frequency
       LIPIDS FXN
       Diverse structures
      Structural: make up cellular membranes that form physical boundary between the inside of cell and surroundings and membranes of internal organelles

      = 10% of dry weight of cell
       Polysaccharides FXN
      sugars

      Structural (such as cellulose and Chitin) and energy storage (such as glycogen and starch)

      = 6 - 7% of dry weight of cell
       Most important FXN of polypeptidesthe function of enzymes as catalysts of chemical RXNS
       Polypeptide: RNA polymeraseLocation: Cytoplasm of bacteria and archaeons, nucleus of eukarya

      FXN: fxns as an enzyme that produces RNA molecules from DNA template.
       Polypeptide: Glycogen phosphorylaseLocated in CYTOPLASM

      FXN: Conversion of glycogen into glucose monomers
       Polypeptide: K+ channelLocated in PLASMA MEMBRANE

      FXN: Passive transport of K+ across the membrane, from an area of high concentration to an area of low concentration.
       Polypeptide: Na+/K+ ATPaseLocated in the PLASMA MEMBRANE

      FXN: Active transport of Na+ and K+ across the membrane, from areas of low concentration to areas of high concentration.
       Polypeptide: FlagellinLocated in BACTERIAL FLAGELLUM

      FXN: monomers polymerize to form flagellum which aids in bacterial motility.
       Polypeptide: FtsZLocation: Associated with plasma membrane of bacteria

      FXN: Key component of cell division machinery.
        Enzyme

      Substrate

      Active Site
       what forms a cell's plasma membrane?

      Polysaccharides and polypeptides can be embedded in a lipid bilayer, forming a cells plasma membrane. This separates the external environment from the interior of the cell.

        Polypeptide/polysaccharide membrane
       What can studying the genetics of microbes teach us about the evolution of life on Earth?
      The very early environment on Earth was drastically different than it is today.
      There was little oxygen in the atmosphere, and the surface of the planet was a soup of chemicals in liquid form.
      This early atmosphere and environment led to the initial synthesis of the first forms of macromolecules (and their use in primitive single-celled life).
        MICROBIAL GENETICS
       How do we age earth and how old is earth predicted to be?

      Through radioisotope analysis Earth is thought to be between 4.5 to 4.6 billion years old.


      microbial fossils: Swartkoppie chert – granular silica – fossilized mats of microbes3.5 billion years old
      fossil record sparse; indirect evidence and scientific method are used to study origins of life--visually not changed from then to now
       Stromatolitelayers of cyanobacteria and minerals    
        STROMATOLITE
       How did the first microbial life arise?
      In the 1950s, a grad student named Stanley Miller formed organic molecules from a primordial soup.
      --Saw clear liquid turn cloud then red--seeing amino acids
      --Wanted to see what earth was going to look like in the beginning.
        Stanley Millers Set up
       Tools Used for studying Microorganisms
      microscopes
      culture techniques
      molecular genetics
      genomics
       Discovery of Microorganisms Robert Hooke
      Robert Hooke (1635-1703) first to discover eukaryotic tree cells
      Looking at a thin slice of cork with a crude microscope
      Life consisted of little boxes – cells
      •Developed Cell theory – all living things are composed of cells
       Discovery of Microorganisms Leeuwenhoek

      Antony van Leeuwenhoek (1632-1723)

      first person to observe and describe microorganisms accurately with a microscope that could magnify 
      50 – 300 times
      Called microbes animalcules
      Studied his teeth scrapings and diarrhea
        Leeuwenhoek's microscope
       Leeuwenhoek describing GIARDIA

      I weighed about 160 pounds…and I ordinarily a-morning have a well-formed stool, but now and then hitherto I have had a looseness, at intervals of 2, 3 or 4 weeks, when I went to stool some 2, 3 or 4 times a day… My excrement being so thin, I was at diverse times persuaded to examine it…I will only say that I have generally seen, in my excrement, many irregular particles of sundry size.  All particles aforementioned lay in a clean and transparent medium wherein I have sometimes also seen animalcules a-moving very prettily.  Their bodies were somewhat longer than broad, and their belly, which was flattened was furnished with sundry little paws, wherewith they made such a stir in the medium and among the globules.

       Archaezoa
      No mitochondria
      Multiple flagella
      Giardia lamblia
      Trichomonas vaginalis (no cyst stage)
        Giardia=proteosome 
       spontaneous generation theory and who discredited the theory?

      Spontaneous generation: living organisms can develop from nonliving or decomposing matter, thought maggots came from nothing.

      The Conflict over Spontaneous Generation

      Francesco Redi (1626-1697) discredited spontaneous generation

      –showed that maggots on decaying meat came from fly eggs

      –His experiment – 3 containers with meat, one covered with paper, one covered with gauze (NO maggots), one open (got maggots)

      Biogenesis=one living thing must produce another living thing.

       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
       Louis Pasteur (1822-1895) disproved spontaneous generation?
      Settled the matter of spontaneous generation in 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
      Biogenesis – Life comes from life
        Louis Pasteur's experiment: a simple yet elegant experiment to disprove spontaneous generation theory in the late 1800's

      Realized the experiment was getting contaminated by air.

      Pasteur filtered air through a cotton plug and then place the cotton in broth. He found the broth became cloudy...Why? 
      ---Micro organisms in cotton that came from air contaminated the broth.
       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
       The Role of Microorganisms in Disease - Germ Theory
      was not immediately obvious

      infectious disease believed to be due to supernatural forces (miasmas – bad vapors)

      establishing connection depended on development of techniques for studying microbes
       Evidence for the Relationship between Microorganisms and Disease
      Agostini Bassi (1773-1856)showed that a disease of silkworms was caused by a fungus *critical finding

      M. J. Berkeley (ca. 1845)demonstrated that the great Potato Blight of Ireland was caused by a water mold

      Heinrich de Bary (1853)showed that smut and rust fungi caused cereal crop diseases
       Evidence for the Relationship between Microorganisms and Disease-Louis Pasteur
      Louis Pasteur (1822-1895)
      demonstrated that microorganisms carried out fermentations
      developed pasteurization – low temperature heating to destroy unwanted microbes in wine and milk – used to reduce spoilage bacteria and kill harmful bacteria – heated to 145F(63C) for 30 min. then cooled, now 161F (72C) for 15 sec
      showed that pébrine disease of silkworms=caused by protozoan
      Pasteurization: heating process to kill organisms and bring back down to cool temperature and inoculate it.
       Evidence for the Relationship between Microorganisms and Disease-Semmelweis & Lister
      I. Semmelweis (1840s) high incidence of purpural fever in obstetrical patients could be reduced by washing hands (autopsy source)
      J. Lister (1827-1912) provided indirect evidence that microorganisms were causal agents of disease
      developed a system of surgery designed to prevent microorganisms from entering wounds as well as methods for treating instruments and surgical dressings using phenol
      his patients had fewer postoperative infections...1st to use PHENOL
       John Snow – 1st Epidemiologist 1854 Cholea outbreak
      Soho neighborhood of London   500 died in 10 days  
       Mapped affected areas    --Caused by microorganism Briocholera--contaminated the water.
      Centered on Broad Street well – sewer pipe was leaking into well
      Convinced officials to remove pump handle
      Interestingly people who worked at a local brewery were unaffected.
       Final proof that microbes cause disease…

      Robert Koch (1843-1910)

      Established relationship between Bacillus anthracis & anthrax
      Used criteria developed by teacher Jacob Henle (1809-1895)
      these criteria now known as Kochs postulates--four determining factors to see if an organism is a causative agent.
      still used today to establish the link between a particular microorganism and a particular disease
       What did Koch discover in terms of the causes of Anthrax and how did this help him
      Robert Koch determined Bacillus anthracis and Mycobacterium tuberculosis were the causes of anthrax and tuberculosis (respectively).
      His work with anthrax helped sheep herders and cattle ranchers avoid costly animal losses.
      The basic rules Koch established made it possible for others to determine which microbes caused which diseases. They are still in use to this day.  HIS LAB DISCOVERED PETRI DISHES
       Koch’s Postulates (guidelines)
      the microorganism must be present in every case of the disease but absent from healthy individuals
      the suspected microorganism must be isolated and grown in a pure culture
      the same disease must result when the isolated microorganism is inoculated into a healthy host
      the same microorganism must be isolated again from the diseased host
       Limitations of Koch’s Postulates (guidelines)
      some organisms cannot be grown in pure culture (e.g. Mycobacterium leprae)
      using humans in completing the postulates is unethical (e.g. Ebola hemorrhagic fever)
      molecular and genetic evidence may be used instead 
       What theory explains when/how eukaryotes appeared
      Endosymbiotic theory: Primitive prokaryotic microbes ingested other microbes, starting a symbiotic relationship, forming the first basic eukaryotes.
      Ingested microbes that could use oxygen for a respiratory process to produce chemical energy became mitochondria.
      Ingested microbes that could fix carbon dioxide into organic molecules using light energy became chloroplasts.
       Endosymbiotic Hypothesis

      • Mitochondria, hydrogenosomes, and chloroplasts are all thought to have evolved from bacterial cells that invaded or were ingested by early ancestors of eukaryotic cells
      • Mitochondria and chloroplasts are very similar to extant bacteria and cyanobacteria, respectively
      • Both mitochondria and chloroplasts contain bacterial DNA and ribosomes
      • Both are similar based on SSU rRNA

       The Development of Techniques for Studying Microbial Pathogens
      Kochs work led to discovery or development of:
      Agar (Fanny Hess): adds to medium in order to solidify polysaccharides.
      Petri dish (Richard Petri)
      nutrient broth and nutrient agar
      methods for isolating microorganisms
      Aseptic technique
       Advantages of using Agar over gelatin as a solidifying agent
      Not broken down by most organisms

      Doesnt melt until it reaches 100C

      Doesnt solidify until it reaches 50C

      Produces a clear product
      Some organisms can degrade gelatin 
       methods for studying viruses (Chamberland)

      Charles Chamberland (1851-1908): developed porcelain bacterial filters used by Ivanoski and Beijerinck to study tobacco mosaic disease

      • determined that extracts from diseased plants had infectious agents present which were smaller than bacteria and passed through the filters
      • infectious agents were eventually shown to be viruses
      • Called filterable particles”: b/c of their nature of going through that porcelain filter.

       methods for studying viruses (Pasteur and Roux)
      Pasteur and Roux
      discovered that incubation of cultures for long intervals between transfers caused pathogens to lose their ability to cause disease – could be used to develop vaccines
      Pasteur and his coworkers
      developed vaccines for chicken cholera, anthrax, and rabies
       Magic Bullet
      Paul Ehrlich (1910) – German Physician
      1st chemotherapy, synthetic drug salvarsan, arsenic derivative, offered salvation from syphilis
       Famous Microbial Disease
      Some microbial diseases have had a profound impact on humanity—e.g., plague=black death killed 1/3 population of the world.
        HOW THE PLAGUE SPREAD
       In 20th century=dramatic drop in U.S. deaths from infectious diseases. How is this possible?
      Where has this reduction in deaths come from?
      Prevention of infection through
      Use of antiseptics (Joseph Lister)
      Sanitation improvements (sewage treatment)
      Food/water safety (pasteurization)
      Personal hygiene improvements
      Vaccination
      Treatment of infections (antibiotics!)
        Smallpox Virus
        Koch’s Postulates applied to tuberculosis
       Immunological Studies
      once established, led to study of host defenses - immunology
      Edward Jenner (ca. 1798)
      used a vaccination procedure to protect individuals from small pox
      Milkmaid with cowpox was immune
      Tested on 8 year old volunteer
      This preceded work establishing role of microorganisms in disease.
      Small pox caused by virus, chinese would take scabs and put them in people's noses and they would get some type of immunity.
       The Birth of Modern Chemotherapy
      1928: Alexander Fleming discovered the first antibiotic.
      He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus.
      1940s: Penicillin was tested clinically and mass produced.

      The colony of penicillia and nothing grew around fungal penecillin...showing an inhibition complex within penecillin.
       What were the earliest molecules?
      original molecule must have fulfilled structural, catalytic, and hereditary functions (these activities are now performed by proteins, DNA, RNA)--if you cut yourself, you can seal a cut.
      ribozymes – Discovered by Thomas Cech in 1981
      RNA molecules that form peptide bonds
      perform cellular work and replication
      earliest cells may have been RNA surrounded by liposomes 
       Ribozymes

      Some RNA molecules have the ability to catalyze reactions (these are known as ribozymes, a combination of ribonucleic acid and enzymes)


      This means RNA could serve the dual purpose of genetic information storage AND catalyzing reactions!

        How Rybozymes worked
       But what about separating interior from exterior?
      A single lipid layer known as a micelle may have been an early form of plasma membrane. (can form spontaneously)
      This could have formed a crude way of separating interior contents from the external environment.

      Micelles=LIPOSOMES that form spontaneously covered RNA molecules.
        Micelle
       So then, the basic idea of how microbial life arose on Earth is:
      Early conditions formed RNA and micelles.

      These came together into a primitive cell using RNA for storing genetic info and coding.

      Primitive cells eventually changed from using RNA to DNA instead for storing their genetic information.
        
      So then, the basic idea of how microbial life arose on Earth is:

        


      Endosymbiotic
      Hypothesis    
       Microbial SPECIES

      eukaryotic species are animals or plants that naturally interbreed
      Bacteria & Archaea DONT reproduce sexually & are referred to as strains

      • Strain consists of descendents of single, pure microbial culture – share stable properties
      • may be biovars, serovars, morphovars, pathovars

      binomial nomenclature – developed by Carolus Linnaeus (1750)

      • genus and species epithet
      • (e.g., Escherichia coli – italics or underline)
      • Can abbreviate after first use (e.g. E. coli)

       What accounts for differences in the rate a disease spreads?
      Humanitys interaction with infectious diseases is always changing.
      Sometimes cultural/economic differences can mean differences in disease rates (lack of malaria in the United States versus Africa).

      Microbes live in parasites and they cause disease....some microbes can develop resistance to antibiotics.
       How old are microbial fossils?

      • Multicellular fossils dating to about 0.5 billion ybp (years before present) have been found—meaning microbes dominated the planet for approximately 3.5 billion years!
      • Some microbial fossil records do exist, largely in fossilized mats discovered in Australia.-Stromatolites

        Fossilized Cyanobacteria
        Fossilized Stromatolites
      -Live in australia
        
      Of all the microbes we can grow in the lab, its estimated there are many more that cant be grown.
        Early microfossils similar to modern filamentous cyanobacteria
      About 3.5 billion years old
          
        

      Endosporeè2nd photo….go into dormant stage when conditions aren’t right

      Circle around the dot in green, means that that organism is sensitive to the antibiotic


      If it is clear, it is affected....they can modify to survive.

       Are Microorganisms good or bad?Depends on their environment w/o them, we could not exist.

      Ex: food born pathogens....very few are pathogens and 99% are beneficial to the environment, they turn back oxygen we need.
       Proteins can beEnzymatic and structural
       Most important FXN of polypeptidesCatalysts of chemical rxns.
       Ignaz Semmelweis storyDoctor had two clinics...one doctors the the other physicians, the physicians would do an autopsy and not wash hands before delivering babies. there were more deaths at physician offices than midwives.
       Why are pure cultures critical to koch's postulatesTo know which organism causes the disease.
       Elemental makeup of important cell moleculesProteins:        CHONS
      Lipids:           CHOP
      Carbohydrates:    CHO
      Nucleic Acids:     CHONP
       Microscopy

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

       Lenses and the Bending of Light

             light is refracted (bent) when passing from one medium to another

             refractive index      a measure of how greatly a substance slows the velocity of light

             direction and magnitude of bending is determined by the refractive indices of the two media forming the interface (i.e., glass and air)

             Glass has a higher refractive index than air

       Lenses

             focus light rays at a specific place called the focal point

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

       How lenses bend light

      (F = focal point, f = focal length)

      The strength of the lens is related to the focal length

      short focal length Þmore magnification

                  Refracted- bent as comes away and focused on focal point- where specimen should be if shorter little f, the higher magnification

       

       Types of Light Microscopes (5)

             Many varieties

            bright-field microscope