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m (ClassVII double stranded DNA with RNA intermediate: Viral multiplication section added)
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**in cytosol (RNA virus except orthomyxovirus)  
 
**in cytosol (RNA virus except orthomyxovirus)  
 
**or in nucleus (DNA viruses except Pox virus)
 
**or in nucleus (DNA viruses except Pox virus)
 +
 +
===Viral Replication===
 +
*''primary cells'' -taken directly from host and grown. may be diluted and placed into new flasks which would be classified as ''secondary cells''
 +
 +
*Transformed cells when cells are suceptible but non permissve to dna containing viruses
 +
*you see changes in
 +
** morphology (rounder)
 +
**   physiology (increased multiplication)
 +
** behavior (lose contact inhibition)
 +
*potential to cause tumors in small mammals
 +
 +
====Single step replication====
 +
two phases
 +
=====Eclipse phase=====
 +
* after infection of cells, virus enters host, uncoats itself.
 +
** virus cannot be seen or recovered from infected cell
 +
* phase ends when first virus particle is observed inside the infected cell
 +
 +
=====Latent phase=====
 +
*time elapsed between the start time to the appearance of first new extracellular virus
 +
*virus accumulates inside the infected cell ready to be released into the medium
 +
*ends when virus is seen extracellularyly
 +
*Eclipse phase is included in latent phase
  
 
==Atypical virus like agents==
 
==Atypical virus like agents==

Revision as of 04:58, 14 March 2008

Contents

Medical Microbiology

Study of microscopic organisms includes

  • Bacteriology Bacteria simplest smallest single celled free living organisms
  • Virology viruses - non cellular parasitic, not living organisms
  • Mycology fungi - microscopic (molds and yeast), macroscopic (mushrooms and puff balls), unicellular and multicellular
  • Protozoology - protozoa, single celled
  • Phycology/Algology - algae - simple aquatic organisms ie seaweeds

Koch's postulates

  1. The same microbe is always associated witha specific disease
  2. this microbe can be recovered and grown in pure culture
  3. The pure culture must cause disease in an experimental animal
  4. The original microbe must be recovered from t he experimental disease

Examples of Problems:

  • Mycobacterium leprae - requres animal host
  • Neisseria gonorrhoeae - only human host
  • Opportunistic pathogens - immunocompromised host

Use of Molecular Postulates

  • Criteria a for determining cause of infections using molecular techniques
  1. Virulence factor: gene or gene product must be found in pathogenic stains but not in non-pathogenic stains
  2. When a virulence factor is introduced into a non-pathogenic strain it should become a pathogenic strain
  3. Genes for virulence must be expressied during the course of disease
  4. Antibodies specific for the virulence gene products should be protective


Classifications of Microogranisms

Organisms:

  • Bacteria, Archaeabacteria and Eukaryotes.
  • Infectious Agents: Viruses, viroids, prions

Bacterial and fungal classification using taxonomy

  • Kingdom, Phylum, Class, Order, Family, Genus, Species
  • Order has suffix of ales, Family has suffix of aceae
  • Biovar - variant strain that differs physiologically or biochemically
  • Serovar - sub division of species
  • Strain - a genetic varient or subtype

Viral Taxonomy

  • Order suffix virales
  • Family suffix viridae
  • Subfamily suffix virinae
  • Genus suffix virus
  • Species individual virus

Bacterial Morphology

Atypical bacteria

Fungi

Viruses

general characterisics

  • infectious agents
  • acellular
  • require hosts for replication
  • termed obligate intracellular parasites


  • exploit machinery of host cell
  • possess no ribosomes nor ATP generating systems
  • replicate independently of host cell chromosome

virus definition

a subcellular agent consisting of a core of nucleic acid surrounded by a protein coat that must use the metabolic machinery of a living host to replicate and produce more viral particules

viron

A term for complete infectious viral particle (extracellular form)

size

  • ultramicroscopic- smallest infectous agent <.2micrometers)
  • require electron microscope to visualize
  • animal viruses largest .4 microm in length
  • smallest .02 microm diameter

viral ultra structure

  • composition is simple by diverse varying in size shape and chemical composition
  • has a covering and a central core
  • covering -capsid and envelope(but not in all viruses)
  • central core - nucleic acid and various proteins
  • capsid found in all viruses
  • capsid protects the genome
  • protein shell surrounds nucleic acid sometimes referred to as nucleocapsid (capsid and nucleic acid)
  • composed of identifcal individual protein molecules known as protomers
  • protomers organize aggregate themselves into capsosmers polypeptides

**advantage: reduces the need for excessive genetic information and promotes self assembly requiring no ATP or additional enzymes there are two types of capsid symmetry helical and icosahedral

helical capsid

  • a viron with helical symmetry appears rod shaped . Capsomers are arranged into a coild -> helix 3d shape
  • diameter is determined by size and packaging of the protomers
  • length is determined by length of NA

iscosahedral capsid

  • a viron with icosahedral symmetry appears approximately spherical
  • shape and dimensions depend on protomers (3d)
  • in general a 20 sided polygon (12 spaced corners with a capsomer at each corner) with two capsomer types
  • triangular hexons composes the flat faces of 6 capsomers
  • round pentons compose the corners of 5 capsomers
  • there are variable capsomer #'s
  • poliovirus 32 (12 pentons , 20 hexons) adenovirus 252 (12 pentons , 240 hexons)

capsid function

  1. provides the nucleic acid protection from digestion by enzymes
  2. contains special sites on its surface allowing attachment of the virion to a host cell
  3. provides proteins that enable the virion to penetrate the host cell membrane
  4. in some to inject the infectious nucleic acid into the cell's cytoplasm

viral envelope

  • surrounds nucleocapsid of many viruses but not all
  • a protein phospholiipid layer of unique composition
  • lipids from host cell membrane
  • proteins are virus specific modified viral proteins not host membrane proteins

note sensitivity

  • presence of an envelope makes the viral particle sensitive to lipid dissolving agents (ether) dessication (influcenting survival time in the environment) detergents acids etc

envelope -> adds instability

types of envelope proteins

  • matrix proteins link envelope to capsid
  • stabilize virus and mediates interaction between capsid proteins and envelope
  • surface proteins exposed capsid proteins or envelope
  • glycoproteins sometimes reffered to as spikes or peplomers
  • requred for attachement of virus to host cell (antigenic determinants)
example of exposed surface proteins
  • influenza virus has 2 types
  • haemagglutinin (surface glycoprotein)
  • attaches to sialic acid residues of host cell surface, agglutinates RBCs
  • Neuroaminidase (surface spike) dissolves neuraminic acid (cytoplasmic membrane of mammalian cells) aids in releasing virus from host cell
  • please note not all virusses possess an envelope
  • if lacking - virus is termed a naked virus as opposed to an enveloped virus

nucleoprotein core

  • viral nucleic acid (NA) - this is either DNA or RNA but not both
  • viral genomes are small
  • largest known bacteriophage - 670kbp
  • note bacteria 1000-5000kbp
  • some genomes are so small that they only contain 4 genes (hep b virus) .. herpes contains hundreds
  • Is so small becuses viruses use host cell's nucleic acid and protein and energy so only need genes to encode for proteins of own envelope , capsid and nucleoprotein core

Viral genome types

  • there are a variety of viral genome types

dna viruses (either double ds or single stranded)

  • most we deal with are double stranded
  • for example
    • poxviridae
    • herpesviridae
    • adenoviridae
    • hepadnaviridae
    • papoviridae and polymaviruses

rna viruses

  • learn mostly single stranded ones
  • positive (+) stranded rna - genomes of the same polarity as mRNA
  • ie Picornaviridae and calciviridae
  • Negative (-) stranded RNA genomes of opposite polarity to mRNA
  • ie Rhabdoviridae
  • Unique Ambisense genomes (both positive and negative stranded RNA)
  • ie arenaviridae and bunyaviridae

nucleoproteins

  • found within the core
  • stabilize nucleic acid during replication or have enzymatic function
  • majority required for replication strategies

Polymerases (synth of dna 7 rna)

  • dna -dependent RNA polymerase (poxvirus)
  • RNA dependent DNA polymerase (RNA tumor viruses)
  • RNA dependent RNA polymerase (RNA viruses)

Reverse transcriptase

DNA copy from RNA genome (HIV)

VIRAL classification

ICTV standards

  1. type of nucleic acid
  2. symmetry of capsid
  3. presence/absense of envelope
  4. size of virus particle

baltimore system

  • production of mRNA during infection
  • compliments ICTV
  • viral replication strategies
  • placement of viruses in 7 arbitary groups
classI double stranded DNA
  • some replicate in nucleus
  • some replicate in cytoplasm
classII single stranded(+) sense DNA
  • replications occurs in the nucleus
  • formation of (-) sense strand
  • serves as template for +strand RNA and DNA synthesis
class III double stranded RNA
  • have segmented genomes
  • each genome segment transcribed separately to produce monocistronic mRNAs
classIV single stranded + sense RNA
polycistronic mRNA
  • hep A genome RNA = mRNA
  • naked RNA is infectious , no viron particle associated polymerase
  • translation-> formation of polyprotein product
  • cleaved to form mature proteins
complex transcription
  • two or more rounds of translation necessary to produce genomic RNA
Class V single stranded - sense RNA
  • must have viron particle RNA directd RNA polymerase
segmented
  • first step transcription of (-) sense rna genome by viron RNA dependant RNA polymerase to produce monocistronic mRNAs
  • serve as the template for genome replication
non-segmented
  • as above monocistronic mRNA are produced
class VI Single stranded + sense RNA with DNA intermediate in life cycle (retroviruses)
  • genome + sense but unique doploid)

vdoes not serve as mRNA but as template for reverse transcription

ClassVII double stranded DNA with RNA intermediate
  • relies on reverse transcription
  • occurs inside particle on maturation
  • on infection of new cell
  • first event is repair of gapped genome then transcription

Viral multiplication

  1. Adsorption attachment
  2. Penetration
  3. Uncoating
  4. Replication of macromolecules (DNA,RNA and Proteins)
  5. Assembly of viral structural components
  6. Release of mature virus from the host

Adsorption

aka Attachment

  • virus comes at specific binding site of suseptible host cell.
  • Must be aligned
  • Attachment is instantaneous

Penetration

  1. Endocytosis- Energy dependant. Naked and Enveloped viruses may be endocytosed by host cell into an endocytic vesicle which releases the viral capsid into the cell cytoplasm
  2. Fusion - Membrane enveloped fusion . most viruses use this.

Uncoating

  • involves removal of viral proteins
  • preparation for transcription/translation

Transcription and/or Translation

synthesis of viral mRNA and translation of viral proteins from new mRNA or parental RNA (transfecting viruses)

Replication

newly synthesized proteins (DNA or RNA dependant polymerases) initate steps for viral genome biosynthesis

Assembly/Maturation

  • assembly of structural proteins and genome into virions happens:
    • in cytosol (RNA virus except orthomyxovirus)
    • or in nucleus (DNA viruses except Pox virus)

Viral Replication

  • primary cells -taken directly from host and grown. may be diluted and placed into new flasks which would be classified as secondary cells
  • Transformed cells when cells are suceptible but non permissve to dna containing viruses
  • you see changes in
    • morphology (rounder)
    • physiology (increased multiplication)
    • behavior (lose contact inhibition)
  • potential to cause tumors in small mammals

Single step replication

two phases

Eclipse phase
  • after infection of cells, virus enters host, uncoats itself.
    • virus cannot be seen or recovered from infected cell
  • phase ends when first virus particle is observed inside the infected cell
Latent phase
  • time elapsed between the start time to the appearance of first new extracellular virus
  • virus accumulates inside the infected cell ready to be released into the medium
  • ends when virus is seen extracellularyly
  • Eclipse phase is included in latent phase

Atypical virus like agents

  • defective- complete virion but unable to replicate without helper virus
  • pseudovirons contain host DNA not viral, infectious but no replication

Prions

  • subviral particle , no nucleic acid
  • slow infection, long incubation period (months-years)
  • transmissible spongiform encephalopathies 1950's scrapie (hseep) kuru (and now BSE bovine spongiform encephalopathy

vproion sialoglycoprotein called PrP 27-30

  • Prp gene chromosome 20
  • Prp Protein (33-35) noninfectious -> Prp (27-30)
  • link to human disease
  • neurgenerative crytzfel-jacob disease transmissible spongiform encephalopahty progressive degeneration of the brain
  • nvCJD (new variant CJD)
  • suspect for alzheimers, multiple sclerosis
  • transmission horizontal (person to person, formites, eating contaminated meat)

viroids

  • infectious agents/subviral particle
  • small circular ss RNA with/without capsid or envelope
  • mainly cause plant diseases recently identified hep D as a viroid
  • hep d is enclose in a hep b virus capsid
    • must be simulataneously infected with both hep b and hep D viroid

bacterial viruses

  • bacteriophage/phage is a viral agent of bacteria

structure

  • phage head : protein capsid (icosahedral symmetry) which surrounds nucleic acid (majority ds DNA)
  • tail: attached prtein (helical in structure which may have base end plate with spikes (pins and tail fibers which allows injection of nucleic acid in bacterial cell

Comparison

Character Bacteria Mycoplasma Chlamydiae Rickettsiae Fungi Viruses
DNA Both Both Both Both Both Both
Obligate intracellular pathogen No No Yes Yes Maybe Yes
Peptidoglycan cellwall Yes No Yes (1 stage) Yes No No
Growth on artifical media Yes Yes No No Yes No
Contain ribosomes Yes Yes Yes Yes Yes No

Micobial gowth and multiplication

  • Physical and chemical requirements are required to grow and multiply
  • Physical requirements such as temperature osmotic pressure and pH
  • Chemical requirements Carbon Nitrogen Sulfur phosphorus trace elements oxygen organic growth factors
  • Survival of bacteria in a hostile environment requires that there must be a continous macromolecular synthesis and genome replication. -> increase in cell mass and in bacterial genome necessitiates that the cell mst divide to maintain its physical size and the size of its genome
  • Therefore one bacterial cell divides into 2 daughter cells by asexual process known as binary fission
  • The time required for a single cell to divide into 2 daughter cells is termed as doubling time
  • doubling time is deined as formation of new daughter bacterial cells from parent cell. its growth to propersize due to replication fo bacterial macromolecules and eventual division into two cells

Sample doubling times :

  • Bacillus stearothermophilus 11min
  • Escherichia coli 20 min
  • Staphylococcus aureus 28min
  • Lactovacillus acidophilus 60-80 min
  • mycobacterium tuberculosis 360 minutes
  • treponema pallidum (causative agent of syphyllis) 1980 minutes

growth curve

in a closed system where nutrients and space are finite no removal of waste products

lag phase

newly inoculated cells need adjustment to the new environment (nutrients temp ph) 2) no cell divisiion 3) very few bacterial cells population is sparse/dilute

exponential (log) phase

1) cell adjustment is complete metabolism and replication at full speed growth at geometric / logarithmic rate cells reach maximum rate of cell division (this continues as long as nutrients and environment is favorable)

stationary phase

1) population reaches maximum numbers ate of cell inhibition (death) = rate of multiplication factors: nutrient depletion, ph changes, accumulation of waste, reduced O2 (can induce endospores of bacillus and clostridium species)

death

1) decline in growth rate 2) death in geometric proportions causes : depletion of nutrients, O2, excretion of toxic waste products, increased density of cells (limited space) factors same as stationary phase+ release of lytic enzymes

measurement of viable bacteria

serial dilution: dilute concentrated samble 10 folds and meansure enumerated bacteria in each plate count

plate count use for enumeration of viable bacteria (CFU colony forming unit) . A fraction of each dilution is placed on agar plate and counted. Assume each colony is formed by one bacterium and total # of viable bacteria = total number of colonies. Total viable count= # of colonies x sample size (.1) and the dilution factor.

Filtration; for water testing and highly diluted samples Turbitidy for blood cultures

Physical factors affecting growth

-temperature- whether a bacteria multiplies slowly or luxuriantly at certain temperatures 3 groups

  1. minimum - lowest temp at which bacteria can survive and permit microbial growth and metabolism
  2. maximum - highest temp at which bacteria can survive and permit microbial growth and metabolism
  3. optimum - narrow range of temp that promotes the fastest growth of a bacterial species
Min Max Optimum
Ecoli 15-20 45 37


Psychrophile

psychrophilic opt temp~ 4 celcius capable of growth at 0 degrees celcius not involved in human infections found in snow fields polar ice deep ocean examples: psydomonas, flavobacterium, alcaligenes, achromobacte species

Facultative psychrophile

grow slowly in cold condition but optimial above 20c staph aureus c. monocytogenes contamination of food/dairy


Mesophile

opt temp 20-40c capable of growth 10-50 group containing human pathogens (30-37) example: e. coli

Thermophile

optimum temp 60 cel. capable of growth 45-85 celcius incapable of growth at usual body temp not involved in human infections found volcano, direct exposure to sun

Osmotic Pressure

AKA Water activity

  • all bacteria require water for growth and reproductions . It is an essential solvent involved in all biochemical reactions and indirectly maintain osmotic pressure
  • increase of salts leads to a decrease in the rate of growth , cell swells , replication halts
  • aw = index of free available water= atmospheric measure , relative humidity = rh
  • rh= 1.00 aw
  • therefore 90% rh = .9 Aw
  • absorption and solution factors reduce availabilty of free water (decreased Aw)

pure distilled water (Aw =1) Saturated solution of NaCl (Aw=.8) Seawater NaCla = 3%

  • Most bacteria have an active metabolism at Aw > .0
  • Foods that have more free water will spoil more quickly as opposed to foods which have lower Aw (which would have less water0
  • Lower Aw -> slow growth
  • Below aw of .09 -> Bacteria unable to grow
  • Exceptions to this
    • Xerotolerant: lower Aw
    • Fungi can grow at Aw of .6
    • Yeasts ~ .6
    • Salt tolerant bacteria, Halophiles (high solute, low Aw)


Effects of pH

Growth rates are influenced by pH values

Oxygen Requirements

  • limting factor in bacterial growth because O2 has limited solubiity in water
  • Enzymes are needed to reduce oxygen into water as well as ROI byproducts
  1. Catalase 4H2O2 -> 3H2O + 2O2
  2. Peroxidase H2O2 + NADH + H+ -> 2H2O + NAD+
  3. Superoxide dismutase 2O2- + 2H+ -> H2O2 + 2

Based on oxygen requirements

Obigate aerobes

  • Totally dependant on O2
  • Need at least 1atm
  • have catalase and SO dismutase

Microaerophiles

Grow in presence of O2 but only tolerate 4% of I2

  • possess enzymes but growth will be inhibited if toxic products accumulate

Obligate anaerobes

Grow only in absense of O2

  • Do not possess detoxifying enzymes
  • Low levels of O2 -> LETHAL

Examples

  1. Clostridum species
  2. Bacteroides species

Capnophiles

  • grow better in high CO2 but tolerate O2

Faculative anaerobe

  • can grow in with / without O2 present
  • grow best under aerobic conditions
  • use aerobic respiration when O2 is present

example: Enterobacteriacea

Bacterial Enzymes related to O2

Microorganism Type Catalase SO Dismutase
Aerobe + +
Facultative anaerobe + +
Microaerophile - +
Obligate Anaerobe - -

Growth Location

Growth location based on Gaseous requirement
[  ]  [  ]  [  ]  [  ]  [  ]
 --    --    --    --    --
|  |  |..|  |  |  |  |  |..|
|  |  |..|  |  |  |  |  |..|
|  |  |  |  |..|  |  |  |..|
|  |  |  |  |..|  |  |  |..|
|  |  |  |  |  |  |  |  |..|
|  |  |  |  |  |  |..|  |..|
|  |  |  |  |  |  |..|  |..|
 --    --    --    --    --
 1     2     3     4     5
  1. uninoculated
  2. obligate aerobe
  3. microaerophile
  4. obligate anerobe
  5. facultative anerobe


Culture media

Developed to isolate and grow bacteria

  • Types :
  • Chemically defined media
  • Complex media
    • chemicals + complex organic compoinds added to facilitate bacterial growth
  • Anaerobic growth media
    • known as Reduced medium
    • reducing agents added which remove any free oxygen from medium
    • adds reducing medium (thioglycolate cystine or ascorbate to remove O2
  • Selective media
    • contain nutrients and inhibitiong agents for unwanted bacteria
    • addition of specific C or energy source, adjust pH, increase osmotic pressure, adjust O2 tension
    • examples Salmonella-Shigella agar, mannitol salt agar
  • Enrichment media
    • special nutrients are added to make it 'tasty' for bacteria
  • All purpose media
    • nutrient agar and broth support growth of most microbes
  • Enriched media
    • Also contains additional nutrition ie: blood agar
  • Transport medium
    • Used to preserve microbes for transit following initial procurement from patient
  • Differential Medium
    • more than one type of organism can grow but speration is based on growth pattern
    • examples:
      1. MacConkey agar - enterobacteriaceae
      2. Eosin-methylene blue (EMB) E coli Enterbacter aerogenes
      3. Blood Agar - differential for streptococci only

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