Review: What type of microscopy is this?

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Review What type of microscopy is this?
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Review What type of microscopy is this?
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Review What type of microscopy is this?
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Review What type of microscopy is this?
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Review What type of bacteria is this?
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Review What type of bacteria is this?
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Culture Methods
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Culturing Microbes

• Detection by culture or infectivity assays is
  preferred
• demonstrates that the target microbes are alive
  and capable of multiplication or replication.
• From a public health and risk assessment
  standpoint, microbial pathogen assays based on
  infectious units are the most relevant and
  interpretable ones

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Culturing Bacterial Pathogens

• Continued interest and use because of newly
  recognized, newly appreciated and evolving agents
  
• Ability to culture some bacterial pathogens goes
  back more than a century
• Salmonella and Vibrio species
• Culturing bacterial pathogens from water,
  wastewater and biosolids remains technologically
  underdeveloped
• Has not advanced greatly beyond the application
  of methods used in clinical diagnostic and/or
  food bacteriology
• Greater need for more rapid culture methods
• Combine culture with molecular detection

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Culturing Bacteria Pathogens

• Salmonella, Shigella, Campylobacters and Vibrios
  culture methods little changed beyond efforts to
  improve recoveries using modified pre-enrichment
  and enrichment broths and differential and
  selective agars
• For some other bacterial pathogens
• e.g., enterohemorrhagic strains of Escherichia
  coli (O157H7) culturing from water a challenge
  many other, non-pathogenic strains of E. coli.
• select for their growth based on distinctive
  biochemical or other properties to facilitate
  separation from the other, non-target strains
• e.g., sorbitol-MacConkey Agar for E. coli O157H7
• Most E. coli ferment D-sorbitol most E. coli
  O157H7 do not
• Sorbitol-negative E. coli O157H7 colonies are
  colorless
• Non-O157H7 colonies are pink.

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Culture or Infectivity Assays for Bacteria

• Traditional approach
• pre-enrich and/or enrich using non-selective and
  then selective broth media, or
• grow colonies on membrane filters
• Then
• Transfer to differential and selective agars
• Recover presumptive positive colonies
• Biochemical, metabolic and other physiological
  testing
• Serological or other immunochemical typing and
  identification (agglutination, enzyme
  immunoassay, etc.)
• Other characterization phage typing, nucleic
  acid analyses, virulence tests (cell cultures,
  animal ileal loops, animal infection, etc.)

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Enrichment Cultures

• Observe for growth by turbidity, clearing, gas
  production, color change, etc.
• Score as presence-absence (positive or negative)
• Quantify using replicate and different volumes to
  compute a Most Probable Number
• Example
• 5 x 10 ml
• 5 x 1 ml
• 5 x 0.1 ml

Left negative Right positive (color change)
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Bacterial Culture

• Often 2 step
• Presumption
• Confirmation
• All in one test
• Chromogenic media
• Enzymatic test (e.g. ?-glucoronidase activity on
  MUG)

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Basic Bacterial Techniques

• Streak Plate
• For isolation of single colony
• Spread Plate
• Pour Plate
• MPN liquid assay

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Problems in Culture Methods for Bacterial
Pathogens

• Inefficient growth (low plating efficiency),
• Slow growth rates
• Overgrowth by other non-target bacteria.
• Efforts to improve culture and reduce or
  eliminate non-target bacteria
• antibiotics
• physical (heat) treatments
• chemical (acid) treatments
• specialized plating e.g., dual media plating

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Problems in Culturing Bacterial Pathogens

• Inability of typical culture methods now in use
  to detect or distinguish
• pathogenic from non-pathogenic strains
• sources of pathogens (microbial source
  tracking)
• newly emerging pathogenic strains
• evolutionary processes and mechanisms
• the role of environmental change in selection for
  or emergence of new pathogenic strains
• Antibiotic and disinfectant resistant strains

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Pathogenic Bacteria For Which Culture Methods Are
Underdeveloped for Food and Water

• Pathogenic E. coli
• Campylobacter jejuni other Campylobacters
• Yersinia enterocolytica,
• Aeromonas hydrophila,
• Helicobacter pylori,
• Legionella species
• Mycobacterium avium-intracellulare
• Shigella
• Better developed
• Salmonella species
• Some pathogenic E. coli O157H7

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Detection of Stressed, Injured and
Viable-But-Nonculturable (VBNC) Bacteria

• Waterborne bacterial pathogens and indicators are
  often physiologically altered/stressed and not
  efficiently cultured using standard selective and
  differential media
• Heavy metals, disinfectants, pH extremes, other
  environmental stressors
• Causes considerable underestimation of the
  concentrations of these bacteria in water and
  therefore, underestimation of their risks to
  human health
• Stressed, injured and VBNC bacteria may still be
  fully infectious for humans and other animal
  hosts
• (there is disagreement on this point)
• Repair and resuscitation methods improve the
  detection of viable and potentially cultural
  bacteria, but, these methods are rarely used to
  detect pathogens in drinking water.

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Assay Methods for Viruses

• Electron Microscopy (EM) and Immune EM
• Insensitive (gt1,000,000 particles/ml)
• OK for clinical but not environmental virology
• Animal Infectivity
• Slow, cumbersome, expensive, ethical
  considerations
• Culture or infectivity
• Now widely used in environmental virology
• Cytopathogenic effects
• Growth, but no cytopathogenic effects
• detect viral antigens or nucleic acids
• Immunoassays
• insensitive for direct detection
• Amplify viruses in cell cultures
• Nucleic acid assays
• insensitive for direct detection by hybridization
• Amplify in cell culture or in vitro (PCR or
  RT-PCR)

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Quantifying Human Virus Infectivity is a Challenge

• Some infect only humans
• Some infect certain experimental animals, too
• Some infect experimental animals and cell
  cultures
• Different ratios of infectivity to virions
  (particles)
• 1 infectious unit 1 virus particles
• some bacteriophages)
• 1 infectious unit 100 virions
• some cell culture adapted viruses
• 1 infectious unit 10,000-100,000 virions
• many wild-type or field viruses

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Detection Of Viral Pathogens by Culture

• Viruses are obligate intracellular parasites
• Some viruses can be propagated or cultured in
  susceptible hosts
• whole animals
• mammalian cells grown in culture
• Quantify viruses in animals using quantal methods
  (e.g., Most Probable Number or MPN) in eggs
  using pock assays on egg membranes (smallpox
  and vaccinia initially enumerated this way)
• Virus assays in cell cultures by quantal (e.g.,
  MPN) or enumerative methods (plaque or local
  lesion assays)

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Virus Detection in Cell Culture

• Some viruses (some enteroviruses, reoviruses,
  adenoviruses and astroviruses) propagate in
  susceptible host cell cultures and produce
  morphologically distinct cytopathogenic effects
  (CPE).

Virus plaques in a cell layer overlaid with agar
medium and stained with a vital dye
Infected Cell Culture with CPE
Uninfected Cell Culture
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Challenges in Assaying Viral Infectivity

• Host susceptibility and variability in host
  susceptibility
• Type of host cells and cell culture assay
  methods
• Plaque (enumerative) assays
• Quantal (liquid culture) assays
• Quantal assay endpoints
• Cytopathogenic effects visible changes in cells
• Immunodetection or nucleic acid detection
• Animal bioassays used if no cell culture assay
  available
• Virus titer often increases with serial passages
  in hosts

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Estimating Viral DoseRelationship of
Infectivity to Virus Particle Count

• As little a one or a few intact, functional virus
  particles are capable of causing infection in a
  susceptible host.
• Ratios of virus particles to infectious units are
  highly variable and are subject to change
• Passage of viruses in susceptible host cells
  reduces the ratio of virus particles to
  infectious units
• rotavirus
• initial ratio 50,000 virus particles/infect.
  unit
• after cell culture passage 100
  particles/infect. Unit
• Norwalk Virus appears to be infectious at doses
  corresponding to as little as a few virus
  particles.

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Challenges in Assaying Viral Infectivity

• Some viruses (some enteroviruses, adenoviruses,
  rotaviruses, astroviruses and hepatitis A virus)
  grow poorly or slowly in cell cultures and
  produce little or no CPE.
• Greater detection with additional analytical
  techniques
• Viral antigens
• Immunofluorescence assays, enzyme immunoassays,
  radioimmunoassays, etc.
• Viral nucleic acid assays hybridization and/or
  amplification
• Combined cell culture RT-PCR demonstrates
  presence of greater numbers of infectious viruses
  than CPE alone
• Post-disinfection, more viruses are detected
  than by CPE

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Detection of Hepatitis A Virus in Cell Culture by
Radioimmunofocus Assay
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Detection of Protozoan Parasites by Culture

• Environmental forms of some protozoan parasites,
  such as spores and oocysts, are culturable in
  susceptible host cells
• Culture Cryptosporidium parvum oocysts and some
  microsporidia spores in mammalian cell cultures
  observe living stages
• Culture free-living amoebas (Naegleria spp. and
  Acanthamoeba spp.) on lawns of host bacteria,
  such as E. coli, on nonnutrient agar they form
  local lesions.
• For other waterborne parasites, such as Giardia
  lamblia and Cyclospora cayatenensis, culture from
  the environmental stage (the cyst or oocyst) is
  still not possible

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Detection of Protozoan Parasites by Culture

• Spores of some microsporidia (Encephalitozoon
  intestinalis) and the oocysts of Cryptosporidium
  parvum can be cultured in mammalian host cells
  where spores germinate or oocysts excyst and
  active stages of the organisms can proliferate.
• Living stages detected (after immunofluorescent
  or other staining) and quantified score positive
  and negative microscope fields or cell areas
  (slide wells), or count numbers of foci of living
  stages or discrete living stages.
• Express concentrations MPNs or other units, such
  as numbers of live stages.
• Detection of living stages also possible by FISH,
  PCR or immunoblotting
• Facilitates molecular characterization

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C. parvum Oocysts
Immunofocus of Living Stages in MDCK Cells with
C3C3-FITC Antibody
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Pathogen Detection by Biochemical Methods

• Enzymatic activities unique to target microbe
• Signature Biolipid Analysis
• Detection of unique biolipids by
  gas-chromatography, mass spectrometry and other
  advanced organic analytical methods
• Extract and purify from cells
• Analyze
• Other biochemical markers unique to a specific
  pathogen or class of pathogens.

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Examples of Other Biochemical Techniques

• BIOLOG (carbon/nitrogen/sulfur utilization
  profiles)
• API Biostrips
• VITEK and VITEK II
• Enzyme activity assays
• Colormetric or Fluorescence reporting
• FAME-fatty acid methyl ester
• FTIR spectroscopy

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General Biochemical Targets

• Adenylate Kinase/ATP Luminescence
• Headspace pressure/gas analysis
• CO2 production
• Electrical conductance/impedance
• UV 260/280
• Excites amino acids with conjugated double bonds
  (e.g. trytophan, tyrosine, phenylalanine)
• Longer wavelengths (Nicotinamides/Riboflavins)
• NAD(P) 325-354 excitation 480 peak fluorescence
• Riboflavin 380-400 excitation 520 peak
  fluorescence

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Light Adsorption/Fluorescence

• Examples of Stand-off/DTW Systems
• BAWS system (UV/visible absorbance and
  Fluorescence)
• FLAPS system (near UV instead of Far to improve
  signal to noise ratio
• LIDAR and UV-LIF
• UVRR or Raman Resonance
• Raman Spectroscopy
• LIBS

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Terminology

• Biosensor
• ACPLA