Review: What type of microscopy
is this?
Hint: Trick Question
Review: What type of microscopy
is this?
Review: What type of microscopy
is this?
Review: What type of microscopy
is this?
Review: What type of bacteria is
this?
Review: What type of bacteria is
this?
Culture Methods
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
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
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 (O157:H7):
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, nontarget strains
• e.g., sorbitol-MacConkey Agar for E. coli O157:H7
– Most E. coli ferment D-sorbitol; most E. coli O157:H7 do not;
– Sorbitol-negative E. coli O157:H7 colonies are colorless
– Non-O157:H7 colonies are pink.
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.)
Enrichment Cultures
• Observe for growth by
turbidity, clearing, gas
production, color change,
etc.
• Score as presenceabsence (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)
Bacterial Culture
• Often 2 step
– Presumption
– Confirmation
• All in one test
– Chromogenic media
– Enzymatic test (e.g. Β-glucoronidase activity
on MUG)
Basic Bacterial Techniques
• Streak Plate
– For isolation of single colony
• Spread Plate
• Pour Plate
• MPN liquid assay
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
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
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 O157:H7
Detection of Stressed, Injured and Viable-ButNonculturable (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.
Assay Methods for Viruses
• Electron Microscopy (EM) and Immune EM
– Insensitive (>1,000,000 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)
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
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)
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).
Uninfected Cell
Culture
Infected Cell Culture with
CPE
Virus plaques in a cell
layer overlaid with agar
medium and stained with
a vital dye
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
Estimating Viral Dose:
Relationship 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.
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
Detection of Hepatitis A Virus in Cell Culture by
Radioimmunofocus Assay
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
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
C. parvum Oocysts
Immunofocus of Living Stages: in MDCK Cells with C3C3-FITC Antibody
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.
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
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
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
Terminology
• Biosensor
• ACPLA