1. Methods for Detection of Microbial Contaminants – Part I
ENVR 421
Mark D. Sobsey

2. Detecting Pathogens and
Indicators in the Environment

3. Detection of Pathogenic Microbes in Water
Three main steps:
(1) recovery and concentration,
(2) purification and separation, and
(3) assay and characterization.

4. Microbial Methods for Pathogen Detection
Initial sampling, concentration, or recovery methods
Efficient recovery of low numbers from waters
One of the greatest challenges for environmental detection
Pathogen detection and isolation methods
Modified methods from clinical microbiology
Must overcome environmental inhibitors
Pathogen confirmation and characterization
Where did the fecal waste come from?? (source attribution and source tracking)

5. Initial Recovery and Concentration of Pathogens from Water
Sedimentation by Centrifugation
Bacteria and Parasites: differential centrifugation
several thousand times gravity for several minutes to tens of minutes
Blood cell separator; continuous flow centrifugation and particle accumulation
being used for parasites; previously used for bacteria
Viruses: ultracentrifugation
50-100,000 x gravity for several hours
Recover sedimented microbes in a small volume of aqueous solution

6. Membrane and other microporous filters
Filtration: Bacteria
Membrane and other microporous filters
Filter 100s-1000s of ml of water through cellulose ester, fiberglass, nylon polycarbonate, diatomaceous earth or other filters
Apply membrane filters to agar medium and incubate to get colonies
Place filters in liquid culture medium to culture bacteria

7. Filtration: Parasites
Absolute or nominal pore size filters, 1-several micrometer pore size
Polypropylene, cotton, et.c. yarn-wound cartridge filters
Polycarbonate, absolute pore size disk filters
Polysulfone, pleated capsule filters
Spinning cartridge and hollow fiber ultrafilters
Cellulose acetate, absolute pore size, circular disks
Others
Recover retained parasites by elution (washing) or recovery of retentate water containing particles

8. Filtration: Viruses Ultrafiltration: 1,000-100,000 MWCO
Viruses are retained by size exclusion
Hollow fiber, spiral cartridge, multiple sheets, flat disks, etc
polysulfones, cellulose ester, etc.
tangential flow to minimize clogging
Recover viruses in retentate; facilitate by elution of filter medium

9. Filters to Recover and Concentrate Microbes from Water

10. Filtration: Viruses
Adsorbent filters; pore size of filters larger than viruses; viruses retained by adsorption
electrostatic and hydrophobic interactions
negatively charged cellulose esters, fiberglass
must acidify water and add multivalent cations
Electropositive filters:
charge-modified fiberglass as disks or pleated cartridges
fiberglass filter disks one coats with precipitated aluminum or iron salts in their own laboratory, or
positively-charged natural quartz fabricated into fiberglass that one packs into a column to make an adsorbent filter

11. Cuno 1 MDS Virosorb Electropositive Filter (flat disk - cartridge)
NOTE: course texture
Flat Disk – filter holder
Filter Material
Cartridge – filter holder
Filter material used as double layers
Cartridge filter pleated to increase surface area

12. 5 – 6 (depending on strain)
How it works?? – Electrostatic and Hydrophobic Interactions: Virus Adsorption
Electropositive filter >>> at ambient pH, viruses are negatively charged
Isoelectric point – pH where there is no net charge on a particle or surface
Hydrophobic interactions >>> hydrophobic areas on the filter surface that enhance viral adsorption
Virus
Isoelectric Point
Poliovirus
7.0
Coliphage MS2
3.9
Coliphage PRD1
4.2
Coliphage Q
5.3
Coliphage X174
6.6
Norovirus
5 – 6 (depending on strain)
Adenovirus
4.55 (hexon)
4.69 (penton)
7.07 (fiber)

13. How it works?? – Electrostatic and Hydrophobic Interactions: Virus Elution
Eluting solutions at higher pH (typically pH 9.5) surpass the isoelectric point of the filter > both filter and virus have net negative (-) charge
Virus and filter repels each other releasing viruses into solution
Negatively charged constituents within the eluting solution that may compete for adsorption sites on the filter surface
enhanced by particles with high isoelectric points that remain negatively charged if the pH of the eluting solution is raised
a contributing factor for elution with beef extract/glycine
positively charged eluting solutions may compete with the filter surface for the negatively charged virus particles
a contributing factor for elution with alternative eluting solutions

14. Virus Elution from Adsorbent Filters
Elute adsorbed viruses with alkaline organic buffer solutions:
Beef extract
Amino acids
Others
Beef extract less compatible with nucleic acid detection methods

15. Viruses: precipitate with polyethylene glycol or aluminum hydroxide
Initial Recovery and Concentration of Pathogens from Water by Chemical Precipitation Methods
Viruses: precipitate with polyethylene glycol or aluminum hydroxide
resuspend PEG precipitate in aqueous buffer
dissolve aluminum floc in dilute acid solution
both have been used as second-step concentration and purification methods
Parasites: precipitate with calcium carbonate
dissolve precipitate in dilute sulfamic acid

16. Secondary Concentration: PEG Precipitation
Polyethylene glycol (C2H6O2)
General mode of action of a precipitation reagent is the binding of water
Used along with NaCl > essentially “salting out” protein particles
Rapid, inexpensive, non-destructive to viruses
Gentle precipitation at neutral pH

17. Other Primary Recovery and Concentration Methods
Minerals, such as iron oxide and talc; used to adsorb viruses
Synthetic resins: ion exchange and adsorbent
Other granular media: glass beads and sand
Less widely used; less reliable, cumbersome; uncertain elution, desorption, exchange efficiencies

18. Separation and Purification Methods
Purification, separation and concentration of target microbes in primary sample or sample concentrate
Separate target microbes from other particles and from solutes
Reduce sample size (further concentrate)
Variety of physical, chemical and immunochemical methods:
Sedimentation and flotation (primarily parasites)
Precipitation (viruses)
Filtration (all classes)
Immunomagnetic separation or IMS (all classes)
Flow cytometry (bacteria and parasites); an analytical method, too

19. Assay Methods for Waterborne Pathogens
culture or infectivity
viability or activity measurements
immunoassays
nucleic acid assays
microscopic examinations

20. Culturing Waterborne 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

21. Traditional Approach: Culture or Infectivity Assays for Bacteria
pre-enrich and/or enrich using non-selective and then selective broth media, or
grow colonies on membrane filters
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 and animal ileal loop assays for pathophysiologic response, animal infection, etc.)

22. Enrichment Cultures Cultures may have characteristic appearance
Color change
Other phenomena
E.g., stormy fermentation
Cultures may require additional measurement to confirm a positive result
Subculture
Apply other analytical measurement
Immunoassay
Nucleic acid assay

23. Culturing Waterborne Bacteria 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,
Culturing bacterial pathogens from water remains technologically underdeveloped
Has not advanced greatly beyond the adaptation of methods used in clinical diagnostic and/or food bacteriology

24. Culturing Waterborne 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 is a challenge due to relative abundance of other, non-pathogenic strains of E. coli.
select for their growth based on unique biochemical or other properties to facilitate their separation from the other, non-target strains
e.g., sorbitol-MacConkey Agar for E. coli O157:H7

25. Waterborne Pathogenic Bacteria For Which Culture Methods Are Underdeveloped
Campylobacter jejuni; other Campylobacters
Yersinia enterocolytica,
Aeromonas hydrophila,
Helicobacter pylori,
Legionella species
Mycobacterium avium-intracellulare
Shigella
Better developed:
Salmonella

26. Problems in Culture Methods for Bacterial Pathogens in Water
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:
Dual media plating
Biochemicals with chromogenic reaction products

27. Problems in Culturing Bacterial Pathogens in Water
Inability of typical culture methods now in use to detect or distinguish:
pathogenic from non-pathogenic strains
the sources of pathogens
newly emerging pathogenic strains
evolutionary processes and mechanisms
the role of environmental change in selection for or emergence of new pathogenic strains

28. 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
Causes great underestimation of true concentrations in water and other samples
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 to improve the detection of viable and potentially cultural bacteria
Such methods are rarely used to detect pathogens in drinking water; more so in foods

29. Detection Of Viral Pathogens by Culture
Viruses are obligate intracellular parasites,
many enteric viruses can be propagated or cultured in susceptible hosts
whole animals
mammalian cells grown in culture
Quantify viruses in animals and cells using quantal methods (e.g., Most Probable Number or MPN)
Virus assays in cell cultures by quantal (e.g., MPN) or enumerative methods (plaque or local lesion assays)

30. Enteric Virus Detection in Cell Culture
Some viruses (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

31. Enteric Virus Detection in Cell Culture
Other viruses (some enteroviruses, adenoviruses, rotaviruses, astroviruses and hepatitis A virus) grow poorly or slowly in cell cultures and produce little or no CPE.
Detection of these viruses requires the used of additional analytical techniques directed at detecting viral antigens (immunofluorescence assay, enzyme immunoassays and radioimmunoassays) and nucleic acid (nucleic acid hybridization or amplification assays).

32. Detection of Hepatitis A Virus in Cell Culture by Radioimmunoassay

33. Viruses Not Detected in Cell Culture
Enteroviruses, caliciviruses (noroviruses) parvoviruses, coronaviruses (some), picobirnaviruses and hepatitis E virus can not be propagated in any known cell cultures.
Not detectable in water unless an alternative analytical method, such as nucleic acid amplification by PCR or RT-PCR, is applied directly to concentrated samples.

34. Detection of Protozoan Parasites by Culture
Environmental forms of some protozoan parasites, such as spores and oocysts, are culturable in susceptible host cells
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) recovered from water is still not possible

35. 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 also possible by PCR or immunoblotting
Facilitates molecular characterization

36. Progress in Detection of Protozoan Parasites by Culture
Oocysts of Cryptosporidium parvum and spores of some microsporidia (Encephalitozoon intestinalis) infect mammalian host cells:
Spores germinate and oocysts excyst
Active stages of the organisms proliferate
Detect and quantify (after immunofluorescent or other staining)
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 as MPNs or other units based numbers of live stages, numbers of infectious foci or number of positive microscope fields
Detect by NA methods (PCR, FISH, etc.)
Facilitates molecular characterization
Immunofocus of C. parvum Living Stages: in MDCK Cells with C3C3-FITC Antibody

37. Combined Cell Culture and Nucleic Acid Detection and Amplification of Waterborne Pathogens
Inoculate sample into susceptible host cell cultures
incubated to allow the viruses or parasites to infect the cells and proliferate.
After producing enough nucleic acid, extract and either hybridize directly with a gene probe or further amplify by PCR or RT-PCR
Facilitates detection of infectious but non-cytopathogenic viral and protozoan pathogens able to proliferate in cell cultures.
Reduces incubation time to detect pathogen nucleic acid.
Facilitates molecular or other methods of characterization

38. Detection of Waterborne Pathogens by Viability or Activity Assays
Assay bacteria for viability or activity by combining microscopic examination with chemical treatments to detect activity or "viability".
measure enzymatic activities, such as dehydrogenase, esterase, protease, lipase, amylase, etc.
Example: tetrazolium dye (INT) reduction:
2-[p-iodophenyl]-3-[p-nitrophenyl]-5-phenyltetrazolium Cl (measures dehydrogenase activity).
Reduction of tetrazolium dye leads to precipitation of reduced products in the bacterial cells that are seen microscopically as dark crystals.

39. Progress in Detection of Waterborne Bacteria by Viability or Activity Assays
Combine activity measurement and immunochemical assay (for specific bacteria).
Combine fluorescent antibody (FA) (for detection of specific bacterium or group) with enzymatic or other activity measurement
Use image analysis tools to improve detection and quantitation
Flow cytometry
Computer-aided laser scanning of cells or colonies on filters

40. Detection of Waterborne Bacterial Pathogens by Viability or Activity Assays
Combine methods for bacterial detection in water, such as activity measurement and immunochemical assay (for specific bacteria).
Example "FAINT”: combines fluorescent antibody (FA) (for detection of specific bacterium or group) with tetrazolium dye reduction (INT)
Look for INT crystals in cells specifically stained with fluorescent antibodies

41. Viability or Activity Assays for Protozoan Cysts and Oocysts
Example: Stain with DAPI (the fluorogenic stain 4',6‑diamidino‑2‑phenylindole; taken up by live oocysts and propidium iodide (PI; taken up by dead oocysts).
Viable Cryptosporidium oocysts are DAPI-positive and PI-negative
Non-viable oocysts are DAPI-negative and PI-positive
Alternative stains may be more reliable
Viability staining is often poorly associated with infectivity; detects inactivated cysts and oocysts
Detects cysts and oocysts inactivated by
UV and chemical disinfection

42. C. parvum oocysts
Dual stain : DAPI (blue) and propidium iodide (red)

43. Detecting Active or Viable Pathogens Using Nucleic Acid Targets
Detect short-lived nucleic acids present in only viable/infectious microbes:
ribosomal RNA
messenger RNA
genomic RNA of viruses (large amplicons)
Detect pathogen nucleic acid by fluorescent in-situ hybridization (FISH)
applied to bacteria, protozoan cysts and oocysts, as well as viruses in infected cell cultures
(see pictures in later slides)