Presentation on theme: "Indicator Microorganisms – Chapter 23"— Presentation transcript:
1 Indicator Microorganisms – Chapter 23 ObjectivesBe able to define what an indicator microorganism is and why they are usedBe able to list the criteria for an ideal indicator organismBe able to list at least three different types of indicatorsBe able to describe standard detection methods used to measure indicatorsUnderstand the difference between Water Quality Standards and Water Quality Guidelines
2 What Is an Indicator Microorganism? A nonpathogenic microorganism whose presence suggests the presence of enteric pathogensIndicator organisms are used because pathogens themselves are frequently difficult to detect in drinking water and wastewaterlow numbers (but still above MID)difficult, time consuming, or expensive to culture
3 U.S. Public Health Services adopted coliforms as indicators of fecal contamination of drinking water in 1914coliforms are bacteria that live in the intestines of warm-blooded animals and are excreted in high numbers in fecesindicate fecal contamination of drinking waterpresence demonstrates a breakdown of wastewater treatment processesThe food industry uses other indicator microorganisms to evaluate the efficiency of food processing
4 Criteria for an Ideal Indicator Organism Should be useful for all types of water (drinking water, wastewater, recreational water, sea water)Should be present whenever enteric pathogens are present, and absent when pathogens are absentShould survive longer in the environment than the toughest enteric pathogenShould not grow in waterDetection protocols should be easy and inexpensiveDensity of indicator microorganisms should correlate with the degree of fecal pollutionShould be a member of the normal intestinal microflora of warm-blooded animals
5 Types of Indicators Total coliforms Fecal coliforms Fecal Streptococci Anaerobic bacteriaBacteriophage
6 Estimated levels of indicator organisms in raw sewage Organism CFU/100ml .ColiformsFecal coliformsFecal streptococciEntercocciClostridium perfringensStaphylococcusPseudomonas aeruginosaAcid-fast bacteriaColiphagesBacteroides
7 Total coliformsMost commonly used indicator for: drinking water, wastewater treatment, shellfish harvesting water, and recreational waterAerobic or facultatively anaerobicGram negativenon-spore-formingrod-shapedgas production during lactose fermentation within 48 hours at 35°CExamples: Escherichia, Citrobacter, Klebsiella and EnterobacterHigh numbers (2 x 109 per capita per day) in human AND animal feces< 1 coliform per 100 mL drinking water ensures the prevention of bacterial waterborne disease outbreaks
8 DrawbacksColiforms may grow in aquatic environments, particularly if organic matter levels and temperatures are elevatedColiforms may form biofilms in drinking water distribution systems – this is a problem because, for example, E. coli is 2400 times more resistant to chlorine in a biofilm than when planktonicColiforms may recover from disinfectant injuryGrowth of heterotrophic bacteria on media selective for coliforms can mask coliform population in water (occurs when heterotrophic counts exceed 500/mL)More vulnerable to disinfection and environmental trauma than enteric viruses or parasitesDo not necessarily indicate fecal contamination
9 100 10 l m Coliforms 10 10 g 1 n / i n i i l a o 1 10 m c e . r E a Regrowth of coliforms and E. coli in sewage effluents after inactivation with 5 mg/L chlorine100107lmColiforms10106g1n/inDie-off rate depends onamount/type of organicmatter present and thewater temperaturecan lead tofalse positivesiilao1105mce.rEa0.1104riorestcm0.01103arBofi%l0.001o102E. coliC0.0001101Time (days)
10 Fecal coliforms Subgroup of total coliforms Able to ferment lactose and produce both acid and gas at 44.5°C in 24 hoursInclude Escherichia and Klebsiella, which are exclusively fecal in origin (perhaps. . .)Drawbackssame drawbacks as for total coliformsindicates fecal contamination for sure, but can’t distinguish between animal and human fecescan survive and grow for extended periods of time in tropical watersmay be natural inhabitants of these waters!
11 Fecal Streptococci do not multiply in water are more resistant to stress/disinfectionlast longer in the environmentused as indicators of enteric viruses, and gastroenteritis for swimmersMembers of the lactic acid bacteriaGram positive, non-motile, non-spore-forming, aerotolerant anaerobic bacteria that ferment sugars to lactic acidFC/FS ratio - ratio of fecal coliform counts to fecal strep countsFC/FS >4 : fecal contamination of human originFC/FS < 0.7: fecal contamination of animal originThis relationship is only valid for recent fecal contamination (within the last 24 hours)
12 Anaerobic bacteria Clostridium perfringens – one example Gram positive, anaerobic spore-forming rod-shaped bacteriumSpores are heat resistant (can survive 75°C for 15 min), resist disinfection, can remain viable in the environment for a long timeMay be used as indicator of resistant pathogens (viruses, parasites), past fecal contamination, or tracing fecal contamination in a marine environmentDrawbacksA common soil bacterium; may not necessarily indicate fecal contaminationPathogenic (causes gas gangrene if it infects wounds, produces enterotoxin in small intestine causing gastroenteritis)Anaerobic culture is difficult
13 Bacteriophage Coliphage – one example bacteriophage that infect coliforms, particularly E. colisimilar to enteric viruses in size, morphology, and performance in environmentfound in higher numbers than enteric viruses in wastewater and other watersrapid and easy detection methods availablesurvive for 7 days in shellfish without increasing in numbersroutinely used as indicator microorganisms to determine the effectiveness of wastewater treatment processesresistant to disinfection
14 Most Probable Number (MPN) Detection Methodsgas production+ gas productionMost Probable Number (MPN)Used to detect coliformsThis test consists of two to three steps:Presumptive testConfirming testCompleted testPresumptive test: dilute water sampleInoculate 3 or 5 tubes of lauryl sulfate-tryptose-lactose broth containing upside-down Durham tubes with water dilutionsIncubate at 35°C for 48 hoursDetermine number of tubes at each dilution that are positive for gas production (contain bubble in Durham tube)
16 Confirming test – select a positive tube and inoculate a Levines EMB agar and Endo Agar plate Completed test – inoculate a colony back into MPN media and confirm acid and gas production. (Not always performed)Levines EMB agarEndo Agar+-+-Coliforms produce“nucleated” coloniesColiforms and surroundingmedium turn red
17 What would you do to detect fecal coliforms instead of coliforms ???? Drawback to MPN test: HPC can outcompete coliforms and fecal coliforms for nutrients in the environment and mask their detection by this method.
18 Membrane Filter TestUsed to detect coliformsFilter 100 mL water through a 0.45 m filterIncubate filter on pad soaked with a differential medium (Endo medium; contains lactose and Basic Fuchsin dye) at 35°C for hoursCount colonies that grow on filtercoliforms will be dark red with metallic gold sheenTo enumerate Fecal Streptococci, grow on Streptococcus agar at 37°C for 24 hours. Fecal streptococci reduce 2,4,5-triphenyltetrazolium chloride to formazan, which makes colonies appear redMuch quicker and easier than MPN method
20 Presence-Absence Tests, e.g., Colilert Test Qualitative NOT quantitativeUsed to detect total coliforms and E. coliAdd packet of salts and nutrients to water sample and incubate 24 hoursTotal coliforms can convert o-nitrophenyl--D-galactopyranoside (ONPG) to yellow nitrophenol with -galactosidaseE. coli can metabolize 4-methylumbelliferone glucuronide (MUG) to a molecule that fluoresces under UV light with glucuronidaseMay not detect up to 1/3 of E. coli strains (including pathogenic ones!)Broth and agar plate techniques involving ONPG and MUG also exist
22 Heterotrophic Plate Counts (HPC) Enumeration of all aerobic and facultative anaerobic chemoheterotrophs in waterincludes Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acinetobacter, Proteus, Alcaligenes, and MoraxellaVaries from 1 to 104 CFU/mL, and depends on temperature, residual chlorine concentration, and availability of organic nutrientsIndicates general quality of water (particularly levels of organic matter in water)HPC > 500 CFU/mL indicates poor water quality
23 Plaque AssayUsed to detect bacteriophageFilter phage from water with charged membrane filterElute with beef extract, pH 9.0Flocculate solids (including phage) with HCl.Centrifuge. Remove supernatant and resuspend pellet in beef extract. Neutralize solution.Inoculate 4 mL loose (0.7%) agar with host bacterial culture and 100 L phage concentrate.Pour loose agar onto a solid agar plate. Incubate for 8-18 hoursHost bacteria will form lawn on plate. Bacteriophage will lyse small holes in the lawn (plaques)Count plaques and compare to the volume of filtered water to determine bacteriophage population in the water sample
24 Water Quality Standards and Guidelines Regulated at both the Federal and State levelsWater quality standards are legally enforceable!!
25 Authority Standards . U.S. E.P.A. California Safe Drinking Water Act coliforms/100mlClean Water ActWastewater discharges fecal coliforms/100 mlSewage sludge <1000 fecal coliforms/4 g<3 Salmonella/4 g<1 enteric virus/4 g<1 helmintha ova/4 gCaliforniaWastewater reclamation <2.2 MPN coliformsfor irrigationArizonaWastewater reclamation fecal coliforms/100mlfor irrigation of golf enteric virus/40 Lcourses No detectable Giardia/40 L
26 Guidelines for Recreational Water Quality Standards Water Quality Criteria and GuidelinesComprise recommendations for acceptable levels of indicator microorganismsNOT legally enforceable!!!Guidelines for Recreational Water Quality StandardsRegimeCountry (samples/time) Criteria or standardU.S.EPA /30 days fecal coliforms/100ml<10% to exceed 400/mlFresh water33 enterocci/100 ml126 fecal coliforms/100 mlMarine water35 enterococci/100 ml