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Basic Water MicrobiologyBasic Water Microbiology
and Microscopyand Microscopy
Tom O’Connor, PE
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Discovery of the Microbial WorldDiscovery of the Microbial World
Antonj van Leeuwenhoek
1632-1723
“I have had several gentlewomen in myhouse, who were keen on seeing the littleeels in vinegar; but some of them were so
disgusted at the spectacle, that theyvowed never to use vinegar again.
But what if one should tell such people infuture that there are more animals living
in the scum on the teeth in a man’s mouth,than there are men in a whole kingdom?”
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LeeuwenhoekLeeuwenhoek’’ss Microscope Microscope
50 to 300X magnification “animalcules,” or little animals
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Scanning Electron Scanning Electron MicroscpeMicroscpe
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Scanning Electron Scanning Electron MicroscpeMicroscpe
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Compound Light MicroscopeCompound Light Microscope
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Compound Light MicroscopeCompound Light Microscope
• 100 to 1,000X magnification
• flat specimens, often filters
• transmitted light / epifluorescence
• phase contrast / differential interference contrast (DIC)
• algae, bacteria, protozoans
• particles, floc
• plant performance evaluations
• turbidity correlations
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Transmitted Light toTransmitted Light to
EpifluorescenceEpifluorescence
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Lake WaterLake Water
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FlocFloc
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Filtered WaterFiltered Water
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Organisms Rinsed from Filter SandOrganisms Rinsed from Filter Sand
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GACGAC
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GACGAC
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GACGAC
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GACGAC
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GACGAC
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GACGAC
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Stereo and Computer ScopesStereo and Computer Scopes
Intel QX310, 60, 200X
Zeiss6-150X
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Stereo and Computer ScopesStereo and Computer Scopes
• three-dimensional specimens
• top- and bottom-illumination
• protozoans, macroinvertebrates
• media from filters, ion exchange
• GAC, PAC
• pipes
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Examination of Old SandExamination of Old Sand
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Examination of Old SandExamination of Old Sand
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Examination of Old SandExamination of Old Sand
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Examination of Old SandExamination of Old Sand
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Examination of Old SandExamination of Old Sand
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Used CarbonUsed Carbon
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Used CarbonUsed Carbon
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Stereo ScopeStereo Scope
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Pitting CorrosionPitting Corrosion
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The PitThe Pit
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Pit Pit PicsPics
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Filtration Spectrum (Filtration Spectrum (jpgjpg))
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Microbes of Concern inMicrobes of Concern in
Water & WW TreatmentWater & WW Treatment
• Bacteria
• Virus
• Algae
• Protozoans
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TaxonomyTaxonomyorganization of speciesorganization of species
KINGDOM_
PHYLUM_
CLASS_
ORDER_
FAMILY_
GENUS_
SPECIES
Kings Play Chess On Fine Glass Stools
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Five KingdomsFive Kingdoms
Animals Plants Fungi Monera Protoctists
(bacteria) (algae, protozoans)
Viruses are not typically considered to be living organismsdue to their inability to replicate without a host cell
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BacteriaBacteria
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Pin PointPin Point
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Classification of BacteriaClassification of BacteriaBergey’s Manual
Archaebacteria
Aerobic
Anaerobic
Eubacteria
Autotrophic bacteria
Phototrophic
Purple bacteria
Green bacteria
Chemotrophic
Nitrifiers
Sulfur oxidizers
Fe / Mn oxidizers
Methane oxiders
Heterotrophic bacteria
Gram-negative
Aerobic
Facultatively anaerobic
Anaerobic
Gram-positive
Mycobacteria
Bacteria with complex structures
Actinomycetes
Stalked and budding bacteria
Sheathed bacteria
Gliding and creeping bacteria
Spirochaetes
Mycoplasmas
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MorphologyMorphologyFancy Word for ‘Shape’
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Types of BacteriaTypes of Bacteria
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Cell WallCell Wall
• selective permeability
• protein !”pumps”
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Gram-positive/negativeGram-positive/negative
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FoodFood
Autotrophs (self-nourishing)
require water, CO2 , inorganic salts, energy source
Heterotrophssaprophytic—absorb nutrients through cell membrane
holozoic—eat, digest, and absorb particulate food
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OxygenOxygen
Aerobes
utilize oxygen in respiration
Obligate Anerobes
quickly killed by oxygen
Facultative Anerobes
can take it or leave it
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ReproductionReproduction
Asexually (fission)
a cell divides into two cells
Kinetics (speed)
Cells can divide every 20 minutes
One cell ! 8 hours ! 12,000,000 cells
Inhibitors
• lack of food
• accumulation of waste products
• predators
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Survival - DormancySurvival - Dormancy
Dormancy during Dryout
• loses water
• shrinks
• becomes inactive
• waits patiently for water
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Survival - SporesSurvival - Spores• cell retreats in times of unfavorable conditions (dryness, temp, disinfectant)
• forms new, thicker cell wall within old one
• when favorable conditions reappear, spore absorbs water, breaks out ofinner shell, returns to normal
• Anthrax bacilli can survive 30 years in spore form
• most pathogenic (disease-causing) bacteria do not form spores
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AilmentsAilmentsAssociated with BacteriaAssociated with Bacteria
typhoid fever ! Bacillus typhosus
diarrhea ! Escherichia coli
Legionnaire’s disease ! Legionella
Leptospirosis ! Leptospirea
salmonellosis, paratyphoid ! Salmonella
bacillary dysentary (Shigellosis) ! Shigella
cholera ! Vibrio cholerae
plague ! Yersinia
There are over 3,000 species of bacteria;
only a handful are pathogenic (disease-causing)
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Chlorine Disinfectants:Chlorine Disinfectants:
Effectiveness and ResistanceEffectiveness and Resistance
LEAST RESISTANT MOST RESISTANT
Bacteria Virus Protozoan Cysts Some Bacterial Spores
LEAST EFFECTIVE MOST EFFECTIVE
R-NHCl NH2Cl NHCl2 OCl- HOCl Cl2
LEAST RESISTANT MOST RESISTANT
Bacteria Virus Protozoan Cysts Some Bacterial Spores
LEAST EFFECTIVE MOST EFFECTIVE
R-NHCl NH2Cl NHCl2 OCl- HOCl Cl2
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Bacterial JobsBacterial Jobs
Fermentation: enzymatic anaerobicbreakdown of carbohydrates
Putrefaction: enzymatic anaerobicbreakdown of proteins and amino acids
Stench—nitrogen-and sulfur-containingcompounds produced during putrefaction
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NitrogenNitrogen
CycleCycle
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NitrifierNitrifier
NH4+ + 2O2 ! NO3
-"+ 2H
+ + H2O
1 mg N/l + 4.6 mg O/l 1 mg N/l Acid Production
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HPC & HPC & ColiformColiform
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Bacterial RemovalBacterial Removal
During TreatmentDuring Treatment
Vast majority of cellremoval occursduring settling
Sand filters areineffective at
planktonic cellremoval
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Bacterial RemovalBacterial Removal
During TreatmentDuring Treatment
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RegrowthRegrowth During During
DistributionDistribution
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RegrowthRegrowth During Distribution During Distribution
PLANT FINISHED WATER DISTRIBUTION SYSTEM
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VirusesViruses
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Vir
use
sV
iru
se
s
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Viruses - Plant and BacterialViruses - Plant and Bacterial
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Viruses - AnimalViruses - Animal
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Virus StructureVirus Structure
CORE: nucleic acid
(single- or double-
stranded DNA or RNA)
CAPSID:
protective protein coat
ENVELOPE:
lipoproteins or lipids
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PhagePhage“one that eats”
Head contains
DNA
Legs attach to
bacterium
!0.2 "m long
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BacteriophageBacteriophage Micrograph Micrograph
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BacteriophageBacteriophage
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Six-step ReproductionSix-step Reproduction
Adsorption: Adsorption: attach to receptor sites on host cellattach to receptor sites on host cell
Penetration: Penetration: injection of nucleic material (phages) orinjection of nucleic material (phages) or
complete cell wall penetration (animal viruses)complete cell wall penetration (animal viruses)
Eclipse: Eclipse: host host proteolyticproteolytic enzymes strip protein coat enzymes strip protein coat
((capsidcapsid))
Replication: Replication: virusvirus’’ nucleic acid replicates and nucleic acid replicates and
synthesizes viral proteinssynthesizes viral proteins
Maturation: Maturation: nucleic acid and protein coat are assemblednucleic acid and protein coat are assembled
Release: Release: rupture of hostrupture of host’’s cell walls cell wall
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BacteriophageBacteriophage Reproduction Reproduction
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Phage InvadersPhage Invaders
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BacteriophageBacteriophage Cycle Cycle
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DNA InjectionDNA Injection
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HIVHIV
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AilmentsAilments
Associated with VirusesAssociated with Viruses
Polio (Poliovirus)
Meningitis (Coxsackievirus)
Conjunctivitis (Andenovirus)
Meningitis, epidemic exanthem, infantile diarrhea (ECHO Virus)
Hepatitis (Hepatitis A and B virus)
Gastroenteritis (Reovirus, Rotavirus, Norwalk agent)
Also: colds, flu, fever, rash, eye infection, respiratory illness,smallpox, measles, mumps, herpes, AIDS, cold sores, warts
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Effect of pH on VirusesEffect of pH on Viruses
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Inactivation of VirusesInactivation of Viruses
• pH > 11
• Chlorine• 0.5 ppm, pH 7.8, 2°C, 3 to 60 min. contact time !99.99% kill
• free chlorine 50 times more effective than chloramines
• Ultraviolet Light
• Ozone (very effective)
• Heat (60°C)
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AlgaeAlgae
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Algae Color PlateAlgae Color PlateStandard MethodsStandard Methods
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DiatomsDiatoms
Asterionella Pinnularia
Navicula
SynedraTabellaria
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DiatomsDiatoms
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VolvoxVolvox and and SynuraSynura
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DinoflagellateDinoflagellate
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Plankton & Plankton & ActinomycetesActinomycetes
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Floating SolidsFloating Solids
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Micrograph of Floating SolidsMicrograph of Floating Solids
Green crystals - calcium carbonateGreen crystals - calcium carbonate
Orange strands - algal filaments (Orange strands - algal filaments (OscillatoriaOscillatoria))
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Lime Softening SolidsLime Softening Solids
Calcium carbonate crystals (green)
Algal cells (orange)
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Algae, Bacteria, CaCOAlgae, Bacteria, CaCO33
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RotationRotation
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RollRoll
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SplitSplit
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Protozoan, Algae, BacteriaProtozoan, Algae, Bacteria
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Bacterial Chains & FilamentsBacterial Chains & Filaments
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Bacterial MotionBacterial Motion
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More Chains and FilamentsMore Chains and Filaments
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ChroococcusChroococcus
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Nematodes in the MatNematodes in the Mat
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ProtozoansProtozoans
Cryptosporidium
• 40,000 species
• single-celled
• up to several mm long
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ParameciumParamecium
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Paramecium ciliaParamecium cilia
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Didinium
catches, eats a
Paramecium
Life in theLife in the
Food ChainFood Chain
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AmebaAmeba
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TardigradeTardigrade(Water Bear)
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AilmentsAilments
Attributed to Attributed to ProtozoansProtozoans
amoebic dysentary (Entamoeba histolytica)
giardiasis (Giardia lamblia)
giant roundworm (Ascaris lumbricoides)
cryptosporidiosis (Cryptosporidium)
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Lines of DefenseLines of Defense
• Source Water Protection
• Physical Removal
• Kill / Inactivation
• Disinfectant Residual
• Maintain Integrity of Distribution and Storage