Applied Environmental Microbiology

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Water Purification and Sanitary Analysis

• Microbial containment candidates– potential pathogens that can survive in water

and represent severe health risks

• Water purification– critical link in controlling waterborne disease

Problem Microbes• Not consistently removed by coagulation, rapid sand

filtration, and disinfection processes– Giardia intestinalis

• slow sand filters effectively remove Giardia cysts

– Cryptosporidium• small protozoan that escape usual purification schemes

– Cyclosporan• protozoan that causes diarrhea

– viruses• up to 99.9% removed by usual purification; may not be

sufficient protection

Water Purification• Water held with high levels of suspended material

sedimentation basin where large particles settle out

• Partially clarified water mixed with chemicals such as alum and lime and settling basin

– more material precipitates out in coagulation or flocculation process

• removes microbes, organic matter, toxic contaminants and suspended fine particles

• Water rapid sand filters physically trap fine particles and flocs

• Water treated with disinfectant

– chlorine

• concern about disinfection by-products (DBPs) such as trihalomethanes (THMs) which may be carcinogens

– ozone

Water Purification

• EPA has developed regulations called the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 rule)

– sets maximum containment level goal (MCLG) for specific pathogens

• health goals set at a level at which no known or anticipated adverse effects on health of persons occur and which allows an adequate margin on safety

Water Purification

• Precise treatment depends on level of contamination but generally involves filtration techniques– slow sand filters

• water passed over bed of sand with microbial layer (biofilm) cover the surface of each sand grain

• waterborne microbes are removed by adhesion to biofilm

– aeration and disinfection also used

Sanitary Analysis of Waters

• Based on detecting indicator organisms

– indicate fecal contamination of water supplies

– indicate possible contamination by human pathogens

“Ideal” Indicator Organism

• Suitable for analysis of all types of water• Present whenever enteric pathogens are present• Survives longer than hardiest enteric pathogen• Does not reproduce in contaminated water• Its level in water reflects degree of fecal pollution• Detected by highly specific test

– test easy to do and sensitive

• Harmless to humans

Two Commonly Used Indicators

• Coliforms

• Fecal streptococci (enterococci)

– increasingly used to test brackish and marine water

• Other:

– Fecal coliforms

– Coliphage

Coliforms

• Facultative anaerobic, Gram-negative, non-

endospore forming, rod-shaped bacteria that

ferment lactose with gas formation within 48

hours at 35° C

• Traditional method of detection is multiple-

tube fermentation test (MPN)

– Presumptive, Confirmed and Completed tests

• Fecal coliforms – same definition but are

incubated at 44.5 C

Other Tests for Indicator Organisms

• Membrane filtration technique• Presence-absence (P-A) test• Defined substrate tests• Molecular analysis

Membrane Filtration Technique1. water passed through filter

2. filter placed on surface of growth medium (usually ENDO agar – selective and differential for GNRs like EMB)

3. incubate

4. count colonies (green sheen or pink mucoid with dark centers) Used to detect total coliforms, fecal streptococci, and fecal coliforms (from intestines of warm-blooded animals)

Presence-Absence Test

• Modification of MPN

• Uses larger water sample (100 ml)

• Sample added to lactose containing medium– contains pH indicator to detect acid production

• Based on assumption that no indicator organisms should be present in 100 ml of water

• Detects total coliforms and fecal coliforms

Defined Substrate Tests• Tests for both total coliforms

and E. coli; e.g., Colilert

• 100 ml sample added to medium containing ONPG and MUG

• Produces a fluorescent product

• Other indicator microorganisms are fecal enterococci– used for brackish and fresh

water