Water Lab Test 1

FOV diameter =

Field # / objective mag

• objective mag is actual microscope (ie 40x)

• field # is next to eye piece

Total magnification =

optical x objective

• ex 10 × 40 = 400

Examples of coliforms

bacteria in warm blooded animal intestines that is excreted in feces

Examples:

• Escherichia spp.,

• Citrobacter spp.,

• Enterobacter spp., and

• Klebsiella spp.

Colilert 24 Test results

Clear = clear of coliforms and E. coli

Yellow = coliforms, must use 6 watt 365nm UV to test for E. coli

E. coli is a violation of the revised total coliform

Enzymes of colilert test

• The hydrolysis of ONPG by Beta-D-galactosidase, common in

  coliforms, produces o-nitrophenol yielding a distinct yellow color.

• E. coli also produce the enzyme β-D-glucuronidase which hydrolyzes MUG and produces the fluorescent molecule 4-methyl-umbelliferone.

There are two common HPC methods accepted by the Environmental Protection Agency

the Spread Plate Method and the Enzyme Substrate Method (i.e. SimPlate)

Spread Plate method (HPC) agar

• R2A agar (less nutrients that promote water bacteria better)

• 
  

The Enzyme Substrate Method uses a medium in which

substrates are hydrolyzed by multiple microbial enzymes, causing the release of 4-methylumbelliferone which fluoresces when exposed to UV light. The number of blue fluorescing wells corresponds to a most probable number (MPN) of bacteria in the sample.

HPC counts shoiuld not be higher than

500 CFU/ml

(counting between 30-300 colonies)

Free chlorine and its typical levels

• Refers to both hypochlorous acid (HOCl) and the hypochlorite (OCl–) ion or bleach

• Typical levels of free chlorine in drinking water are 0.2 - 2.0 mg/L.

MRDL for chlorine

The EPA has established the maximum residual disinfectant level (MRDL) for chlorine at 4.0 mg/L or 4 ppm as an annual average.

Combined chlorine

Refers to chloramines, which form when chlorine molecules combine with ammonia or organic nitrogen (eg. monochloramine, dichloramine, and trichloramine)

Total chlorine

sum of free chlorine and combined chlorine

Hach test quantifies; regeant

The Hach test quantifies Free and Total chlorine, and the Combined chlorine is determined mathematically. The reagent n,n-diethyl-p-phenylenediamine (DPD) is added to the water sample and is oxidized by free chlorine species or tri-iodide in the water sample to produce a pink color (Figure 1). Free chlorine is measured via direct reaction with DPD

Total chlorine result in Hach test

3 reactions take place in the Total Chlorine assay. 1) the Free Chlorine direct reaction with DPD to form a pink color. 2) Combined chlorine (chloramine) reacts with iodide to form tri-iodide (I3-). 3) Tri-iodide reactions directly with DPD to produce a pink color.

The concentration of combined chlorine is

the difference between Total and Free chlorine

• Free chlorine can bind to microbes

  • combined chlorine is partially bound to nitrogen samples and cannot be used to disinfect

• If free chlorine is added to a water sample and quickly becomes combined chlorine, then there is a lot of contamination is the water

Sodium thiosulfate

used to neautrlaize chlroine and stop antimicrobial activity

Common water purification methods

1. aeration

2. coagulation

3. sedimentation

4. filtration

5. disinfection

aeration

Aeration is the process of introducing air to a given system. With respect to water treatment, aeration facilitates the removal of dissolved gases, the oxidation of dissolved metals, and the “stripping” of volatile organic chemicals (VOC). The following are undesirable chemicals in water:

• VOCs - benzene, trichloroethylene, dichloroethylene, and perchloroethylene

• Carbon dioxide

• Hydrogen sulfide (rotten-egg odor)

• Methane (flammable)

• Iron (will stain clothes and fixtures)

• Manganese (black stains)

• Various chemicals causing taste and odor

Coagulation / flocculation

Coagulation/flocculation is, “the effect produced by the addition of a chemical to a colloidal dispersion resulting in particle destabilization by the reduction of the forces tending to keep the particles apart” (Water Quality and Treatment, 1971). Chemicals with a positive charge (such as alum) are added to neutralize particles with a negative charge. This process makes particles not repel each other by charge and thereby allows small particles to stick together to form flocs

Sedimentation

is the process by which particles, such as flocs, settle to the bottom of a reservoir (river, lake, holding tank, beaker, soda bottle, etc.) due to their weight and gravity

Filtration

is the process of removing particles such as dust, parasites, bacteria, viruses and chemicals by passing the water sample through materials of various compositions (e.g., sand, gravel, and charcoal) and pore sizes. Filters are mainly classified as either gravity or pressure filters. Filtration depends on several chemical and physical processes, the most important being adsorption

Disinfection

The purpose of disinfection is to kill disease-causing microorganisms. With respect to water treatment, the targets are intestinal and fecal bacteria, viruses and protozoa, such as Giardia and cryptosporidium. Methods of disinfection include chlorination, ozonation and treatment with UV light

Coliform bacteria belong to the family Enterobacteriaceae and are defined as

• 1. all aerobic and facultative anaerobic

  • Gram-negative

  • non-spore forming bacilli

  • ferment lactose with gas production within 48 hours at 35°C,

• all aerobic and many facultative anaerobic

  • Gram-negative,

  • non-spore-forming,

  • rod-shaped bacteria that develop a red colony with a metallic sheen within 24 h at 35°C on an Endo-type medium containing lactose

Fecal coliforms are more fecal-specific in origin and are found in

the intestines and feces of humans and other warm-blooded animals. They are capable of producing gas from lactose at 44.5°C ± 0.5° C. Fecal coliforms include both pathogenic and non-pathogenic bacteria. The most common fecal coliform is Escherichia coli. Unless additional testing is performed, Fecal Coliforms are assumed to be E. coli for regulatory purposes.

Fecal streptococci are (gram…)

• Gram-positive

• catalase-negative

• non-spore forming cocci that grow at 35°C in a medium containing bile salts and sodium azide

• Cells can hydrolyze esculin

• Fecal streptococci also occur in the digestive systems of humans and other warm-blooded animals

Enterococci are a subgroup within the fecal streptococci group and are typically

more human-specific.

• They can grow in the presence of 6.5% NaCl at 45°C.

• Examples include Enterococcus faecalis, E. faecium, E. avium and E. gallinarum.

• Due to their ability to survive in salt water, they are considered the best indicator of public health in saltwater systems.

Steps for MTF

The multiple tube fermentation (MTF) method includes three steps: the presumptive, confirmed, and completed tests. During the presumptive test, a moderately selective medium (eg. lauryl tryptose broth) is used to test for growth of coliforms.

MTF results (presumptive)

Growth and gas production are recorded as a positive result.

Because it is possible for non-coliforms (e.g. Clostridium or Bacillus) to grow and be false positives, all positive tubes are subjected to a confirmed test wherein the positive tubes are inoculated into a more selective medium (e.g., brilliant green lactose broth and/or EC Broth).

MTF mre selective media

the more selective medium is used to eliminate organisms except true coliforms or fecal coliforms. Positive tubes from the confirmed tests are used to streak onto highly selective agar (complete tests). After incubation, subsequent colonies can be evaluated via multiple tests to identify coliforms. The results of the MTF technique are used to calculate the most probable number (MPN) of microorganisms in order to assess whether or not there is a public health risk.

MPN is

is a statistical estimate of the mean number of coliforms in the sample.

There are two main assumptions of the MPN method

1) the organisms are distributed randomly throughout the liquid (i.e. there is no tendency for organisms clumping together or repelling each other- an organism is equally likely to be found in any part of the liquid), and 2) each sample that is inoculated into the culture medium is certain to exhibit growth (i.e. positive result) whenever the sample contains one or more organisms

Recreational Waters freshwater standards

Based on a statistically sufficient number of samples (generally not less than 5 samples equally spaced over a 30-day period), the geometric mean of the indicated bacterial densities should not exceed one or the other of the following:

E. coli: 126 per 100 ml; or

Enterococci: 33 per 100 ml

Recreational Waters Marine water standards

Based on a statistically sufficient number of samples (generally not less than 5 samples equally spaced over a 30-day period), the geometric mean of the enterococci densities should not exceed 35 per 100 ml.

Oklahoma primary criteria

E. coli (EC): 126 per 100 mL

Enterococci (EN): 33 per 100 mL

this is primary criteria

Oklahoma secondary criteria

E coli:

• 630 per 100 ml monthy

• lake: 1175 per 100 ml

• 2030 per 100 ml other waterbodies

Entero

• 165 per 100 ml monthly

• 305 per 100 ml lake

• 540 per 10 ml other

Field Blank

These should be collected at 10 percent of your sample sites along with the regular samples. Sterile water in sterilized containers should be sent out with selected samplers. At a predetermined sample site, the sampler fills the usual sample container with this sterile water. This is labeled as a regular sample, but with a special notation (such as a "B") that indicates it is a field blank. It is then analyzed with the regular samples. Lab analysis should result in "0" bacteria counts for all blanks. Blanks are used to identify errors or contamination in sample collection and analysis.

Internal Field Duplicates

These should be collected at 10 percent of your sampling sites along with the regular samples. A field duplicate is a duplicate stream sample collected at the same time and at the same place either by the same sampler or by another sampler. This is labeled as a regular sample, but with a special notation (such as a "D") that indicates it is a duplicate. It is then analyzed with the regular samples. Lab analysis should result in comparable bacteria counts per 100 mL for duplicates and regular samples collected at the same site. Duplicates are used to estimate sampling and laboratory analysis precision.

External Field duplicates

An external field duplicate is a duplicate stream sample collected and processed by an independent (e.g., professional) sampler or team at the same place at the same time as regular stream samples. It is used to estimate sampling and laboratory analysis precision.

Membrane filtration (MF) can be used to measure

the quality of recreational, shellfish growing, ambient and potable waters. Membrane filtration provides a direct count of bacteria in water based on the development of colonies on a membrane filter.

Membrane filtration for fecal colifrmos

Red/magenta colonies are counted after incubation. Further tests can be conducted to verify that the colonies are indeed of fecal origin by determining that they are: 1) EC gas positive, 2) indole positive, 3) oxidase negative and, 4) do not grow on citrate medium

Membrane filtration for enterococci

Colonies with blue halo formation are counted after incubation. Further tests can be conducted to verify that the colonies are enterococci by determining if they: 1) grow on brain heart infusion (BHI) plates and broth at 45°C, 2) grown in BHIB broth with 6.5% NaCl, 3) stain Gram positive, and 4) hydrolyze esculin

Waterborne

Illness caused by ingestion of or recreation in water contaminated with microorganisms

• Drinking water/ingestion of contaminated foods

Transmission of waterborne diseases

fecal-oral transmission

• F-diagram (World Health Organization)

Fecal transmission F diagram

Early 1900s water treament

• waterborne diseases were finally controlled at an acceptable level in industrializing (affluent) countries

• Filtration and chlorination

• better dispoal

• milk pas

• food prep and storage

Miasma theory

that communicable diseases arose from “bad air” produced by decaying organic matter detected by smell

• Remove miasma rather than quarantine

• Replaced by germ theory of disease in 1800s (i.e. disease is caused by microorganisms

1500-1800s London

• Shallow public well supply

• wealthy homes had pipes

• Waste thrown into cesspools

• 1800s mid made sewer systems

• waste in river thames

• miasma made this worse

John Snow cholera

• John Snow, M.D., in London documented the 1848 cholera epidemic and established the first sound epidemiologic basis for development of the "theory of contagion".

1854 cholera outbreak London

• Death rates in London to 200-300 per week

• John Snow documented where cholera victims obtained their water and

hypothesized that water source was contaminated, e.g., the point source

• Many victims used the Broad street pump

• Solution: remove the handle on the pump! (Sept 8, 1854; stopped the outbreak)

Germ theory

• Louis Pasteur (1822-1895) showed microorganisms arise from other microorganisms

• Robert Koch: 1882 showed that the tubercle bacillus caused tuberculosis and in 1884 found the cholera bacillus, Vibrio cholerae

• Turned the tide against miasma theory

• Pathogens or “germs” can cause disease

Chicago water (EPA stuff)

• 1871-1872 typhoid epidemic

• 100,000’s cases, 10,000’s deaths

• Drinking water from Lake Michigan but sewage from Chicago River was flowing into Lake Michigan

• Solution: 28-mile sanitation canal reversed the flow of the river.

Dead lake Erie caused

Federal Water Pollution Control Act of 1948

– 1st major US law to address water pollution

– Hard to enforce

• Environmental Protection Agency established in 1969 by President Nixon

• Public Law 92-500: Federal Water Pollution Control Act Amendments of 1972 (Clean Water Act)

Outbreaks in Affluent Countries:

• Wastewater contamination

• Inadequate knowledge of source water hazards

• Extreme weather (heavy precipitation and runoff)

• Filtration failures

• Distribution failures

• Livestock and wildlife fecal contamination

• Plant maintenance or treatment process changes

4 activities needed water levels

drinking - 5 liters

sanitation - 20 liters

bathing - 15 liters

foo prep - 10 liters

Water is scare even ocean why

Available water not potable – saline

• Water not evenly distributed

• Water is tied up in hydrologic cycle

• Water crosses borders

• Available water not potable – microbes

Most of the world get its water from

rivers and lakes

water per person

about 11 million liters

• 1.5-2 million each year

20% of all freshwater in

lake Baikal in russia

20% of all freshwater in (USA)

great lakes

Types of human impact water

• green water - lievstock, crops, and forestry

• glue water - water available for coonsumption

• grey water - water erquire to assimilarre pollution

• land distrubance

• climate change

water footprint

indicates the water required to sustain a population.

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Virtual water

volume needed to produce a commodity or service

daily water use in USA

drinking - 5.7 liters

washing, toilet, etc: 150 liters

USA - 380 liters

4100-5500 liters per day

Water borne vs water related

Water based vs water washed

Six types of E coli

1. Shiga toxin producing

2. Enterpathogenic

3. Enterotoxogenic

4. Enteroinvasive

5. Enteroaggregative

6. Diffusely adherent

Shiga toxin producing E coli (STEC) (VTEC)

• O157:H7

• 36% of infections

• Fecal-oral

• attaches to intestinal epithelial cells

• 3-4 days after exposure

• Shiga toxin (all STEC have this)

• LEE patho island

• alpha-hemolysin

• water diarrhea and cramping

• bloody diarrhea

• hemolytic uremic syndrome (10%)

• Thrombotic thrombocytopenia

• 
  

Enteropathogenic E coli EPEC

• infantile diarrhea

• LEE

• now uncommon but only in less than 2 yp poor babies

• Low mortailty

• Watery diarrhea

• vomiting

• low grade fever

• Fecal-Oral entry, adheres to intestinal

  mucosa, causes A/E lesions, and extensively

  affects the digestive-absorptive enzyme

  system, causing nutrient malabsorption

Enterotoxic E coli ETEC

• travelers diarrhea

• 380k, mostly kids dying

• Fecal oral

• water diarrhea, cramps, nausea

• small intestine, pili

Enteroinvasive E coli (EIEC)

• Fecal oral, epitlieal colon and destruction of those

• Bacillary dystemery

• cramps, diarrhea, vomiting, fever, chills, stool- blood and mucus

• 0 ddeaths

• virulence plasmid

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EAEC Enteroaggregative E coli

• persistent watery diarrhea in less developed countries

• Fecal-Oral entry, form biofilm on small intestinal mucosa, induce cytotoxic effects

Diffusely adherent E coli (DAEC)

• causation unknown

• low mortiality

• duration unknown

• watery diarrhea but not sure, may be asymptomatic

• Fecal-Oral entry, diffuse adherence to

  epithelial cells (thin, patchy biofilm)

Locus of enterocyte effacement (LEE)

• Gene region: prophage region in this case

• Entero: cell of interstinal lining

• Effacement: erase from a surface

1. Cells attach to enterocytes and form microcolonies

2. Villi below colony are eroded

3. Stimulate reorganization of cytoskeleton immediately below attached bacterial cells

LEE mechanism

• Form a characteristic pedestal (A/E lesion; attaching and effacing lesion) LEE encodes eae and tir

• eae: intimin – causes a signal cascade in mammalian cells to rearrange cytoskeleton

• tir: receptor for intimin; the bacteria insert a receptor onto the mammalian cells to receive intimin and cause the expression signal cascade

Shiga toxin

• Encoded by stx gene (two types with many subtypes based on sequence)

• stx2a associated with worst symptoms

• shiga toxin (1 A protein and 5 B proteins)

• B proteins attach to host vascular cells, stimulating internalization (endocytosis)

• Inside the host cell, the A subunit is released and cleaves the 28S rRNA of ribosomes, inhibiting protein synthesis, causing cell death

• Toxin can move from intestines to kidney; can cause kidney failure and neurological complications

Indicator microbes should

Exist in high numbers in the human intestine and feces

• Absent in uncontaminated waters

• Not be pathogenic to humans

• Be easily, reliably and cheaply detected in environmental waters

• Other important criteria include:

– not multiply outside the enteric environment

– in greater numbers than pathogens in the environment

– should have a similar die-off behavior as the pathogens

– if human fecal pollution needs to be distinguished from animal pollution, the indicator should not be very common in the intestine of farm and domestic animals

Detection of E coli colilert

• beta galactosidease - yellow (cleaves ONPG)

• beta glcuronidase metabolizes MUG giving fluroescnce

Detection of E coli memebrane filtration

Cultivation

– Selective Medium (many)

– Example: Modified mTEC: nutrients and high temperature selective for E. coli; strains possessing the enzyme beta-D-glucuronidase break down Chromagen (5-Bromo-6-Chloro-3-Indoyl-beta-D-Glucuronide) producing red or magenta colonie

• Serotype by immunological (antibody) approaches and PCR

methods

Total coliforms

Fecal (thermotolerant) Coliforms

Fecal Streptococci and Fecal

Enterococci

Drinking water regs