Mod 8

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mush

Cards (39)

  • Importance of Water
    Drink for your health, prevents dehydration, helps body keep normal temperature, lubricate and cushion joints, protect spinal cord and other sensitive tissues, get rid of wastes
  • Sources of Water
    • Surface water
    • Ground water
    • Rain water
  • Surface Water

    • Derived from streams, brooks, lakes, ponds and river, always contaminated by surface run-off, polluted by uncontrolled disposal of human excreta, domestic & industrial waste
  • Ground Water
    • Obtained from wells & springs, largest source of water, untreated, non-saline ground water is biologically pure from organisms
  • Rain Water
    • Basically free from impurities, contamination may occur at the collection and storage points
  • Protective Measures for Drinking Water
    • Washing clothes or bathing from source of drinking water is prohibited within 25 meters
    • No source of water should be constructed within 25 meters from any source of pollution
    • No radioactive materials shall be stored within 25 meters
    • Any physical connection between distribution system of a public water supply system to any other water supply is not allowed
    • Installation of booster pump is not allowed where low water pressure prevails
  • Water Sampling
    1. Location of Sampling
    2. Considerations for Storage of Water Sample
  • Sample Collection from a Tap or Pump Outlet

    1. Clean the tap
    2. Open the tap
    3. Sterilize the tap (optional)
    4. Open the tap prior to sampling
    5. Open a sterilized bottle
    6. Fill the bottle
    7. Stopper or cap the bottle
  • Sample Collection from a Water Source or Reservoir

    1. Open the sterilized bottle
    2. Fill the bottle
    3. Stopper or cap the bottle
  • Sample Collection from Dug Wells and Similar Sources

    1. Prepare a bottle
    2. Attach bottle to string
    3. Lower the bottle
    4. Fill the bottle
    5. Raise the bottle
    6. Stopper or cap the bottle
  • Parameters for Water Analysis
    • Physical
    • Chemical
    • Bacteriological
    • Radiological
  • Physical Parameters
    • Color and palatability,
    • Turbidity - impurities in suspension,
    • Color – imparted by substances present in solution,
    • Taste & odor – expressed only qualitatively
  • Chemical Parameters
    • pH, Hardness – due to increased Ca & Mg, Total solid content – total mineral impurities present
  • Biological Parameters

    • Index of pollution, Presence of organisms responsible for odor and taste,
    • Bacteriological testing – most important single test to find out if water is potentially dangerous; detects indicator organisms
  • Radiological Parameters

    • Testing is done when there is a reason to suspect their presence,
    • Naturally occurring radionuclides in rocks and soils,
    • Principal source of radium and radon
  • Aesthetic Parameters for Water Analysis
    • Turbidity
    • Color
    • Taste & odor
  • Indicator Organisms
    • Total coliforms
    • Thermotolerant coliforms
    • Escherichia coli
    • Fecal streptococci
  • Total Coliforms

    Gram-negative, rod-shaped bacteria capable of growth in the presence of bile salts or other surface-active agents with similar growth-inhibiting properties, ferment lactose at 35–37°C with the production of acid, gas, and aldehyde within 24–48 hours, oxidase-negative, non-spore-forming, display β-galactosidase activity, long been recognized as a suitable microbial indicator of drinking-water quality, are easy to detect and enumerate in water, include Escherichia, Citrobacter, Enterobacter, and Klebsiella
  • Thermotolerant Coliforms

    Coliform organisms that are able to ferment lactose at 44–45°C, the group includes the genus Escherichia and some species of Klebsiella, Enterobacter, and Citrobacter, may also originate from organically enriched water such as industrial effluents or from decaying plant materials and soils, regrowth in the distribution system is unlikely unless sufficient bacterial nutrients are present, unsuitable materials are in contact with the treated water, the water temperature is above 13°C, and there is no free residual chlorine
  • Escherichia coli
    Belongs to family Enterobacteriaceae, contains enzymes such as β-galactosidase and β-glucuronidase, grows at 44– 45°C on complex media, ferments lactose and mannitol with the production of acid and gas, and produces indole from tryptophan, does not produce oxidase or hydrolyse urea, found in sewage, treated effluents, and all natural waters and soils subject to recent fecal contamination, whether from humans, wild animals, or agricultural activity, presence in water sample must not be ignored, because the presumption remains that the water has been fecally-contaminated and that treatment has been ineffective
  • Fecal Streptococci
    Generally present in the feces of humans and animals, they belong to the genera Enterococcus and Streptococcus, most of Enterococcus species; S. bovis and S. equinus for streptococcus, are of fecal origin, generally be regarded as specific indicators of human fecal pollution for most practical purposes, rarely multiply in polluted water, but more persistent than E. coli and coliforms, use as additional indicators for treatment efficiency and quality control
  • Principal Analytical Techniques

    • Presence – Absence Tests
    • Membrane – Filtration Method (MF)
    • Multiple – Tube Or Most Probable Number Method (MPN)
  • Presence –absence Test

    Presumptively detects for coliforms in water, based on the principle that coliforms and other pollution indicator organisms should not be present in a 100ml water sample, one test sample, 100ml, is inoculated into a single culture bottle to obtain qualitative information on the presence or absence of coliforms based on the presence or absence of lactose fermentation
  • Membrane Filter Technique

    An effective, accepted technique for testing fluid samples for microbiological contamination, involves less preparation than many traditional methods and is one of a few methods that will allow the isolation and enumeration of microorganisms, this technique is inappropriate for waters that is very turbid would cause the filter to become blocked, procedure: introduce sample into filtration assembly, apply vacuum to draw sample through membrane filter, transfer filter to culture medium, incubate, count colonies
  • Multiple-tube Method or MPN

    A series of tubes containing a suitable selective broth culture medium is inoculated with test portions of a water sample, coliforms if present in water utilizes the lactose present in the medium to produce acid and gas, the presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium, the number of total coliforms is determined by counting the number of tubes giving positive reaction with standard statistical tables, MPN test is performed in three stages: presumptive test, confirmatory test, completed test
  • Colony forming units (CFU)

    Units used to express the results when visually identifiable colonies are formed and counted
  • Multiple-tube Method or MPN

    1. A series of tubes containing a suitable selective broth culture medium is inoculated with test portions of a water sample
    2. Coliforms if present in water utilizes the lactose present in the medium to produce acid and gas
    3. The presence of acid is indicated by the color change of the medium
    4. The presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium
    5. The number of total coliforms is determined by counting the number of tubes giving positive reaction with standard statistical tables
  • MPN test
    1. Presumptive test
    2. Confirmatory test
    3. Completed test
  • Materials and Supplies
    • Lauryl tryptose broth
    • Brilliant green lactose bile broth (BGLB)
    • EC broth
    • Eosin methylene blue (EMB) agar plate
    • Nutrient agar slant
    • Water sample bottle
    • Durham tubes, 6 x 30 mm
    • Micro pipet
    • Incubator
    • Serologic pipet and rubber bulb
    • Test tubes
    • Inoculating loop
    • Gram staining kit
  • Multiple-tube/Most Probable Number Method Presumptive Test
    1. Remove the cap from the sample bottle
    2. With the stopper in position, shake the bottle vigorously to achieve a homogeneous dispersion of bacteria
    3. With a sterile 10-ml pipette, inoculate 10ml of the sample into each of five tubes containing 10ml of presumptive broth
    4. Add 50ml of sample to a tube containing 50ml of presumptive broth
    5. Incubate the tubes at 35°C to 37°C for 24 hours
    6. Examine each tube for the presence of gas
    7. If none is visible, gently shake the tube; if any effervescence is observed, the tube should be considered positive
    8. Record the number of positive tubes after 24 hours
    9. Reincubate negative tubes for a further 24-hour period
    10. At the end of this period, check the tubes again for gas production
    11. Gas production at the end of either 24- or 48-hours' incubation is presumed to be due to the presence of coliforms in the sample
    12. Record the number of positive tubes after 48 hours
  • Brilliant Green Lactose Bile (BGLB) broth
    Used to confirm total coliforms<|>The basal medium is composed of peptone, which contains 2% bile, and brilliant green dye<|>Bile is inhibitory to gram-positive microorganisms<|>Brilliant green dye inhibits selected gram-negative bacilli<|>Lactose-fermenting organisms resistant to these inhibitors are detected by the production of gas
  • E. coli medium (EC)

    Used to confirm fecal coliforms<|>The medium consists of a buffered lactose broth with casein peptone and bile salts<|>Lactose serves as a source of fermentable carbohydrate<|>Casein peptone source of nutrients<|>Bile salts inhibitory agents toward gram-positive cocci and spore formers<|>Lactose-fermenting capable of growing in the presence of the bile salts ferments lactose resulting in gas production within the durham tube
  • Multiple-tube/Most Probable Number Method Confirmatory Test
    1. Using a pipet, transfer 3 - 6 μl from each presumptive positive tube into two tubes containing respectively BGLB and EC broth
    2. Incubate them for 48 hours at 35 to 37°C for total coliforms (BGLB broth) or for 24 hours at 44 ± 0.5 °C for fecal coliforms (E. coli medium)
    3. At the end of incubation, examine each broth tube for growth and the presence of gas in the Durham tube
    4. Record the result
    5. Confirmatory tests positive for growth, and gas production on BGLB tubes show the presence of coliforms
    6. Growth and gas production on EC broth shows the presence of E. coli
  • Multiple-tube/Most Probable Number Method Completed Test
    1. Streak one or more eosin methylene blue (EMB) agar plates from each tube of brilliant green lactose bile broth showing gas
    2. Incubate the inoculated plates at 34.5 to 35.5°C for 22 to 26 hours
    3. Observe for bacterial growth and note the macroscopic characteristics
    4. Pick one or more typical well-isolated colonies, if no typical colonies are present, pick two or more colonies most likely to consist of organisms of the coliform group
    5. Inoculate into a nutrient agar slant
    6. Incubate inoculated media at 34.5 to 35.5°C for 22 to 26 hours
    7. Perform Gram stain smear from growth on nutrient agar slant
    8. Presence of Gram-negative, non-spore-forming, bacilli constitute a satisfactory completed test result
  • For treated water, where one 50-ml and five 10-ml portions are inoculated, the MPN can be found from the test results by means of the table below
  • Comparison of Multiple fermentation tube (MPN) techniques and Membrane filter technique
    • MPN techniques are slower, require 48 hours for a positive presumptive positive, more labor-intensive, require more culture medium and glassware, more sensitive, result obtained indirectly by statistical approximation (low precision), not readily adaptable for use in the field
    • Membrane filter technique is more rapid, quantitative results in about 18 hours, less labor-intensive, requires less culture medium and glassware, less sensitive, results obtained directly by colony count (high precision), readily adapted for use in the field
  • Procedure for a supposed treated drinking water sample

    1. Check if proper storage during sample transport was met
    2. Distribute appropriate sample volume to corresponding number of tubes containing presumptive media
    3. Inoculate 10ml of the sample into each of five tubes containing 10ml of presumptive broth
    4. Add 50ml of sample to a tube containing 50ml of presumptive broth
    5. Incubate the tubes at 35°C to 37°C for 24 hours
    6. Examine each tube for the presence of gas
    7. If none is visible, gently shake the tube; if any effervescence is observed, the tube should be considered positive
    8. Record the number of positive tubes after 24 hours
    9. Reincubate negative tubes for a further 24-hour period
    10. At the end of this period, check the tubes again for gas production
    11. Gas production at the end of either 24- or 48-hours' incubation is presumed to be due to the presence of coliforms in the sample
    12. Record the number of positive tubes after 48 hours
    13. Using a pipet, transfer 3 - 6 μl from each presumptive positive tube into two tubes containing respectively BGLB and EC broth
    14. Incubate them for 48 hours at 35 to 37°C for total coliforms (BGLB broth) or for 24 hours at 44 ± 0.5 °C for fecal coliforms (E. coli medium)
    15. At the end of incubation, examine each broth tube for growth and the presence of gas in the Durham tube
    16. Record the result
    17. Confirmatory tests positive for growth, and gas production on BGLB tubes show the presence of coliforms
    18. Growth and gas production on EC broth shows the presence of E. coli
    19. Streak one or more eosin methylene blue (EMB) agar plates from each tube of brilliant green lactose bile broth showing gas
    20. Incubate the inoculated plates at 34.5 to 35.5°C for 22 to 26 hours
    21. Observe for bacterial growth and note the macroscopic characteristics
    22. Pick one or more typical well-isolated colonies, if no typical colonies are present, pick two or more colonies most likely to consist of organisms of the coliform group
    23. Inoculate into a nutrient agar slant
    24. Incubate inoculated media at 34.5 to 35.5°C for 22 to 26 hours
    25. Perform Gram stain smear from growth on nutrient agar slant
    26. Presence of Gram-negative, non-spore-forming, bacilli constitute a satisfactory completed test result
  • To test clean glassware for an alkaline or acid residue, add a few drops of 0.04% bromothymol blue (BTB) or other pH indicator to at least five (5) Petri dishes, ten (10) test tubes and ten (10) Durham tubes washed weekly, chosen randomly. Observe the color reaction. BTB should be blue-green (in the neutral range). It changes to yellow in acidic conditions. At least 3 out of 5 Petri dishes and 7 out of 10 each for test tubes and Durham tubes should test blue-green in BTB. Otherwise, rewash the whole batch and repeat pH check.
  • To prepare 0.04% bromothymol blue indicator solution, add 16 mL 0.01N NaOH to 0.1 g BTB and dilute to 250 mL with distilled water. Place in an amber glass container and store at room temperature.