Lab 11

• Most of the large number of microbes found in water bodies do not cause human diseases
• Water bodies may be contaminated with fecal matter carried away by water in runoff or sewer that often empty into rivers and lakes
• Sewage treatment plant effluent, animal feces off sidewalks, improperly maintained septic systems may discharge pathogens into lakes, streams or underlying groundwater
• Manure can contribute pathogens to surface water via runoff and erosion
• These bodies of water may in turn empty into the ocean and pose health risks for people swimming, bathing, and playing in the water near the shores
• Escherichia coli


  Nonpathogenic intestinal organism used as an indicator of fecal contamination
• Enterococcus faecalis


  Nonpathogenic intestinal organism used as an indicator of fecal contamination
• Characteristics that make E. coli and E. faecalis good sewage indicators of fecal contamination are: (1) they are normally not present in water or soil, (2) they are relatively easy to identify, and (3) they survive a little longer in water than enteric pathogens
• E. coli


  • Gram-negative, non-spore-forming rod
  • Classified as a coliform
  • Considered an indicator of fecal contamination
• E. faecalis


  • Gram-positive coccus
  • Not a coliform
  • Considered a fecal indicator
• Coliform is a gram-negative non-spore-forming rod and is a facultative anaerobe that ferments lactose to produce gas
• Escherichia coli and Enterobacter aerogenes fit the definition of a coliform, but only E. coli is considered an indicator of fecal contamination, not E. aerogenes
• Most Probable Number (MPN) method is routinely used method in enumeration of coliforms
• Enterococcus faecalis, even though a fecal indicator, is not a coliform. Therefore, a completely different set of tests must be used to detect its presence
• Bacterial water quality standards are based on estimates that ensure a low risk of illness in people
• The U.S. Environmental Protection Agency (U.S. EPA) estimates that, at concentrations of 126 E. coli per 100 ml, 8 out of every 1,000 swimmers will become ill
• When bacterial levels rise to unacceptable levels at beaches, an advisory or closing will be issued
• California's coast offers year-round recreation and attracts more than 175 million visitors annually from all over the world
• Monitoring programs indicate that beach pollution often requires posting and closure of beaches for swimming and recreation
• The environmental group, Heal the Bay, has consistently ranked Moonlight Beach in Encinitas, CA, poorly
• The source of pollution causing the poor grades at Moonlight Beach was water from Cottonwood Creek, which empties into Pacific Ocean at the beach
• Polluted urban runoff from the city of Encinitas was channeled in to the creek, which subsequently polluted the beach
• When other water quality improvement measures failed to produce significant results, the city opted to install the UV disinfection system to treat the water in the creek in 2003
• According to the data provided by the state of California, there were 12 closures at Moonlight beach in 2006 compared to 3 beach closures in 2005 and 5 beach closures in 2004
• This was a big jump considering the fact that beach closures at Moonlight beach have been low after the installation of an ultraviolet radiation treatment facility to treat the water from Cottonwood Creek that discharges into the ocean
• Many places that are suspected to be out of compliance are not monitored due to perceived high cost and complexity of equipment involved in a local monitoring program
• Citizen-based volunteer monitoring programs have been developed in several states, such as Iowa, and have been used for purposes of preliminary screening of local waters for identifying problem areas
• Since E. coli enumeration methods generally require expensive media and equipment not available to volunteers, a convenient and inexpensive method of E. coli enumeration is needed for such programs
• Compact DryTM EC plates


  Intended for enumerating E. coli in food, dairy products, and environmental waters
• Compact Dry procedure


  1. Inoculate by pipetting 1ml of sample directly to the center of a dry tray well

  2. Sample will automatically diffuse across the surface by capillary action to form a gel

  3. Incubate at 35-37°C for 24 hours

  4. Count blue colonies for E. coli count, count all pink-purple and blue colonies for total coliform count
• MPN (Most Probable Number) method


  Statistical estimation method that utilizes multiple tubes to determine bacterial numbers in water samples within 95% confidence limits
• MPN procedure


  1. Presumptive test: Inoculate series of 9 tubes of lactose broth with water to see if it contains any lactose-fermenting bacteria that produce gas

  2. Confirmed test: Inoculate EMB agar plates from positive (gas-producing) tubes to see if the organisms are Gram-negative
• Presumptive Test (day 1)


  1. Set up 3 DSLB and 6 SSLB tubes

  2. Mix the bottle of water to be tested by shaking 25 times

  3. Transfer 10 ml of water to each of the DSLB tubes

  4. Transfer 1 ml of water to each of the middle set of three tubes, and 0.1 ml to each of the last three SSLB tubes

  5. Incubate the tubes at 35° - 37°C for 24 hours

  6. Examine the tubes and record the number of tubes in each set that has 10% gas or more

  7. Determine the MPN by referring to the MPN table
• Confirmed Test (day 2)


  1. Take one of the presumptive positive tube (yellow with gas) from day 1

  2. Streak an EMB and a MAC with a loopful of this growth and incubate for 24 hours
• EMB agar


  • Selective and differential medium commonly used for differentiation of enteric bacteria
  • Contains two stains, eosin and methylene blue, and lactose sugar
  • Methylene blue dye inhibits the growth of Gram-positive bacteria and helps differentiate between lactose fermenters and non-fermenters
  • Lactose fermentation produces acids, which lower the pH and encourages dye absorption by the colonies, which are now colored purple-black
  • Lactose non-fermenters may increase the pH by deamination of proteins, ensuring that the dye is not absorbed and the colonies show up as light colored or colorless
• Colonies on EMB agar


  • E. coli - small dark colonies with a distinctive metallic green sheen
  • Enterobacter aerogenes - large colonies with dark centers and lighter rims
• Differentiation on EMB agar is not completely reliable, as E. coli is a more reliable sewage indicator than E. aerogenes since it is not normally present in soil
• MacConkey Agar (MAC)


  • Differential and selective medium commonly used for differentiation of enteric bacteria
  • Contains crystal violet and bile salts that inhibit the growth of gram-positive organisms, allowing for the selection and isolation of gram-negative bacteria
  • Enteric bacteria that have the ability to ferment lactose can be detected using lactose and a pH indicator neutral red
• Completed Test (Day 3)


  1. Pick an isolated colony from the EMB agar plate that truly matches the definition of a coliform and use that colony for inoculation of TSA

  2. Inoculate a TSA slant and a tube of lactose broth

  3. Incubate for 24 hours at 37°C

  4. Observe for gas production on lactose broth

  5. Perform a Gram stain on a growth on the TSA slant
• If gas is produced in the lactose broth and a slide from the slant reveals a Gram-negative non-spore-forming rod, we can be certain that we have a coliform