Heterotrophic translated means "mixed eaters." Coliforms usually eat (metabolize) lactose and produce acid and gas. To first isolate coliforms, the lab inoculates the medium with 100 mL of the water sample. If some heterotrophs or "mixed eaters" are present, they will grow but will not produce acid, determined by a yellow color, or produce gas, indicated by bubbles in the medium, they may grow faster and cover the presence of coliforms. If the Lab finds large numbers of bacteria that grow in the lactose medium but do not produce acid or gas, this indicates that the heterotrophic bacteria present may have masked the presence of the coliforms.

The Lab will then send a replacement bottle and ask for another sample. When this sample gets to the lab we then use different media referred to as Defined Substrate Media (DSM) to keep the heterotrophs from interfering with the growth of the coliforms. Following are some ways to keep the heterotrophs under control.

• Keep the free chlorine residual above 1.5 mg/L.
• Do not allow water to stagnate. Keep water flow conditions optimal for your size of distribution system. Make adjustments to the distribution system if necessary to keep the water flowing.
• Collect at locations where a reasonable amount of water is being used. Do not sample vacant buildings. Make sure that your distribution system does not have any dead ends, if at all possible. Do not sample if the resident has been gone or on vacation for over a week or two. If you need to collect a water sample in the morning, wait until the owner has used some water or run the water an extra length of time. Do not collect water in a public building that has been vacant since five PM the previous day, especially on upper floors.
• Cut time in transit by collecting in the afternoon or as close to the time that the postal truck leaves your post office for the distribution point. If you use a courier service follow the same procedure.
• Do not collect in the morning and leave the sample in your truck or a warm area for a number of hours. This will increase the growth of bacteria. The sample should be iced when collected and also when sent or delivered to the EM Laboratory. If you use a courier service, get your receipt and check the drop off point to verify that the samples have been picked up.
• If there is a site that is repeatedly positive for interfering (heterotrophic bacteria) a remedy may be to increase the temperature of the hot water to above 160 ° F and flush the water line and faucet for up to five minutes. Then collect the sample after flushing the faucet with cold water for the normal four to five minutes.
• There are five prominent failures of the distribution system, breakthroughs from:
  1. treatment pools in treatment plants,
  2. regrowth from biofilm,
  3. organics in water,
  4. ingress of contaminated water, possibly from an upstream break in distribution system, or cross connections, condition of plumbing, (customer plumbing may be important),
  5. closed valves creating a dead end.

  Try to eliminate these failures.