Steps to speed up sludge testing

Many laboratories throughout the UK are switching their E.Coli sludge testing procedures from traditional most probable number (MPN) methods to IDEXX’s rapid microbiological test, Colilert-18/Quanti-Tray/2000. The principal drivers behind the trend are reduced sample handling time, the ability to provide completed results in
18 hours, compared to a possible three-four days, and the method’s recent inclusion in the Drinking Water Inspectorate’s (DWI) The Microbiology of Sewage Sludge (2003) part three: Methods for the isolation and enumeration of Escherichia Coli.

Contract laboratory ALcontrol, in Rotherham, and South West Water are just two of the laboratories which have recently adopted the new method for E.Coli testing. As one of Europe’s leading analytical services organisations, ALcontrol currently carries out up to 100 sludge tests per week at its laboratory on behalf of Yorkshire Water. ALcontrol had previously employed a MPN test, which used a multiple tube fermentation process, involving various stages of dilution, pipetting and as many as 15 separate test tubes per sample.

The company switched to Quanti-Tray/2000 because it involves less effort and gives completed results in 18 hours. Using the Quanti-Tray/2000 method, ALcontrol takes a suitable aliquot of the sludge sample, which is then diluted to 100ml with RO water and homogenised. Colilert-18 reagent is then added to the sample and dissolved before being poured into the Quanti-Tray/2000, a multi-welled, disposable vessel that holds a 100ml sample. The mixture is automatically distributed and sealed into 97 wells by the Quanti-Tray Sealer in less than 20 seconds. The sealed tray is then incubated for 18 hours and the coloured wells are counted. A table converts the number of yellow wells to a coliform count and yellow/fluorescent wells to an E.Coli count. Quanti-Tray/2000 eliminates subjective colony interpretation and time-consuming confirmation. In most instances, ALcontrol reports the results using electronic data transfer, giving E.Coli counts for 1g of both wet and dry samples. ALcontrol’s chief scientist Clive Thompson commented: “The MPN test we used previously was extremely laborious, involved multiple stages of preparation and an extensive amount of laboratory equipment.

With a total hands-on time of less than 1min per sample Quanti-Tray/2000 is faster, simpler and extremely cost-effective for E.Coli analysis.” South West Water conducts 4,000 E.Coli sludge tests per annum on both wet and dry samples. The Exeter laboratory, which employs 20 microbiologists, regularly samples 24 sites in rotation over a fortnightly period, with five pots of sludge taken from each site. As with ALcontrol, South West Water had previously used a MPN test but decided to conduct a validation study of the Quanti-Tray/2000 method for submission to UKAS. Samples were run in triplicate for digested, limed, pressed, thermally dried and composted sludge.

As the data in Table 1 indicates, the Quanti-Tray/2000 method compared well with traditional methods for sludge analysis. John Watson, senior scientist at South West Water, said: “The Colilert-18/Quanti-Tray 2000 test is a lot quicker than our current MPN methods, providing results the
following day, whereas previously they may have taken three-four days.

“We have also found the microbiological test is a lot simpler, saving our technicians significant time and effort.” IDEXX European sales manager Steve Morris added: “With the inclusion of our method in the DWI’s Blue Book for Sludge Analysis, we anticipate many new UK labs will rapidly adopt Quanti-Tray/2000. Its popularity has been clearly illustrated in the drinking water market – now we can bring customers those same benefits for sludge testing.” The Quanti-Tray/2000 test protocol for sewage sludge is as follows:

• add 10g of sample to a sterile, 100ml Colilert vessel containing 90ml of sterile diluent,
  
• this mixture is transferred
  to a stomacher bag and homogenized for 1min, then transferred back to the vessel and mechanically shaken for one hour,
  
• a series of 100 fold dilutions are prepared from the homogenized sample. Each of these dilutions will contain in 100ml: 0.1g, 0.001g and 0.00001g of the original
  sample – A. 0.01ml of homogenized sample added to 99ml of sterile water in a 100ml Colilert vessel = 0.1g of original sample. B. 0.10ml of the homogenized sample into 9.9 ml of sterile water. Vortex. C. 0.01ml of solution in part B into 99ml of sterile water in a 100ml Colilert vessel = 0.001g of original sample. D. 0.10ml of solution in part B into 9.9ml of sterile water. Vortex. Then add 0.01ml of this solution to 99ml of sterile water in a 100ml Colilert vessel = 0.00001g of original sample,
  
• the contents of one Colilert-18 blister pack are added to each of the three 100ml dilutions. Each 100ml sample is poured into a Quanti-Tray and sealed immediately using the IDEXX Quanti-Tray Sealer,
  
• Quanti-Trays are incubated ‘wells down’ at 37°C for
  18 hours,
  
• Quanti-Tray results are read and multiplied by the appropriate dilution.
  The Colilert-18 test is based on defined substrate technology, which is a patented method of detecting bacteria through the effect of their constitutive enzymes on chromogenically and fluorogenically labelled, specific substrates. Colilert-18 detects coliforms and E.Coli through the activity of the enzymes beta-D-galactosidase and beta-D-glucuronidase respectively, which are generally accepted as being primary characteristics of these organisms. Colilert-18 contains a specific substrate for each of these enzymes. The Quanti-Tray is a multi-welled, disposable vessel that holds a 100ml sample, mixed with Colilert-18 reagent. The mixture is automatically distributed and sealed into 51 wells by a Quanti-Tray Sealer in less than 20 seconds.

The sealed tray is then incubated for 18 hours and the coloured wells are counted.
A table converts the number of yellow wells to a coliform count and yellow/fluorescent wells to an E.Coli count. The Quanti-Tray eliminates subjective colony interpretation and time-consuming confirmation. Quanti-Tray/2000 is a 97 well tray designed for raw water and wastewater testing.

The necessity for manual dilutions of heavily contaminated water samples is greatly reduced because the tray’s maximum counting range of one-2,419 colony forming units is more than 20 times that of membrane filtration