Laboratory testing and reporting
Water can be tested by laboratories in three main ways. These are a filter-based protocol, where 100ml sample is typically tested, a most probable number (MPN) based test method where a number of smaller volumes are independently tested, and by direct plating onto culture media. The filter-based method passes water through a porous membrane with pores are that are too small to allow bacteria through; thereby concentrating the bacteria in a large volume of water on one side of the membrane. The membrane is then placed on top of a solid growth media to allow single cells to grow into visible colonies which can be counted. Pictures of typical membranes after culture is shown as Figure 1.
Figure 1 A set of typical incubated water filter membranes from a fast flowing overland river before ultraviolet light treatment (A) and after treatment (B)
The MPN method is considered to be old-fashioned compared with the filter method. The MPN method however is still useful if the water sample has a large amount of dissolved solids which makes filtration difficult. It is important to note that the MPN method is a statistical estimate of the bacterial numbers within a sample rather than the absolute count made by the filter method. In brief, when undertaking MPN, small volumes of supplied sample are diluted by x10, x100, x1,000, x10,000 and x100,000. A number of volumes (typically 3 or 5) are removed from each of the dilutions and then added to tubes of liquid growth media. After incubation, the number of tubes containing growth for each dilution used in conjunction with a set of lookup tables to estimate the statistically most probable number of bacteria contained within the original sample. It is usual for laboratories to report an MPN test result as MPN CFU/100mls rather than CFU/100mls.
Direct plating is commonly used for a count of total mesophilic aerobes contained in poor-quality water. The techniques is simple and simply involves spreading small quantities of either neat or diluted water directly onto the surface of an agar plate or mixing the sample into molten agar and allowing it to set. Figure 2 shows a set of plates after sufficient incubation to allow single organisms to grow up to visible colonies for counting.
Figure 2 A Petri dish showing the range of different bacterial colony morphologies typically observed after incubation of a high dilution (1,000,000 times diluted) sample during a total mesophilic aerobic count laboratory test. Figure yyB A less diluted portion of water from the same sample (1,000 times diluted) which contains too many individual colonies to count.
Irrespective of the test method used, water test results are normally always reported as CFU per 100mls (because it’s rare to find a single bacteria in 1 ml of good-quality water). When food is tested by a laboratory, a 25g sample is typically used for the test and the result is reported as the number of colony forming units per gram (CFU/g) of food.
Interpretation of test results
There are no statutory criteria for irrigation water quality in the UK. Given that there are a number of sets of guidance for acceptable indicator numbers, growers should decide which standard they want to adopt. In a number of cases, retailer requirements will dictate which standard is adopted. At periodic intervals determined by growers (once per month is considered a reasonable initial test frequency), water samples should be collected and sent for testing. In order to form a picture of how the water microbiology changes over time, many grower technical managers find plotting a trend of test results to be of benefit. A trend of historical test results makes it easy to see if an atypically high result has been recorded. A typical plot of test results for a surface river is shown as Figure 3.
Figure 3 A trend of faecal coliform numbers in river water abstracted for the irrigation of RTE
Out of specification results
If test results are out of specification, a number of approaches can be attempted to try and help bring the water back into specification. The course of action will be largely dictated by the finances available to the grower and the seriousness of the contamination problem. Often the simplest course of action is to switch to an alternative water supply which meets the specification. Although expensive, mains water is considered to be the highest quality irrigation. It is not considered good practice to dilute out-of-specification water with mains water to bring it back into specification because the high test result indicates a potential problem with the water source that will still exist after dilution. The safest course of action is to treat the water by buying (or short-term leasing) an ultraviolet light water treatment unit or a reverse osmosis filtration device. Alternatively, chemical treatments such as the use of a reactive oxygen-based purifier (e.g. ozone or hypochlorite) can be added to cost-effectively reduce the bacterial population of smaller volumes of water before application. It is acknowledged that each of these courses of actions has a financial impact for growers. However, overall, the general strategy of fairly frequent monitoring of water sources with action (and expense) only when test results go out of specification; is the most cost-effective strategy for dully diligent growers interested in producing microbiologically safe crops. Although it will not change the status of the water, many growers switch to more frequent sampling and testing if they have an out of specification result because it provides more information on how the irrigation water quality is changing during the period of concern.
Important issues relating to laboratories and results interpretation
When trending test results over time, there are a number of considerations to be made. It is important that the laboratory does not change the test method it uses to count indicators or detect pathogens. A test report for a good-quality laboratory will always contain a test method reference, and this reference should be checked to make sure it’s the same as previous test before trending new data with historical data. If a single water sample is tested using two different test methods, the two results will be different (sometimes very different). Similarly, because test results are sensitive to the equipment used to measure liquids, the brand and type of growth media used and numerous other factors, changing the test laboratory can also result in large changes in the test results. Furthermore, if the same test method is used, but the tests are undertaken in different laboratories, it is not good practice to mix different lab’s results on a single trend graph. If the testing laboratory or testing method is changed, a new trend graph should be started.
Good-quality laboratories will have determined the detection limits for all of the microbiological tests that they undertake. Consequently these laboratories will not report low numerical test results not as 0 CFU/100ml. If the detection limit of the test result is 10 CFU per 100mls, and no bacteria grew during the test, the laboratory will report the result as <10 CFU/ 100mls; acknowledging that the test method in use is unable to detect below 10 CFU/100mls. It is standard practice when trending microbiological results to use half the limit of detection for any low counts reported. Thus a result of <10 CFU/ 100mls would be trended as 5 CFU/ 100mls. The same approach and value would be used for the calculation of any averages from multiple sample tests.