Accuracy_and_Precision

Title Accuracy and Precision Introduction We can never say a measurement is perfect. All measurements have some error associated with them. The best we can do is to come as close as possible within the limitations of the measuring instruments. In microbiology, two terms often used somewhat interchangeably to describe measurement errors are precision and accuracy. The Accuracy of a measurement system is the degree of closeness of measurements from a standard or true value of the quantity being measured. The Precision of a measurement system, can be called repeatability or reproducibility, is the degree to which repeated measurements under the same conditions get the same results each time. Figure [ 1 ]: Accuracy indicates proximity of measurement results to the true value, precision to the repeatability or reproducibility of the measurement It was asked in this practical to understand the meaning of accuracy when working with numbers of organisms and also to explain why it is important to be accurate and precise within a microbiological practical. Discussion A measurement system is called valid if it is both accurate and precise. Failure to achieve either accuracy or precision requirements is enough to constitute a failed test or calibration. Calibration is the act of checking or adjusting (by comparison with a standard) the accuracy of a measuring instrument. The precision instruments that can be used are many and for different purposes as well. In microbiology the most used ones are: micropipettes, balances/scales and incubators, just to give an example. There are high standards pre-established to make sure that calibrated equipment meets accuracy and precision requirements. When these requirements are not met actions have to be taken to determine if there was any adverse effect on the product quality. The faulty equipment would have to be either repaired and/or recalibrated and three particular areas would have to be checked: the product design or validation process data or parameters; the quality of the existing components, being processed or finished products; appropriate corrective action taken (example: recall the products if there not suitable for consumption). That is way regular verification checks for accuracy and precision are essential. It has been proposed that microbiology laboratories introduce a measurement uncertainty for organism counts. One method of achieving this involves studying replicate counts from subsamples. This will estimate the variation of organisms in different subsamples, and the different characteristics of strains of organisms within the species or group being counted. A standard deviation is a statistical measure of the precision for a series of repetitive measurements. It measures how widely spread data points are: if the data values are all equal to each other, then the standard deviation is zero. If a high proportion of data points lie near the mean value, then the standard deviation is small. Any experiment that yields data with a low standard deviation, is said to have high precision. If a high proportion of the data points lie far from the mean value, then the standard deviation is large. Any experiment that yields data with a high standard deviation, is said to have low precision. Microorganisms can be observed by direct method, which consists of microscopic observation with the aid of dyes. Due to enormous difficulty operating and long runtime of procedures, the direct method for verification of micro-organisms, is rarely used, although in some situations can be especially helpful when comparing different methodologies. The limitation of this method is the difficulty to distinguish living organisms from the dead ones. Based on these facts the Serial dilution technique and the counting of colony forming units under specific parameters assuming that each cell or spore form a colony is the one used most frequently. It is possible to perform the count of viable microorganisms cells through the pour-plate method respecting the optimum conditions that the cells needs, e.g. appropriate culture medium, temperature, oxygen and time necessary to support their growth and allow the final counting of colonies formed. In the pour-plate method, after preparing the dilutions, these are inoculated in quantities of 1.0 ml into sterile petri dishes, using two plates for each dilution. After placing the material (sample under study) on the petri dish, add 10 to 20 ml of molten agar in rotating movements. They must be incubated at room temperature and conditions which should be best, placed upside down in the incubator. After incubation, perform the count, using membrane filtration. From the set of plates incubated, the best are those that have 30 to 300 colonies, which will facilitate counting and provide a more exact approximation of microbial population. Get the average number of colonies and multiply it itself by the dilution factor. The result is expressed as CFU/ml. An example of a serial dilution is shown here: Materials used: * 4 Serial dilution tubes * Peptone water * 1ml pipette * Incubator * Bunsen Burner * 10 sterile petri dishes * Bacterial Stock/sample * Sterile loop Method: To count the total number of microorganisms proceed as follows: a) Check the plates and bottles for dilution are named and placed in a ordered way ( 1(initial stock), 10-1, 10-2, 10-3, 10-4) b) Using Aseptic technique, gently invert the bottle so as not to damage the cells and transfer 1ml from the full strength stock and transfer it to the bottle labelled 10-1, that should contain 9 ml of peptone water, this is a dilution of 1:10 or 10-1 c) Shake the bottle of 10-1 dilution and withdraw with a pipette 1 ml of the same vial, and transfer it to the bottle labelled 10-2 dilution that must contain 9 ml of peptone water, this is a dilution of 1:100 or 10-2. d) Shake the bottle of 10-2 dilution and withdraw with a pipette 1 ml of the same vial and transfer it to the bottle labelled 10-3 dilution that must contain 9 ml of peptone water, this is a dilution of 1:1000 or 10-3. e) Repeat the above to get a dilution of 1:10000 or 10-4 of the original sample f) Using aseptic technique pour approximately 20ml, warmed at 50ºC, molten agar into each of the 10 plates. Carefully move it up and down, left to right and diagonally three times, to help it spread evenly over the plate. g) Take 1ml from the stock sample (bottle labelled n1) and place it on plates named full strength stock sample A & full strength stock sample B. h) Transfer, with a pipette, 1ml of the 10-1 dilution to the petri dishes named: 10-1 Dilution, sample A and to the 10-1 Dilution sample B. i) Transfer, with a pipette, 1ml of the dilution to the petri dishes named: 10-2 Dilution, sample A and to the 10-2 Dilution sample B. j) Transfer, with a pipette, 1ml of the dilution to the petri dishes named: 10-3 Dilution, sample A and to the 10-3 Dilution sample B. k) Transfer, with a pipette, 1ml of the dilution to the petri dishes named: 10-4 Dilution, sample A and to the 10-4 Dilution sample B. l) Allow the plates to set and leave them inverted to avoid the formation of condensation water on the surface of the agar plate. Transfer the Petri dishes for incubation at 33ºC for 3 days. Results: As soon as possible (2 two 3 days in a real situation or a week in practice) examine the plates and find the dilutions containing the easiest numbers of organisms to count e.g. between 30 -300 colonies. Calculate the number of colony forming units (CFU/ml) in the original sample. Record the amount of growth. Describe the colonies if any is present. Usually counts are recorded for the first dilution which satisfies a rule defining what is an acceptable range of counts( 25-250 or 30-300). When the counts are below the acceptable range there are called zero counts. It means that the number of CFU/ml is not enough to give a reliable reading. CoNclusioN Serial dilution is still one of the cheapest, more reliable methods to count microorganisms and probably the most known/used by microbiological laboratories. As long as calibration and precision checks are performed on a regular basis and the results recorded on a log book, everything machine or precision should be running smoothly on the process that is being used thus allowing the company/quality control to know almost for sure that the final product will be inside the normal requirements. It will also allow it to see if it is not inside those standard requirements and take appropriate action. References * Quality Digest (17/12/2008), Defining Accuracy and Precision, retrieved on the 22/01/2011, from website: http://www.qualitydigest.com/inside/fda-compliance-article/defining-accuracy-and-precision.html * Wikipedia (2011), retrieved on the 22/01/2011, from website: http://en.wikipedia.org/wiki/Accuracy_and_precision * Martin A. Hamilton and Albert E. Parker(22/09/2010), Testing Surface Disinfectants, retrieved on the 23/01/2011 from website: http://www.biofilm.montana.edu/files/CBE/documents/KSA-SM-06.pdf