White Paper16-4102

IntroductionA comparative evaluation study for Total Plate Count (TPC) versus the o�cial ISO method was recently com-pleted. The rapid microbial method of interest was the MOCON GreenLight microbial detection system. This evaluation was conducted with the valuable support of AMUL Dairy (Kaira District Co-operative Milk Producers’ Union Ltd, Anand, India; who arranged to supply the raw cow and bu�alo milk for the testing.

The goal of the testing was to assess the ability of the GreenLight method to correlate to the reference method, in this case ISO4833:2013 (Total Viable Count) for the assessment of raw milk as received at AMUL Dairy in Anand, India.

The protocol used for the study was to sample raw cow and bu�alo milk from tankers arriving at the AMUL dairy, then transport the samples to the Hemshell laboratory in Vadodara. Both the GreenLight and the reference method would test the samples and a comparison established over many samples.

Eventually, 64 samples were compared over a period of approximately 60 days in order to develop a reproduc-ibility study across many variables.

Analysis of the results shows a reasonable correlation to the reference method for the GreenLight system of 0.7076. A subset of the data produced later in the study revealed correlation to the reference method of 0.975.

The possibilities for improvement and further work are listed in the later sections of this report.

Sampling and TransportThe visiting tankers arriving at AMUL dairy carried combined volumes of raw cow and bu�alo milk with unknown mix ratios from day to day. Estimates of the ratio were not kept. A sample volume of 25mL was taken into a sterile container and packed in a cold storage container with ice, then transported to the Hemetek lab.

The total samples received and tested were as follows:

Comparative TestingFor the reference method, the laboratory obtained agar plates that conformed to the standard. The GreenLight system was received from MOCON, Inc. and installed in a temperature controlled area.

GreenLight is an oxygen sensing system that relies on a unique polymer sensor integrated into a test vial. Read-ings are made optically using the GreenLight model 930-2 reader by �lling the 2.0mL test vial with a prepared sample and running a resident software program to observe oxygen depletion of aerobic organisms. Green-Light is able to provide TPC results ten or more times faster than the reference methods and requires less sample preparation.

MethodsSummary protocol:1. 10mL of milk sample was diluted with 90 mL sterile Bu�ered Peptone Water (BPW) in sterile container.

2. Pipette 2.0mL of resulting diluent into GreenLight APCheckTM vial. a. Place APCheck vial into GreenLight system and run at 35C until results are displayed on the connected laptop computer.

Minneapolis, MN 55428 USAPhone: 763.493.6370Fax: 763.493.6358www.microbialdetection.com

Comparative Test of MOCON® GreenLight® System Versus Applicable Reference Methodfor Microbial Testing of Raw Milk

Date(s)

30-Jul-13

1-Aug-13

6-Aug-13 to 9-Aug-13

13-Aug-13

22-Aug-13

10-Sep-13

16-Sep-13

27-Sep-13

28-Sep-13

Total

Number of Samples

10

10

6

5

8

5

5

10

5

64

07.30.2014

Authors: Alan Traylor, Daniel B. Smith (MOCON, Inc.); Bency Joseph (Hemetek Techno Pty.); Ms. Bency Kathayat ;Ms. Alpa Irani (Amul Dairy-Kaira District Co-operative Milk Producers’ Union Ltd); D. G. Shah Ph.D (Consultant)

Continued...

3. Pipette 1.0mL of diluent onto prepared agar plate.

4. Dilute 1.0mL of diluent with 9mL of BPW and repeat in order to create a dilution set (6 dilutions). a. Plate serial dilutions.5. Invert and incubate agar plates for 48 hours at 35C

Note: All plate counts were tested in duplicate.

Results1. The combined data from all runs of GreenLight and plate count were used to develop a correlation curve of the format t=-m. (TPC) + T, where t= GreenLight time-to-result, m= the GreenLight gain, TPC= the reference plate count, T= the GreenLight o�set. a. Several experimental curves were derived from subsets of the data in order to determine the best overall �t. The most reliable (highest) correlation number was found in the later trials run from September 16-28. This was probably due to increased con�dence in all stages of samples, transport and testing process. b. The calibration curve was use to determine TPC values for GreeLight and produce di�er- ence tables in comparison to the reference counts. i. We present here the overall data and the last set of data from test in Septem- ber 2013.

2. A key addition to the data analysis is the inclusion of negative control data point in order to provide a data point corresponding to log ) CFU/mL.

3. All data

This data is derived from the calibration equation as t= -0.6773 (log TPC) + 6.2032

4. September data

This is the data and calibration we would choose as the basis for production testing of tankers arriving at AMUL. The correlcation is expressed as t= -0.6463 (log TPC) + 6.1989

Conclusions1. The GreenLight system was observed to give oxygen depletion responses una�ected by variations in the milk matrix, such as time of day or type of animal, when prepared in a single dilution in bu�ered media (BPW)

2. All samples produced a de�ned time-to-result on the GreenLight system (averaging 1.6 hours) versus 72 hours time-to-result for the ISO reference method

3. The overall sample statistics for all 64 samples were: a. The overall correlation for GreenLight to plate count was R²=0.7076

4. The statistics for reduced number of samples; SEP 16-28 were: a. The reduced data set of tests run at the later stages of the trial returned a much improved correlation factor of 0.975 and reduced variance compared to the reference method

Minneapolis, MN 55428 USAPhone: 763.493.6370Fax: 763.493.6358www.microbialdetection.com

07.30.2014

Method

GreenLight

Reference Method

Ratio

Mean

6.84

6.84

1

3-sigma

2.37

1.43

1.65

Method

GreenLight

Reference Method

Ratio

Mean

6.60

6.60

1

3-sigma

1.47

2.71

0.54

White Paper 16-4102

White Paper 16-4102

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5. The sources of variability in the GreenLight data are most likely caused by the di�erences from sample to sample of the type of milk (cow, bu�alo) and the source site of the sample as well as the day and tanker number. Therefore, this study can be viewed as a broad reproducibility study of the method across many variables. By reducing the number of sample variables, the correlation factor will be increased such that in-plant repeatability of measurement can improve on existing technologies and methods.

Further Work1. There is an expressed interest in the use of the Green Light system to test raw milk in an environment with fewer resources compared to a food microbiological laboratory. a. GreenLight can test milk without the need for any additives or bu�ers but the respiration responses of the resident micro�ora appear, from other trials, to create more variability in results. b. In order to set up GreenLight to test with “neat” raw milk the calibrations tests would be re-run and the resulting protocol adhered to rigidly in production testing. c. It is not necessary to precisely copy incubation or dilution parameters between GreenLight and the reference method. It is, however, important that the calibration protocols and the actual production test protocols are the same. d. The opportunity presents itself to run the agar plate count data (TPC) and GreenLight at a higher incubation such as 37C rather than the ISO standard temperature of 30C in order to test in realistic ambient conditions for India. i. The GreenLight incubation tempera- ture can be programmed in order to control temperature above the ambient lab temperature but less than 40C

Minneapolis, MN 55428 USAPhone: 763.493.6370Fax: 763.493.6358www.microbialdetection.com

07.30.2014