CERTIFICATIONAOAC Research Institute
Performance Tested MethodsSMCertificate No.

012002The AOAC Research Institute hereby certifies the method known as:

PixeeMoTM

manufactured by
AFI Corporation
2rd Fl, SR Bldg Umeshin
6-7-5 Nishitenma
Kita-ku, Osaka 530-0047 JAPANThis method has been evaluated in the AOAC Research Institute Performance Tested MethodsSM Program and found to perform as stated in the applicability of themethod. This certificate indicates an AOAC Research Institute Certification Mark License Agreement has been executed which authorizes the manufacturer to display theAOAC Research Institute Performance Tested Methods SM certification mark on the above-mentioned method for the period below. Renewal may be granted by theExpiration Date under the rules stated in the licensing agreement.

Scott Coates, Senior Director
Signature for AOAC Research InstituteIssue Date

November 30, 2023

Expiration Date

December 31, 2024

2275 Research Blvd., Ste. 300, Rockville, Maryland, USA Telephone: +1-301-924-7077 Fax: +1-301-924-7089
Internet e-mail: aoacri@aoac.org * World Wide Web Site: http://www.aoac.org

AUTHORSYoshikazu Wakizaka, Takayuki Itoi, Masayo Takano, Eiko Kato, Yusei
Sato, Satoshi Morita, and Takaharu Enjoji

SUBMITTING COMPANY
AFI Corporation
2rd Fl, SR Bldg Umeshin
6-7-5 Nishitenma
Kita-ku, Osaka 530-0047 JAPANMETHOD NAME
PixeeMoTM

CATALOG NUMBERS
ELS-002, ELC121 and ELB100N

INDEPENDENT LABORATORY
Japan Food Research Laboratories
52-1 Motoyoyogi-cho
Shibuya-ku, Tokyo 151-0062 JapanAPPLICABILITY OF METHOD
Analyte – Aerobic Bacteria
Matrixes – Drinking Water
Performance claims - Performance of the PixeeMoTM method isequivalent to that of the Standard Method for the Examination ofWater and Wastewater (SMEWW) 9060, Samples (2) and SMEWW
9215, Heterotrophic Plate Count, Part B – Pour Plate Method (3) forenumeration of aerobic bacteria in drinking water.

ORIGINAL CERTIFICATION DATEJanuary 14, 2020

CERTIFICATION RENEWAL RECORD
Renewed annually through December 2024.

METHOD MODIFICATION RECORD
1. September 2022 Level 1

SUMMARY OF MODIFICATION
1. Corporate address change from Sakyo-ku, Kyoto Japan to Kitaku, Osaka Japan.

Under this AOAC Performance Tested MethodsSM License Number, 012002
this method is distributed by:NONE

Under this AOAC Performance Tested MethodsSM License Number, 012002
this method is distributed as:NONE

PRINCIPLE OF THE METHOD (1)PixeeMoTM method consists of feeding a liquid sample to a microchannel chip, capturing bacteria contained in the sample, observing microscopically, andcounting the captured bacteria. “Fluid, electric filtering and sorting technology (FES)” is used for capturing bacteria. FES is a selection technology that integrates
electrical measurement and fluid control. A sample is fed into an appropriately designed microchannel, AC electric field is generated by the electrode in themicrochannel, and the electric force acts to attract bacteria when passing over the electrode. This method uses the intracellular ion homeostasis of the bacteria,
therefore intact cellular membrane is required for capture on the electrodes, implying only viable organisms will be captured. To perform the method, the water
sample is first centrifuged, then the water replaced with buffer, and the sample is then loaded into a syringe, which is placed on the instrument and brought into
contact with the chip. The software controls the settings and operation of the instrument for drinking water analysis. While the syringe plunger expels liquid at aconstant flow rate for a specified time period, the voltage supply is turned on and microbes are captured on the electrodes. At the end of the collection period,
digital images are taken and analyzed by software to determine the microbial concentration. By collecting the bacteria on the electrode, it is possible to directly
observe and count the bacteria within one or two fields of view. With this method, the bacterial concentration of a sample is determined by counting thecaptured bacteria from 1 mL of sample fed through the microchip. Feeding time is approximately 17 min, therefore it is possible to determine the bacterial
concentration of a sample within 1 hour.

AFI Corporation, PixeeMoTM, AOAC Performance Tested MethodsSM Certification Number 012002
DISCUSSION OF THE VALIDATION STUDY (1)The method of SMEWW 9215B takes 7 days to get the results. On the other hand, the PixeeMo method can provide the results within 1 hour because incubationis unnecessary. Obtaining the results on the day of testing is a notable advantage of the PixeeMo method. Furthermore, the test procedure using AFI PixeeMo isquite simple and easy.
The maximum repeatability standard deviation of the PixeeMo method was 14.8%. The difference of mean log10 values between the PixeeMo and SMEWW
9215B methods ranged from -0.015 to 0.258. Similar results have been obtained in the independent laboratory study. However, there was a slight difference
only from the lower confidence limit for the difference of means between the two methods at the low contamination level by the independent laboratory. On
the other hand, the scatter plot of both methods indicated a high correlation between the two methods from the developer and the independent laboratory.
Overall, there is no statistically significant difference between the PixeeMoTM and SMEWW 9215B methods.
In the product consistency and stability study, chips and buffers within the expiration date are considered to have no change in performance. In the robustness
study, three parameters (centrifugation of sample, sample conductivity and output voltage of Channel 2) were varied, it was confirmed that there was no effectwithin the expected range. As a result of comparing the measurement results of the 3 PixeeMo instruments by analysis of variance, there is no significant
difference among the data of 3 PixeeMo instruments.

Table 1. Method comparison data summary and statistics (1)SMEWW 9215B

PixeeMoTM

95% CIb

Matrix (organism)Cont.
levelN

Drinking watere(naturallycontaminated)

Low

5

Medium

5High

5

3.755

0.139

3.7

4.012

0.023

0.6

0.258

0.069

0.446

Drinking waterf

Low

51.806

0.039

2.2

2.205

0.077

3.5

-0.399-0.506-0.292Medium

5

2.989

0.106

3.5

3.142

0.111

3.5

-0.153-0.343

0.039

High5

3.937

0.091

2.3

4.119

0.172

4.2

-0.182-0.4230.059(naturallycontaminated)

Meansr

RSDr, %

Meansr

RSDr, %

1.761

0.260

14.8

1.704

0.028

1.7

3.006

0.189

6.3

2.992

0.025

0.8

DOMa

LCLc

UCLd

-0.057-0.3620.248-0.015-0.221

0.191

aDOM = Difference of MeansbCI = Confidence Interval for DOM

cLCL = Lower confidence limit for DOM
dUCL = Upper confidence limit for DOM

eMethod Developer dataf

Independent Laboratory data

REFERENCES CITED
1.
Wakizaka, Y., Itoi, T., Takano, M., Kato, E., Sato, Y., Morita, S, and Enjoji, T., AFI Corp. PixeeMoTM for Enumeration of Aerobic Bacteria in Drinking Water, AOAC
Performance Tested MethodsSM certification number 012002.
2.
Standard Method for the Examination of Water and Wastewater (2017) Method 9060, Samples, American Public Health Association (Washington, D.C.),
American Water Works Association (Denver, CO), and Water Environment Federation (Alexandria, VA),
https://www.standardmethods.org/doi/10.2105/SMWW.2882.184
3.
Standard Method for the Examination of Water and Wastewater (2017) Method 9215, Heterotrophic Plate Count, Part B – Pour Plate Method, American Public
Health Association (Washington, D.C.), American Water Works Association (Denver, CO), and Water Environment Federation (Alexandria, VA),
https://www.standardmethods.org/doi/abs/10.2105/SMWW.2882.188