Metagenomic Analysis Protocol (MAP) is a Nalco Water patented approach that allows for detection, quantification, and identification of microbial DNA present in the papermaking process, sheet defects, deposits, incoming water, and other materials. MAP delivers an unprecedented level of detection and precision in troubleshooting microbial-based problems. MAP provides advantages over traditional monitoring approaches by detecting all microorganisms, and breaking them down into problem-causing categories.

• The benefits of MAP Monitoring Technology:

• Detection and quantification of problematic microorganisms that may be missed using traditional monitoring approaches.

• Identification of contamination “hot zones”.

• Targeted treatment to eliminate problematic microbes and the problems they cause.

• The MAPExpress (MAPE) Test Kit is used to prepare and stabilize mill samples before shipping to Nalco Water’s Customer Analytical Laboratory for the final analysis and interpretation.

MAPE TEST KIT – DNA STABILIZATION ON-SITEThe MAPE Test kit is used to prepare paper process water samples before shipping to Nalco Water’s customer analytical laboratory for the final analysis and interpretation. MAP is a patented approach that allows for detection, quantification, and identification of microbial DNA present in the papermaking process, sheet defects, deposits, and other materials. MAP allows an unprecedented level of detection and precision in troubleshooting microbial based problems. MAP provides

advantages over traditional monitoring approaches by detecting all microorganisms and breaking them down into problem-causing categories.

The MAPE kit is used to extract and stabilize microbial DNA in paper process water. The vortex and vortex adapter combine to make a unique tool that lyses (breaks open) the bacterial cell to release the DNA that is stabilized by the reagent. The sample is added to the processing tubes that are prefilled with glass beads. When shaken vigorously in the vortex adapter, the bacterial cell is broken open by chemical and physical action and the DNA is stabilized. Do not remove the beads, they aid in DNA extraction from the cells. Simply add the sample to the processing tubes, add the lysis-stabilization solution, tighten the lid and insert the tube into the vortex adapter. Place the adapter on the vortex and turn it on for 10 min at the maximum shaking speed (10).

Stabilizing the sample immediately after collection eliminates any introduction of variability before samples are analyzed and completely stops any further changes to the microbial population. Once processed, the samples can be sent immediately for analysis or held for several days and sent as a batch. The processing vials are the shipping containers and the final volume of the prepared sample is only 2 ml which cuts shipping weight and reduces the opportunity for spills.

ANAYLTICAL PROCEDURE

MAPExpress Test Kit ProtocolAP-156

SAMPLE SUBMISSION AND SHIPPING INSTRUCTIONS FOR USE WITH MAPE KIT

Types of samples that CAN be processed with MAPE Kit:

• Incoming fresh water

• Paper process water

• Pulp

• Effluent water

• Uncooked Starch slurries

• Cooked Starch slurries

• Low viscosity process additives, as long as they are water soluble

Types of samples that CANNOT be processed with MAPE Kit:

• Retention Aids

• Coating Pigments

• Direct Dyes and Pigments

• Latex

• OBA

• Hydrophobic substances

Contact Paper RD&E if your sample is not specifically listed.

General sampling points:

• incoming fresh water

• headbox

• broke

• machine chest

• shower water

• pulp

• additives

DO NOT PROCESS COATING SAMPLES USING MAPE KIT!

Shipping Instructions:

1. Form 619 and Form 323A must be included with all samples.

2. CLEARLY LABEL ALL MAP SAMPLES WITH REQUESTED ANALYSIS: MAP

3. Send samples to:

Attn: Sample Receiving – MAP TESTING

NALCO Water

1601 West Diehl Road

Naperville, IL 60563

4. Send an email to Liliya Lund and Elisa Luth (llund@nalco.com, emluth@nalco.com) with the following information:

a. Account name and location

b. # of samples being submitted

c. List of sample points

d. package tracking number

5. Additional details on shipping samples can be found in Connections:

https://connections.nalco.com/sites/Offerings/Services/AnalyticalServices/Pages/default.aspx

SUPPLIES REQUIRED:For a complete MAPE Test kit, order all of the following items from Nalco’s Equipment Catalog:

Description (Part No.):

Vortex Analog Mixer 500-P9502.88

Vortex Adapter 500-P9503.88

Xpedition Lysis/Stabilization Solution, 40 ml 460-S0590.71

Pipettor, Single Channel, 100-1000 μL 500-P3756.88

Tips, Pipet, 100-1000μL, 96/Rack 500-P3757.88

Glass Bead Mixing Tubes, 50/pk 500-P9500.88

1 2

1000μL

3 4

5 6

10

7

11

8

12

Collect sample into a clean container.

Add 750 μL of Xpedition Lysis/Stabilization Solution to tube with sample.

Shake tubes in the vortex adapter for 10 minutes.

Mix sample well before pipetting and label sample tubes.

Aseptically cut pipette tip for thicker samples (broke, headbox, machine chest, etc.).

Place 1000 μL of sample into the tube with glass beads. If airbubbles are present, tap the tube gently on the counter to release them.

Replace cap back on tube, and screw it on tightly.

Remove tubes from the vortex adapter and store in the refrigerator at 4 °C until ready for shipment.

Horizontally insert tubes into adapter with caps facing center. Be sure to balance tubes.

Tubes should be shipped in a padded shipping envelope or box with Form 619 and Form 323A

Secure adapter on vortexer. Set speed control on the vortexer to the maximum value, 10.

Attn: Sample Receiving

NALCO Water

MAP Testing

1601 West Diehl Road

Naperville, IL 60563

9

4°COR

40°F

Nalco Water, an Ecolab CompanyNorth America: Headquarters – 1601 West Diehl Road • Naperville, Illinois 60563 • USA Nalco Champion – 7705 Highway 90-A • Sugar Land, Texas 77487 • USAEurope: Richtistrasse 7 • 8304 Wallisellen • SwitzerlandAsia Pacific: 2 International Business Park • #02-20 The Strategy Tower 2 • Singapore 609930Latin America: Av. das Nações Unidas 17.891 • 6° Andar 04795-100 • São Paulo • SP • Brazil

nalco.ecolab.com

NALCO Water and the logo are Trademarks of Ecolab USA Inc.©2017 Ecolab USA Inc. All Rights Reserved 12/17 AP-156

DATA INTERPRETATION:The results reported are measured using a DNA-based assay (qPCR). This technique is able to measure the quantity of microbial cells present in a sample and has a higher level of detection than traditional plating methods. As such, the amount of organisms present is calculated with the use of a standard and reported as cells/g or cells/mL. These units are not the same as CFU/mL. qPCR does not differentiate between live or dead organisms. The presence of microbial DNA provides no information about the viability of species detected. However, even the presence of non-viable/dead DNA indicates that organisms were present and there might be an unidentified source of contamination to the process (or something like that).In uncoated freesheet graphics grades, 105 cells/g is a typical background level of organisms in a sheet from a well-treated process. In sheet defects that have been analyzed where there was a major microbial contribution to the defect, the level of bacteria detected in and around the defect is expected to be >107 cells/g.

Please refer to the Metagenomic Analysis Protocol (MAP) Interpretation Guide for more information.

Organism Target Groups:

Total bacteria – All viable and nonviable bacteria present in the sample.

Primary biofilm-formers – A group of bacteria that attach to a clean surface and create a base layer for other bacteria and debris to adhere to.

Adaptive biofilm-former – A bacterium that has developed a tolerance to oxidant-based biocontrol programs. Typically found in fresh water and the environment.

Filamentous biofilm-formers – A group of fresh water bacteria that can form filaments and biofilms. Typically used as an indicator of fresh water quality.

Nitrifying bacteria - Bacteria that oxidize ammonia to nitrate. Used as indicators of performance of monochloramine-based fresh water treatment programs.

Sulfate-reducing bacteria – Anaerobic organisms that reduce sulfate to hydrogen sulfide (H2S).

Fungi – All fungal species present in the sample. Not quantitative; represented with a “low, medium, high” scale.

CAUTION:1. MAP is an advanced problem-solving tool. It is not

meant to replace routine, on-site microbiological testing.

2. Samples that arrive on Monday will not be analyzed.

3. If samples are not addressed to Sample Receiving with requested MAP analysis they may not be analyzed due to excessive storage times that can impact results.

4. Not following the MAPE protocol exactly in regards to mixing speed and time, leads to low yield of DNA and artificially low results. (See graph below)