1. TOX-SPOT Rapid Detection Of Water Toxicity On-Site Test Protocol - Step By Step Version 2.0

2. TOX-SPOT Kits Content Refill Kit Reagents: 1. freeze dried bacteria vials 2. freeze dried buffers vials: Negative Control (Pro-Metal; Pro-Organic) Positive Control (Pro-Metal; Pro-Organic) Sample 1 (Pro-Metal; Pro-Organic) Sample 2 (Pro-metal; Pro-Organic) 3. Hydration Buffer 4. Empty test tubes Accessories: 5. Portable luminometer 6. Pipettor (0.1-1ml) 7. Tips 8. Timer 9. Portable incubator 10. Carrying case 2 13 4 6 5 7 8 9 10

3. Step 1- Preparations Turn portable incubator on by connecting to cigarette plug in the car. Place 8 black-capped plastic tubes inside incubator

4. Step 1- Preparations (cont.) In the designated foam slots, place 8 freeze-dried vials marked as follows: M-NC,M-PC,M-S1,M-S1 O-NC,O-PC,O-S1,O-S2 Collect samples from suspected water source (river, lake, drinking water tap, etc.).

5. Step 2 - Hydrate Bacteria & Incubate Place a blue tip on the end of the pipettor and hydrate one vial of freeze-dried bacteria with 0.9ml Hydration Buffer. Place in designated slot in incubator and leave for 10 minutes at 30°C.

6. Step 3 - Add Clean Water Withdraw from clean water container 0.9ml with pipettor and dispense into each of the control vials: O-NC O-PC M-NC M-PC Mix well the contents of each vial by vigorous flicking with index finger

7. Step 4 - Add Samples Withdraw from Samples containers 0.9ml with pipettor and dispense into each of the Sample vials: O-S1 O-S2 M-S1 M-S2 Mix well the contents of each vial by vigorous flicking with index finger

8. Step 5 - Biosensor Addition After exactly 10 minutes of incubation, remove the hydrated bacteria (Biosensor) and mix vigorously with finger. Using the pipettor (set at 100 microliter), rapidly dispense exactly 0.1 ml into each of the 8 vials.

9. Step 6 - Liquid Transfer Carefully and quickly, pour the content of each vial into its parallel empty plastic tube in the incubator.

10. Step 7 - Mixing & Incubation Place the black cap back on each plastic tube and mix contents by shaking. Incubate all 8 tubes (holding bacteria, buffer, and water) in the incubator for 15 minutes.

11. Step 8 - Read Results Using the orange holder, pick up one tube at a time and insert into luminometer in the following order: M-NC M-PC M-S1 M-S2 O-NC O-PC O-S1 O-S2

12. Step 9- Analyze Data Record the light level obtained in each tube. Use the provided Excel module to automatically analyze the data. NC (Negative Control) is defined as 100% baseline. PC (Positive Control) should be at least 50% of NC. If Samples (S1, S2) reading indicates >50% change in light compared to NC = TOXICITY ALERT Data is stored in the luminometer for future download to PC.

13. Excel Module - Data Analysis Example