TOX-SPOT

Rapid Detection Of Water Toxicity On-Site

Test Protocol - Step By Step

Version 2.0

TOX-SPOT Kit’s 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

65

7

8

9

10

Step 1- Preparations

Turn portable incubator on by connecting to cigarette plug in the car.

Place 8 black-capped plastic tubes inside incubator

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.).

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.

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

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

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.

Step 6 - Liquid Transfer

Carefully and quickly, pour the content of each vial into its parallel empty plastic tube in the incubator.

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.

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

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 .

Excel Module - Data Analysis Example

TOX-SPOT - Test Protocol Results

Minimum: 400

Enter the values displayed on the luminometer in the corresponding white cells:

Metal Organic Comments

(M) (O) (M) (O)

Negative Control (NC) 500 400 100% 100% Base line

Positive Control (PC) 100 150 80% 63%

Sample 1 (S1) 251 201 50% 50%

Sample 2 (S2) 270 210 46% 48%

Note: Inhibitory Concentration (light reduction) of 50% and above indicates toxicity !!

Date: 17-Jan-10

Comment:

Tester Name:

Signature:

IC - Inhibitory Concentration (%)

Kikkoman PD-20Select Luminometer type: