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Section 11 Luminescent Bacteria Toxicity
Test: Screening and LIMIT measure
The Luminescent Bacteria Toxicity (LBT) Test screening and LIMIT measure procedures use the luminometer. Before doing either procedure:
• Read
section 3.1, Overview on page 13 .
• Do the procedures in section 3.2, Prepare the luminometer for use on page 14 .
• Print color copies of the Screening Luminescence Results Sheet to use in the field
(refer to page 64) from www.Hach.com.
This chapter describes the LBT screening luminescence procedure and LIMIT measure procedure and contains the procedure steps.
The screening luminescence procedure and LIMIT measure procedure are used in the field or in an emergency situation when a rapid assessment of the inhibitory effects of a sample is necessary. The screening luminescence procedure and LIMIT measure procedure are a simplified test procedure that uses the same reagents according to ISO 11348 but at ambient conditions.
The LBT screening luminescence procedure and LIMIT measure procedure are done the same with two exceptions:
• Different options on the luminometer are selected to measure the sample dilutions.
• Different options on the luminometer are selected to show or send results.
• A LIMIT value is set by the user using the luminometer for the LIMIT measure procedure.
A column is added to the LIMIT measure test results that shows whether the percent inhibition measured for each sample dilution is above or below the LIMIT value (percent inhibition) set by the user on the luminometer.
Do the LBT screening luminescence procedure to do a toxicity screening measure.
Do the LBT LIMIT measure procedure to do a toxicity limit measure.
11.1 Overview
The screening luminescence procedure or LIMIT measure procedure is done to identify if a sample is free of any inhibitory effects on the luminescent bacteria or, if an inhibition is expected, to make an inhibitory or risk assessment of the sample.
Therefore, the user should measure dilutions of a sample and the percent inhibition of the dilution steps to get more information about the severity of the inhibitory effects.
In one run, the sample is measured in three different concentrations: 20% sample,
50% sample and 80% sample. The inhibitory effect of each sample dilution on the
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luminescent bacteria is measured by the luminometer and is shown as percent inhibition.
Due to the nature of the simplified procedure and because the test is done at ambient temperatures, the results may be different if compared directly with results for the same sample using the LBT measurement luminescence (ISO 11348) procedure.
11.2 Accuracy
The error or standard deviation of the test is the sum of the error introduced to the test by all components, the ambient and all manipulations. The higher the degree of variation, the higher the total error.
A Luminescent Bacteria Toxicity Test done strictly according to ISO 11348 has a better precision (lower CV (coefficient of variation)) than a LBT simplified luminescence screening procedure or LIMIT measure procedure under field conditions.
For screening measurements and LIMIT measurements, the measurement CV is
7% in the middle of the measuring range of 10-90% inhibition. In practice, samples that shows results of +/-15% inhibition in the 80% sample concentration have no affect in the Luminescent Bacteria Toxicity Test.
If higher precision or lower CV is needed, do the LBT measurement luminescence procedure under more controlled conditions in a lab using additional accessories like a LUMIStherm temperature controlled incubator (LTV053).
11.3 Reagent description
The Luminescent Bacteria Toxicity Test reagent contains living luminescent bacteria that have been grown under optimal conditions, harvested and lyophilized
(freeze-dried). The reagent is a freeze-dried preparation of a specially selected strain of the marine bacterium Vibrio fischeri (formerly known as Photobacterium phosphoreum, NRRL number B-11177). A vial of reagent contains roughly one hundred million test organisms.
Refer to section Appendix A, Luminescent bacteria risks on page 125
for bacteria risk information.
11.3.1 Quality assurance test
The standards specify that certain validity criteria must be met for the reagent.
Accordingly, a test must be done for each batch of bacteria that is prepared in-house or moved in. The quality certificate delivered with each package of luminescent bacteria reagent by HACH-LANGE GmbH guarantees compliance with the stipulated validity criteria.
To make sure that the test operates correctly on site, the user does control measurements with the standard solutions (refer to the ISO standard procedure).
The necessary information about standard substances, test concentrations and
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sources of supply is contained in the quality certificate that comes with every box of luminescent bacteria reagent.
The standard stock solutions should be prepared with 2% NaCl solution. The pH of the sample should not be adjusted. Prepare the standard solution such that 0.5 mL of standard solution and 0.5 mL of bacteria solution gives the above mentioned final test concentration. Check in duplicate whether those standard tests give 20–80 % inhibition after 30 minutes of exposure time at 15 °C.
11.4 Reagent storage and preservation
The freeze-dried reagent can be kept at -18 °C until the expiration date shown on the package.
Tubes that contain thawed but not reactivated freeze-dried luminescent bacteria can be frozen again and kept on stock.
The reagent can be transported or shipped up to 7 days at no more than 25 °C.
11.5 Prepare the reagent
Prepare the Luminescent Bacteria Toxicity Test reagent in the field using the procedure in this section.
The amount of light made by the luminescent bacteria is affected by the temperature at which the reagent is reconstituted. The luminescent bacteria and reconstitution solution must be mixed as cold as possible at refrigerator temperatures (3 to 8 °C). If the temperature is higher, the amount of initial light made by the bacteria will be lower.
11.6 Prepare the stock suspension using the LCK491 reagent
Prepare the stock suspension by adding the reconstitution solution to the freeze-fried bacteria reagent. The reconstitution solution rehydrates the bacteria reagent.
Reconstitution solution is specially made non-toxic ultra pure water. Do not make reconstitution solution or use substitutes.
The dry reagent can be kept at ambient temperatures (not higher than 25 °C) up to
5 days without cooling. Make sure that reactivation conditions are as cool as possible.
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The stock suspension can be kept in a refrigerator as long as the validity criteria are met (typically up to 4 hours).
This procedure is temperature sensitive.
1. Remove the luminescent bacteria test reagent from the freezer.
Remove the reconstitution solution and Diluent from refrigerator.
2. Put the frozen luminescent bacteria reagent, refrigerated reconstitution solution and
Diluent in a cool box that contains thermal packs if possible.
3. In the field, remove the cap from the reconstitution solution bottle.
4. Remove the foil seal and rubber stopper from the reagent bottle.
5. Set the 1.0-5.0 mL pipette to 1.0 mL.
6. Put the end of the
1.0-5.0 mL pipette in to a clean pipette tip.
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7. Put the tip of the pipette in to the reconstitution solution and slowly pull in
1.0 mL.
8. Put the tip of the pipette in to the luminescent bacteria reagent bottle and slowly dispense the solution in to the reagent.
9. Put the rubber stopper in the reagent bottle. Swirl the reagent bottle to mix.
10. Cool the reagent for 5 minutes in the cool box.
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11.7 Prepare the test suspension
Prepare the test suspension (stock suspension and Diluent mixture) by doing the procedure in this section.
The Diluent is made according to ISO11348-3 and makes sure that the test is not negatively affected by the presence of potassium (K+) and magnesia (Mg2+) ions in the sample. The Diluent is a specially made non-toxic 2% sodium chloride (NaCl) solution that contains potassium and magnesia ions.
The marine bacterium in the reagent requires the osmotic protection that is given by the 2% NaCl in the Diluent. The potassium and magnesium in the Diluent stabilize the light made over time. This stabilization helps keep high negative inhibitions from getting with samples that contain potassium and magnesium ions.
Do not make Diluent or use substitutes.
1. Remove the Diluent from the cool box.
Remove the cap from the
Diluent bottle.
2. Put 14.0 mL of Diluent at refrigerator temperature in the reaction vessel using the pipette.
3. Remove the stock suspension (rehydrated reagent) from the cool box.
Remove the rubber stopper from the reagent bottle.
4. Set the 1.0-5.0 mL pipette to 1.0 mL.
5. Put 1 mL of stock suspension at refrigerator temperature in to a clean reaction vessel using the pipette.
6. Put the cap on the reaction vessel and shake to mix thoroughly.
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7. Wait 15 minutes.
8. Remove the pipette tip from the pipette and put in the waste bag.
Put the pipette in the storage case.
11.8 Sample collection, storage and preservation
The test can be used with samples of municipal and industrial waste water, aqueous eluates from soil and waste, aqueous solutions of pure chemicals and with surface, well and water of other sources.
Collect samples in clean glass bottles.
Keep samples in the dark at 0 to 5 °C for no longer than 2 days.
Freeze and store samples at -18 °C for not longer than to 2 months. Record preservation activities.
Before use, defrost samples completely. Homogenize the defrosted samples.
11.9 Interferences
Samples interferences can inhibit the light made by luminescent bacteria.
Interfering substances
Chlorine
High oxygen consumption pH
Interference levels and treatments
Changes the viability of the bacterial reagent. Chlorine is toxic to the bacteria.
To remove chlorine from a sample, add one powder pillow of sodium thiosulfate (Hach 1436369 dechlorination agent) to 20 mL of sample and wait for 10 minutes.
Causes light inhibition that is not caused by toxicity pH-related light inhibition may occur if the pH is below 6.0 or above
8.0. The pH of the sample must be within 7 +/- 0.2 pH units of the standard.
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Interfering substances
Sodium chloride
Temperature
Turbidity and color
Interference levels and treatments
A sodium chlorine (NaCl) concentrations of less than 15 g/L or more than 50 g/L (or their osmolarity equivalents) in a sample will cause osmosis-related light inhibition.
The addition of solid NaCl to the sample (2% final concentration), prevents osmosis-related light inhibition of samples of low or unknown NaCl concentrations.
This biological test is strongly temperature-dependent.
ISO 11348 requires that the test is done under temperature controlled conditions at 15 °C using a appropriate thermostat (i.e.
LUMIStherm, LTV053).
Causes high-bias results due to physical absorption or scattering of light.
Use color correction cuvettes (accessories) in a separate test according to ISO 11348 or dilute the samples (i.e. 25% or 50%) before testing in the screening measure to remove the interference.
11.10 Prepare the sample
To prepare the sample for testing:
1.
If the sample is turbid, either:
• Filter the sample with a modified polysulfone filter
Before using other filter materials, test the filter material with 2% NaCl first to make sure that the filter material can be used with the Luminescent Bacteria
Toxicity Test. Check the acceptable filters in the ISO method.
Note: Do not use a cellulose nitrate or a cellulose acetate filter. The use of cellulose nitrate or cellulose acetate filters can cause light inhibition that is not caused by the sample.
• Let the sample sediment for 1 hour, or
• Centrifuge the sample (e.g., 10 minutes at 5.000 g)
2.
Check the pH level. Adjust the sample to pH 6 to 8 using HCl or NaOH. Use a strength of HCl or NaOH that does not change the volume of the sample by more than 5% in total.
3.
Add one spoon of solid NaCl (LCX058) and dissolve it in 7 mL of sample. The concentration of salt in the test should not exceed 35 g/L.
Note: Do not add NaCl to the sample if the salt concentration of the sample is more than 20 g/L (guide value: conductivity of 35 mS/cm).
Note: The salt content of the sample should not exceed 50 g/L. This corresponds to a conductivity of about 70 mS/cm without taking other conductive compounds in to account.
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Solid NaCl is used to change the sample osmolarity to a value that is correct for the marine bacterium used in the test.
4.
If the sample has a high toxicity, carry out a preliminary dilution of the sample with 2% NaCl solution. Select a preliminary dilution from the levels 1:2, 1:4, 1:8,
1:16, etc. to make sure of a continuous dilution series using the dilution procedure of the manufacturer.
11.11 Prepare the test tubes
At the end of this procedure, the test tubes contain the percent sample dilutions shown in
.
1 Test suspension
2 2% NaCl solution
Figure 3 Screening dilution series
3 Sample
1. Put four test tubes in the test tube stand.
2. Set the 0.2 - 1.0 mL pipette to 0.2 mL.
3. Put the end of the pipette in to a clean pipette tip.
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4. Put the tip of the pipette in to the test suspension and slowly pull in 0.2 mL.
5. Slowly dispense the test suspension in to the test tube in position 1.
until all four test tubes contain 0.2 mL of test suspension
7. Set the 0.2 - 1.0 mL pipette to 0.8 mL.
8. Put the tip of the pipette in to the 2% NaCl and slowly pull in 0.8 mL.
9. Slowly dispense the 2%
NaCl solution in to the test tube in position 1.
10. Set the 0.2 - 1.0 mL pipette to 0.6 mL.
11. Put the tip of the pipette in to the 2% NaCl and slowly pull in 0.6 mL.
12. Slowly dispense the 2%
NaCl solution in to the test tube in position 2.
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13. Set the 0.2 - 1.0 mL pipette to 0.3 mL.
14. Put the tip of the pipette in to the 2% NaCl and slowly pull in 0.3 mL.
15. Slowly dispense the 2%
NaCl solution in to the test tube in position 3.
Note: No 2% NaCl is put in the test tube in position 4.
16. Set the 0.2 - 1.0 mL pipette to 0.2 mL.
17. Set the timer clock for
15 minutes (contact time).
18. Put the tip of the pipette in to the sample and slowly pull in 0.2 mL.
19. Slowly dispense the sample in to the test tube in position 2. Start the timer.
Note: No sample is put in to the test tube in position 1. Test tube 1 is the non-toxic reference.
20. Set the 0.2 - 1.0 mL pipette to 0.5 mL.
21. Put the tip of the pipette in to the sample and slowly pull in 0.5 mL.
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22. Slowly dispense the sample in to the test tube in position 3.
23. Set the 0.2 - 1.0 mL pipette to 0.8 mL.
24. Put the tip of the pipette in to the sample and slowly pull in 0.8 mL.
25. Slowly dispense the sample in to the test tube in position 4.
26. Remove the pipette tip from the pipette and put in the waste bag.
Put the pipette in the storage case.
11.12 Measure the toxicity of the sample dilutions
The Luminescent Bacteria Toxicity Test is a biological test method and the result is therefore strongly temperature-dependent. Record the temperature at which the test was done. The results of tests done at different temperatures can not be compared directly.
A non-toxic reference is added to the test suspension during the test and measured. The reference measurement is used to compensate for changes in light levels from the luminescent bacteria. The light levels change with time.
In some instances, if reconstitution is done at the optimum temperature and the test is carried out at 20 °C, the initial light made by the bacteria can be more than 1000
Eclox light units. This causes the error Detector Overload. If an error occurs, change the measurement range from the 0–1000 range to the 0–2000 range and do the readings again (refer to
Set the measurement range on page 16
).
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To measure the toxicity of the sample dilutions:
1.
Push
ON
(green button) for several seconds to apply power to the luminometer.
When the built-in tests are done, push PROCEED. The Main Menu is shown.
2.
Select Luminescent Bacteria Test and push ENTER.
3.
Select Measure and push ENTER.
4.
To do the screening luminescence procedure:
a.
Select Screening Luminescence and push ENTER.
b. Select one option that is shown:
• To measure luminescence and save the results on the luminometer, select
Screen and Save and push ENTER.
• To measure luminescence and manually record the measuring values on paper, select Screen without Saving and push ENTER.
• To measure the luminescence and send the results to a PC, start LUMISsoft on the computer, start the test on LUMISsoft, and when Please select LSoft at the luminometer is shown, select Measure Luminescence and Send to PC and push ENTER. The luminometer must be connected to a computer (refer to
Connect the luminometer to a computer on page 21 ).
• To measure the luminescence and print the results on a printer, select
Screening and Send to Printer and push ENTER. The luminometer must be
connected to a printer (refer to Connect the luminometer to a printer on page 19 ).
5.
To do the LIMIT measure procedure:
a.
Select LIMIT Measure and push ENTER.
b. Select Set LIMIT Value and push ENTER.
c.
Push CHANGE to set the LIMIT value.
d. Push STORE to save the LIMIT value shown.
e.
Select one option that is shown:
• To measure luminescence and save the results on the luminometer, select
LIMIT Measure and Save and push ENTER.
• To measure luminescence and manually record the measuring values on paper, select LIMIT Measure without Saving and push ENTER.
• To measure the luminescence and send the results to a PC, start LUMISsoft on the computer, start the test on LUMISsoft, and when Please select LSoft at the
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Luminometer is shown, select LIMIT Measure and Send to PC and push
• To measure the luminescence and print the results on a printer, select LIMIT
Measure and Send to Printer and push ENTER. The luminometer must be connected to a printer (refer to
Connect the luminometer to a printer on page 19
).
6. Open the luminometer lid and make sure a sample is not in the cell. Close the lid.
7. Push PROCEED to show the test status. When the cell tests are done, push PROCEED again.
8. Wait until the timer clock completes 15 minutes.
9. Open the luminometer lid.
10. Put the test tube in position 1 (non-toxic blank) in to the black test tube holder in the luminometer cell.
11. Close the luminometer lid. Push MEASURE.
When the measurement is complete (approximately
15 seconds), the luminometer shows the relative light units measured.
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12. Open the lid of the luminometer.
Remove the test tube from the luminometer and put it back in the LUMIStherm.
13. Do steps 22 to 24 again to measure the three other test tubes in the correct order (2, 3, and then 4).
The Inhibit% and rel. units are shown on the screen.
Record the Inhibit% and rel. units values for each sample dilution on the
Screening Luminescence
Results Sheet.
14. Use the color chart on the Screening
Luminescence Results
Sheet to identify which sample dilutions are toxic
(red) and which are non-toxic (green).
Note: The more of your results in the red zone, the stronger are the inhibitory affects of the sample, the more critical is the sample.
15. Put the solution in the test tubes in to the waste bottle.
16. Put the test tubes in to the waste bag.
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Luminescent Bacteria Toxicity Test - Screening Luminescence Results
Sample: _______________________________________________________
Date: _______________ Time: _______________
Operator: ______________________________________________________
Comments:
2
3
4
Order 1st 2nd
% sample
3rd % inhib.
Rel. units
Tube
1
Test susp.
(mL)
0.2
2%
NaCl
(mL)
0.8
Sample
(mL) no
0.2
0.2
0.2
0.6
0.3
no
0.2
0.5
0.8
Sample
Conc.
Non-toxic reference
20%
50%
80%
Procedure:
1. Add 1.0 mL of reconstitution solution to the reagent. Swirl to mix. Wait 5 minutes.
2. Add 1.0 mL of stock suspension to 10 mL of
Diluent. Shake to mix. Wait
15 minutes.
3. Add one spoon of solid
NaCl to 7 mL of sample.
4. Fill four test tubes with the test suspension, 2% NaCl, and sample in the order shown in the table. Start the timer after adding the first sample volume. Set the timer for 15 minutes.
5. Push
ON
(green button) on the luminometer. Go to the
Screening Luminescence
Menu.
6. Measure test tube 1
(non-toxic reference).
7. Measure the sample tubes in the order 2, 3 and then 4.
Record the results in the table.
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11.13 Show or send previous results
To show previous results on the luminometer, do the procedure in this section for the type of procedure done.
To send previous results to a computer:
Note: At this stage, the results can not be sent to the LUMISsoft 4.
1.
Do the steps in
Connect the luminometer to a computer on page 21
.
2.
Start LUMISsoft.
3.
In LUMISsoft, select Transfer, Options, Interface Protocol, Connect.
4.
Do the procedure in this section for the type of procedure done.
and then do the procedure in this section for the type of procedure done.
11.13.1 Description of screening luminescence results
Reference (non-toxic) luminescent measurements are saved as R1 to Rx. The counter starts with R1 every time the storage is erased from the luminometer.
Sample luminescent measurements are done after a reference luminescent measurement is done. Sample luminescent measurements are saved as S1 to Sx.
The luminometer records reference and sample measurements and then calculates the percent inhibition value for each sample measurement (refer to
).
For example, two different screening luminescence tests have been done. One test with 3 samples or sample dilutions and one test with two samples or sample dilutions. The results of the first test are indicated as R1 with S1,S2 and S3. The results of the second tests are indicated as R2 with S1 and S2.
Figure 4 Example of screening luminescence results
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11.13.2 Description of LIMIT measure results
The LIMIT measure procedure results are recorded the same as the screening luminescence results. The only difference is that the LIMIT measure results include a column that shows whether the percentage inhibition calculated for each sample measurement is above the LIMIT value or below the LIMIT value set by the user as
Figure 5 Example LIMIT measure results
11.13.3 Show or send screening luminescence results
To show or send previous results saved on the luminometer for the screening luminescence procedure:
1.
Push ON to apply power to the luminometer.
The luminometer does built-in tests that make sure that the electronics and software are operating correctly.
2.
When the built-in tests are done, push PROCEED.
The Main Menu is shown.
3.
Select LUMINESCENT BACTERIA TEST and push ENTER.
The Luminescent Bacteria Test Main Menu is shown.
4.
Select Previous Results and push ENTER.
The Previous Results Menu is shown.
5.
Select Show Previous Screenings and push ENTER.
The Previous Screenings Menu is shown.
6.
To show all or send all of the results saved on the luminometer, select one option:
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• To show the results on the luminometer, select Show all (R1 to Rx) and push
ENTER.
• To send the results to the computer, select Send all (R1 and Rx) to PC and push
ENTER.
• To send the results to the printer, select Send all (R1 to Rx) to Printer and push
ENTER.
7.
To show or send a specific range of results saved on the luminometer, select one option:
• To show the results on the luminometer, select Show selection and push
ENTER.
• To send the results to the computer, select Send selection to PC and push
ENTER.
• To send the results to the printer, select Send selection to Printer and push
ENTER.
8.
If an option in step 7 was selected, select the data to be recalled:
a.
Select the starting indicator in the From field. Push SELECT to change the value. Then push PROCEED.
b. Select the ending indicator in the To field. Push SELECT to change the value. Then push SHOW.
9.
Push PROCEED to show more results.
11.13.4 Show or send LIMIT measure results
To show or send previous results saved on the luminometer for the LIMIT measure procedure:
1.
Push ON to apply power to the luminometer.
The luminometer does built-in tests that make sure that the electronics and software are operating correctly.
2.
When the built-in tests are done, push PROCEED.
The Main Menu is shown.
3.
Select LUMINESCENT BACTERIA TEST and push ENTER.
The Luminescent Bacteria Test Main Menu is shown.
4.
Select Previous Results and push ENTER.
The Previous Results Menu is shown.
5.
Select Show Previous LIMITs and push ENTER.
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The Previous LIMITs Menu is shown.
6.
To show all or send all of the results saved on the luminometer, select one option:
• To show the results on the luminometer, select Show all (R1 to Rx) and push
ENTER.
• To send the results to the computer, select Send all (R1 and Rx) to PC and push
ENTER.
• To send the results to the printer, select Send all (R1 to Rx) to Printer and push
ENTER.
7.
To show or send a specific range of results saved on the luminometer, select one option:
• To show the results on the luminometer, select Show selection and push
ENTER.
• To send the results to the computer, select Send selection to PC and push
ENTER.
• To send the results to the printer, select Send selection to Printer and push
ENTER.
8.
If an option in step 7 was selected, select the data to be recalled:
a.
Select the starting value in the From field. Push SELECT to change the starting value. Then push PROCEED.
b. Select the ending value in the To field. Push SELECT to change the ending value. Then push SHOW.
9.
Push PROCEED to show more results.
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