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2886888
Eclox
™
Rapid Response Test Kit
User Manual
01/2018, Edition 5
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Visit us at www.hach.com
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Table of Contents
Section 1 Specifications......................................................................................... 7
Section 2 General information............................................................................... 9
2.1 Safety information ............................................................................................... 9
2.1.1 Use of hazard information......................................................................... 9
2.2 Product overview............................................................................................... 10
2.2.1 Test descriptions ..................................................................................... 10
2.2.2 Test setup ............................................................................................... 12
2.3 Unpack the instrument ...................................................................................... 13
Section 3 Luminometer ........................................................................................ 15
3.1 Overview ........................................................................................................... 15
3.2 Prepare the luminometer for use....................................................................... 16
3.2.1 Test the operation ................................................................................... 16
3.2.2 Erase the results saved on the luminometer........................................... 17
3.2.3 Set the measurement range ................................................................... 18
3.3 Change the default settings .............................................................................. 20
3.3.1 Set the LCD contrast .............................................................................. 20
3.3.2 Set the waiting time and measuring time................................................ 20
3.4 Connect the luminometer to a printer................................................................ 21
3.5 Connect the luminometer to a computer........................................................... 23
3.6 Install LUMISsoft on the computer.................................................................... 23
Section 4 Chemiluminescence Toxicity Test...................................................... 25
4.1 Overview ........................................................................................................... 25
4.2 Prepare the reagents for luminometer calibration and sample testing.............. 25
4.2.1 Prepare CT Reagent 2............................................................................ 26
4.2.2 Prepare CT Reagent 3............................................................................ 26
4.3 Calibrate the luminometer................................................................................. 28
4.4 Measure pollutants in the water sample............................................................ 30
4.5 Show the previous results................................................................................. 33
4.6 Send previous results to a computer................................................................. 33
Section 5 EZ Arsenic Test Kit .............................................................................. 35
5.1 Test preparation ................................................................................................ 35
5.2 EZ Arsenic, 0–500 ppb (0, 10, 25, 50, 100, 250, 500) ...................................... 35
5.3 EZ Arsenic, 0–4000 ppb (0, 35, 75, 175, 1500, 4000) ..................................... 37
5.4 Interferences ..................................................................................................... 37
5.4.1 Optional procedure for removing sulfide ................................................. 38
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Section 6 Pesticide/Nerve Agent Test..................................................................39
6.1 Pesticide/Nerve Agent procedure ......................................................................39
Section 7 Free and Total Chlorine Test................................................................41
7.1 Test preparation .................................................................................................41
7.2 Free or total chlorine procedure, 0–3.4 mg/L Cl2 ..............................................41
Section 8 Color Test ..............................................................................................43
8.1 Test preparation .................................................................................................43
8.2 Color, apparent (0–100 APHA platinum cobalt color units) ...............................43
8.3 Color, apparent (0–500 APHA platinum cobalt color units) ...............................45
Section 9 Pocket Pro™+ Multi 2 Tester ...............................................................47
9.1 pH or TDS measurement...................................................................................47
9.2 pH calibration.....................................................................................................49
9.3 TDS/Conductivity calibration..............................................................................50
Section 10 Luminescent Bacteria Toxicity Test: Screening/LIMIT measure ....53
10.1 Overview..........................................................................................................53
10.2 Accuracy..........................................................................................................54
10.3 Reagent description.........................................................................................54
10.3.1 Quality assurance test...........................................................................54
10.4 Reagent storage and preservation ..................................................................55
10.5 Prepare the reagent.........................................................................................55
10.6 Prepare the stock suspension using the LCK491 reagent...............................55
10.7 Prepare the test suspension............................................................................58
10.8 Sample collection, storage and preservation...................................................59
10.9 Interferences....................................................................................................59
10.10 Prepare the sample .......................................................................................60
10.11 Prepare the test tubes....................................................................................61
10.12 Measure the toxicity of the sample dilutions ..................................................64
10.13 Show or send previous results.......................................................................69
10.13.1 Description of screening luminescence results ...................................69
10.13.2 Description of LIMIT measure results..................................................70
10.13.3 Show or send screening luminescence results ...................................70
10.13.4 Show or send LIMIT measure results..................................................71
Section 11 Luminescent Bacteria Toxicity Test ISO 11348 part 3 .....................73
11.1 Overview..........................................................................................................73
11.2 Accuracy ..........................................................................................................74
11.3 Thermostat and PC software requirements .....................................................74
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11.4 Reagent description ........................................................................................ 74
11.5 Reagent storage and preservation .................................................................. 75
11.6 Prepare the reagent ........................................................................................ 75
11.6.1 Prepare the stock suspension using the LCK491 reagent.................... 75
11.6.2 Prepare the test suspension ................................................................. 77
11.7 Sample collection, storage and preservation .................................................. 83
11.8 Interferences ................................................................................................... 83
11.9 Prepare the sample......................................................................................... 84
11.10 Prepare the dilutions series........................................................................... 85
11.10.1 Prepare a 9 dilution series (D 2 values and higher) ............................ 85
11.10.2 Prepare a 3 dilution series (D 2 values and higher) ............................ 89
11.10.3 Prepare a 9 dilution series (D 1 values and higher) ............................ 93
11.10.4 Prepare a 3 dilutions series (D 1 values and higher) .......................... 98
11.11 Measure the light intensity of the test suspension....................................... 101
11.12 Measure the light intensity of the test suspension after the sample dilutions are
added............................................................................................................. 106
11.13 Show or send previous results .................................................................... 108
Section 12 Maintenance ......................................................................................111
12.1 General maintenance.....................................................................................111
12.1.1 Cleaning the kit ....................................................................................111
12.1.2 Cleaning the luminometer....................................................................111
12.2 Decontamination ............................................................................................111
12.3 Battery replacement...................................................................................... 112
12.3.1 Luminometer battery replacement ...................................................... 112
Section 13 Troubleshooting............................................................................... 115
Section 14 Replacement parts and accessories .............................................. 117
Luminescent bacteria risks 121
Risk specifications ................................................................................................ 121
Biosafety Level 1 information
..................................................................................................... 121
Disposal ............................................................................................................... 122
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Section 1 Specifications
Specifications are subject to change without notice.
General
Dimensions 520 x 450 x 215 mm (20.5 x 17.5 x 8.5 in.)
Weight 9 kg (20 lb), fully loaded
Temperature Tested from –20 to 55 °C (–4 to 131 °F)
Modes
Chemical Tests
Certification CE marked
Chemiluminescence Toxicity Testing
Luminescent Bacteria Toxicity Testing
Arsenic, Chemiluminescence Toxicity, Chlorine, Color,
Nerve Agents, Pesticides, pH, Total Dissolved Solids
(Conductivity)
Luminometer—Water and chemical warfare agent resistant
Weight 1.4 kg (3.09 lb), including batteries
Dimensions 230 x 77 x 125 mm (9.1 x 3 x 4.92 in.)
Temperature Tested from –20 to 55 °C
Battery Type 4 AA alkaline, at least 250 test per set of batteries
Display Graphical LCD display with backlight for low light conditions
Up to 60 test results recorded in full detail
Up to 100 luminescent measurements for Luminescent
Data Logging
Download Capability RS232
Power
Bacteria Toxicity Test
Up to 100 screening results for Luminescent Bacteria
Toxicity Test
Battery powered alkaline cell, lithium cell, AA
CAUTION: For quality and safety reasons, only alkaline
batteries should be used with this instrument. Use of other
batteries may reduce the functioning of and/or damage the
instrument electronics by overloading the electronics, or,
depending on the battery type, can cause fire or an
explosion.
Warranty One year
Light detection for the
Luminescent Bacteria
Toxicity Test
Arsenic
Range 0 to 4 mg/L
Limit of detection 0.01 mg/L
Two decades in two different ranges:
20 to 1000 relative units (default mode)
20 to 2000 relative units
Precision: 2% coefficient of variation
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Chlorine (free)
Range 0 to 3.4 mg/L
Chlorine (total)
Range 0 to 3.4 mg/L
Color
Range 0 to 100, 0 to 500 APHA Platinum-Cobalt Color units
Pocket Pro™+ Multi 2 Tester
pH: 0.0 to 14.0 pH
Range
Accuracy TDS: ± 1%
Operating temperature 0 to 50 °C
Battery life 250 hours with backlight continuously on
Enclosure IP67 Rated, waterproof (immersible), dust proof
Warranty
TDS: Auto-ranging (0.0 to 99.9 ppm, 100 to 999 ppm, 1.00 to
10.00 ppt)
1 year for the tester and 6 months for replacement sensor
for manufacturing faults only. Damage from use is not
covered.
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Section 2 General information
In no event will the manufacturer be liable for direct, indirect, special, incidental or
consequential damages resulting from any defect or omission in this manual. The
manufacturer reserves the right to make changes in this manual and the products it
describes at any time, without notice or obligation. Revised editions are found on
the manufacturer’s website.
2.1 Safety information
Important Note: The manufacturer is not responsible for any damages due to misapplication
or misuse of this product including, without limitation, direct, incidental and consequential
damages, and disclaims such damages to the full extent permitted under applicable law. The
user is solely responsible to identify critical application risks and install appropriate
mechanisms to protect processes during a possible equipment malfunction.
Please read this entire manual before unpacking, setting up, or operating this
equipment. Pay attention to all danger and caution statements. Failure to do so
could result in serious injury to the operator or damage to the equipment.
Make sure that the protection provided by this equipment is not impaired. Do not use
or install this equipment in any manner other than that specified in this manual.
CAUTION: Chemical exposure hazard. Obey laboratory safety procedures and wear all
of the personal protective equipment appropriate to the chemicals that are handled.
Refer to the current safety data sheets (MSDS/SDS) for safety protocols.
2.1.1 Use of hazard information
DANGER
Indicates a potentially or imminently hazardous situation which, if not avoided, will
result in death or serious injury.
WAR NING
Indicates a potentially or imminently hazardous situation which, if not avoided, could
result in death or serious injury.
CAUTION
Indicates a potentially hazardous situation that may result in minor or moderate injury.
Important Note: Indicates a situation which, if not avoided, may cause damage to the
instrument. Information that requires special emphasis.
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2.1.2 Precautionary labels
Read all labels and tags attached to the instrument. Personal injury or damage to
the instrument could occur if not observed. A symbol on the instrument is
referenced in the manual with a precautionary statement.
This is the safety alert symbol. Obey all safety messages that follow this symbol
to avoid potential injury. If on the instrument, refer to the instruction manual for
operation or safety information.
Electrical equipment marked with this symbol may not be disposed of in
European domestic or public disposal systems. Return old or end-of-life
equipment to the manufacturer for disposal at no charge to the user.
This symbol indicates that a risk of electrical shock and/or electrocution exists.
This symbol indicates the need for protective eye wear.
2.2 Product overview
The Eclox™ Rapid Response Test Kit is used to do first line water testing. The kit is
a generic qualitative test that gives a broad indication of water quality. To do a proper
toxicity test, identify a baseline using the product in the waters to be tested.
The kit can be used to:
• Compare and prioritize possible source waters that might be used in a purification
process to make drinking water.
• Give information to an operator to help the operator identify the correct water
treatment process for the quality of the source water available.
• Be a regular quality assurance test on the drinking water made or the water given
for drinking and, if applicable, on the source water.
Other configurations are available, including the Eclox ‘toxicity only’ kit and the
luminometer by itself.
2.2.1 Test descriptions
Eight tests are included in the Eclox™ Rapid Response Test Kit:
• Chemiluminescence Toxicity Test—shows the toxicity of the water sample. This
test uses a plant enzyme, which creates light (chemiluminescence) when mixed
with other reagents. Pollutants in the water sample prevent this reaction, which
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reduces the amount of light that is created. The more pollutant that is in the
sample, the less light that is created. The light made by the sample water is
compared to a pure water reference and the percentage inhibition of the light
made is measured and made known.
• Arsenic Test—measures the arsenic content of the water sample. Arsenic is a
common poison and industrial pollutant. Arsenic is also in chemical warfare (CW)
agents, such as Lewisite. The result of this test can be read in mg/L from a
comparison chart.
• Pesticide/Nerve Agent Test—gives a YES/NO answer if pesticide/nerve agents
are in the water sample.
• Chlorine Test—measures how much free chlorine is in the water sample and
gives the results in mg/L. Systems using monochloramines may choose to
monitor total chlorine.
Chlorine is frequently used to disinfect water for human consumption. The
quantity of chlorine used must be carefully controlled and the free residual
concentration of the chlorine in the water gives a useful means of monitoring the
effectiveness of the water treatment done. Chlorinated water can, however, cause
damage to the Reverse Osmosis (RO) filter of water purification equipment and
should not be used as a source water in a RO type of process.
•Color Test—a comparison test that compares the water sample with a calibrated
gradient color disc. The results are read in platinum cobalt (Pt-Co) color units.
Color in water may be caused by the presence of natural metallic ions (iron and
manganese), peat materials, plankton, weeds and industrial wastes.
• Total Dissolved Solids (TDS) Test—measures the level of dissolved solids in the
water sample. TDS and the conductivity of a sample are related. The TDS is
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approximately 0.7 of the conductivity result (µS/cm
).
• pH Test—measures the pH level of the water sample.
• Luminescent Bacteria Toxicity Test (optional)—a biotest that measures the
toxicity of environmental samples. Toxicity is a biological or biochemical sum
parameter that cannot be measured by chemical analysis. Toxicity is a measure of
the effect of a sample on living organisms, biological systems and enzymes. Other
biotests such as fish, daphnia and algae tests are more complex and, because
other biotests use higher living organisms, are also controversial. In the practice
of environmental analysis, the Luminescent Bacteria Toxicity Test has shown to be
fast, simple, reliable and sensitive.
The Luminescent Bacteria Toxicity Test uses natural bacteria that make light.
Toxic samples decrease the amount of light the bacteria make. The more toxic the
sample, the less light the bacteria make. The amount of light that is made by the
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bacteria after exposure to a sample is compared to the amount of light made by
the bacteria after exposure to a control to identify the percent inhibition value of
the sample. The control contains no sample but a non-toxic reagent blank (2%
NaCl solution).
The reagent set is sold independently.
The Luminescent Bacteria Toxicity (LBT) Test can be done using either the:
• Measurement Luminescence procedure—used in the lab when a thorough
assessment of the inhibitory effects of a sample is necessary. Use the LBT
measurement luminescence procedure if the test needs to be done according to
ISO 11348.
• Screening Luminescence procedure—used in the field or in an emergency
situation when a rapid assessment of the inhibitory effects of a sample is
necessary. The LBT screening luminescence procedure is a simplified test
procedure that uses the same reagents according to ISO 11348 but at ambient
conditions.
• LIMIT measure procedure—the same as the screening luminescence
procedure. However, the LIMIT measure procedure lets the user set a LIMIT
value on the luminometer. The LIMIT value is used by the luminometer to
include in the test results whether the percent inhibition is above or below the
LIMIT value.
2.2.2 Test setup
There are three basic operations to be done when using the kit:
• Pre-deployment checks—complete before starting on a series of tests. Refer to
the Quick Start Guide (28878-88) on the lid of the case.
• Luminometer test—test the operation of the luminometer before
Chemiluminescence Toxicity Tests or Luminescent Bacteria Toxicity Tests are
done.
• Luminometer calibration—calibrate the luminometer each day before
Chemiluminescence Toxicity Tests are done.
• Measure samples—measure the samples with the tests.
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2.3 Unpack the instrument
Remove the Eclox Rapid Response Test Kit from the shipping carton and check it
for any visible damage. If any items are missing or damaged, contact the
manufacturer or a sales representative immediately.
• Carrying case • pH 4.01 SINGLET™
• Blue pipette, 1000 µL • pH 7 SINGLET™
• Yellow pipette, 100 µL • Waste bag with zipper
• Pocket Pro™+ Multi 2 Tester • Waste bottle, 250 mL
• Color comparator box • Beaker, 50 mL
• Color viewing tubes, plastic (2x) • Pesticide/Nerve Agent Test Strips
• Longpath viewing adapter • Pesticide test clip
• Color discs (2x) • Luminometer
• EZ Arsenic Reagent 1 Powder Pillows • Serial cable for luminometer
• EZ Arsenic Reagent 2 Powder Pillows • Batteries, AA, Alkaline
• EZ Arsenic Test Strips • Test record pad
• Arsenic reaction bottle with cap • Cuvette and pipette tip set, blue
• DPD Free Chlorine Powder Pillows • Cuvette and pipette tip set, yellow
• DPD Total Chlorine Powder Pillows • Cuvette holder
• Sample cell, plastic • LUMISsoft installation CD
• Sodium chloride, 85.47 mg/L (100 mL) • Chemiluminescence Reagent Set
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Section 3 Luminometer
3.1 Overview
The Eclox luminometer is used with the Chemiluminescence Toxicity Test and the
Luminescent Bacteria Toxicity Test to measure and record relative light units made
by the reagents when exposed to samples.
The Eclox luminometer is made for use under extreme field conditions. The Eclox
luminometer components are rugged, easy to use and reliable (refer to Figure 1).
Figure 1 Luminometer components
1. Lanyard 5. Non-slip feet
2. Battery compartment 6. Cell lid
3. Battery compartment screws 7. Function keys (Figure 2)
4. Label 8. Display
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Figure 2 Function keys
Item
number
1 Soft key Does the action on the display directly above the key.
2 Soft key Does the action on the display directly above the key.
3 Back light button Illuminates the display.
4 Off button Removes power to the instrument.
5 On button Applies power to the instrument.
Description Function
3.2 Prepare the luminometer for use
Do the procedures in this section before each deployment to prepare the
luminometer for use.
3.2.1 Test the operation
Do this procedure to make sure that the luminometer is operating correctly. If the
luminometer passes all the tests done in this procedure, it is operating correctly.
To test the operation of the luminometer:
1. Open the hinged cell lid of the luminometer and make sure that a sample is not
in the cell.
2. Remove the black test tube holder from the cell. Make sure that the cell is clean
and free from debris.
3. Put the black test tube holder into the cell and close the cell lid.
4. Push ON (green button) for several seconds to apply power to the instrument. If
the instrument does not energize, replace the batteries (refer to section 12.3 on
page 112).
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The instrument does built-in tests that make sure that the electronics and
software are operating correctly.
5. Make sure that all tests show a PASS on the display. If an error is shown on the
display, refer to Troubleshooting on page 115.
6. Push PROCEED.
The Main Menu is shown.
7. Select either ECLOX or Luminescent Bacteria Test and push ENTER. Either
option can be selected for this procedure.
8. Check the battery level symbol at the top right corner of the display. Make sure
that at least two level bars are shown. If two or more bars are not shown,
replace the batteries in the instrument and go back to step 4.
9. Select System Tests and push ENTER.
The System Tests Menu is shown.
10. Push ENTER to select Check Signal Level.
The Signal Level screen is shown.
11. Push PROCEED to do a cell zeroing test. When the instrument has passed the
test, the Signal Level screen is shown again.
12. Push and hold TEST. Make sure that the signal level shown is above the
minimum and below the maximum. If the signal level is not above the minimum,
contact the manufacturer for technical support.
13. Push and hold the back light button. Make sure that the instrument display light
comes on.
14. Push and hold QUIT for a few seconds.
The Systems Test Menu screen is shown.
15. Select Return to the ECLOX Main Menu (or LBT Main Menu) and push ENTER.
3.2.2 Erase the results saved on the luminometer
1. Push ON (green button) for several seconds to apply power to the instrument.
The instrument 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. To erase all the Chemiluminescence Toxicity Test measurements, select
ECLOX and push ENTER.
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4. To erase all the Luminescent Bacteria Test measurements, select Luminescent
Bacteria Test and push ENTER.
5. Select Set-Up and push ENTER.
The Set-Up Menu is shown.
6. Select Clear All Measurements and push ENTER. Push YES to confirm.
All saved measurements on the luminometer are erased.
7. Push PROCEED.
The Set-Up Menu is shown.
3.2.3 Set the measurement range
Set the luminometer measurement range to 0–1000 light units (normal use) or
0–2000 light units (measurement of sea water samples).
If the measuring value is marked with an * (e.g., 1020*) or the lumiometer shows
Detector Overload, the measurement is above the set measurement range. If this
occurs, change the measurement range to 0–2000 light units and do the reading
again.
Statistical research of each measurement range has shown that the standard
deviation of the 0–2000 range is less than the standard deviaton of the 0–1000
range and that the precision at the 0–2000 range may be better. In comparison
studies of each range, the phenol standardization check showed equal results to
the expected 50% inhibition range.
Non-polluted sea water samples “enhance” the signal (give a higher light inhibition
of approximately -40%). As the sea water becomes more polluted the percentage
inhibition increases (towards 0%) and then goes positive (e.g., 10%). Sea water
which is very polluted gives a signal similar to that of fresh water which is very
polluted (e.g., 70–100% light inhibition).
3.2.3.1 Eclox chemiluminescence test
To show or change the measurement range for the Eclox chemiluminescence test:
1. Push ON (green button) to apply power to the instrument.
The instrument 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 ECLOX and push ENTER.
The ECLOX Main Menu is shown.
4. Select Set-Up and push ENTER.
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The Set-up Menu is shown.
5. Select Set Measurement Range and push ENTER.
The current range is shown.
6. Push YES to confirm.
7. To change the range, push CHANGE
8. Push STORE to save the change.
The Set-up Menu is shown.
3.2.3.2 Luminescent Bacteria Test
If the measurement range is set to the 0–2000 light units and the measuring value
is marked with an * (e.g., 2010*) or the lumiometer shows Detector Overload, the
measurement is above the set measurement range. If this occurs, dilute the
bacterial stock suspension with Diluent and do the reading again.
To show or change the measurement range for the Luminescent Bacteria Test:
1. Push ON (green button) to apply power to the instrument.
The instrument 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 LBT Main Menu is shown.
4. Select Set-Up and push ENTER.
The LBT Set-Up Menu is shown.
5. Select Set Measurement Range and push ENTER.
The current range is shown.
6. Push YES to confirm.
7. To change the range, push CHANGE
8. Push STORE to save the change.
The Set-up Menu is shown.
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3.3 Change the default settings
3.3.1 Set the LCD contrast
The luminometer comes from the factory with the LCD contrast set correctly. Do this
procedure to increase the luminometer LCD contrast for low light conditions.
1. Push ON for several seconds to apply power to the instrument.
The instrument 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 ECLOX or Luminescent Bacteria Test and push ENTER. Either option
can be selected for this procedure.
4. Select Set-Up and push ENTER.
The Set-up Menu is shown.
5. Select Set Screen Contrast and push ENTER.
The Set Contrast screen is shown.
6. Push DOWN or UP to change the contrast. The screen shows the contrast level
with a Max/Min indicator bar.
7. Push DOWN and UP at the same time to save the changes.
The Set-up Menu is shown.
3.3.2 Set the waiting time and measuring time
This procedure only applies to the Luminescent Bacteria Test.
The measurement of the light intensity of luminescent bacteria is divided up into
two parts:
•Waiting time—the amount of time the luminometer waits (after the test tube is put
in, the lid is closed and MEASURE is pushed) before measuring the light intensity
from the test tube. The luminometer needs to wait a few seconds to compensate
for the high light level of the open lid.
• Measuring time—the amount of time the sample is measured by the
luminometer.
Note: There is no need to change the default settings of 8 seconds waiting time or 7 seconds
measuring time unless HACH or HACH-LANGE customer service asks the user to do so.
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To show or change the waiting time and measuring time:
1. Push ON (green button) to apply power to the instrument.
The instrument 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.
4. Select Set-Up and push ENTER.
The Set-up Menu is shown.
5. To show or change the waiting time:
a. Select Set Waiting Time and push ENTER.
The current settings are shown.
b. To change the waiting time, push CHANGE.
c. Push STORE to save the change.
The Set-up Menu is shown.
6. To show or change the measuring time:
a. Select Set Measuring Time and push ENTER.
The current settings are shown.
b. To change the measuring time, push CHANGE.
c. Push STORE to save the change.
The Set-up Menu is shown.
3.4 Connect the luminometer to a printer
Luminometer measurements can be sent to a printer either during the test or after
the test is done.
To connect the luminometer to a printer:
1. Pull out the plug that is attached to the lanyard.
2. If using a DPU-414 thermal printer, turn off the printer.
3. Connect the RS232 serial interface cable to the luminometer.
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4. Put an adapter (DB9, 3 wires, male-male, 2-3, 3-2, 5-5. Cross over, not straight)
on the other end of the RS232 serial interface cable.
5. Connect the RS232 serial interface cable to the printer.
6. If using a DPU-414 thermal printer:
a. Configure the printer (refer to the printer manual for more information):
DIP switch Switch Position Setting
1 Off Input = Serial
2 On Printing speed = High
3 On Auto loading = On
DIP SW1
DIP SW2
4 Off Auto LF = Off
5 On Setting command = Enable
6Off Printing
7 On Density
8 On = 100%
1 On Printing columns = 40
2 On User font backup = On
3 On Character select = Normal
4 On Zero = Normal
5 On International
6 On Character
7Off Set
8 Off = England
1 On Data length = 8 bits
2 On Parity settings = No
DIP SW3
b. When Continue? is shown, push ON-LINE SW.
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3 On Parity condition = Odd
4 Off Busy control = XON/XOFF
5Off Baud
6On Rate
7 On Select
8 On = 9600 bps
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c. When Write? is shown, push PAPER FEED SW.
d. Turn on the printer.
7. If not using a DPU-414 thermal printer, configure the printer:
Option Setting
Data length 8 bits
Parity setting No
Parity condition Odd
Busy control XON/XOFF
Baud rate 9600 bps
3.5 Connect the luminometer to a computer
To connect the luminometer to a computer:
1. Install the LUMISsoft software on the computer (refer to Install LUMISsoft on
the computer on page 23).
2. Pull out the plug that is attached to the lanyard.
3. Connect the RS232 serial interface cable to the luminometer.
4. Connect the other end of the RS232 serial interface cable to the computer.
3.6 Install LUMISsoft on the computer
Install LUMISsoft on a computer by doing the instructions on the CD cover. A
shortcut for LUMISsoft is added to the desktop during installation.
In the lab, LUMISsoft is used to automatically get LBT Measurement Luminescence
procedure results from the luminometer during the test and put the values into
LUMISsoft. LUMISsoft then does calculations according to ISO 11348.
LUMISsoft is also used to send previous results that are saved on the luminometer
to a computer as a text file. The results can then be shown in graphical and tabular
format using Microsoft Excel
on the luminometer into LUMISsoft to do calculations.
®
. The user can also manually put test results shown
23
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Section 4 Chemiluminescence Toxicity
Test
The Chemiluminescence Toxicity Test uses the luminometer. Before doing the
Chemiluminescence Toxicity Test, read section 3.1, Overview on page 15 and do
the procedures in section 3.2, Prepare the luminometer for use on page 16.
4.1 Overview
The Chemiluminescence Toxicity Test and Luminescent Bacteria Toxicity Test both
show the inhibitory effects of a sample. However, the Chemiluminescence Toxicity
Test reagents are more rugged than the Luminescent Bacteria Toxicity Tests
reagent and can be used under conditions where the Luminescent Bacteria Toxicity
Tests reagent cannot be used.
The Chemiluminescence Toxicity Test reagents are stable for months even if stored
under higher ambient temperatures up to 40 °C (Table 1). The Luminescent
Bacteria Toxicity Tests reagent cannot be stored under those conditions.
4.2 Prepare the reagents for luminometer calibration and
sample testing
Prepare the chemiluminescence test (CT) Reagents 2 and 3 at the beginning of
deployment.
The chemiluminescence test (CT) Reagents 2 and 3 are temperature sensitive and
degrade at high temperatures. For long-term storage, store the reagents in their
stable forms. On the first day of testing, prepare the reagents for routine use.
Diluted reagents are stable for 72 hours. The life of the reagents is longer if the
reagents are kept cool (e.g., in a refrigerator) and in a dark place. Before use, let
the reagents get to ambient temperature.
Table 1 Chemiluminescence test reagent stability
Reagent
Reagent 1 12 to 18 months 1 year
Reagent 2 (stable form) 12 to 18 months 6 months
Reagent 2 (diluted form) 12 to 18 months 72 hours
Reagent 3 (stable form) 12 to 18 months 4 months
Refrigerated in
a dark place
Raised Temperatures
(+40 °C)
Reagent 3 (diluted form) 12 to 18 months 72 hours
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4.2.1 Prepare CT Reagent 2
1. Remove the CT
Reagent 2 buffer and CT
Reagent 2 caps.
Note: Do not open the bottles
in heavy winds. The reagent in
the CT Reagent 2 is small.
Note: Do not touch the
reagent.
2. Carefully put all of the
CT Reagent 2 buffer into
the CT Reagent 2 bottle.
4.2.2 Prepare CT Reagent 3
3. Put the caps back on
the bottles. Shake the CT
Reagent 2 bottle for 30
seconds. Let dissolve for
10 minutes before use.
1. Remove the CT
Reagent 3 concentrate and
CT Reagent 3 caps.
Note: Make sure that the
batch number for CT Reagent
3 is the same as the batch
number used for CT Reagent
2.
26
2. Push the end of the pipet
into a clean 100 µL yellow
pipet tip and remove from
the box.
3. Push in the operating
button on the top of the
pipet to the stop.
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4. Put the tip in the CT
Reagent 3 concentrate
1 cm below the surface.
Slowly release the
operating button to pull in
the concentrate.
7. Put the cap on CT
Reagent 3.
5. Put the tip into CT
Reagent 3 and dispense
the liquid by gently pushing
in the operating button.
Put the tip into the liquid
and then remove from the
liquid.
6. Remove the tip from
the pipet and put in the
waste bag.
Put the pipet in the
storage case.
Turn over the CT Reagent
3 bottle several times to
mix the solution.
Note: An ice chest can be
used in the field to extend the
life of the reagent.
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4.3 Calibrate the luminometer
Calibrate the luminometer before doing the Chemiluminescence Toxicity Test and
after preparing the reagents.
The luminometer needs to be calibrated with every new batch of
chemiluminescence reagents.
To calibrate the luminometer:
1. Push ON (green button) 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 ECLOX and push ENTER.
The ECLOX Main Menu is shown.
4. Push ENTER to select Measure.
The Measure Menu is shown.
5. Select Measure Reference and push ENTER.
6. Open the luminometer lid and make sure a sample is not in the cell. Then close
the lid.
7. Push PROCEED.
The test status is shown. The test may go a few minutes before it is done.
8. When all the cell tests are done, push PROCEED.
9. Open the lid of the Cuvette and 1000 µL Pipette Tip Set.
10. Put one cuvette in the black cuvette holder.
11. Place a blue pipette tip on the blue pipette.
12. Completely push in the operating button on the pipette to the stop and put the
pipette tip in deionized water about 1 cm below the surface.
13. Release the operating button slowly to pull in the deionized water into the
pipette.
14. Touch the pipette tip against the side of the deionized water bottle to remove
any drops from the outside of the tip.
15. Place the pipette tip into the cuvette and dispense the liquid into the cuvette by
gently pushing in the operating button to the stop.
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16. Put the pipette tip into the cuvette and remove the pipette from the cuvette to
remove any drops from the outside of the tip.
17. Remove the lids from the CT Reagents 1, 2 and 3.
18. Put a yellow pipette tip on to the yellow pipette.
19. Put 100 µL of CT Reagent 1, 2 and 3 into the cuvette using the pipette. Use a
new pipette tip for each reagent.
20. Open the lid of the luminometer.
21. Lift the cuvette from the holder and gently tap the cuvette two times to mix the
solution.
22. Immediately put the cuvette into the luminometer cell and close the lid.
23. Push PROCEED.
The luminometer automatically starts measuring. After four minutes, the screen
count down timer displays DONE.
24. When the measurement is complete, remove the cuvette from the luminometer.
25. Put the solution in the cuvette into the waste bottle.
26. Put the cuvette into the waste bag.
27. If the reference is between 300 and 900, the calibration is complete.
28. If the reference is not between 300 and 900, push PROCEED and do the
calibration procedure again.
Note: New reagents may give a signal over 900. If the signal is 900 or over, change the
measurement range to the 0–2000 range. Do not throw away the reagent set. If the signal is
below 300, the reagents are probably unusable due to temperature sensitivity and new ones
are required.
Note: If the reagent baseline is reading over 1000 or the luminometer shows Detector
Overload, change the measurement range to the 0–2000 range and continue. There is no
need to throw away the chemiluminescent reagents.
29. If the signal is below 300 again, add another 100 µL of CT Reagent 3 to the
cuvette and do the calibration procedure again.
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4.4 Measure pollutants in the water sample
Start with fresh reference everyday and with each new reagent set.
If the measured light units for the reagents are over 900, change the measurement
range to the 0–2000 range and continue (refer to section 3.2.3, Set the
measurement range on page 18).
1. Fill the beaker with
50 mL of sample water.
2. If more than 0.4 mg/L
chlorine is present,
neutralize the sample by
adding two drops of
pre-conditioner reagent to
the sample beaker.
Note: Two drops of
pre-conditioner reagent can
neutralize up to 15 mg/L of
chlorine.
3. 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.
4. Select ECLOX and push
ENTER.
Select Measure and push
ENTER.
Select Measure Sample
and push ENTER.
30
5. Open the luminometer
lid and make sure that a
sample is not in the cell.
Close the lid.
6. Push PROCEED to
show the test status.
When the cell tests are
done, push PROCEED
again.
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7. Put one cuvette from the
Cuvettes and 1000 µL
Pipet Tip Set into the black
cuvette holder.
8. Put a blue pipette tip on
the blue pipet.
9. Push in the operating
button on the pipet to the
stop.
10. Put the tip in the
sample water 1 cm below
the surface. Slowly release
the operating button to pull
in the sample.
13. Put a yellow pipet tip on
the yellow pipet.
Use a new pipet tip for
each reagent.
11. Put the tip into the
cuvette and dispense the
liquid by gently pushing in
the operating button.
14. Do steps 9 to 12 to put
100 µL of CT Reagents 1, 2
and 3 into the cuvette.
12. Remove the tip from
the pipet and put in the
waste bag. Put the pipet in
the storage case.
15. Open the luminometer
lid. Remove the cuvette
from the cuvette holder.
Gently tap the cuvette twice
to mix the solution. Put the
cuvette in the luminometer
cell.
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16. Close the lid. Push
PROCEED.
The luminometer
automatically starts
measuring. After four
minutes, the screen timer
shows DONE.
17. The Inhib% is shown
on the screen. Record the
Inhib% value and graph on
the Test Record Sheet.
Note: For sea water samples,
the graph may appear higher
than the reference and the
Inhib% may be negative.
18. Push PROCEED to go
back to the Measure Menu.
19. Remove the cuvette
from the luminometer cell.
Put the solution into the
waste bottle. Put the
cuvette into the waste bag.
20. Sign the Test Record
Sheet.
Put the sample from the
beaker in the wastewater
drain using local operating
procedures.
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4.5 Show the previous results
Up to 60 previous results (samples plus references) and graphs can be saved on the
luminometer and then shown later on the luminometer.
To save previous results to a computer, refer to Send previous results to a
computer on page 33.
To show previous results saved on the luminometer:
1. Push ON (green button) for several seconds 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 ECLOX and push ENTER.
4. Select Previous Results and push ENTER.
The Previous Results Menu is shown.
5. To show previous results:
a. Select Recall Results and push ENTER.
b. Push MORE to show more results.
c. Push QUIT to go back to the Previous Results Menu.
6. To show previous graphs:
a. Select Recall Graphs and push ENTER.
b. Push UP to move through the saved samples.
c. Push SELECT when the required graph number is shown to select a
graph.
d. Push SELECT again on the last selected graph number to select multiple
graphs.
4.6 Send previous results to a computer
To send previous results to a computer:
1. Do the steps in Connect the luminometer to a computer on page 23.
2. Start LUMISsoft.
3. In LUMISsoft, select Transfer, Options, Interface Protocol, Connect.
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4. Push ON (green button) for several seconds to apply power to the luminometer.
The luminometer does built-in tests that make sure that the electronics and
software are operating correctly.
5. When the built-in tests are done, push PROCEED.
The Main Menu is shown.
6. Select ECLOX and push ENTER.
7. Select Previous Results and push ENTER.
The Previous Results Menu is shown.
8. Select Download to PC and push ENTER.
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Section 5 EZ Arsenic Test Kit
DANGER: Hydrogen and arsine gasses are generated during the test. Work in a
well-ventilated area away from open flames and other sources of ignition. Review
Material Safety Data Sheets for safe handling, storage and disposal information.
5.1 Test preparation
• For samples with sulfide greater than 15 ppb, follow Optional procedure for
removing sulfide on page 38 before performing the test.
• Do not expose reacted strips to direct sunlight. Reaction products are
photosensitive and may turn dark.
• Do not allow test strips to touch the reaction vessel solution. Test strips react with
gases, not solution.
• Orient the test strip pad paper side down and centered over the hole in the black
cap so the generated gases can make good contact with the pad.
• Two samples may be analyzed simultaneously with this kit.
5.2 EZ Arsenic, 0–500 ppb (0, 10, 25, 50, 100, 250, 500)
1. Insert a test strip into the
cap so the pad completely
covers the small opening.
Close the flap and press to
secure.
2. Fill the reaction bottle
with sample to the fill line
(50 mL).
3. Add one Reagent #1
and one Reagent #2
powder pillow to the
sample.
See Interferences on
page 37.
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4. Immediately attach the
cap to the reaction bottle.
Swirl continuously for 60
seconds.
Do not shake or invert or
allow sample to get on
the strip.
Wait 20 minutes. Swirl
twice during the reaction
period.
5. Remove the test strip
and immediately compare
the developed color to the
chart on the test strip bottle
(0–500 ppb row).
Read strips in the shade.
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5.3 EZ Arsenic, 0–4000 ppb (0, 35, 75, 175, 1500, 4000)
1. Insert a test strip into the
cap so the pad completely
covers the small opening.
Close the flap and press to
secure.
4. Immediately attach the
cap to the reaction bottle.
Swirl continuously for 60
seconds.
Do not shake or invert or
allow sample to get on
the strip.
Wait 20 minutes.
Swirl twice during the
reaction period.
2. Fill the square
measuring vial to the top
with sample (9.6 mL). Pour
the sample into the reaction
bottle.
5. Remove the test strip
and immediately compare
the developed color to the
chart on the test strip bottle
(0–4000 ppb row). Read
strips in the shade.
3. Add one Reagent #1
and one Reagent #2
powder pillow to the
sample.
See Interferences on
page 37.
5.4 Interferences
Ion or Substance Concentration
Sulfide >15 ppb
Selenium > 1 ppm
Table 2 Interfering substances
1
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Table 2 Interfering substances
Ion or Substance Concentration
Antimony > 250 ppb
Tellurium Likely to interfere, but not tested.
< pH 5. Do not acid-preserve samples. If
Acidity
Nitric acid
1
See section 5.4.1 on page 38 for information on removing sulfide.
samples are below pH 5, adjust pH to
between 5 and 6 before beginning test.
Interferes with the reduction step. Do not use
samples preserved with nitric acid because
low results will be observed. If samples must
be preserved, use HCl or sulfamic acid to
adjust sample to pH 2. Adjust to pH 5–7
before running the test.
Table 3 Non-interfering substances
Ion or substance Concentration
Hardness 1000 ppm as CaCO
Alkalinity 1000 ppm as CaCO
Iron 100 ppm
Temperature 10 to 40 °C (50 to 104 °F)
3
3
5.4.1 Optional procedure for removing sulfide
If a rotten egg smell is detected after adding reagent #1, sulfide is present at interfering levels.
Complete the following steps to remove the sulfide before beginning the test procedure:
1. Tear off a small piece of cotton and form a ball the size of a pea.
2. Saturate the cotton with a few drops of lead acetate. Squeeze the excess liquid out of the
cotton, leaving it damp.
3. Press the saturated cotton ball into the small opening of the reaction bottle cap from the
bottom. Be sure that the cotton is firmly in place and that a gap remains between the
cotton and the top surface of the cap.
4. Insert the test strip as detailed in step 1 of the 0–500 or 0–4000 ppb test procedure and
continue with the test.
Note: The lead acetate must not touch the test strip!
Always wear gloves or wash hands thoroughly after handling lead acetate.
Visit us at www.hach.com
Page 39
Section 6 Pesticide/Nerve Agent Test
6.1 Pesticide/Nerve Agent procedure
1. Remove one pesticide
strip from the storage case.
Open the foil packet on the
notched side. Remove the
contents. Keep the strip
and the foil, but put the
wadding in the bag.
4. Remove the pesticide
strip from the sample
beaker.
2. The pesticide strip has a
white disc at one end and a
larger pink disc at the other
end. Fold back the
protective film covering the
white disc only.
5. Remove the protective
film covering the pink disc.
Fold the strip in half along
the perforations and push
the white disc against the
pink disc.
3. Put the white
disc in the beaker that
contains the sample water
for at least one minute.
6. Put the strip in the
pesticide clip and put the
strip/clip back into the foil
packet. Keep the foil
packet warm by holding it
under the armpit (outside
of clothes) for three to
four minutes.
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7. Open the strip and look
at the color of the smaller
disc. For the best results,
hold the strip against
something white so the
color development is easier
to see.
8. There are two possible
results:
A white disc indicates
POSTIVE–pesticides or
nerve agent are present.
A blue disc (matching blue
or darker blue than larger
disc) indicates
NEGATIVE–no pesticide or
nerve agent present.
9. Do the test again if a
positive result is seen or
compare with a test from
a known clean water
sample.
10. Record the results.
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Section 7 Free and Total Chlorine Test
7.1 Test preparation
Important Note: This product has not been evaluated to test for chlorine and
chloramines in medical applications in the United States.
• Analyze samples immediately after collection.
• Put the color disc on the center pin in the color comparator box (numbers to the
front).
• Use sunlight or a lamp as a light source to find the color match with the color
comparator box.
• Rinse the tubes with sample before the test. Rinse the tubes with deionized water
after the test.
• If the color match is between two segments, use the value that is in the middle of
the two segments.
• If the color disc becomes wet internally, pull apart the flat plastic sides to open the
color disc. Remove the thin inner disc. Dry all parts with a soft cloth. Assemble
when fully dry.
• Undissolved reagent does not have an effect on test accuracy.
• For free chlorine, read the result immediately after the reagent is added to prevent
interference from monochloramine. If the sample contains 3.0 mg/L
monochloramine, the free chlorine result increases each minute by 0.1 mg/L.
7.2 Free or total chlorine procedure, 0–3.4 mg/L Cl
2
1. Fill a tube to the first line
(5 mL) with sample. This is
the blank.
2. Put the tube into the left
opening of the color
comparator box.
3. Fill another tube to the
first line (5-mL) with
sample.
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4. Add one DPD (Free or
Total) Chlorine Powder
Pillow to the second tube.
5. Swirl to mix. A pink color
develops.
For free chlorine, read the
result within 1 minute.
For total chlorine, wait 3
minutes. Read the result
within 6 minutes.
6. Put the second tube
into the color comparator
box.
7. Hold the color
comparator box in front of a
light source. Turn the color
disc to find the color match.
8. Read the result in mg/L
in the scale window.
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Section 8 Color Test
8.1 Test preparation
• Put the color disc on the center pin in the color comparator box (numbers to the
front).
• Use sunlight or a lamp as a light source to find the color match with the color
comparator box.
• Rinse the tubes with sample before the test. Rinse the tubes with deionized water
after the test.
• If the color match is between two segments, use the value that is in the middle of
the two segments.
• If the color disc becomes wet internally, pull apart the flat plastic sides to open the
color disc. Remove the thin inner disc. Dry all parts with a soft cloth. Assemble
when fully dry.
• The long-path adapter for the low range test shows the color in the tubes from top
to bottom. Make sure the light source is above the tubes during the color match.
8.2 Color, apparent (0–100 APHA platinum cobalt color units)
1. Install the longpath
adapter in the color
comparator box.
2. Fill a tube to the top line
with deionized water or
water that has no color.
3. Put the tube into left
opening of the color
comparator box.
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4. Fill a second tube to the
top line with sample.
Put the second tube into
the color comparator box.
5. Hold the color
comparator box below a
light source. Turn the color
disc to find the color match.
6. Read the results in
platinum cobalt color units
in the scale window.
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8.3 Color, apparent (0–500 APHA platinum cobalt color units)
1. If installed, remove the
longpath adapter.
4. Fill a second tube to the
first line (5-mL) with
sample. Put the second
tube into the color
comparator box.
2. Fill a tube to the first line
(5 mL) with deionized water
or water that has no color.
5. Hold the color
comparator box in front of a
light source. Turn the color
disc to find the color match.
3. Put the tube into the
left opening of the color
comparator box.
6. Read the value in the
scale window.
Multiply the value by 5 to
get the result in platinum
cobalt color units.
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Section 9 Pocket Pro™+ Multi 2 Tester
9.1 pH or TDS measurement
1. Push the
Power/Backlight key to set
the power to on. Remove
the sensor cap.
4. Add the sample to the fill
line of the sensor cap.
2. Rinse the sensor and the
sensor cap with deionized
water. Dry with a no-lint
cloth.
5. Put the sensor fully into
the sensor cap. The tester
reads the value.
Air bubbles under the
probe tip when submerged
can cause slow
stabilization or error in
measurement. Shake the
tester from side to side to
remove air bubbles.
3. Push and hold the
Lock/Parameter key to
select pH or TDS as the
measurement mode.
6. When the display is
stable, read the value.
To stabilize and keep the
value, push the
Lock/Parameter key.
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7. Push the
Power/Backlight key to set
the power to off.
8. Rinse the sensor and the
sensor cap with deionized
water. Dry with a no-lint
cloth.
9. For faster response
and longer test life, put
several drops of
deionized water in the
sensor cap. This keeps
the glass bulb from
becoming dry. Put the
sensor cap on the sensor.
Note: To extend electrode life, soak the electrode tip in tap water for a few minutes each week.
Note: Refer to the documentation supplied with the Pocket Pro+ Multi 2 Tester to do
conductivity, salinity and temperature procedures.
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9.2 pH calibration
1. Push the
Power/Backlight key to set
the power to on. Remove
the sensor cap.
4. Pour a pH 7.00 buffer to
the fill line of the sensor
cap.
2. Rinse the sensor and the
sensor cap with deionized
water. Dry with a no-lint
cloth.
5. Put the sensor fully into
the sensor cap. The tester
reads the buffer value.
Air bubbles under the
probe tip when submerged
can cause slow
stabilization or error in
measurement. Shake the
tester from side to side to
remove air bubbles.
3. Push and hold the
Lock/Parameter key until
“pH” shows as the
measurement mode.
6. Push the
Calibration/Settings key
to start the calibration.
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7. Wait for the value to
stabilize, then push the
Calibration/Settings key to
save the buffer value.
8. Optional: Do steps 2
through 7 againto measure
a pH 4.00 and/or a pH
10.00 buffer.
9.3 TDS/Conductivity calibration
9. Push and hold the
Calibration/Settings key
to exit.
1. Push the
Power/Backlight key to set
the power to on. Remove
the sensor cap.
50
2. Rinse the sensor and the
sensor cap with deionized
water. Dry with a no-lint
cloth.
3. Push and hold the
Lock/Parameter key until
“Cond” shows as the
measurement mode.
Page 51
4. Pour a 1413 µS/cm
conductivity standard to
the fill line on the sensor
cap.
5. Put the sensor fully into
the sensor cap. The tester
will read the standard
value.
Air bubbles under the
probe tip when submerged
can cause slow
stabilization or error in
measurement. Shake the
tester from side to side to
remove air bubbles.
6. Push the
Calibration/Settings key
to start the calibration.
7. Wait for the value to
stabilize, then push the
Calibration/Settings key to
keep the standard value.
8. The display shows
“END” when the calibration
is complete.
9. To measure a second
standard (147 µS/cm), do
step 2, then steps 4
through 7 again.
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Section 10 Luminescent Bacteria Toxicity
Test: Screening/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 15.
• Do the procedures in section 3.2, Prepare the luminometer for use on page 16.
• Print color copies of the Screening Luminescence Results Sheet to use in the field
(refer to page 68) 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.
10.1 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.
10.2 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).
10.3 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 121 for bacteria
risk information.
10.3.1 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.
10.4 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.
10.5 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.
10.6 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.
4. Remove the foil seal and
rubber stopper from the
reagent bottle.
2. Put the frozen
luminescent bacteria
reagent, refrigerated
reconstitution solution and
Diluent in a cool box that
contains thermal packs if
possible.
5. Set the 1.0-5.0 mL
pipette to 1.0 mL.
3. In the field, remove the
cap from the reconstitution
solution bottle.
6. Put the end of the
1.0-5.0 mL pipette into a
clean pipette tip.
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7. Put the tip of the pipette
into the reconstitution
solution and slowly pull in
1.0 mL.
10. Cool the reagent for 5
minutes in the cool box.
8. Put the tip of the pipette
into the luminescent
bacteria reagent bottle and
slowly dispense the
solution into the reagent.
9. Put the rubber stopper in
the reagent bottle. Swirl the
reagent bottle to mix.
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10.7 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.
4. Set the 1.0-5.0 mL
pipette to 1.0 mL.
2. Put 14.0 mL of Diluent at
refrigerator temperature in
the reaction vessel using
the pipette.
5. Put 1 mL of stock
suspension at refrigerator
temperature into a clean
reaction vessel using the
pipette.
3. Remove the stock
suspension (rehydrated
reagent) from the cool box.
Remove the rubber
stopper from the reagent
bottle.
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.
10.8 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.
10.9 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
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.
Interference levels and treatments
10.10 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% intotal.
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 into 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.
10.11 Prepare the test tubes
At the end of this procedure, the test tubes contain the percent sample dilutions
shown in Figure 3.
Figure 3 Screening dilution series
1 Test suspension 3 Sample
2 2% NaCl solution
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
into a clean pipette tip.
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4. Put the tip of the pipette
into the test suspension
and slowly pull in 0.2 mL.
5. Slowly dispense the test
suspension into the test
tube in position 1.
6. Do steps 4 and 5 again
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.
10. Set the 0.2 - 1.0 mL
pipette to 0.6 mL.
8. Put the tip of the pipette
into the 2% NaCl and
slowly pull in 0.8 mL.
11. Put the tip of the pipette
into the 2% NaCl and
slowly pull in 0.6 mL.
9. Slowly dispense the 2%
NaCl solution into the test
tube in position 1.
12. Slowly dispense the 2%
NaCl solution into 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
into the 2% NaCl and
slowly pull in 0.3 mL.
15. Slowly dispense the 2%
NaCl solution into 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.
19. Slowly dispense the
sample into the test tube in
position 2. Start the timer.
Note: No sample is put into
the test tube in position 1. Test
tube 1 is the non-toxic
reference.
17. Set the timer clock for
15 minutes (contact time).
20. Set the 0.2 - 1.0 mL
pipette to 0.5 mL.
18. Put the tip of the pipette
into the sample and slowly
pull in 0.2 mL.
21. Put the tip of the pipette
into the sample and slowly
pull in 0.5 mL.
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22. Slowly dispense the
sample into 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
into the sample and slowly
pull in 0.8 mL.
25. Slowly dispense the
sample into 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.
10.12 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 cannot 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 18).
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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 23).
• 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 21).
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
ENTER. The luminometer must be connected to a computer (refer to Connect
the luminometer to a computer on page 23).
• 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 21).
6. Open the luminometer
lid and make sure a sample
is not in the cell. Close the
lid.
9. Open the luminometer
lid.
7. Push PROCEED to
show the test status. When
the cell tests are done,
push PROCEED again.
10. Put the test tube in
position 1 (non-toxic blank)
into the black test tube
holder in the luminometer
cell.
8. Wait until the timer clock
completes 15 minutes.
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 into the waste
bottle.
16. Put the test tubes into
the waste bag.
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Luminescent Bacteria Toxicity Test - Screening Luminescence Results
% sample
% inhibition
Sample: _______________________________________________________
Date: _______________ Time: _______________
Operator: ______________________________________________________
Comments:
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.
Order 1st 2nd 3rd %
Tube Test
susp.
(mL)
10.2 0.8no
2 0.2 0.6 0.2 20%
3 0.2 0.3 0.5 50%
4 0.2 no 0.8 80%
2%
NaCl
(mL)
Sample
(mL)
inhib.
Rel.
units
Non-toxic
reference
Sample
Conc.
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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10.13 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 cannot be sent to the LUMISsoft 4.
1. Do the steps in Connect the luminometer to a computer on page 23.
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.
To send previous results to a printer, do the steps in Connect the luminometer to a
printer on page 21 and then do the procedure in this section for the type of
procedure done.
10.13.1 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 Figure 4).
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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10.13.2 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
shown in Figure 5.
Figure 5 Example LIMIT measure results
10.13.3 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.
10.13.4 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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Section 11 Luminescent Bacteria Toxicity
Test ISO 11348 part 3
The Luminescent Bacteria Toxicity (LBT) Test uses the luminometer. Before doing
the procedure, read section 3.1, Overview on page 15 and do the procedures in
section 3.2, Prepare the luminometer for use on page 16.
This chapter describes the LBT Test measurement luminescence procedure and
contains the procedure steps. Use the LBT Test measurement luminescence
procedure if the test needs to be done according to ISO 11348 part 3. The Eclox
LBT Test measurement luminescence procedure meets the criteria of validation of
ISO 11348-3.
11.1 Overview
The test criterion is luminescence which is measured after a contact time of 15 or
30 minutes (optionally 5 minutes at 15 °C) taking into account a correction factor
(fK). The correction factor is a measure of intensity change of control samples
during the exposure time (refer to the ISO standard procedure).
The luminometer measurements are in relative light units. The luminometer
measurements are used by the LUMISsoft computer program or custom made
calculations to calculate percent inhibition, LID, EC20 and EC50 values.
• Percent inhibition—the percentage of light made by the bacteria that is inhibited
by the sample. The higher the percentage inhibition of the light emission, the more
harmful the sample is to the bacteria and the higher the toxicity level of the
sample.
•LID—first dilution value of a sample that causes less than 20% inhibition. The
higher the LID, the more harmful the sample is to the bacteria.
•EC20 or EC50—the concentration of a sample that causes exactly 20 or 50%
inhibition. The lower the EC-value, the more harmful the sample is to the bacteria.
The linear measuring range is between 10% and 90% inhibition. Refer to ISO
11348 for more detailed information on the Luminescent Bacteria Toxicity Test.
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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 simplified screening test
under field conditions.
The total error for the test is typically lower than 20%.
11.3 Thermostat and PC software requirements
ISO 11348 states that the measuring luminometer must have a 15 °C temperature
controlled measuring well. The Eclox does not have a temperature controlled
measuring cell.
According to ISO 11348 optional accessories that should be used in the lab include:
• LTV053 LUMIStherm, 230V, thermostat to 15 °C
• LZV093 LUMISsoft 4 PC software
11.4 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 121 for bacteria
risk information.
The standards stipulate that certain validity criteria must be complied with for the
reagent. Accordingly, a test is done for each batch of bacteria that is prepared
in-house or bought 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, do control measurements with
the standard solutions (refer to the ISO standard procedure). The necessary
information about standard substances, test concentrations and sources of supply is
contained in the quality certificate that comes with every box of luminescent bacteria
reagent.
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11.5 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.
11.6 Prepare the reagent
Prepare the Luminescent Bacteria Toxicity Test reagent not more than 4 hours
before testing according to ISO 11348 as done 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.
11.6.1 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 stock suspension can be kept in a refrigerator (+8 °C) without being diluted
with Diluent as long as the validity criteria are met. Typically up to 4 hours. The
sensitivity spectrum of reactivated bacteria may shift as time elapses.
If the reagent is to be used 90 minutes or more after reconstitution, periodically
monitor the performance of the reagent with a suitable standard to show changes in
sensitivity.
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This procedure is temperature sensitive.
1. Remove the
luminescent bacteria test
reagent from the freezer.
Remove the reconstitution
solution from refrigerator.
4. Set the 1.0-5.0 mL
pipette to 1.0 mL.
2. Remove the cap from
the reconstitution solution
bottle.
5. Put the end of the pipette
into a clean pipette tip.
3. Remove the foil seal and
rubber stopper from the
reagent bottle.
6. Put the tip of the pipette
into the reconstitution
solution and slowly pull in
1.0 mL.
7. Put the tip of the pipette
into the luminescent
bacteria reagent bottle.
Quickly dispense the
solution into the reagent.
76
8. Put the rubber stopper in
the reagent bottle. Swirl the
reagent bottle to mix.
9. Cool the sample for at
least 15 minutes in a
refrigerator.
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11.6.2 Prepare the test suspension
Prepare enough test suspension (stock suspension and Diluent mixture) to do the
test. Each test tube used for the test is filled with 0.5 mL of test suspension. To
identify the number of test tubes used for a test:
• For D 2 values an higher, add 1 to the number of sample dilution steps to be
measured (e.g. 1 blank + 9 dilutions = 10). Then multiply that number by 2.
• For D 1 values and higher, add 2 to the number of sample dilution steps to be
measured (2 blanks + 3 dilutions = 5). Then multiply that number by 2.
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.
11.6.2.1 Test suspension for D 2 values and higher
Prepare the test suspension for D2 values and higher if the sample is expected to be
toxic.
1. Remove the Diluent
from the cool box.
Remove the cap from the
Diluent bottle.
2. Put 50 parts Diluent
solution (D) at refrigerator
temperature into the
reaction vessel using a
pipette.
3. Remove the stock
suspension (rehydrated
reagent) from the cool box.
Remove the rubber
stopper from the reagent
bottle.
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4. Put 1 part stock
suspension (S) at
refrigerator temperature
into a clean reaction vessel
using a pipette.
5. Put the cap on the
reaction vessel and shake
to mix thoroughly.
6. Put one half of the new,
empty test tubes in Row B
and one half of the test
tubes in Row C of the
LUMIStherm.
For example:
0.2 mL S + 10 mL D
7. Set the 0.2 - 1 mL pipette
to 0.5 mL.
8. Put the end of the
pipette into a clean pipette
tip.
Note: The LUMIStherm
should be operating at 15 °C.
9. Put the tip of the pipette
into the reaction vessel and
slowly pull in 0.5 mL of the
test suspension.
10. Slowly dispense the
test suspension into the
test tube in position B1.
78
11. Do step 9 and 10 again
until each test tube in Row
B and Row C contains
0.5 mL of test suspension.
12. Cool the filled test
tubes in the LUMIStherm at
15 °C for 15 minutes.
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13. Remove the pipette tips
from the pipettes and put in
the waste bag.
Put the pipettes in the
storage case.
11.6.2.2 Test suspension for D 1 values
Prepare the test suspension for D1 values if the sample is probably non-toxic to
measure the sample toxicity using the highest possible sample concentration of 80%
(= D 1).
1. Remove the Diluent
from the refrigerator.
Remove the cap from the
bottle.
2. Put 20 parts Diluent
solution (D) at refrigerator
temperature into the
reaction vessel using a
pipette.
3. Remove the stock
solution (rehydrated
reagent) from the
refrigerator.
Remove the rubber
stopper from the reagent
bottle.
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4. Put 1 part stock
suspension (S) at
refrigerator temperature
into a clean reaction vessel
using a pipette.
For example:
0.1 mL S + 2.0 mL D
5. Put the cap on the
reaction vessel and shake
to mix thoroughly.
Put a “1:20” label on the
reaction vessel.
6. Put 50 parts Diluent
solution (D) at refrigerator
temperature into a clean
reaction vessel using a
pipette.
7. Put 1 part stock
suspension (S) at
refrigerator temperature
into a clean reaction vessel
using a pipette.
For example:
0.2 mL S + 10 mL D
8. Put the cap on the
reaction vessel and shake
to mix thoroughly.
Put a “1:50” label on the
reaction vessel.
9. Put one half of the new,
empty test tubes in Row B
and one half of the test
tubes in Row C of the
LUMIStherm.
Note: The LUMIStherm
should be operating at 15 °C.
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10. Set the 0.2 - 1 mL
pipette to 0.2 mL.
11. Put the end of the
pipette into a clean pipette
tip.
12. Put the tip of the
pipette into the reaction
vessel that contains the
1:20 test suspension and
slowly pull in 0.2 mL of the
test suspension.
13. Slowly dispense the
test suspension into the
test tube in position B1.
14. Do step 12 and 13
again until the test tubes in
position C1, B2 and C2
contain 0.2 mL of test
suspension.
15. Set the 0.2 - 1 mL
pipette to 0.5 mL.
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16. Put the tip of the
pipette into the reaction
vessel that contains the
1:50 test suspension and
slowly pull in 0.5 mL of the
test suspension.
17. Slowly dispense the
test suspension into the
test tube in position B3.
18. Do step 16 and 17
again until each test tube in
Row B and Row C (position
3 and higher) contains 0.5
mL of test suspension.
19. Cool the filled test
tubes in the LUMIStherm at
15 °C for 15 minutes.
20. Remove the pipette tips
from the pipettes and put in
the waste bag.
Put the pipettes in the
storage case.
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11.7 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.
11.8 Interferences
Samples interferences can inhibit the light made by luminescent bacteria.
Interfering
substances
Chlorine
High oxygen
consumption
pH
Sodium chloride
Temperature
Interference levels and treatments
Affects 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.
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).
Turbidity and color
Cause 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.
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11.9 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% intotal.
3. Add solid NaCl to the sample until the concentration in the sample is 2% (w/v).
For example, weigh out 0.3 g of NaCl and dissolve it in 15 mL of sample or
dissolve one spoon of solid NaCl (LCX058) 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 into account.
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.
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11.10 Prepare the dilutions series
Prepare the sample dilutions series using one of the procedures in this section.
The sample dilutions are added to the test suspension later to identify the percent
inhibition of each sample dilution.
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.
11.10.1 Prepare a 9 dilution series (D 2 values and higher)
To make a 9 dilution series according to ISO 11348 of D 2 sample values and higher,
do this procedure.
This procedure makes dilutions ranging from undiluted to a dilution ratio of 1:16 in
Row A. This corresponds to D values of 2 to 32 in the test (Figure 6), as 0.5 mL of
the sample dilution is added to 0.5 mL of the test suspension during the test in Row
B and C. Adding test suspension to the sample dilutions increases the sample
dilutions in row A by a factor of two as final test concentration.
Note: The test tubes in the higher Row A positions are more concentrated. The pipette is
moved from A9 to A2 (higher concentration to lower concentration) when making the dilution
series, so the pipette tip does not need to be replaced during this procedure.
Figure 6 Dilution series - 9 dilutions, D 2 values and higher
Tube Contents
A1 2% NaCl (1.5 mL)
A2, A3 2% NaCl and sample (3.0 mL)
A4 - A9 2% NaCl and sample (1.5 mL)
A10 Sample (1.5 mL)
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1. Put 10 empty test tubes
into Row A of the
LUMIStherm.
2. Set the 1.0 - 5.0 mL
pipette to 1.0 mL.
3. Put the end of the pipette
into a clean pipette tip.
4. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.0 mL.
6. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.5 mL.
Slowly dispense the 2%
NaCl solution into the test
tube in position A9.
7. Slowly dispense the 2%
NaCl solution into the test
tube in position A8.
5. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
8. Do steps 6 and 7 againto
put 1.5 mL of 2% NaCl
solution in each test tube in
positions A7, A6, A5, A4,
A3, A2 and A1.
Note: Do not put 2% NaCl into
the test tube in position A10.
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9. Set the 1.0 - 5.0 mL
pipette to 2.0 mL.
10. Put the tip of the pipette
into the sample and slowly
pull in 2.0 mL.
11. Slowly dispense the
sample into the test tube in
position A9.
12. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
15. Do steps 13 and 14
againto put 1.5 mL of the
sample into the test tube in
position A10.
Figure 7 on page 89
shows the contents of the
test tubes in Row A after
this step is completed.
13. Put the tip of the pipette
into the sample and slowly
pull in 1.5 mL.
16. Pull the solution in A9
into the pipette 2 to 3 times
to mix the sample dilution.
14. Slowly dispense the
sample into the test tube in
position A8.
17. Start making the
sample dilution series in
Row A:
Pull in 1.5 mL of solution
from A9 and put it into A7
using the pipette.
Pull the solution in A7 into
the pipette 2 to 3 times to
mix the sample dilution.
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18. Pull in 1.5 mL of
solution from A7 and put it
into A5 using the pipette.
19. Pull in 1.5 mL of
solution from A5 and put it
into A3 using the pipette.
20. Pull the solution in A8
into the pipette 2 to 3 times
to mix the sample dilution.
Pull the solution in A5 into
the pipette 2 to 3 times to
mix the sample dilution.
21. Pull in 1.5 mL of
solution from A8 and put it
into A6 using the pipette.
Pull the solution in A6 into
the pipette 2 to 3 times to
mix the sample dilution.
Pull the solution in A3 into
the pipette 2 to 3 times to
mix the sample dilution.
22. Pull in 1.5 mL of
solution from A6 and put it
into A4 using the pipette.
Pull the solution in A4 into
the pipette 2 to 3 times to
mix the sample dilution.
23. Pull in 1.5 mL of
solution from A4 and put it
into A2 using the pipette.
Pull the solution in A2 into
the pipette 2 to 3 times to
mix the sample dilution.
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24. Keep the dilution series
at 15 °C for at least 5
minutes to correct the
temperature.
Figure 7 Before the dilution series is started - 9 dilutions, D 2 and higher
1 Sample 2 2% NaCl solution
11.10.2 Prepare a 3 dilution series (D 2 values and higher)
To make a 3 dilution series according to ISO 11348 of D 2 sample values and higher,
do this procedure.
This procedure makes dilutions ranging from undiluted to a dilution ratio of 1:2 in
row A. This corresponds to D values of 2 to 4 (Figure 8) in the test, as 0.5 mL of the
sample dilution is added to 0.5 mL of the test suspension during the test in Row B
and C. Adding test suspension to the sample dilutions increases the sample
dilutions in row A by a factor of two as final test concentration.
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Figure 8 shows the contents of the test tubes in Row A at the end of this procedure
Figure 8 Dilution series - 3 dilutions, D 2 values and higher
1 Sample 2 2% NaCl solution
1. Put 4 empty test tubes
into Row A of the
LUMIStherm.
4. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.0 mL.
2. Set the 1.0 - 5.0 mL
pipette to 1.0 mL.
5. Slowly dispense the 2%
NaCl solution into the test
tube in position A3.
3. Put the end of the pipette
into a clean pipette tip.
6. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
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7. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.5 mL.
8. Slowly dispense the 2%
NaCl solution into the test
tube in position A2.
9. Do steps 7 and 8 againto
put 1.5 mL of 2% NaCl
solution into the test tube in
position A1.
10. Set the 1.0 - 5.0 mL
pipette to 2.0 mL.
13. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
11. Put the tip of the pipette
into the sample and slowly
pull in 2.0 mL.
14. Put the tip of the pipette
into the sample and slowly
pull in 1.5 mL.
12. Slowly dispense the
sample into the test tube in
position A3.
15. Slowly dispense the
sample into the test tube in
position A4.
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16. Do steps 14 and 15
againto put 1.5 mL of the
sample into the test tube in
position A2.
17. Keep the dilution series
at 15 °C for at least 5
minutes to correct the
temperature.
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11.10.3 Prepare a 9 dilution series (D 1 values and higher)
To make a 9 dilution series according to ISO 11348 of D 1 sample values and higher,
do this procedure.
This procedure makes dilutions ranging from undiluted to a dilution ratio of 1:8 in
Row A. This corresponds to D values of 1 to 16 (Figure 9) in the test, as 0.5 mL of
the sample dilution is added to 0.5 mL of the test suspension during the test in Row
B and C. Adding test suspension to the sample dilutions increases the sample
dilutions in row A by a factor of two as final test concentration.
Note: The test tubes in the higher Row A positions are more concentrated. The pipette is
moved from A9 to A4 (higher concentration to lower concentration) when making the dilution
series, so the pipette tip does not need to be replaced during this procedure.
Figure 9 Dilution series - 9 dilutions, D 1 values and higher
Tube Contents
A1 2% NaCl (2.0 mL)
A2 Sample (2.0 mL)
A3 2% NaCl (1.5 mL)
A4, A5 2% NaCl and sample (3.0 mL)
A6 - A9 2% NaCl and sample (1.5 mL)
A10 Sample (1.5 mL)
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1. Put 10 empty test tubes
into Row A of the
LUMIStherm.
2. Set the 1.0 - 5.0 mL
pipette to 1.0 mL.
3. Put the end of the pipette
into a clean pipette tip.
4. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.0 mL.
7. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 2.0 mL.
5. Slowly dispense the 2%
NaCl solution into the test
tube in position A9.
8. Slowly dispense the 2%
NaCl solution into the test
tube in position A1.
6. Set the 1.0 - 5.0 mL
pipette to 2.0 mL.
9. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
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10. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.5 mL.
11. Slowly dispense the 2%
NaCl solution into the test
tube in position A8.
12. Do steps 10 and 11
againto put 1.5 mL of 2%
NaCl solution in each test
tube in positions A7, A6,
A5, A4 and A3 .
13. Set the 1.0 - 5.0 mL
pipette to 2.0 mL.
16. Put the tip of the pipette
into the sample and slowly
pull in 2.0 mL.
14. Put the tip of the pipette
into the sample and slowly
pull in 2.0 mL.
17. Slowly dispense the
sample into the test tube in
position A2.
15. Slowly dispense the
sample into the test tube in
position A9.
18. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
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19. Put the tip of the pipette
into the sample and slowly
pull in 1.5 mL.
20. Slowly dispense the
sample into the test tube in
position A8.
21. Do steps 19 and 20
againto put 1.5 mL of the
sample into the test tube in
position A10.
Figure 10 on page 97
shows the contents of the
test tubes in Row A after
this step is completed.
22. Pull the solution in A9
into the pipette 2 to 3 times
to mix the sample dilution.
23. Start making the
sample dilution series in
Row A:
Pull in 1.5 mL of solution
from A9 and put it into A7
using the pipette.
Pull in the solution in A7
into the pipette 2 to 3 times
to mix the sample dilution.
24. Pull in 1.5 mL of
solution from A7 and put it
into A5 using the pipette.
Pull in the solution in A5
into the pipette 2 to 3 times
to mix the sample dilution.
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25. Pull the solution in A8
into the pipette 2 to 3 times
to mix the sample dilution.
26. Pull in 1.5 mL of
solution from A8 and put it
into A6 using the pipette.
27. Pull in 1.5 mL of
solution from A6 and put it
into A4 using the pipette.
28. Keep the dilution series
at 15 °C for at least 5
minutes to correct the
temperature.
Pull in the solution in A6
into the pipette 2 to 3 times
to mix the sample dilution.
Pull in the solution in A4
into the pipette 2 to 3 times
to mix the sample dilution.
Figure 10 Before the dilution series is started - 9 dilutions, D 1 and
1 Sample 2 2% NaCl solution
higher
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11.10.4 Prepare a 3 dilutions series (D 1 values and higher)
To make a 3 dilution series according to ISO 11348 of D 1 sample values and higher,
do this procedure.
This procedure makes dilutions ranging from undiluted to a dilution ratio of 1:1.5 in
Row A. This corresponds to D values of 2 to 3 (Figure 11) in the test, as 0.5 mL of
the sample dilution is added to 0.5 mL of the test suspension during the test in Row
B and C. Adding test suspension to the sample dilutions increases the sample
dilutions in row A by a factor of two as final test concentration.
Figure 11 shows the contents of the test tubes in Row A at the end of this
procedure
Figure 11 Dilution series - 3 dilutions, D 1 values and higher
1 Sample 2 2% NaCl solution
1. Put 5 empty test tubes
into Row A of the
LUMIStherm.
2. Set the 1.0 - 5.0 mL
pipette to 1.0 mL.
3. Put the end of the pipette
into a clean pipette tip.
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4. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.0 mL.
5. Slowly dispense the 2%
NaCl solution into the test
tube in position A4.
6. Set the 1.0 - 5.0 mL
pipette to 1.5 mL.
7. Put the tip of the pipette
into the 2% NaCl solution
and slowly pull in 1.5 mL.
10. Put the tip of the pipette
into the sample and slowly
pull in 1.5 mL.
8. Slowly dispense the 2%
NaCl solution into the test
tube in position A3.
11. Slowly dispense the
sample into the test tube in
position A5.
9. Do steps 7 and 8 againto
put 1.5 mL of 2% NaCl
solution into the test tube in
position A1.
12. Set the 1.0 - 5.0 mL
pipette to 2.0 mL.
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13. Put the tip of the pipette
into the sample and slowly
pull in 2.0 mL.
16. Keep the dilution series
at 15 °C for at least 5
minutes to correct the
temperature.
14. Slowly dispense the
sample into the test tube in
position A4.
15. Do steps 13 and 14
againto put 2.0 mL of
sample into the test tube in
position A2.
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