algaltoxkit f slideshowbiotoxicity.org/images/toxicity pdf/algatoxkitf_test_presentation.pdf ·...
TRANSCRIPT
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ALGALTOXKIT F
Test procedure
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1
PREPARATION OF
ALGAL CULTURING MEDIUM
- VOLUMETRIC FLASK (1 liter)
- VIALS WITH NUTRIENT STOCK
SOLUTIONS A (2 vials), B, C, D
- DISTILLED (or deionized) WATER
A B C D
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2
TRANSFER 10 ML FROM ONE OF
THE TWO “NUTRIENT STOCK A” VIALS
IN ± 800 ML DISTILLED WATER
IN THE 1 LITER VOLUMETRIC FLASK
A
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3
TRANSFER 1 ML FROM THE
NUTRIENT STOCK VIALS
B, C AND D INTO THE 1 LITER
VOLUMETRIC FLASK.B
C D
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4
- FILL THE FLASK TO THE 1 LITER MARK
WITH DEIONIZED WATER
- STOPPER THE FLASK AND SHAKE
THOROUGHLY TO HOMOGENIZE
THE CONTENTS
- AERATE THE ALGAL CULTURING
MEDIUM FOR AT LEAST 30 MINUTES
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5
ADJUST THE pH
(if necessary)
TO 8,1 ± 0,2
(with either 1 M HCl
or 1 M NaOH)
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6
DE-IMMOBILIZATION OF THE ALGAE
TAKE ONE TUBE CONTAINING ALGAL BEADS AND POUR OUT THE LIQUID
TAKE CARE NOT TO ELIMINATE ANY OF THE ALGAL BEADS DURING THE PROCESS !!
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7
OPEN THE VIAL “MATRIX DISSOLVING MEDIUM” AND TRANSFER 5 ML
TO THE TUBE WITH ALGAL BEADS
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8
CAP THE TUBE AND SHAKE VIGOROUSLY TO DISSOLVE THE ALGINATE MATRIX
OF THE ALGAL BEADS, PREFERABLY WITH THE AID OF A VORTEX SHAKER
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9
CONTINUE THE SHAKING UNTIL THE ALGAL BEADS ARE TOTALLY DISSOLVED
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10CENTRIFUGE THE TUBE FOR 10 MINUTES AT 3000 RPM IN A CONVENTIONAL LAB CENTRIFUGE
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11
POUR OUT THE SUPERNATANT FROM THE TUBE
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12- ADD 10 ML DISTILLED WATER TO THE TUBE
- CAP AND SHAKE THE TUBE TO RESUSPEND THE ALGAE
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13CENTRIFUGE THE TUBE AGAIN AT 3000 RPM FOR 10 MINUTES
AND THEN POUR OUT THE SUPERNATANT
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14
- ADD 10 ML ALGAL CULTURING
MEDIUM TO THE TUBE
- CAP THE TUBE AND SHAKE TO
RESUSPEND THE ALGAE
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15
PREPARATION OF CONCENTRATED ALGAL INOCULUM
TRANSFER THE ALGAL SUSPENSION FROM THE TUBE INTO A 25 ML CALIBRATED FLASK
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16- ADD ALGAL CULTURING MEDIUM TO THE 25 ML MARK OF THE FLASK
- STOPPER THE FLASK AND SHAKE TO HOMOGENIZE THE ALGAL SUSPENSION
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17
- PUT 25 ML ALGAL CULTURING MEDIUM IN THE CALIBRATION LONG CELL
AND CLOSE THE CELL WITH THE LID
- PLACE THE CELL IN THE SPECTROPHOTOMETER
25 ml
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18
ZERO-CALIBRATE THE INSTRUMENT AT A WAVELENGTH OF 670 NM
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19
TRANSFER THE 25 ML ALGAL SUSPENSION INTO THE ALGAL STOCK CELL
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20
CLOSE THE ALGAL STOCK CELL WITH THE LID
AND SHAKE TO DISTRIBUTE THE ALGAE EVENLY
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21PUT THE ALGAL STOCK CELL IN THE SPECTROPHOTOMETER
AND READ THE OPTICAL DENSITY (OD1) AFTER 10 SECONDS
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22
- WITH THE AID OF THE REGRESSION FORMULA CALCULATE THE NUMBER
OF ALGAE N1 CORRESPONDING TO THE MEASURED OD1 IN THE ALGAL STOCK CELL
- WITH N2 = 1.106 ALGAE/ML, CALCULATE FROM THE N1/N2 RATIO THE DILUTION FACTOR
NEEDED TO REACH OD2 (corresponding to 1.106 algae/ml)
- TAKE THE OD/N
(optical density/algal number)
SHEET
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23
POUR THE 25 ML ALGAL
SUSPENSION FROM THE
ALGAL STOCK CELL INTO
A 100 ML FLASK
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24
ADD THE CALCULATED VOLUME OF ALGAL CULTURING MEDIUM
TO THE FLASK, TO MAKE UP A SUSPENSION OF 1.106 ALGAL CELLS / ML
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25
PREPARATION OF THE
TOXICANT DILUTION SERIES
TAKE SIX 200 ML CAILBRATED
FLASKS AND LABEL THEM FROM
C0 TO C5
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26
TO ELIMINATE TURBIDITY,
SAMPLES MUST BE
FILTERED BEFORE TESTING
(e.g. over a membrane filter
of 0.45 µm),
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27PUT 100 ML ALGAL CULTURING MEDIUM
IN THE 200 ML FLASKS C0, C2, C3, C4 AND C5
100 ml
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28
FILL FLASK C1
TO THE 200 ML MARK
WITH THE FILTERED SAMPLE
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29- ADD 2 ML “NUTRIENT STOCK SOLUTION A” AND 0.2 ML OF NUTRIENT STOCK SOLUTIONS
B, C AND D TO FLASK C1
- STOPPER THE FLASK AND SHAKE TO MIX THE CONTENTS
A
B
C D
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30- POUR 100 ML SAMPLE FROM FLASK C1 INTO A GRADUATED CYLINDER
AND TRANSFER THIS VOLUME INTO FLASK C2 TO MAKE THE FIRST 1:1 DILUTION
- STOPPER FLASK C2 AND SHAKE TO HOMOGENIZE THE CONTENTS
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31
REPEAT THE FORMER
DILUTION PROCEDURE
FOR THE OTHER FLASKS
(i.e.. 100 ml from C2 to C3, etc.)
Highest
concentration
Lowest
concentration
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32
REMOVE AND DISCARD
100 ML SOLUTION FROM
FLASK C5 TO HAVE 100 ML
SOLUTIONS IN EACH FLASK
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33- TAKE THE FLASK CONTAINING THE 1.106/ML ALGAL SUSPENSION AND SHAKE IT GENTLY
- ADD 1 ML ALGAL SUSPENSION TO EACH OF THE 6 FLASKS C0 TO C5, IN ORDER TO
OBTAIN AN INITIAL ALGAL CONCENTRATION OF 1.104 CELLS/ML IN EACH FLASK
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34
TRANSFER OF THE
ALGAE-TOXICANT
DILUTIONS INTO
THE TEST VIALS
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35- LABEL ALL THE LONG CELLS ON THEIR LID (3 replicates per dilution)
- AFTER THOROUGH SHAKING, TRANSFER 25 ML ALGAE-TOXICANT DILUTION
FROM EACH FLASK INTO A GRADUATED CYLINDER, FOR FURTHER TRANSFER
INTO THE CORRESPONDING LONG CELL (3 replicates per toxicant dilution)
25 ml
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36- REDISTRIBUTE THE LONG CELLS IN THE HOLDING TRAY IN A RANDOM WAY
- LIFT UP THE LIDS OF THE CELLS A LITTLE AT ONE SIDE, AND SLIDE
THE PLASTIC STRIP OVER THE OPEN PART OF THE LONG CELLS
TO KEEP THEM SLIGHTLY OPEN DURING THE INCUBATION PERIOD
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37INCUBATE THE HOLDING TRAY WITH THE LONG CELLS FOR 72h IN AN INCUBATOR
AT 23°C ± 2 °C, WITH CONTINUOUS ILLUMINATION:
- SIDEWAY ILLUMINATION = 10000 LUX
- OR BOTTOM ILLUMINATION = 3000-4000 LUX
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38
SCORING OF THE RESULTS
THE OD OF THE ALGAL SUSPENSIONS SHALL BE MEASURED EACH DAY DURING THE 3 DAYS
OF THE TEST, I.E. AFTER 24h, 48h AND 72h EXPOSURE TO THE TOXICANT
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39
ZERO-CALIBRATE THE
SPECTROPHOTOMETER
PRIOR TO THE DAILY MEASUREMENT
OF THE OD IN THE LONG CELLS,
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40
IMMEDIATELY BEFORE MEASURING THE OD IN A LONG CELL, CLOSE THE CELL,
TURN IT UPSIDE DOWN AND SHAKE GENTLY TO RESUSPEND THE ALGAE EVENLY
turn closed long cell upside down to shake
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41
- SCORE THE DAILY OD RESULT OF EACH LONG CELL ON THE “RESULTS SHEET”
- PERFORM THE DATA TREATMENT OF THE RESULTS WITH AN APPROPRIATE PROGRAMME