microtox acute toxicity test - coastal · pdf filepage 3 ©1998 azur environmental. all...

©1998 AZUR Environmental. All rights reserved. Microtox, Mutatox and MicrotoxOmni and the AZUR logo are tradmarks or registered trademarks of AZUR Environmental.

Microtox Acute Toxicity TestPRINCIPLE OF OPERATIONThe test exposes luminescent organisms in MicrotoxAcute Reagent to aqueous samples, and measures theincrease or decrease in light output by the testorganisms.Reagent contains living luminescent bacteria thathave been grown under optimal conditions, harvested,and then lyophilized (freeze-dried). The lyophilizedbacteria are rehydrated with Reconstitution Solutionto provide a ready-to-use suspension of organisms.The test system measures the light output of theluminescent bacteria after they have been challengedby a sample and compares it to the light output of acontrol (reagent blank) that contains no sample. Adifference in light output (between the sample and thecontrol) is attributed to the effect of the sample on theorganisms.

PRECISIONEach test cuvette contains roughly a millionindividual test organisms that are challenged by the

test sample. Variations among individual organismsbecome statistically insignificant. The systemmeasures a single parameter, the simultaneous lightoutput of all of the organisms.Each batch of Reagent, containing lyophilized testorganisms, is prepared under conditions within veryrigidly controlled limits.

TIME AND TEMPERATUREDifferent chemicals affect living organisms atdifferent rates, reflecting differences in mechanism ofaction.For some classes of chemicals, the effect on lightoutput is complete in 5 minutes.For other classes of chemicals, the light output is stilldecreasing rapidly at 5 minutes. In these cases, 15-minute data may be more reliable.We recommend that 5-minute and 15-minute data betaken routinely when dealing with unknown samples.Some laboratories elect to collect data routinely atfive, fifteen and thirty minutes. The software will


Microtox Acute Toxicity Testautomatically process data taken at any one, two, orthree times you select.The temperature during exposure to various materialswill affect the response of the living organisms. Forthe Microtox Acute Toxicity Test procedures theIncubator Wells and READ Well temperature is15°C.

CONTROL (BLANK)Reagent Control is required for each test and is runconcurrently with the test. Light levels normallychange with time, for reasons other than thebioreactivity of the test sample.Use of the Reagent Control allows the operator tocompensate for some of these variables when reducingthe data. In all variations of the Basic Test procedure,the responses of all the test cuvettes are normalized tothat of the Control test response, which compensates forerrors up to 20% in the 10 µL pipetting of reagent.

SUPPLIES & ACCESSORIESTesting requires several special items in addition tothose commonly found in testing laboratories.

REAGENTThe Microtox Acute Toxicity Test Reagent isspecially formulated for bioreactivity testing withsensitivity to a broad range of toxicants.The reagent is a freeze-dried preparation of aspecially selected strain of the marine bacteriumVibrio fischeri (formerly known as Photobacteriumphosphoreum, NRRL number B-11177). A vial ofreagent contains roughly one hundred million testorganisms.Shelf life is one year when stored at minus (-) 20ºC to(-) 25ºC. Do Not store the Reagent below minus (-)25ºC. Storage at normal refrigerator temperaturegreatly reduces shelf-life.Reagent should not be stored in a self-defrostingfreezer, which defrosts by warming up periodically.Use the reagent within one to two hours after


Microtox Acute Toxicity Testreconstitution. The sensitivity of the reagent isessentially unchanged for 1-2 hours afterreconstitution. Changes in sensitivity may becomesignificant after that time for some samples.Some laboratories reconstitute a vial of Reagent at thebeginning of the day, store it in the REAGENT Well,then use the rehydrated reagent as needed through theday until it is consumed.For many applications, this is acceptable practice, butif Reagent is to be used 90 minutes or more afterreconstitution, its performance should be monitoredperiodically with a standard such as PHENOL, toindicate changing sensitivity.

RECONSTITUTION SOLUTIONReconstitution Solution is specially prepared nontoxicUltra Pure Water. Shelf life is one year when stored atroom temperature.

DILUENTThe Diluent is a specially prepared nontoxic2% Sodium chloride (NaCl) solution, used fordiluting the sample and the reagent.The marine bacterium in the reagent requires osmoticprotection that is provided by the 2% NaCl.Shelf life is one year when stored at roomtemperature.

OASOAS (Osmotic Adjusting Solution) is a specially pre-pared nontoxic 22% Sodium chloride (NaCl) solution,used to adjust the osmotic pressure of the sample toapproximately 2% NaCl.To adjust a sample osmotically, add one part of OAS toten parts of sample.

(X mL sample x 0.1 = mL OAS)Example:

2.5 mL sample x 0.1 = 0.25 mL OAS


Microtox Acute Toxicity TestThe sample can also be osmotically adjusted by theaddition of solid AR grade NaCl to a finalconcentration of 2.0%.Shelf life is one year when stored at roomtemperature.

DO NOT PREPARE SOLUTIONSDo not make Diluent or OAS yourself or use

substitutes. The production ofuncontaminated solutions is difficult, andsome laboratories have created problems

using their own Diluent and OsmoticAdjustment Solution.

CUVETTESCuvettes are used to contain samples, controls, andReagent during testing. They are nontoxic anddisposable.Used cuvettes cannot reliably be cleaned for reuse.Traces of detergent or sample contaminants interferewith later tests. The risk of interference fromcontamination is unacceptably high.

PIPETTORS AND PIPETTOR TIPSTest protocols require repeated precise transfer ofsmall amounts of liquid, as little as 10µL. Highprecision adjustable micro-pipettors are necessary. Itis highly recommended to have and use the followingpipettors:

10 - 100 µL adjustable volume pipettor0.25 - 2.5 mL adjustable volume pipettor

repeat pipettor

Sample Collection and PreparationSAMPLE COLLECTIONUse new clean, borosilicate, screw cap containers (30to 50 mL) with Teflon® lined caps. Fill the containercompletely to the top with sample, leaving NOairspace.Completely filling the container helps keep volatilematerial in solution.


Microtox Acute Toxicity TestSAMPLE STORAGETest the sample as soon as possible after collection.If testing is delayed, store samples at normalrefrigerator temperature.The toxicity of the sample can change with time, sotesting the sample within 1-2 hours after collection isbest, but this is not always possible. Try to test therefrigerated sample within 24-48 hours after collection.

COLOURED SAMPLEPerform the test protocol, and determine the sample’sICXX (ECXX). Check the sample concentration at theICXX (ECXX) for visible colour. If it contains obviouscolour, perform the Colour Correction Protocol.

TURBID SAMPLEIf the sample is turbid and this is objectionable,centrifuge the sample at an adequate speed and timeto remove the turbidity.The turbidity is objectionable when the toxicity fromthe turbid material is not wanted. If the toxicity isdesired from the material that causes turbity, do not

centrifuge the sample. If the sample is too turbid toperform a Basic Test use the Solid-Phase Test.

CHLORINE CONTENTThe Reagent (organism) is sensitive to chlorine asare all microorganisms.Municipal Water Treatment (drinking water) orWaste Water Treatment Plants have a commonproblem with the water they are producing, that isbacterial contamination. Chlorination of the wateris generally used to solve this problem. Chlorine istoxic: it is used for killing microorganisms.Most of the time when either a Municipal WaterTreatment or Waste Water Treatment Plant wantsto check for acute toxicity, they do not want toknow the effect of chlorination.Collect the samples before chlorination, unless youare testing for the effect of chlorination. Do notcollect a chlorinated sample for testing, unless youwant to know the effect of chlorination or can notcollect a sample that is not chlorinated. If knowingthe effect of chlorination is desired, it is


Microtox Acute Toxicity Testrecommended to test the sample just beforechlorination and just after chlorination.When the effect of chlorination is desired or cannot be avoided, dechlorinate the sample withSodium Thiosulfate.

Sodium Thiosulfate DechlorinationTo dechlorinate a sample add 1 part of SodiumThiosulfate Stock Solution to 100 parts of Sample.Example: 100 µL Sodium Thiosulfate StockSolution added to 10 mL Sample.Final Sodium Thiosulfate concentration in thesample is 100 mg/L.

Sodium Thiosulfate Stock Solution Preparation 1. Weight out 1.0 g Sodium Thiosulfate

(Na2S2O3). 2. Add 100.0 mL Diluent.

The Sodium Thiosulfate will last for 1-2 monthswhen stored in the refrigerator.

SAMPLE pH 1. Measure the pH of the sample, and record the pH

as part of the information in the sampleDescription.The bacterial Reagent is sensitive to pH. Thereis a minimal pH effect between 6.0 and 8.0.When the pH is higher than 8.0 or lower than6.0, and the sample has buffering capacity theeffect can be dramatic. Example: distilled wateradjusted to pH 5.0 with HCl, has NO pH effectbecause there are not enough hydrogen ions toaffect the pH of the Reagent. Another sample atthe same pH (5.0) may show considerabletoxicity if it does have buffering capacity.

2. If the pH of the sample is below 6.0 or above 8.0and pH adjustment is required, adjust the pH asshown below.

Adjustment is required when the toxiceffect of pH is not wanted.

A. If the pH is below 6.0, adjust the pH to 6.0with NaOH. If over-titration occurs, discardsample and start again.

B. If the pH is above 8.0, adjust the pH to 8.0


Microtox Acute Toxicity Testwith HCl. If over-titration occurs, discardsample and start again.When the sample is below pH 6.0, titrating thesample above 6.0 may precipitate the sample,thus changing the effective toxicity.When the sample is above pH 8.0, titrating thesample below 8.0 may precipitate the sample,thus changing the effective toxicity.When the sample has been over-titrated, backtitrating may not resolubilize the sample, ifprecipitation occurred.Use a 5 Normal (5N) acid or base for coarsepH adjustment of a sample with a high/strongbuffering capacity to minimize sampledilution.Use a 0.5 Normal (0.5N) acid or base whenthe sample has a low/weak buffering capacity.

PURPOSE OF A STANDARDTesting a standard (reference toxicant), whose testresults are well characterized, confirms yourunderstanding of a test protocol and checks theperformance of the complete test system (e.g.analyzer, Reagent, Diluent and ReconstitutionSolution).The 3-Basic Test Protocol is the best procedure fortesting "Standards," as it provides the highestconfidence level, precision and flexibility.When testing "Standards" never except an IC50

derived from extrapolated data, always retest usingthe appropriate "Standard" dilutions.


Microtox Acute Toxicity TestPHENOL STANDARD

Phenol IC50 5 minutes = 13 to 26 mg/L 1. Weigh out ~50 mg (~0.050 g) of crystalline

Phenol, add it to a 500 milliliter (mL) ambervolumetric flask.Do not try to weigh out the exact amount ofsample, calculate sample concentration for theamount weighed out.If an amber volumetric flask is not available,cover the entire flask with aluminum foil toprotect the Phenol Standard from light.

2. Add Diluent to the 500 mL mark on thevolumetric flask.

3. Seal the flask, and mix well by inverting thevolumetric flask.

4. Label the flask and store at normal refrigeratortemperature (2-8°C).The Phenol standard will last for 3-4 monthswhen stored in this manner.

ZINC SULFATE STANDARDIC50 15 minutes

ZnSO4.7H2O = 3 to 10 mg/LZn++ = 0.6 to 2.2 mg/L 1. Weigh out ~ 50 mg (~ 0.050 g) of ZnSO4.7H2O,

add it to a 500 milliliter (mL) volumetric flask.Do not try to weigh out the exact amount ofsample; calculate sample concentration for theamount weighed out.

2. Add Diluent to the 500 mL mark on thevolumetric flask.

3. Seal the flask and mix well by inverting thevolumetric flask.

4. Label the flask and store at normal refrigeratortemperature (2-8°C).The Zinc sulfate standard will last for 3-4 monthswhen stored in this manner.


Microtox Acute Toxicity TestMicrotox Acute Toxicity:True Deviation or Human Error?At times it is very difficult to tell whether a largeconfidence range is the product of human error ortrue deviation. In such a case it is best to retest thesample using a Duplicate Test procedure. If therepeated results show the same type of dose-response curve, it is probably a true deviation. Ifthey do not, it is probably human error. Humanerror is suggested when the placement of gammason the dose-response curve of the Microtox AcuteTest Data Report is random or skewed. Error is alsoindicated when the results of tests on duplicateconcentrations of a sample do not agree with oneanother.

An example of human error is shown below.

21

4

3

68

7

In this example it is easy to see that thishuman error, as the test is in duplicate (1&2,3&4, 5&6, 7&8), and the duplicates do notagree with each other.


Microtox Acute Toxicity TestTrue deviation from theory is suggested whenrepeated tests show the same curvature. In this casethe data are good, but the statistical information (e.g.residual variance, 95% confidence factor, correlationcoefficient) will indicate that the fit to a straight lineis poor, as in the case of human error.

21

43

687

In this example it is easy to see that thisTrue Deviation, as the test is in duplicate(1&2, 3&4, 5&6, 7&8), and the duplicatesclosely agree with each other.

Microtox Acute Toxicity Test


Microtox Acute Toxicity:Precision and BiasQuality data are produced when test procedures arefollowed as stated. When the highest confidence andprecision are required, duplicate testing methods arerecommended. In duplicate testing, data quality isimproved through cross comparison. The mostcommon source of error will be that due to operatorerror. Errors are most likely to occur during samplepreparation, salinity adjustment, sample dilution,reagent dilution, sample transfer and mixing steps,and data interpretation and resulting calculations. Useof the proper equipment and development of theappropriate skills required for using the testequipment are necessities in producing quality data.

Duplicate test results are unacceptable if the lightmeasurements at any duplicate concentration aredifferent by more than 20% of either value. This islikely to occur when improper transfer (pipettingerrors), dilution, and mixing procedures have beenused.



Microtox Acute Toxicity:Data InterpretationLight Level-Time Response DifferencesDifferent chemicals affect the organisms in MicrotoxAcute Test Reagent at different rates, producingdistinctly different Light Level-Time Responsecurves. Three types of Light Level-Time Responsecurves for the Microtox Acute Test Reagent typicallyoccur.The three following Light Level-Time Responsecurves indicate why multiple light readings (It) at 5,15 and sometimes 30 minutes, are recommended fortesting samples whose average characteristics are notwell documented.Phenol Light Level-Time Response curve.Phenol, for example, completes its action veryrapidly. Light output drops sharply, then levels off, orrises slightly over time thereafter.

0 5 time (minutes)

Ligh

t Lev

el

Phenol Light Level-Time Response curve.

conc. A

conc. B



0 5 10 15 time (minutes)

0 5 time (minutes)

Ligh

t Lev

el

Ligh

t Lev

el

Most Common Light Level-Time Response Curve.

This Light Level-Time Response curve is typical oforganic compounds. The light output drops somewhatmore gradually than for phenol, then levels off.

Heavy metals Light Level-Time Response Curve

This Light Level-Time Response curve is typical ofmany heavy metal compounds. The decay rate of thelight is essentially constant for an extended period oftime, and depends on concentration. Response curvesof this type indicate why multiple light readings (It) at5, 15 and 30 minutes, are recommended.

conc. B

conc. B

conc. A

conc. A



Which Incubation Time to Report forMicrotox Acute Toxicity TestsAssume that two assay times have been used for asingle sample, one 5 minute, 15 minute. Both timeswill always be available for reference, but forsimplicity you probably want to select a single assaytime to represent the results of testing this sample.Which assay time is appropriate to report?

For most samples, the 5 and 15 minute IC50s and thequality of data will be about equal. In such cases the 5minute data are usually used in reports.

When the sample contains certain metal compounds,the 15 minute data will show considerable increase inbioreactivity response. If this is the case, and if the95% confidence range is comparable to that for the 5minute data, report the 15 minute data.

Do whatever satisfies your report requirements. Someorganizations require that all data be reported. In anycase, it is appropriate to denote the time (5 or 15minutes) with the data actually presented. In someinstances inhibition can occur at 15 minutes whichmay not be seen at 5 minutes.



Microtox Acute Toxicity:Difficult Samples Made EasyIn several situations, samples may require specialhandling.

1. SAMPLES THAT HAVE KNOWNSPECIAL REQUIREMENTSSome samples are already saline, requiringomission of the osmotic adjustment step; othersare coloured, others have unusual physicalcharacteristics. Such samples are characteristic ofcertain industries and activities and variousmanufacturing situationsThis manual contains a number of specialprotocols (variations on the fundamental MicrotoxAcute Test protocols) designed specifically fordealing with samples known to require specialhandling. Examine these special protocolscarefully, and select one that serves yourparticular need.

2. SAMPLES THAT AREEXCEPTIONALLY TOXICSome samples are so bioreactive that the tests younormally run show complete light loss at allconcentrations.Retest the sample, using the Extended (13Dilution) Basic Test Protocol. With adequatedilution, the ECXX of any highly toxic samplecan be found.

3. SAMPLES THAT ARE OFEXCEPTIONALLY LOW TOXICITYThe toxicity of some samples is extremely low,but important to know. When the toxicity levelsare very low, they may be confused with thebackground “noise” produced by irregularities inpipetting.Run a 90% Basic Test protocol, in duplicate,using two controls in each set. When the 90%Basic Test protocol does not provide ECxx, run aComparison Test or Inhibition Test.



4. SAMPLES THAT HAVE AN EXTREMELYSTEEP DOSE-RESPONSE CURVESome test samples show a dose-response curve sosteep that the light level drops from very high tovery low from one sample concentration to thenext, providing too few useful data points forcalculation of ECXX.Complete light loss may occur at allconcentrations in an Abbreviated Basic TestProtocol. After a second test with a primarydilution, all concentrations may show no light lossat all. A trial-and-error search for the narrowrange in which the change occurs can be time-consuming.This sort of data has a dose-response curve sosteep at times that it cannot be plotted by thesystem.Use narrower (1:1.5) serial dilutions.

5. SAMPLES THAT HAVE A NEGATIVE SLOPESome test samples show a negative slope (reversedose-response curve). The light level appears toincrease with increase in sample concentration.This condition may be caused by one or more ofthe following:

(1) The sample dilutions were made in reversesequence.

(2) The It light readings were read in reversesequence.

(3) The most common cause is background noise(pipetting error) which by chance forms anegative slope. Background noise can be morenoticeable with low toxicity samples and the90% Basic Test protocol.Run the test protocol used, in duplicate, usingtwo controls with higher sample concentrationif possible. During the test be very careful inpipetting, making the sample dilutions andreading the light levels.



6. SAMPLES THAT HAVE AFLAT DOSE-RESPONSE CURVESome samples show dose-response curves so flat(at 1:2 dilution ratio) that the data points may beconfused with background (pipetting) noise, andcalculation of ICXX may require extrapolation to adistant point, widening the confidence rangebeyond acceptable range limits.Rerun the test, using the Extended (9 dilutions)Basic Test.

7. THE ECXX IS DETERMINED BYEXTRAPOLATIONSome samples are so bioreactive, or so slightlybioreactive, at the range of concentrations testedthat all of the data points fall on one side of theICXX, which can only be determined byextrapolation. This is always less desirable than anICXX bracketed by data points.If the gammas are all larger than that

corresponding to the ICXX of interest, all gammasgreater than 1.0 for IC50, retest the sample using aprimary dilution testing lower concentrations. Theproblem with extrapolation to determine ICXX isthat the sample may be nonlinear at lower orhigher concentrations.If the gammas are all smaller than thatcorresponding to ICXX (gammas less than 1.0 forIC50) retest using a more concentrated sample.



Microtox Acute Toxicity:Common Questions and AnswersThese common Questions & Answers provide insightinto the decisions and practices that produce highquality data.

Which Test Procedure Should I use?The MicrotoxOmni Wizard is the best source forselecting which is the best test procedure to use bysample type.

What Effect Does the Samples pH, Free Chlo-rine and Colour/Turbidity and Have on the TestResults?All of the above may have an effect on the test results.Please see Microtox Acute Toxicity Fundamentalspages 5 through 7 for further information.

The Light Level Readings Did Not Enter Into theSoftware, What is Wrong?There is a communications problem between theanalyzer and the computer. Check the following: • Make sure you have good cable connection

between the analyzer and computer, this is themost common problem.

• Make sure the cable is good, a bad cable is thesecond most common problem.

If this is the first time the analyzer was connectedto a computer also check:

• Make sure the computer cable is connected to acomputer communications port (COM1, COM2etc).

• If the computer has more than one communicationport make sure the software is set to the correctcommunications port. The software default settingis set for COM1.



The Microtox Acute Toxicity Test Work Just Finefor the Reference Toxicant (Phenol or Zincsulfate) and for Influent Samples but for EffluentSample the Microtox Did Not Work, What is theProblem?When first getting started in performing toxicity testsit is generally more difficult to understand the datafrom low to nontoxic samples. The information belowshould help. • The MicrotoxOmni software calculates an ICxx

(ECxx) with minimum and maximum effectswhen three usable gamma’s are calculated fromthe data.

• The MicrotoxOmni software calculates an ICxx(ECxx) without minimum and maximum effectswhen two usable gamma’s are calculated from thedata. A minimum of three usable data points isrequired to calculate statistical data.

• The MicrotoxOmni software calculates Percentwhen only one usable gamma is calculated fromthe data.

• When no results are provided the sample showedno toxicity at the concentrations tested. If theinitial sample concentration can be increased,retest the sample using a higher sample concentra-tion. If the highest sample concentration testedwas 81.9% to 100% (using the 81.9% Basic Test,Comparison Test Inhibition Test or the WholeEffluent Test) the sample is just nontoxic.

What is Toxicity Units (TU)?Toxicity Units (TU) = 100 ÷ IC

50 (EC

50)

When Should I Use Toxicity Units?It is easier to detect a deviation from normal whentrend monitoring Toxicity Units of a sample site withhigh toxicity such as Wastewater Treatment Plantinfluent. And the Windows 95 MicrotoxOmni soft-ware has Trend Monitoring capabilities.



What is Trend Monitoring and When Should IUse It?Windows 95 MicrotoxOmni software has TrendMonitoring capabilities that will determine the normalvariation of a sample site and will detect and indicate(reveal) any deviation from this norm.

Duplicate tests. What are they, and when wouldthey be used?The use of duplicates provides additional data to checkthe quality of the test (operator pipetting), and providesa larger context in which to evaluate the data.

How many sample dilutions should be used?When dealing with well-characterized samples, foursample dilutions that have at least one dilution oneither side of the ICXX are enough. In that case, youwill typically be testing samples from the same sourceon a regular basis, looking for significant changes intoxicity levels.Tests using more dilutions are important if you are

testing samples with which you have no previousexperience, and you don’t know what to expect.For instance, if you run a test with just a few dilutionsof a very toxic sample, every concentration may causecomplete loss of light output from the reagent. Thisindicates that the sample has detectable toxicity, butdoes not allow calculation of ICXX, because the fewsample dilutions used were in the wrong range.

Examples of tests in this manual use 1:2 serialdilutions of the sample. Wouldn’t a higher ratioprovide a greater range of concentrations?Yes, but it reduces the resolution of the test. The 1:2dilutions are a good standard compromise betweenrange and resolution, and work well for most samples.



How long can the frozen Microtox Acute TestReagent be stored before it is used?The shelf-life of the reagent after it leaves the factoryis at least one year under proper storage conditions.The expiration date of the reagent is printed on thelabel on the reagent vial. Make a point of using up thereagent with earlier expiration dates before openingnew boxes of reagent with later expiration dates. Firstin/first out.

The M500 Analyzer normally does not show theabsolute amount of light emitted by test reagent.It shows relative values. How can I tell if thefresh reagent always produces the same amountof light?The reagent does not always produce the sameamount of light, and the question is of little practicalimportance. The system measures the percent of lightlost in response to change in toxicity, which isconsistent, no matter what the original light level.Light levels may be different for several reasons:

a) The light output of different batches of reagentvaries.

b) Poor reconstitution technique reduces thepotential light output of the reagent.

c) The light output will change with the age (shelflife) of the reagent.

These variations are unimportant, because youcalibrate the analyzer to the reagent at the beginningof each test (when you press SET), and the graduallight loss or gain during a test is measured, andaccounted for in data reduction calculations of theBasic Test Procedure.In general, light output is not the primaryconsideration in evaluating the quality of the reagent.More important is the response of the reagent tovarious toxic materials.



How long can the reagent be used afterreconstitution?For at least an 2 hours after reconstitution, theorganisms respond consistently to a wide spectrum oftoxic materials. After that time, the response maybegin to change. The reagent does not just becomegenerally “less sensitive.” Its response to differentchemicals may either increase or decrease, changingthe response spectrum.The useful life of the reagent after reconstitution isnominally one hour, but in practice, the useful life ofthe reagent may be several hours. Many laboratoriesreconstitute a vial of reagent for use all morning orafternoon. The conditions under which you useMicrotox Acute Test Reagent beyond the initial hourwill depend on your need for precision andverifiability of results.For instance, if you intend to use the data for routinemonitoring, and data quality requirements may not becritical, the use of reagent over several hours may beperfectly acceptable. If unusual or suspicious results

are seen, these results could be confirmed with testsusing a “fresh” bottle of reagent.Well-characterized samples can be tested repeatedlywith reagent of increasing age, so that the “normal”response of the reagent at any age is well known. ThePhenol Standard is useful for this. It is also helpful torun several tests on one or more well characterizedstable samples of known toxicity.To establish baseline information, you may also electto test each new lot of Microtox Acute Test Reagentwith a standard such as Phenol over a period of hoursafter reconstitution, so you know what the normalresponse curve is over time.Once again, the decision on how long thereconstituted reagent may be used is based on whatinformation the test is intended to produce.



The protocols call for transfer of only 10 µl of reagentmixture to the test cuvettes. Can’t we increase thatto 20 µl or 30µl, as long as we do it every time?No, don’t increase the amount of reagent.

How can we measure and improve pipettingskills?The Microtox Acute Test requires transfer of smallamounts of liquid, as little as 10µl at a time, with anassortment of micropipettors. Pipetting is an ordinarylaboratory skill and usually improves with repetition.

To check your reagent pipetting consistency:

Using an analytical balance, transfer 10 µL of water intoa teared cuvette and weigh the amount of water pipetted.Repeat this 10 times and calculate the mean.

How can we calibrate a pipettor?Using an analytical balance, weigh the amount ofwater transfered into a tared cuvette. Repeat this 10times and calculate the mean.

Sometimes several or all of the sample cuvettesproduce negative gammas. Does that alwaysindicate a stimulatory effect of the sample?Not always. The effect may also be produced by pipettingerror.

microtox acute toxicity test - coastal · pdf filepage 3 ©1998 azur environmental. all...

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