PROCEDURE FOR CALCULATING TIME-MORTALITY CURVES BY THE BOTTLE
By Jack Petersen
Insecticide resistance is a threat to the success of any mosquito
control program.Early detection and proper
management decisions are essential to keeping this problem in check.One method of determining the response of adult
mosquitoes to a given pesticide is the bottle bioassay.
A standard dose is used to discriminate between insects susceptible
to an insecticide and those that are resistant.The
bottle bioassay is used as an indicator of the insects response to that dose,
specifically, how many minutes it takes to kill all the test mosquitoes.
250 ml Wheaton bottles
Pipettes, glass, 1 ml calibrated in hundreths
Chemical(s) to be tested
clean, dry bottles.If possible, heat bottles
to 350 degrees F in an oven for 15-20 min. before use.Allow the bottles to return to room temperature.
stock solutions.If stock solutions are
already made and have been refrigerated, make sure you allow enough time for them to come
to room temperature before use.
insecticides are given as appendices in another part of this procedural guide.Once the stock solution is prepared, it can be
stored in the refrigerator in light-proof bottles until needed.
bottles.Make sure you mark the cap as well
so you can keep individual bottles and their caps together.This is important because you will be coating the entire bottle, including the
inside of the cap.To ensure the bottle has
the proper dosage you must keep the cap with its respective bottle.
Preparing the Bottle.
the bottle 1 ml of A.C.S. acetone plus the active ingredient (A.I.) to be tested.The precise amount of A. I. to be added to each
bottle depends on the chemical.See Table 1.
one or more control bottles that contain 1 ml of acetone, but no active ingredient.These bottles are an important test of proper
Make sure the
bottle cap is secure so that no acetone evaporates until you are ready for it to do so.
bottle gently so that the acetone evenly coats the bottom.
turning the bottle while inclining the bottle so that the sides are evenly coated.Do this slowly and carefully, looking for streaks.The interior of the bottle must be evenly coated.
bottle so that the acetone puddles in the shoulder.Rotate the bottle several times to distribute the
Turn the bottle
completely up side down.Did any acetone
escape?If it did, reject that bottle and
start over again with another bottle.
Coat the inside
of the cap.
Turn the bottle
on its side and lay it on a flat surface.
Gently vent the
bottle by unscrewing the cap.You should hear
an audible pssst!
Now roll the
bottle to keep the insecticide evenly distributed on the walls while the acetone
procedure with all the bottles until no liquid acetone is visible.
bottles to dry with lids off for at least two (2) hours before proceeding.
bottles in a dark, cool, dry place
mosquitoes into each bottle.Use a number you
can easily count with accuracy as the mosquitoes fly about.It works best to collect all of the mosquitoes for one bottle in a mouth aspirator
and introduce them all at once with a gentle puff of air.Try to avoid adding excessive moisture from your breath.A mechanical aspirator works best.
bottle to be sure all mosquitoes survived the transfer process.If you find any dead mosquitoes, record the
number.You will need to substract this
number from each timed reading.
Start a timer
and record every 15 minutes how many mosquitoes are dead or alive (whichever is easier to
count).Continue until all mosquitoes are
dead.In some cases, when mosquitoes are
resistant to the pesticide, it may be necessary to set an arbitrary maximum time limit
such as five hours.
counts on the data sheet that accompanies this procedure.
all mosquitoes are dead or a pre-determined time has expired.
Count the total
number of mosquitoes in each bottle and calculate the percent mortality for each 15 minute
percent mortality (y axis) against time (x axis) using a logarithmic scale for the percent
test mosquitoes with baseline date established for susceptible mosquitoes of the same
When you are
finished with your bottles or the pesticide is too degraded to use any longer, triple
rinse them with acetone, wash them with warm soapy water and rinse.Place them in an oven to thoroughly dry before
using them again.
If you are
uncertain whether the bottles are completely clean, introduce some susceptible mosquitoes
into the bottles after you dry them.The
mosquitoes should not die in three hours or less.If
they do, clean the bottles again.
2002;Revised:26 April 2002; Revised 10 February 2003; Revised 12 November 2004;
Revised 25 May 2007; :Revised 9 March 2012