The overall goal of this procedure, is to accurately measure the survival time of Brachionus Rotifers. By offsetting possible deleterious effects of maternal generations. This method can help answer key questions in aging research.
By providing a reliable way to measure the survival time of rotifers. The main advantage of this technique is that it is reproduceable, and have high statistical power. To begin, to 4.5 liters of distilled or deionized dilution water, add 454 millimolar sodium chloride, 26 millimolar magnesium chloride, 27 millimolar magnesium sulfate, ten millimolar potassium chloride, and ten millimolar calcium chloride.
Prepare a 0.48 molar stock solution of sodium bicarbonate. And add 25 milliliters of it to the salt solution just prepared. Prepare a 0.4 molar stock solution of sodium bromide.
And add ten milliliters of it, to the salt solution. Then, with distilled water, bring up the salt solution to five liters. And, using a 0.45 micrometer filter membrane, filter the solution.
To culture laboratory raised, or wild captured rotifers, place them in a sterile 100 milliliter beaker, of artificial sea water. And culture them at 25 degrees Celsius. According to the methods of Snell, culture dietary micro algae species, in artificial sea water.
Insuring that the same lot of micro algae are used in all experimental groups. Maintain a stock population of rotifers in a batch culture by feeding them every two days. And changing the medium every week.
Many neonates and adults bearing two to three eggs, can be observed when rotifers are in optimal conditions. To synchronize rotifers by pre-culture, select a single rotifer from the stock population. And culture it in artificial sea water in a 100 milliliter beaker as just described for about two weeks.
After the incubation, collect egg bearing rotifers from the sub-population. Culture them as a single cohort by placing approximately 50 rotifers per milliliter of sea water in the wells of a six well plate, and feed them ad libitum. When the eggs have hatched, transfer neonates to newly prepared culture medium.
Repeat the culturing and transferring of neonates over two to three generations. To measure survival time, place neonates hatched within a three hour period of time, in the wells of 24 or 48 well plates containing one milliliter of artificial sea water. At 12 or 24 hour intervals, transfer the rotifers to newly prepared culture medium.
Record the number of offspring, and whether each individual is dead or alive. Recording the rotifer as dead when the cilia movement of the corona has completely stopped. When experimental rotifers are actively reproducing, remove neonates since they grow rapidly and are sometimes difficult to distinguish from experimental rotifers.
Carry out data analysis according to the text protocol. This figure shows survival curves from two replicates of poorly synchronized rotifer populations. Fed either ad libitum, shown as A-L, or every other day shown as Intermittent Fasting, I-F.
Median survival was 13 and 18 days respectively, in the two groups. Although it is well known that intermittent fasting extends the life span of the rotifer, the differences were not statically significant. In this experiment, rotifer conditions were optimal and well synchronized.
And the animals tended to survive until the later phase of the experiment. Median survival was 13 days for AL rotifers, and 20 days for the IF groups. Which was statically significant.
After watching this video, you should have a good understanding of how to synchronize rotifer conditions. We hope that this protocol enhances of additive rotifers, of the model organism and aging research.
