Biology Question #1361
Michael, a 17 year old male from Winnipeg asks on March 29, 2003,
I've been doing a science fair project testing the effectiveness of Lactaid(TM) lactase. From a competely pristine Lactaid bottle, I transferred half into a clear sterile test tube, left half in the opaque original package, and tested the effectiveness of each. Almost immediately after the transfer, it was clear that the transferred lactase had a rapid drop in productivity, only about half of its original reaction rate. What could possibly cause this? Light (unlikely since both the lactase samples were incubated in a waterbath for 3 hours and exposed to sunlight)? Difference in plastic? The airspace in the tubes? Surface tension? Could you please suggest a possible explanation or refer me to somebody who might have one?
Here are some details of the experiment.
We are using Lactaid liquid droplets which have no additives other than glycerin and water.
Second, we just transferred that Lactaid normally, with a sterile-tipped micropipeter from the original Lactaid container into the other test tube which is why the result is so bizzare. This isn't the first time we've noticed the results either.
Each time we do a series of experiments, we start with a new bottle of lactaid. So, the lactase was transferred from the new original package to the test tube. After two days of refridgeration, we tested the test tube lactase and the packaged lactase and noticed the significant drop off in productivity. We monitored this productivity for 3 weeks and it remained consistent.
the two samples; they are treated exactly the same:
-refridgerated at 4 celcius before runs
-taken out of the fridge for no more than 5 minutes to conduct runs
-both are combined by micropipeter with a lactose and water solution (675 micro litres/200 mL dH2O+10g lactose solution) in a 250mL Erlenmeyer flask
-the solution is incubated for 3 hours in a waterbath (which is exposed to light)
-1.0g yeast is added to each flask and it is incubated for 10 min to activate it
-the gas production in the lactase flasks is measured against a positive glucose control (we expect a 2:1 gas production ratio for glucose:(hydrolyzed lactose))
Thus far, we have found that the ration of glucose:(original lactase):(transferred lactase) is 4:2:1. This result has been consistent and was noticed soon after the transfer.
One more important detail that we haven't been able to make anything of...about nine days into a series of experiments with a given Lactaid sample, I noticed that the lactase from the original package was a darker colour than the test tube sample. That doesn't mean that the colour change wasn't there all along, but I never noticed it so I can't say for sure that it was.
The Lactaid bottle comes with a dropper. Through that dropper, we transferred the 7mL into the test tube.
The test tube is a 14mL sterile plastic test tube. I believe it's made of polyethylene, but we haven't been able to contact the company so far, so we're not sure. It's radius may be a couple of centimetres (rough estimate).
The test tube isn't stoppered, it's capped. The cap seems like a fairly tight seal and it's made of the same stuff as the tube itself. In the fridge, the test tube stands up in a test tube rack.
We only use the micropipetter for the lactase in the test tube. The tip is clean (previously unused), but I'm not sure how to describe our technique otherwise. We just transfer the 675 microL into the flasks normally (the pipette's range is 100-1000 microL). I think I may have confused you about the pipette. It's not one of those big plastic ones where it's hard to be accurate. It's one (possibly Fischer brand (not sure)) where you change the tip each time, so it's not possible that we're getting left over junk in our sample. The tips are stored in a closed case in a cabinet. There are many slots, and one tip fits in each slot.
To perform the final gas test:
1)Make a solution in six flasks (250mL Erlenmeyer), each containing 200mL dH2O and 10g of their respective sugars. Five of these flasks should contain lactose and one should contain glucose.
2)To 2 of the lactose flasks, add 675microL of lactase from the original lactaid container. To 2 of the other lactose flasks, add 675microL of lactase from the test tube.
3)Incubate each flask in a waterbath at 40 celcius for 3 hours.
4)Add active dry yeast to each of the flasks and incubate in the waterbath for 10 min.
5)The gas testing apparatus consists of 1 25ml inverted graduated cylinder filled with water/flask. Each of the flasks is stoppered and water sealed. The stopper is connected to a tube which is connected to the grad cylinder. After the 10 min, this setup is done for each of the 6 flasks.
6)The C02 produced by fermented glucose displaces the water in the cylinder (to equalize the pressure in the passage tube). We monitor this gas production for 20min (after 20 min, the yeast have produced to much gas (it can no longer be measured by etch marks on the grad cylinder)) and then disconnect the apparatus.
As mentioned, we have a positive and negative control, glucose and plain lactose respectively, which ensure that the production is normal. For every single measurement so far, the production has been normal. The only anomoly is the lack of gas production in the test tube lactase flasks.
assay as described. If it does not have reduced activity, it is
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