So there haven't been much new recently, but some updates.
If you remember, last time we discussed agar plates, we did a test with a bunch of plates where we UV exposed half, and used the others straight from the box. It seems that all of them did not have contamination. This good, as we have plates we can use, but leaves the question of contamination wide open. Currently, the theory is that there might be days where the air in the lab is cleaner? or maybe certain batches of plates are better sterilized. In any event, to be on the safe side, we had the A/C people come in and clean the air-conditioner filters (they were supposed to do it before we moved in, but apparently didn't).
Another problem we had was plate drying. We switched to using the 30C hot-room (two floors up), and we do not have any dryness problems. We are trying to check with the incubator supplier if there is something we can fix in the incubator, or we will replace it by a less aggressive one.
On other fronts, we had a microscope training on Tuesday. I will expand on how the microscope is built and how it works in a future entry, as we need more rounds of photography for that. I am going on a one week vacation and so will report on progress when I return.
Showing posts with label incubator. Show all posts
Showing posts with label incubator. Show all posts
Friday, January 22, 2010
Sunday, January 17, 2010
Double agar troubles
Although most of the recent posts where about receiving new equipment and plans to buy additional ones, we also have to push the science forward. One of the things that worries the last few days is getting reliable agar plates for growing yeast strains.
The troubles come in two different areas. I wrote before that we have contamination in plates. Well, we still have contamination in the plates we pour. Moreover, even when we do not have contamination, the plates start drying when we incubate them. For example, here the agar dried and then cracked.
In another plate, it didn't crack, but we see it drying toward one corner (the agar is thinner).
We know that the square plates are prone to water loss (much larger perimeter than round plates). For this reason we seal the plates either in plastic wrap (aka Saran wrap) or inside plastic bags. The two plates above were in the incubator for two days, one with plastic wrap and the other inside a plastic bag and both show bad signs of dryness.
We believe that part of the problem is the aggressive air circulation in our incubator. We called the company and they claimed that there is no way to turn down the fan speed. We now need to consider how to address this. One possible solution is to humidify the incubator by putting a container with water. If this will help, we might consider a more intelligent way of ensuring that it is humid.
The problem of contamination is a serious one. Since we want to grow libraries that will serve as resource for further examinations, having contaminants grow on the same plates is an issue. Since we first noticed the problem we talked with other yeast people in the building and they report similar problems. All of them recommended shutting down the A/C circulation before pouring plates. Apparently the air supply introduces yeast and bacteria from outside the building into the lab.
One of the debates in yeast sterile techniques is whether to use flame. On the one hand, working with a flame you sterilize the opening of the bottle before you close it, reducing chances of contamination. On the other hand, an open flame heats air, which in turn goes up, leading to a circulation of air toward the area you are working in.
To test the effectiveness of different techniques, Ayelet tried a test where she compared pouring inside a biological hood, without flame sterilization and with flame sterilization. The results are inconclusive (small numbers) , but it seems that flame sterilization is a bit worse. It also seems that with A/C off the total amount of contamination was down. Surprisingly, even working inside a hood we got contaminants. This experience might be due to the fact that while waiting for the hood the agar cooled a bit (although it was in a sealed autoclaved flask).
To make sure that the source of contamination is the air and not the plates (that are supposed to be sterile), we tried today another round. This time we exposed half of the plates to U/V (using the Singer RoToR as a UV "oven") and the other half without exposure.
We need to wait another day to see how this batch fared. In the mean time, the number of plates we throw is alarming, and so we hope to solve these issues soon.
The troubles come in two different areas. I wrote before that we have contamination in plates. Well, we still have contamination in the plates we pour. Moreover, even when we do not have contamination, the plates start drying when we incubate them. For example, here the agar dried and then cracked.
In another plate, it didn't crack, but we see it drying toward one corner (the agar is thinner).
We know that the square plates are prone to water loss (much larger perimeter than round plates). For this reason we seal the plates either in plastic wrap (aka Saran wrap) or inside plastic bags. The two plates above were in the incubator for two days, one with plastic wrap and the other inside a plastic bag and both show bad signs of dryness.
We believe that part of the problem is the aggressive air circulation in our incubator. We called the company and they claimed that there is no way to turn down the fan speed. We now need to consider how to address this. One possible solution is to humidify the incubator by putting a container with water. If this will help, we might consider a more intelligent way of ensuring that it is humid.
The problem of contamination is a serious one. Since we want to grow libraries that will serve as resource for further examinations, having contaminants grow on the same plates is an issue. Since we first noticed the problem we talked with other yeast people in the building and they report similar problems. All of them recommended shutting down the A/C circulation before pouring plates. Apparently the air supply introduces yeast and bacteria from outside the building into the lab.
One of the debates in yeast sterile techniques is whether to use flame. On the one hand, working with a flame you sterilize the opening of the bottle before you close it, reducing chances of contamination. On the other hand, an open flame heats air, which in turn goes up, leading to a circulation of air toward the area you are working in.
To test the effectiveness of different techniques, Ayelet tried a test where she compared pouring inside a biological hood, without flame sterilization and with flame sterilization. The results are inconclusive (small numbers) , but it seems that flame sterilization is a bit worse. It also seems that with A/C off the total amount of contamination was down. Surprisingly, even working inside a hood we got contaminants. This experience might be due to the fact that while waiting for the hood the agar cooled a bit (although it was in a sealed autoclaved flask).
To make sure that the source of contamination is the air and not the plates (that are supposed to be sterile), we tried today another round. This time we exposed half of the plates to U/V (using the Singer RoToR as a UV "oven") and the other half without exposure.
We need to wait another day to see how this batch fared. In the mean time, the number of plates we throw is alarming, and so we hope to solve these issues soon.
Monday, January 4, 2010
Spectrophotometer, OD, and yeast growth
As you recall one of our new toys is a spectrophotometer. How does it work? You can check the entry on Wikipedia, but idea is very simple. A light beam of a known wavelength is passed through a small cuvette (fancy name for a container) that holds a fixed volume of sample. On the other side it hits a detector that measures the amount of light that has passed through the sample.
The amount of light that has been absorbed is informative on the content of the sample. Certain wavelengths for example are absorbed by poly-nucleotide and thus can report on DNA/RNA content. In our case, we use 600nm wavelength which is absorbed by cells. Thus, the amount of "lost" light intensity reflects the amount of cells.
As you might guess once you have sufficiently "dense" liquid, no light will pass through, and you cannot really learn much from the measurement. Thus, it is important to understand at what values the measurement is linear in the number of cells.
Today Avital and Ayelet tested the new spectrophotometer by performing serial dilution of dense culture of yeast cells to see how they vary. If we are in a linear range, than a dilution of 1:1 should reduce the loss of light (reported value) by 1/2. They report that they get a linear range from about 0.65 OD (units of Optical Density) and downwards.
On the cool side, we found out that the spectrophotometer is based on batteries and can be moved to the bench you are working on, which is really bonus.
In addition, they used the new incubator to perform a growth curve experiment. They seeded yeast cells in different media and grew them in the incubator.
Then they measured the OD every hour using the spectrophotometer. During exponential grow we expect that every XX minutes the OD will double. The value of XX is the doubling time of the yeast in that media and tells us how "happy" the yeast is there. Hopefully I will load some results in one of the next entries.
The amount of light that has been absorbed is informative on the content of the sample. Certain wavelengths for example are absorbed by poly-nucleotide and thus can report on DNA/RNA content. In our case, we use 600nm wavelength which is absorbed by cells. Thus, the amount of "lost" light intensity reflects the amount of cells.
As you might guess once you have sufficiently "dense" liquid, no light will pass through, and you cannot really learn much from the measurement. Thus, it is important to understand at what values the measurement is linear in the number of cells.
Today Avital and Ayelet tested the new spectrophotometer by performing serial dilution of dense culture of yeast cells to see how they vary. If we are in a linear range, than a dilution of 1:1 should reduce the loss of light (reported value) by 1/2. They report that they get a linear range from about 0.65 OD (units of Optical Density) and downwards.
On the cool side, we found out that the spectrophotometer is based on batteries and can be moved to the bench you are working on, which is really bonus.
In addition, they used the new incubator to perform a growth curve experiment. They seeded yeast cells in different media and grew them in the incubator.
Then they measured the OD every hour using the spectrophotometer. During exponential grow we expect that every XX minutes the OD will double. The value of XX is the doubling time of the yeast in that media and tells us how "happy" the yeast is there. Hopefully I will load some results in one of the next entries.
Sunday, January 3, 2010
Incu Incu Incubator
In the endless list of equipment that one needs to run a yeast lab, the incubator plays a special role. It is crucial for growing yeast! and what would a yeast lab if you cannot grow yeast. After much hesitation we decided to buy a large one that contains both shaking tray and space for revolving wheel. It arrived last week and today it was installed.
After some work (which turned out to happen in unnecessary hard way) we managed to bring the incubator to its designated place and land it there. The tech, after finding out the hard way that only some of the plugs in the wall carry electricity (!), managed to get the incubator working, and now we can keep 30C samples going.
One a less happy note, Ayelet retrieved the plates that we poured last week from the hotroom. Quite a few of them had nasty contaminations.
This means we have to go back to our pouring strategy and figure better sterile technique. We might also consider a sterile chamber for pouring as this is going to be a big issue for us.
After some work (which turned out to happen in unnecessary hard way) we managed to bring the incubator to its designated place and land it there. The tech, after finding out the hard way that only some of the plugs in the wall carry electricity (!), managed to get the incubator working, and now we can keep 30C samples going.
One a less happy note, Ayelet retrieved the plates that we poured last week from the hotroom. Quite a few of them had nasty contaminations.
This means we have to go back to our pouring strategy and figure better sterile technique. We might also consider a sterile chamber for pouring as this is going to be a big issue for us.
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