Just to demonstrate the results of yesterdays demo, here are two plates that we copied yesterday from the same 96-well liquid plate, once into 386 and once into a denser 1536 colonies format (by four iterations).
As you can see, the "holes", we left in the original plate appear in the same places in the replicated ones, except that in the 384 one it is a 2x2 colony hole and in the 1536 it is a 4x4 colony hole.
Another plate is one we copied from a "lawn" of yeast. That lawn dried up in the incubator (it is much drier there than we expected), and so the agar level was uneven. As a result we got stricking colony pattern.
The impressive part is the very uniform and nice colony patterns we see.
You might remember the big crate that we received a while ago. Today Ian from Singer Instruments (UK) showed up to unpack the box and install the Singer RoToR.
At this point we needed to lift the device onto the bench. As an example of good design, it had comfy handles attached to both sides, and so four of us relatively easily lifted the 120kg device up and onto the bench.
Here is a zoom on the handle.
Notice the silver globe. This is one of the important features of the device -- an integrated beer bottle opener. In recent years they added an option also to get instead a corkscrew. We decided to go with the traditional choice :-)
So what exactly is this device all about. The job of the Singer RoToR is to copy multiple colonies between plates. Either agar plates or multi-well liquid plates.
As you can see it has a movable arm (parked on the right here), locations to put plates (color coded, black, blue, red, yellow, and green), and two drawers. On the drawer on the right it can pick up sterile "pads" which are essentially plastic pincushions. It then uses the pins to copy cells from one plate to another (or from one to many, or vice versa). It disposes of the pad in the left into a waste drawer.
While this sounds simple enough, the beauty of the machine is that it is very precise and can copy very dense plates (upto 3074 colonies on a plate) in a precise manner. In addition all the work is done in a sterile environment. When the protective wall is in place, all the operations are done in a space that is U/V sterilized. The device opens the lid, does the copying, and then closes them.
The best way to see this, is to watch a movie. In this movie we asked the robot to copy from 96 wells onto an agar plate in 384 colonies format with four repetitions of each original well.
The result of this process is a dense array of colonies on the agar. In fact, we can see them if we look carefully. Here Drora (a local yeast expert from another lab) shows off the resulting plate.
As part of the ongoing efforts to push the renovations, we need to go over the plans and make sure they fit and do not contain unnecessary elements.
Today we met Itzik and Eyal, who are in charge of the renovations, to go over the electricity plans from the electricity consultant.
We spent an hour and half going over detailed plans, marking missing outlets and removing superfluous ones. We managed to reduce the 200+ sockets (!!) to a more reasonable number. Hopefully this will translate into some savings in the cost of the electricity works.
An alert reader pointed to am error in a previous entry. Although absorbance is one source of optical density, the situation in our case is different. To quote
A solution absorbing at 600 nm has a blue color, the cells do not absorb light at this wavelength. You actually measure the light scattering caused by the cells. The scattered light is not detected by the spectrometer, this is the reason that you measure OD >0.
Thanks!
An additional note regarding the spectrophotometer is that we compared the readings on ours to three others in the institute. To our surprise we discovered that one of them has the same reading as ours, another is slightly off, and the third is way off (almost half the value we measure). We are not sure what to make of this, but it is a point of concern.
As you may recall we are trying to get renovations for a permanent lab. Here are few updates from Itzik who is in charge of the project.
We have detailed plans for A/C, water, and for electricity. The A/C + water plan is within the budget outline. The electricity plan turned out very complex (in terms of number electrical boards, panels, circuits and such) and thus expensive. Itsik is working re-examine our specifications to see if we can make some concessions toward simplifying the details.
The actual renovation (breaking walls, building walls, and such) plans have are waiting for bids from contractors. Tomorrow there is a planned site visit for interested contractors. This means that soon we will see how much they will cost.
The furniture quote is in, and it is about the expected price.
We are still trying to finalize the complete budget for the project and are in contact with the university management to sort out differences between the expected cost and the actual budget.
I am trying to be optimistic that the process is continuing although so far no actual work has been carried out.
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.
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.
