Light box

As you may recall from early posts, we use agar plates for growing and manipulating libraries of yeast strains. We also keep "replicating" copies of these plates to maintain a "live" copy of the libraries.

One of the potential problems is that we mis-copied a plate, or introduced a contamination. One way to check that is to examine the plate pattern. Basically, each library plate has different set of empty spots. This means that we can easily identify plates by these "finger prints". Moreover, a contamination would fill the empty spaces by new colonies. 

Thus, it was clear early on that we need a tool for taking pictures of plates and recording/analyzing the colonies. We could use the standard gel camera we have in the shared room, but that was both unwieldy and exposed our plates (and us) to ethidium bromide (which is used by other labs for dying DNA on gels).

Instead we aimed for a dedicated station for imaging plates. We found some solutions by commercial companies. These however, were very very expensive. And so we decided for a Do It Yourself solution. We consulted with the local electrician, and helped by Meretz, a retired electrician built our own solution.

The box is an old wooden box that was originally for a nikon microscope. The local workshop built us a device to hold the plate in a precise location. Since the plate is asymmetric this ensures that the images are of the correct orientation. We bought a Nikon camera that can have external power supply and remote operation. It is an overkill for the image quality we need, but it does work without battery replacement or manual operation.

As a light source, Meretz suggested using a led strip. He built these leds below the plate holder. This meant that the image colonies showed up as silhouettes.

Image with background light - colonies are dark circle on light background

I wrote a simple program that identifies colonies, their location and their size (which is useful for estimating the fitness of different strains). Eli Peker, who worked as a summer student, wrote a program for uploading images and storing them in a database. This interface allowed different people in the lab to keep track of libraries and compare to previous versions.

Recently, Amit (who deserve his own post soon) suggested we also examine the color and texture of the colonies. This appears to be a known phenotype. However, since the light source was below the colonies we could not use our box. We again contacted the local electricians who installed additional led strips for forground lighting.

"Disco" foreground lights

As you can see, the forground lights turned out to be a bit excessive ("disco" as Amit put it). Now we could make out the color and the texture of the colonies.

Same plate, with foreground lighting

However, now some of these disco lights are reflected in the image and look like small colonies (top part of the image). To deal with this, we added our own DIY screen (paper) to soften the light. 

Foreground lights with diffused screen

Which lead to removal of the annoying reflections on the agar.

Same plate, with diffused foreground lighting

Notice that in this plate there are few white colonies (two small ones) and some that are more yellow. These differences are easier to see if zoom in on a region of the image.

Moreover, some colonies have a different texture, these differences are enhanced when we examine a gray-level version.

For now we use the lightbox to record the plates with different strains. We didn't yet write an automated color classifier (relatively easy) or a texture classifier (a bit harder).

Window sills, cables, and agar plates

News from today.

Our alumnium sub-constructor came in yesterday and installed the two missing windows, and also installed a new nice window sill below them.

The electricity sub-contractor came in last week and finished laying down all the electricity cables. We now have thick green cables running on wireframe shelves along the ceiling and then entering the large panel of tubes to make their way down to the level of the furniture.

In addition they left cables hanging from the ceiling, these will lead into the light bulbs.

The same guys also installed airvents to take airs from the building "fresh air" system into the room (and filter it on the way).

The building people closed off all the tubes and already painted most of them. They also were building a drywall panel to form a wall above the windows.

In addition, you might remember Drorit, who is helping us with chores. Today Ayelet start teaching her to prepare agar plate. She proved to be a very quick learner, so soon we will have help on day to day lab chores.

And as a nice bonus, we got to see a colorful sunset today.

Some lab updates

So its been a while since I had updates from the lab. The main reason is that things are going slow and I don't spend that much time there.

Nonetheless, few updates and some images. We finally got our very own ice-buckets (for some reason our lab-ware supplier had these in backorder for two months).

We are also testing a PCR machine. We got an offer to buy a machine from a reletatively unknown vendor that has good specs. To get a sense for it, we got a demo machine installed in our lab.

One of the nice features of this PCR machine is that has a detachable block, and so it can be either a 96-well block (for a full plate PCR or many reactions) or two 48-well blocks, allowing to run two reactions in parallel.

Next week we will get another machine for a demo, and then we will decide which one to keep.


As part of our work we often have to grow bacteria or yeast in colonies. When starting with very small amount of cells that can grow on the media, we often want to spread them on the plate. The common way of doing so is with a bunch of sterile beads. You pour some onto the agar

and then add the bateria dilluted in water (or liquid media)

and shake well.

After wards we discard the beads into a collection bottle for recycling, and move the plate to incubator for overnight growth.

Starbrusts

The track to learning how to use the tools in the lab leads us with surprising turns. For example, the Singer RoToR is something we thought we understood and start using with reliability. Every time we believe we sorted out the problems we run into new one.

Yesterday, Ayelet decided to copy the GFP-tagged library so that we can have fresh copies to use for picking up strains. What worked fine the previous day, suddenly decided to cause problems. Once again the de-liding arm didn't pick up a plate lid. Unlike the previous event which lead to a cascade of escalating problems, this time the resetting worked fine. But only on the forth attempt (with a complete reset after each one) things went back to normal.

In previous rounds of copying, we had problems with the colonies at the corners of the plate. Our thinking was that the agar is starting to dry and thus is no longer flat. To avoid this problem, Ayelet increased the pressure of the tips onto the agar. This solved the corner problem, but lead to a new one. Since we used fresh plates, the extra pressure on the target plate caused the pins to puncture the agar. Instead of putting cells on the surface, we now have them in small craters. Today, after some growth the colonies look more like starbrusts than the disk shape they should take.

One worry is that copying from these plates will be difficult since the colonies are not growing upwards but rather inwards.

MasterClave (continued)

In the last entry on the MasterClave we skipped the plate pouring step. Today I managed to get some pictures (with the help of Avital) and so we can complete the story.

As you remember, we "cook" the media in the MasterClave. Once the agar is ready, can hook in a hose that sips into the hot agar mix. The hose is pre-sterilized and so does not introduce contamination.

The hose is then hooked into a pump that allows us to pump the media through the hose. The pump is calibrated so that every press on a foot pedal pumps a desired amount of agar.

This means that pouring a stack of plates is done rapidly. We do not need to open the plate completely (for comparison recall that in manual pouring we remove the lid) , and moreover the amount of agar in each plate is uniform and delivered at a constant temperature.

MasterClave

I mentioned briefly this device when it arrived two weeks ago. Today we used and this gives me a chance to tell you more about this device.

When growing microorganisms (bacteria, yeast, etc.) we usually either use a "wet" environment (media) or a "dry" one, where we grow them on agar basis - a jelly substance that contains nutrients. As I learned today, agar is made from seaweed and was originally used to make gelatinous food in the orient, so it is in an essence, jelly.

To make agar plates, we need to prepare a mixture of water, glucose (sugar), nutrients, and, of course,  agar. Each of the components is weighted to make sure we match the recipe.

This mix is then heated to achieve two goals. First, sterilization to ensure that no other microorganism gets to party on the good stuff. And second to melt the agar. When the liquid is cooled down, the molecules will form a network that will stabilize the jelly.

Since we plan on making many agar plates, we equipped ourselves with the MasterClave. A device to make agar plates a piece of cake.

It consists of a pot-like internal chamber where the agar mixture is put. This chamber has an internal blade for mixing the mixture.
 

Once the mixture is in the pot, we install it in the machine. It is secured into place.

After that we can close the lid, and secure it, much like a pressure cooker.

Now the MasterClave heats the mixture to programmed temperature and pressure, and then lets it cool to a pouring temperature --- one that is not too hot to damage the plates, and not to cold enough for the agar to solidify. At this stage we can also add temperature sensitive ingredients (such as antibiotics) and have them mix into the melted agar.


Connected to the MasterClave is pump that allows us to deliver pre-configured amount of agar in each press on foot pedal. I missed the pouring step today (committee meeting), but the results are a nice stack of plates. These need to cool down for a day, and then can be packed and stored for use.

Today we made only a liter of agar, but the MasterClave can make up to 9 liters, which allows for a much more substantial amount of plates.

On an other note, Naomi visited the lab today. We immediately put her to work and had her load a gel. She did not hesitate and did a marvelous job at it.

Quick update(s)

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.

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.

Singer followup

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.

Pouring plates

So today Ayelet and Avital started media preps. Very exciting first steps toward growing our own stuff. We now have our own vintage of YPD, LB, and SC media.

In addition they prepared YPD with ager for plates. I helped pouring some of the agar plates. Yey!

Now we wait to see if these keep sterile over the next few days, or do we need to improve our sterile techniques.