Research Methods: Single Spore Isolation

Friends, family, and colleagues,

As part of my blog, I’d like to share with you, my readers, what I’m actually doing now that I’m settled into the institute. For the sake of my research, I won’t be discussing any results. That’s for once the seminars begin. For the sake of your sanity, I’m also keeping the methods very general, since I want this to reach to everyone. 

In plant pathology, it is said that the journey of a thousand experiments begins with a single spore. Ok, no one in my field says that, I just needed a cheesy introduction. 

The first step when I arrived to JAAS was to take the collection of isolates and culture them from a single spore. This technique is appropriately called the single spore isolation. This is done with most fungi for various reasons. The most important is uniformity. If you have a pure culture (that is, only one organism growing in your plate), there is still no way to ensure that the one organism is a single lineage or if it’s a diverse population. You could have 15 different individuals of the same organism in there, and that could cause some inconsistencies with experiments. Sometimes the fungi is grey or white, next time it’s green! 

This is NOT a pure culture. Some contaminants are clearly present

This is a pure culture. There's only one organism growing in there.
But is it all the same organism or a population of the same organism?

First step: Get spores

It may surprise you that in order to do a single spore isolation, you need spores. Now, that can be challenging for some fungi, since they don’t make spores. I had a labmate who worked on a pathogen called Sclerotium rolfsii that does not produce spores. There are some methods that can be used if that happens. However, that’s a whole different topic that I’m glad to avoid.

For my fungus, it produces two types of spores, asexual (called conidia) and sexual (called ascospores). Each kind of these spores is produced in a separate structure. Conidia are borne on a structure called a pycnidia, whereas ascospores are borne on either a perithecia or a pseudothecia (don’t get too hung up on the names, it has to do with the shape and structure of the fruiting body). For the sake of simplicity, conidia are the ones I want.

This a perithecium with ascospores emerging.

Pycnidia with conidia being released.
Each spore is a clonal copy of its parent

So how do I get these conidia?

The fungus has to be producing these pycnidia in order to get spores. There are a few methods that I use to get said spores. First, adding light to the growth chamber encourages the fungus to grow. Just as plants respond to light, so too will fungi. It isn’t essential for their growth, but it triggers certain responses, including pycnidiation. Adding in UV light also encourages pycnidia to form. Another method is to grow the fungus on a minimal media. If the fungus runs out of food, the next step is to make spores to find a better environment. 

I have spores! So now what?

So once we have spores, we have to count them. The best way is to take some of the spores from a petri plate and scrape them into some sterile water. At high enough concentrations, you can actually measure optical density (i.e. how “blurry” the spores are in water). However, there’s another way if fungus doesn’t produce billions of spores. There is a device called a hemocytometer (sometimes spelled with an "a" hemacytometer). Originally, it was produced to count red blood cells. It is also used to do sperm counts in both animals and humans. Fellas, if you ever go in for fertility testing, there’s a good chance that one of these will be used to assess your sperm.

Thanks Wikipedia!

So, the spore suspension is placed on the hemocytometer, and on the surface, there is a grid pattern. That grid pattern is viewed under a compound microscope. Within each grid is a known volume, typically 0.1µl. That’s one microliter, take your liter of cola and divide it by one million, Farva. We count the number of spores and from that concentration, we can adjust out suspension of spores to a desired amount. I’ll spare you all the math.

A typical grid pattern on a hemacytometer,
The red area (letter A for color blind) is 0.1ul in volume

I did the math! Now what do I do?

Once the spore suspension has been adjusted the suspension is then dropped onto another petri plate. For the sake of simplicity, I’ll go through an example. Say the suspension is adjusted to 1000 spores per 1ml of water, each one of those little droplets is going to be 1µl, which is 1/1000 of 1ml. In theory, there should be 1 spore within that aliquot. This is repeated several times as there is a lot of variance within that suspension. The conidia often come out of the pycnidia in a ribbon and are held together by a gel-like substance; adding a detergent reduces the gelling. Often, it’s called a “spore horn”. Also, this assay does not distinguish between viable cells and dead ones, so more chances are better chances. After the suspension has been dropped, the plate is left alone for 24 hours. 

If you mess with the spores, you sometimes get the horns.

I waited 24 hours, what’s the next step?

After the 24 hours, the plate is observed under the microscope and the spores will have germinated. Now comes the tricky part, an aliquot containing exactly one spore must be found. Given the variance, it’s not uncommon to find one aliquot with 6 germinated spores and the next aliquot to have none. After a bit of patience and luck, one perfect aliquot is found! We use a cork borer to carefully cut the agar chunk out of the original plate and that chunk is then plated onto a fresh, clean plate. After 48 hours, the single spore will have grown to the size where it is detectable to the naked eye. 

I successfully single-spored, now what do I do with it?

Now you can run all of your experiments! 

This is only the first step in the research journey. From start to finish it takes about 3 days to complete, assuming the culture is already creating spores. For the sake of consistency, nothing can be done before the single spore isolation, so it becomes a crucial task if working with fungi. I plan on generally describing some of the other experiments I’m running in the lab. I know I used a lot of technical terms, so feel free to ask for clarification in the comments.

I hope you found this interesting. If you have questions, leave them in the comments.

Mason