Wednesday, July 8, 2009

Fern cultures

Several people have written to me for information relating to fern spore cultures. I am not an expert at this phase of micropropagation and have only a vague familiarity with this type of cultured plants but here is what I do know. Hopefully it is of use to you.

Although there are currently at least 20 different species of ferns which are found here in America, I would venture to say that the same process could easily work with any of them. So without any further wait here is the information.

The fern spores are usually collected in the late autumn from mature fern plants. We will use the domestic “Platycerium superbum” as an example. The spores would naturally have to be sterilized and this is usually accomplished by way of a centrifuging technique. The spores are quickly shaken in a test tube using sterile water with a couple of drops of Tweeen 20 added. If you have access to a centrifuge you could place the tube in it and rotate it at the rate of 2800 RPM for 5 minutes. This will settle the spores to the bottom of the test tube. Next, discard the liquid in the tube and replace it with clean sterile water. Once again shake and let the tube sit at room temperature for a 24 hour period.

The following day re-centrifuge the tube and discard the water again. Now add 10ml of common household bleach. Let the bleach remain in the test tube for approximately 5 minutes. Finally, centrifuge again and discard the solution and place the aseptically clean plant spores into a media as described below.

These spores you will note were planted into the media without a final wash with sterile water. The cultures were then incubated for 16 hours. The temperature should be kept at about 25 degrees C.

This is a slow growth method and your spores will start to germinate and produce prothallus in about three month’s time. You will likely need to sub-divide these early cultures as they start to form. In a future article I shall cover construction of a home made centrifuge as well as an incubator.

This media should be successful for just about any fern cultures that you wish to try it on.

300 mg MS salts
500 mg NaH2PO4
5 mg Inositol
20000 mg Sucrose
8000 mg agar

Copyright @ 2009 Joseph Parish

Tuesday, July 7, 2009

Using the Microwave on your Media

Experimenters are always on the look out for unique and unusual methods by which they can sterilize the particular media that they are employing for their tissue culture work. The time honored home lab process is by the use of a home pressure cooker. Although there is one easier and simpler method that is often overlooked – the use of the family microwave oven.

I have previously covered the use of a microwave oven to sterilize the plastic containers that are used for the process but in this article I am going to provide you with a new insight on using it to sterilize the media itself.

The procedure is essentially very simple. You merely place the container of your selected liquid media into your microwave oven and heat it to just below boiling which should be approximately 4 minutes. What could be easier? Keep firmly in mind that should you forget and allow the media to boil for more then several seconds you will have one heck of a mess to clean up in your microwave.

Several major variables that will affect your procedure are the amount of the media that you will be sterilizing and the maximum power output from your particular microwave. It will more then likely be necessary for you to do a bit of experimenting at first by using different power settings and time for your particular containers. You could fill your vessels with water during your experimental stages in place of actual media. It is usually a lot cheaper using a mixture of water, a little agar, 2 percent sucrose and some tomato juice when doing your tests. The tests if accomplished properly can teach you a lot of valuable information that relates to the problem of contamination and how you best can eliminate it.

You will want to be especially careful if your microwave oven has what is known as “hot spots”. In this case you may need to move your media round slightly to ensure that it sterilizes all of the intended media. You may actually end up increasing your heating time but however you look at it you are still saving a lot of time over the conventional pressure cooker method. A very useful item that you may decide to purchase for use in your microwave is the microwave pressure cooker called the "Micro-Go-Round". It is a microwave turntable made by Nordic. This does an excellent job of microwave tissue culture sterilization. If you are using baby food jars it can sterilize the media right in the jars.

Although this will work well for the home lab you would not want to use it if you eventually go professional as it is simply too small. The unit is 7 inches in diameter and 4 inches high. That’s about 87 cubic inches of useable space. Unfortunately as I mention you could not place too many Petri dishes or tissue flashes in this little marvel. You could place a small plastic rack of some sort in the bottom of it and include a bit of water in order to generate some steam which would exceed 100 degrees C.

Do not place a cap on your containers nor use any sort of aluminum foil as a cover. You can easily use some plastic caps on your culture tubes or a single hole stoppers on culture flasks.

You may be interested in knowing that the process has a tendency to work very well. Media that has been sterilized in this manner has not become cloudy as a result of bacteria and it shows no signs of developing mold even after several weeks of sitting.

The truth of the matter is that you may perhaps contaminate your project as a result of handling your explants as opposed to the media itself being contaminated.

Copyright @ 2009 Joseph Parish

Monday, July 6, 2009

Don’t be a failure statistic

Failing in tissue culture experiments is very easy if you are slack in your procedures and careless when you do your initial explants setups. Some of the major reasons that have been cited for unproductive cultures are as follows:

1. Don’t select a difficult plant to clone initially. Leave that “Hemerocallis” alone and select one of the simpler plants to duplicate. You have plenty of time to work your way up to the more complex plant cloning procedures so take your time.

2. Don’t try and rush to get your explants out of their sterile home and into the open. Give them plenty of time to get accustomed to a new environment. It usually will take several weeks to condition your new explants to the open air life so properly pre-treat them in an enclosed system. Failure to heed this advice could cause contamination of the plant that can not at all be eliminated by the mere surface sterilization and ultimately lead to the destruction of your plant.

3. Make certain that you follow the correct procedures to ensure that you do not induce any sort of contamination. Following all advised procedures that pertain to sterilizing instruments and cleaning the various cutting surfaces properly. Make sure that you use your glove box as it was designed to prevent contamination while you are working. I have actually when first becoming involved in micropropagation used my glove box to grow several of the explants in.

4. Don’t try and rush the procedures. If a specific time is stated for leaving your explants in something such as detergent then by all means make sure that you adhere to that time specified. These are not arbitrary time frames but have been established by many trial and error experiments.

Growing tissue cultured plants can be a lot of fun and is an excellent way to spend your spare time so don’t become one of the failure statistics and make the usual mistakes that most newbie’s tend to make. When in doubt ask someone with a bit more knowledge as it will save you a lot of heartache in the long run.

Copyright @ 2009 Joseph Parish

Sunday, July 5, 2009

Sterilization of TC Containers by Microwave

I generally purchase my tissue culture containers by the case and it goes without saying that they can get rather expensive. The particular ones that I like to use are made out of plastic which tends to make them slightly more affordable then those created from pure glass.

I am especially fond of recycling whenever it is feasibly possible and it seems like such a waste to toss these perfectly good containers into the trash when I am finished with them. In the past the major disadvantage of the plastic containers has been the inability to properly decontaminate them and to be able to reuse it for additional cultures. By being able to do so greatly cuts the cost of the tissue culture containers down to a minimum.

With this thought in mind I decided it was time to reuse these vessels once again. Naturally, they would need to be in a sterile condition in order to prevent contamination of the next series of explants that would be developing within them.

It obviously seemed like a very good idea to use the microwave oven for this exact purpose. The microwave can effectively kill any type of bacteria present and successfully decontaminate the plastic containers for further tissue culture work. Tests have shown that our common home microwave is well capable of decontaminating these containers with only a 3 minute cycle.

Initially cleaning the plastic tissue culture containers is fairly easy. One merely uses their usual dish detergent and washes them well followed by a rinse in fresh water. Generally, this will take most of the residue out of the containers and provide you with a clean container for reuse. It is the sterilization of the containers that used to pose you the greatest difficulty however with the use of the small microwave ovens as described above you are well on your way to an effective means of sterilizing these containers quickly and inexpensively.

So as a home lab user it is now possible to save a lot of money with not having to repurchase your cloning vessels after each and every batch.

Copyright @ 2009 Joseph Parish