Sunday, August 8, 2010

Making your own Petri Dish Medium by Joseph Parish

No one would argue the point with you that the commercial microbial media would produce the ideal growth environment for micropropagation techniques. These commercially purchased medium dishes provide the proper nutrients for optimum growth and sustainability needed for generation of tissue cultures. The major problem encountered here for the home laboratory is the high cost associated with these commercial units.

Since necessity is considered the mother of inventions we can actually create our own homemade gelatin plates for growing tissue cultures in at a fraction of the cost we would spend for the commercial dishes. The homemade medium formula that I am about to discuss can be poured into your selection of sterile Petri dishes as you require. This is a general formula and you should feel free to add your own selection of nutrients as necessary.

As an added bonus in the event that you find the commercial Petri dishes themselves to be a tad expensive, we can also resolve that problem for you. It is a simple matter to substitute foul muffin cup liners for Petri dishes. I have in the past even employed sterilized baby food jars as dishes with a degree of success.

Materials needed for the Medium

  • 4 crushed beef bouillon cubes
  • 4 cups of distilled water
  • Foil muffin cups liners
  • Muffin pans
  • Measuring spoons
  • 4 envelopes of Plain unflavored gelatin
  • 8 teaspoons of Sugar
  • A selection of Ziploc bags

To begin this formula take a medium size sauce pan and mix the 4 envelopes of plain unflavored gelatin together with four cups of distilled water. Add to this 8 teaspoons of sugar and 4 finely crushed bouillon cubes. Slowly bring the mixture to a boil being sure to stirring constantly.

Prepare the foil muffin liners by placing them into the muffin pan. Allow the mixture to cool slightly and then fill the foil muffin cup liners 1/3 to ½ full with the hot gelatin solution. If you are using commercial Petri dishes fill them in a similar manner.

Allow the gelatin to cool to a solid at this time. Refrigerate if you so desire. After the gelatin has become a solid remove the foil muffin liners from the pans. These liners can be placed into a Ziploc bag and refrigerated until needed. Remember at this point they are sterile and you should not touch the surface with your hands.

Use the medium up with 2 to 3 days after making it. The above formula will make anywhere from 25 up to 30 dishes.

Copyright @2010 Joseph Parish

Friday, May 21, 2010

Patents relating to Plant Cloning by Joseph Parish

So you just acquired some beautiful variegated Hostas and are considering propagating them for resale at the local flea market. Perhaps you should think this decision over carefully. Chances are very good that those Hostas are more then likely protected by some sort of copyright or patent.

When cloning any sort of plant one should be careful as to whether the plant in question is a nursery plant that has been legally patented or trademarked. Usually if you take a close look at the plant department in your local agricultural store you will notice a vast number of plants with labels which say “patented” or “trademarked”. At first you just might think this to be one of the silliest things you have ever seen, however think again.

The purpose of patenting plants is to encourage creative designs within the horticulture industry. For many years the plant industries has struggled with issues relating to setting appropriate prices for their research and development efforts. It is believed that the process of trade marking company’s plants provided a greater value for those plants in the marketplace. With this monetary thought in mind the breeders and raisers of plants will expend all efforts towards creating the perfect plant. Without this patent protection it is likely that there would be no new and novel innovation seen within the horticulture industry.

The process for patenting a specific plant is similar to that used for any other sort of item, likewise the rewards are similar. Each time a consumer purchases a patented plant a royalty is provided to the creator of the said plant.

Patenting is a rather costly venture at best and it is likely that one could spend thousands of dollars in an effort to protect their interest. Even then there is no guarantee the consumer will like the particular variety offered thus it is possible that the company will not break even for at least 20 years.

Like any other type of patent, the plant patents prevent anyone from reproducing the species legally unless the proper royalties are paid. These methods of reproduction include cuttings as well as tissue culture germination. You may be wondering how you can determine if a plant is patented. First look at the tag and see if you find a patent number on it. Keep in mind that if the plant is identified as patented you must obtain a license in order to reproduce it.

There is a big difference between a patent and a trademark. A trademark tends to aid the consumer in identifying specific varieties associated with a certain company. This is similar to what the “Big Mac” is to McDonald’s. A trademark only protects the plant’s name and provides no protection what so ever for the cultivar itself.

An interesting side note here is that if you happen to purchase a plant that is trademarked only you may propagate it asexually by cuttings. In fact you are even free to sell the plants but you can not call the plants by the trademarked name.

Although the Patent Office estimates that appropriately 60 percent of the new plants each year ever become patented, you would be best served to approach these issues with a delicate pair of gloves. Patent infringement can become very tacky and drag on for any number of years. Caution is the key word here.

Copyright @2010 Joseph Parish

Sunday, July 19, 2009

Agar Substitutes

Agar is one of those staples of the tissue culture industry we generally can not do without. Agar is a gelatin like substance which is derived from common seaweed. You can find it used in many old fashion and modern desserts such as those found in Japan and are known as anmitsu.

Agar has traditionally been employed universally throughout the world as a medium by which cultures of bacteria or fungus could be readily grown. Usually this product is sold in bulk quantities as a powder and mixed with water for use as it is required. Although agar is the product of choice there are always active amateur scientists that are looking for alternate items to use. I personally have used common grocery store purchased unflavored gelatin as a suitable substitute for agar. It worked very well and I was able to culture quite a variety of plants from it.

Several years back I heard of an individual who did some work on the use of sand in place of actual agar. Naturally I had to try this method out and also found that it too worked as expected.

The procedure for using the sand is to start with the type of sand that is generally employed for a child’s sand box. It is frequently referred to as play sand. Prior to using the sand in any sort of tissue culture protocol you should wash it well to remove any visible contaminates. Next place approximately one ounce of the sand into each of your culture flasks or baby food jars if you are using them for your cultures. Seal the top of the jars with a strip of aluminum foil and place it on a cookie sheet. Your oven should be set for 350 degrees and you should let the jars remain in it for at least one hour.

The jars should be cooled and approximately 30 ml of sterilized medium should be added at this time. The key is to add enough of the medium to saturate the sand but not so much that you cause your explants to float around. Finally you will want to aseptically close your jar with a sterilized lid. I boiled mine and then place them in the oven for a period of time in order to kill off any contaminates.

During the course of your tissue culture experiments if the culture starts to dry out you should then add some additional sterile medium to the container. Naturally you will want to add other items such as nutrients, etc to help with the explants growth. You should at this stage of the game use the sand as if it were the actual agar. If you would normally add a specific ingredient to your agar then add it to the sand.

You should have no problems using the sand in place of the agar and I personally have seen some very interesting results while using it.

Copyright @ 2009 Joseph Parish

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