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Pour Plates for Assaying Phage T4D

We will begin by explaining the media and procedures commonly used to assay coliphage T4D. T4D is a trival name for a phage which will grow inside cells of many strains of Escherichia coli. T4 was the fourth phage in a list of known phages once listed on a blackboard by some scientists. They then put a T as prefix and called the first one T1 and the last T7 was the last one on the list. D stands for Doermann who worked with this phage. Benzer, a professor at Purdue University, was given some T4D and worked with it many years. In his lab the strain evolved and was found to require tryptophan for binding to host cells. Therefore, the Benzer strain was designated as T4B. T4D does not require typtophan for binding to the host cells.

In these early experiments, we will use use Phage T4D, but you can use T4B or other strains. For some strains, minor modifications of the media and methods may be useful.

Top Agar and Bottom Agar

After a T4D phage particle adsorbs to a E coli B and injects its DNA, the DNA causes 200 progeny to be made. about 20 to 25 minutes after infection, the cell opens and oujt come the progeny. Each child phage is like the original and can similarly lyse cells. If this happens to a cell that is embeded in agar. the progeny particles difuse through the agar and attacks any E. coli cell it adsorbs to. Very thin agar lets the progeny difuse faster, but very thiin agar will spill and cause problems.

A nice solution has been found. First make pour bottom agar containing 1% agar in the plate to a depth of 0.5 cm. When the plate has cooled and is solid. Mix 3 drops of bacteria with 3 ml of thin TOP AGAR and pour this onto the bottom agar. Quickly tilt the plate in many directions so the agar covers the entire plate and set it on a level surface to cool. Then invert the plate and incubate 12 to 24 hours at 37 degrees. to one day. You will find the plate is covered with a lawn of bacteria.

Repeat the experiment but this time add a few phage particles to the 3 ml of top agar

Later you can try to modify the standard assay procedures to fit the lequipment and supplies that that you have. You may h

You must grow your bacteriophages on growing bacteria. Therefore you need to know something about bacteria for easier study of phages. Since you will be using many ordinary microbiology tools (petri plates, culture tubes, loops) and methods (streaking, isolation, media preparation), you should look over the bacteria site. Better yet you should isolate some bacteria and culture them. You can then use the bacteria you isolate as hosts for your bacteriophage projects.

While you can grow bacteria potato slices and other kitchen foods without buying anything, the extra speed from using agar and tryptone for phage projects means you might want to consider buying soe basic items.

Basic Items you may Want to Purchase or Beg

Tryptone- trypic digest of milk
Agar - plain agar use 10 grams per liter to get your plates to gel.
Table salt - pure salt from grocery; if solution is cloudy get pure salt.
Aquarium pump to aerate cultures; use screw to regulate air flow
glass

his website will provide a better understanding by giving more details and experiments which teach the details of bacteriophage life cycles, structure, genetics, and other fascinating aspects of phage biology.

There are many routine bacteriological methods which you will use in your study of phages. Please refer to my bacteria website for methods for isolating bacteria, preparing media, and other details. To work with phages it is a great help to have agar, tryptone, a few mL of chloroform, culture tubes, and petri plates. I will try to help you find substitutes, but it may be better to spent $10 on basic supplies. You may also find it worthwhile to buy a few standard bacterial hosts and a few standard phages and famous mutants if you can't find them free. It is possible to repeat some famous experiments without buying anything. If there is enough interest, I will try to make available some cheap phage kits for beginners for $10 to $25.

You may send private e-messages to Dr. Eddleman and he will reply, usually within 24 hours.


First installed January 1999      Revision #1 1999 Jan 4       indbio@disknet.com
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
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