24 October, 1996

Subject: Re: Journal 24 October 1996

Live from the Polar Duke in the Gerlache Strait

Location: 64.04S X 61.47W Wind Speed: 3.1 m/sec

Boat Speed: 0.0 knots Wind Direction: 117.7 degrees

Boat Heading: 220.5 degrees Barometer: 990.3 mb

Humidity: 79.8 % Air Temp.: -2.3 C

Salinity: 33.7 0/00 Water Temp.: -1.2 C

General Weather Conditions: One of the most beautiful days of the entire cruise. A perfect winter day, clear, calm, relatively warm, lots of great ice and a beautiful sunrise and sunset.

Today we filled and deployed the in situ incubation boxes for the last time, the science portion of the cruise come to a halt tomorrow. The boxes did not fail to provide some measure of excitement and entertainment today. There was an awful lot of ice covering the water. The longline that the boxes are strung on became wrapped around an enormous iceberg. It was quite a good show! First they tried ramming the iceberg with the ship to push it out of the way. This didn't work but it made some great noises. Next it was the MO boat to the rescue. Between the MO boat and the Polar Duke using bow and stern thrusters to move

the ice, the orange buoys finally cooperated and moved into formation. All of the gear was retrieved without further complications.

I have been working with Melissa (PhD canidate at Oklahoma State University) on some photoreactivation experiments. In this experiment we are trying to determine if photoreactivation is playing a role in bacterial repair after they undergo certain treatment.

We grew up a culture of bacteria. To do this we took a 0.1 ml sample of water and spread it on culture media in a petri dish. The culture media includes agar (agar comes from seaweeds) and various nutrients for metabolism in bacteria. Since the bacteria we were trying to

isolate live in the Southern Ocean we cultured them in an incubator set to just above freezing.

The organism we isolated was grown in a rich liquid media overnight. The next morning we were ready to set up our experiment. We used plastic bags called whirl-packs that hold about 50ml and made a set of three bags for each treatment: three of each-light, dark, acrylic and mylar. We make three of each sample to increase the amount of data and for statistical purposes. Each whirl-pack was filled with 50 ml of the bacterial culture and put into a UV transparent box on the helo deck. We got the whirl-packs out into the box by 0600. At 1200, the whirlpacks were taken out and put into their appropriate boxes. The packs labeled acrylic went into the acrylic box, mylar into the mylar covered box, light stayed in the UV transparent box and the dark packs were covered in aluminum foil and put into an aquarium tank in a dark room. All of these boxes have water flowing through them act as a water bath so that the temperature of the sample doesn't vary.

At 1800, we collected all of the samples and took them down into the starboard lab. At this point and at 0800 and 2000 for the next three days we did the following:

1. We prepared a direct count slide of the sample. This is done by taking 10ml of the sample and adding some iodine. The iodine kills the bacteria. Than something called DAPI is added to the sample. The DAPI becomes incorporated into the bacterial DNA and will fluoresce when exposed to the fluorescent light in a special type of microscope. This is how the number of bacterial cells can be counted.

2. 10ml of the sample is put into a centrifuge tube. The tubes are spun at high speed for 30 minutes. After 30 minutes the a small pellet collects at the bottom of the tube. The liquid portion is poured off and the pellet is frozen at -80 F. The total cellular protein will be analyzed for the amount of RecA protein back in Melissa's lab in Oklahoma.

3. All of the samples are serially diluted in sterile seawater

and then transferred onto plates (petri dishes with nutrient agar). The bacterial colonies will grow over the next several days. They will be counted and disposed of before we leave the ship. This is to test for the viability of cells.

In between our sessions the bacterial cultures were kept in the dark, therefore repair was probably taking place.

The purpose of this experiment was to investigate how the bacterial repair mechanisms, specifically, RecA protein, respond to different treatments of UV radiation. When Melissa completes the data analysis she will let me know the results. This what I think might happen:

Light Treatment- The bacteria in the whirl-packs that were left in the UV transparent incubation box should have the greatest amount of

damage. The DAPI counts should be the lowest. There are fewer bacterial cells because most of the bacteria dies due to exposure to UV radiation. The plates should should also show very little growth. Some of the plates that we have already analyzed have few or no colonies of bacteria. Apparently the repair mechanisms were not working fast enough to keep up with the damage. The later time points (the samples that we plated out two to three days after the initial timepoint) should have more colonies because more repair should have taken place. The RecA protein that is used to repair DNA damage is the big question mark. I can only guess and I would think that RecA would be highest at the beginning and decrease as more and more DNA was repaired.

Dark- The samples kept in the dark would probably have the highest DAPI counts and the most vigorous cultures after plating. Since they were put into the dark immediately after exposure to UV these would have the least damage and the longest time for repair. DAPI counts and plated cultures counts should increase with later timepoints. Again, the RecA protein is the unknown, I would expect it to be the higest in the first sample and decrease as there was less and less damaged DNA to repair.

Acrylic- Since acrylic screens out UVA and B, I would think that the results would be close to those reached with the dark treatment.

Mylar- Mylar screen out only UVb radiation, therefore I would expect results somewhere in between the light and dark treatments.

Margaret Brumsted

NSF Teacher in Antarctic