The amount of growth on a plate after exposure to UV radiation depends on the initial number of CFU on the plate and the radiation dose received.  To make sure that a uniform number of cells were used to inoculate each plate, we first amplified the number of organisms by incubating the soil samples in nutrient broth.  We then used a pipette to transfer 100 ?L of broth on to each plate.  All of the plates from all our sources were exposed in the same UV hood 47 cm from the 30 W UV bulb to make the radiation dose consistent.  Only the duration of exposure varied.

Our initial plan is outlined here.

We used 0.2 grams of soil from each of two sites from a semi-arid prairie in eastern Kansas to inoculate two tubes of media.  One tube contained plain nutrient broth and the other contained nutrient broth + 6.0% NaCl.  They were incubated at 30º C for 48 hours.

The air samples were collected by exposing three TSA plates and three NA + 7.5% NaCl plates for 40 minutes near the center of Hornbake plaza.  The grounds crew had just finished using string trimmers and there was noticeable airborne dust.  Very light wind from the south kept the particles from dispersing rapidly and blew the dust towards the plates down wind.

Six skin samples were collected by using wetted sterile swabs to directly inoculate three TSA and three NA + 7.5% NaCl plates from skin that is frequently exposed to sunlight.

The most accurate way of determining radiation resistance would be to determine the density of CFU in the media by serial dilution and viable plate counts and to measure the radiation intensity with a sensor.   We did not have access to a UV photometer and we had limited time to complete our project, so we tried to bypass the extra time need to do a viable plate count.   There was no way to control the CFU collected in the air samples and we did not feel that the skin samples were likely to yield our organism so we used those plates to qualitatively assess the D values by exposing the air and skin sample plates to 10, 30 and 60 seconds of UV radiation.  We would use the amount of growth on these plates to adjust the exposure times for the soil samples grown in broth.

We learned from consultation with an experienced user of the UV hood that a plate of susceptible organism, such as E. coli, is killed after a 3-minute exposure at 33 cm from the UV bulb.  Subsequent exposure times for our samples ranged from 2 to 16 minutes with two minutes acting as a nominal control.

After more research (7), we discovered that Micrococcus radiodurans was renamed Deinococcus radiodurans so we changed our media to TGY, a Deinococcus maintenance media.  The colony color of Deinococcus is enhanced by yeast extract.  Later, when our group received soil from three sites from the arid desert near Las Vegas, we used TGY broth to amplify the organisms in the soil.  The TGY broth tubes with 0.2 grams of soil from each desert site were incubated at 30º C for 48 hours in a shaker to maintain high oxygen levels.  TGY plates and NA + 7.5% NaCl plates with 100 ?L of broth were exposed to 5, 10 and 15 minutes of UV radiation and left to grow at 30º C for 7 days since Deinococcus grows slowly.

Only the name of our target organism had changed.  We were still trying to isolate Gram-positive cocci, appearing in pairs or tetrads, strictly aerobic, catylase positive and radiation resistant.

The formulas for the media used are listed below: (1)
 

Nutrient Agar + 7.5% NaCl
Standard recipe on pre-mixed vat of NA powder
75 g NaCl
1 Liter distilled water

TSA plates
    Available pre-poured

6% NaCl Nutrient Broth
    Stock tubes of Nutrient Broth (approx 8.5 mL)
    0.51 g NaCl

TGY Broth
    2.5 g Pancreatic digest of casein
    1.5 g Yeast extract
    0.5 g Glucose
    500 mL distilled water

TGY Agar
    5.0 g Pancreatic digest of casein
    3.0 g Yeast extract
    1.0 g Glucose
    15 g Agar
    1 Liter distilled water