Figure 1 shows the air sample collected on NA + 7.5% NaCl.  After incubating for 48 hours at 30° C there were no colored colonies and the plate was to be discarded.  However, thanks to a second scan of the plates 4 days after plating, a small, pink colony was noted.  Because Deinococcus radiodurans forms pink/red pigmented colonies, we chose that colony for further investigation.  More research revealed that our target organism has a long lag phase and grows very slowly requiring up to a week on rich media for colonies to form. (5)

In Fig. 2, the Gram stain shows Gram positive rods without spores from the lack of clearing in the rods.  An endospore stain did not show spores.  Even though we were searching for cocci, we continued to test this isolate because some Deinococcaceae forms rods.  Pure colonies of the isolated organism were obtained through streak plating and used to grow in nutrient broth.  After incubation for 48 hours, 100 mL aliquots were spread plated on TSA and NA + 7.5% NaCl plates.  Figures 3-5 shows the growth of the organism 7 days after exposure to UV radiation for 5, 10 and 15 minutes.  On all plates (Fig. 3-5), there was growth of pigmented colonies, showing that they are osmotolerant and UV resistant.  Growth around the edge of the plates shows the shielding effect of plastic but there is growth away from the perimeter as well.  In comparison, figures 6-8 shows the growth of E.coli grown overnight on TGY.  E. coli is very susceptible to UV radiation as seen by the graph below in Fig 13.  At two minutes (Fig.6), there is very little growth and even less after four minutes (Fig. 7).  Eight minutes (Fig. 8), killed all the E. coli. A UV dose that kills 99.99% of E. coli has no effect on D. radiodurans.

 

A motility stab showed that the organism is non-motile and red-pigmented growth on the top on the agar shows that this organism is aerobic or a facultative anaerobe.  After growing these rods in TGY broth at 30° C in the shaker, the cells precipitated which also showed that the organism was non-motile. The organism was catalase and oxidase positive, all consistent with Deinococcaceae.

However, we had to conclude that our organism was not a member of Deinococcaceae because more research (5) indicated that the only member of this family that forms rods is Deinobacter grandis and it is Gram negative.  We tried to identify our organism but we were sidetracked by an incorrect assumption that the organism did not sporulate.  Another Gram stain from a seven-day-old colony showed a few spores (Fig. 10).

Using the tests results and observations available, we used a probability matrix for 44 Bacillus species, (8) to conclude that we had been examining Bacillus lentus.

In spite of our spending so much time testing the wrong isolate, we were able to isolate our target organism.  Soil samples from the arid desert outside of Las Vegas, NV were placed in TGY broth and incubated in the shaker for 48 hours at 30° C.  Aliquots of broth were spread plated on TGY and NA + 7.5% NaCl plates and exposed to UV radiation for 5, 10 and 15 minuets.  After seven days of incubation at 30° C, a small, pinpoint, very red colony appeared on the NA + 7.5% NaCl plate (Fig. 10) exposed to 15 minutes of UV radiation.  The Gram stain (Fig. 11) of this organism showed Gram positive cocci.  Not many cocci form spores and the ones that do, do not make red colonies.  This organism grows in aerobic conditions, tolerates high salt concentrations, is Gram positive, is slow growing and survived three times the killing dose of UV radiation for E. coli.  A definitive conclusion cannot be made because there was not enough time to grow the organism for more tests.  We do not know if this organism forms pairs or tetrads because the Gram stain was taken from solid media.  Differentiation between the Deinococcus species requires comparison of the fatty acids in their lipid membranes.


Highlights of Deinococcus radiodurans

The main feature of Deinococcus radiodurans is its great ability to resist ionizing radiation.  It has been called "Conan the Bacterium" and the Guiness Book of World Records calls D. radiodurans the "toughest bacterium" because it can survive 1.5 million rads (3000 times more than a human).  Ionizing radiation causes double stranded breaks in DNA.  About four such breaks are enough to kill a cell but D. radioduranscan survive over 200 double stranded breaks.  Its unusual compartmentalization of the cell is also another distinctive characteristic.  The cell is split up into four sections.  Another thing to note is that this bacterium has two chromosomes.  Inside each compartment there is a copy of each chromosome and plasmid tightly wound into a torus.  Between these compartments are small passageways.  "After about an hour and a half of repair within the ring, the DNA unfolds and migrates to an adjacent compartment where it mingles with the copy of DNA residing there. Then the "regular" repair machinery, common in humans and bacteria alike, comes into play - repair enzymes compare between the two copies of DNA, using each as a template to fix the other. Since the DNA has already been through one phase of repair in which many of the breaks are fixed, this phase can be completed relatively easily." (10)  The entire genome of this organisms has been sequenced and examination of its gene compliment shows that it does not possess any unusual DNA repair mechanisms.  It appears that the tightly coiled ring of DNA is responsible for its ability to put back together shattered DNA. (11)

    
Ecology:

There is no habitat on earth with a natural radiation flux as high as what D. radiodurans can survive.  It seems that the extreme radiation resistance is a byproduct if its adaptation to survive desication.  If placed on a slide and left to sit, dried out, over 80% of the cells will survive after two years.  Various Deinococcus have been found in arid soil, fecal waste of animals, sewage and radioactive waste.  They have also been found in airbourn contaminants, on human skin and in irradiated meat.  They have also been found in granite from Antarctica's Valleys which is similar to the conditions on Mars.  

Significance of Deinococcus Radiodurans:

One important role of these organisms in the environment is their possible use in bioremediation.  Even though there are already many microbes that can clean up toxic waste none can clean up radioactive waste and some heavy metals.  Work is underway to make strains of Deinococcusto cleanup radioactive waste in the soil and groundwater and to transform toxic mercury compounds commonly found at nuclear weapons production sites into less harmful forms. (9) Some strains are already being tested.

NASA is studying D. radiodurans to genetically engineer strains to produce various drugs that humans might need while exploring Mars.  It takes over 16 months to reach Mars with existing propulsion technology and it would be difficult to take all the possible, perishable drugs astronauts might need.  Engineered Deinococcus in suspended animation could be activated if someone became ill while treatment could start with drugs from a small supply kept on hand.  Work is being done on making a D. radiodurans able to be cultivated on Mars to see if it can survive the harsh, extraterrestrial environment.  (10)

Because of its potential to clean up the worst hazardous waste sites, it is important to learn more about this organism and find other similar species with characteristics that may prove to be more utile.  This bacterium may even be key to the exploration of other worlds.