DREAMS-21/22

BIOLOGICAL SAMPLING

Recently, scientists have begun to analyze the biological composition of the upper troposphere and lower stratosphere1

Bacteria, fungal spores, and other microscopic biogenic materials could play an integral role in climate, cloud formation2, and other weather phenomena

Certain scientists have observed possible bacterial adaptations to the high-UV, low pressure, and low nutrient environment of the upper atmosphere 3

…leading them to speculate whether or not these adaptations allow the bacteria to survive independently, forming a novel ecosystem, or “microbiome”

ATMOSPHERIC BACTERIA

In 2012, students at Whitecastle University cultured stratospheric bacteria B. stratophericus to create a biofilm that generates electricity5

A NASA scientist has noted that the adaptations of stratospheric bacteria could be used as a model for terraforming new planets4

Human health implications: the majority of microscopic matter in the atmosphere comes from desert dust, and desertification and development are contributing to this load. Atmospheric pathogenic microbes have the potential to cause numerous health issues6

POSSIBLE APPLICATIONS

Remove agar plates from refrigerator. Allow plates to warm to room temperature for about an hour before

taking the sample. Swab area of interest with sterile swab or inoculating loop. Immediately transfer to agar plate by sweeping the loop/swab across

the surface of the agar. Streak back and forth to distribute bacteria evenly.

Replace cover on dish, tape closed, and label each dish with the bacteria source.

Place upside down in incubator, at 90 F. Incubate for 24-48 hours, checking daily for growth.

COLLECTING SAMPLES: ON THE GROUND

Wearing steri le gloves, prepare 9 nutrient agar plates according to directions included with agar.

With a permanent marker, label the cover and bottom of each agar plate. Label three “fl ight”, three “control”, and three “site”. Number each plate in a set 1-3. Seal them tightly with tape. Leave the control plates in the lab in a refr igerator.

Bring the six plates labeled “site” and “fl ight” in an ice-fi l led cooler to the launch site. An hour before launch, mount to the top the boxes with Velcro the 3 plates labeled “fl ight”.

At some point, remove plate labeled “site 1” from the cooler, and let sit for one hour. Unseal and open for 30 minutes, near the “fl ight” plates, for 30 minutes. Minimize the amount of contact with team members.

At the last possible moment before launch, unseal and open the plates labeled "fl ight".

Immediately after the bal loon lands on the ground, seal the fl ight plates.

For the fi rst bal loon landing, place the precooled “site 2” plate near the landing point and let sit for several minutes. Do the same with the “site 3” plate at the second bal loon landing.

Place al l plates back in cooler or storage. Incubate al l plates at 37°C overnight. Check the every day for growth. Incubate for up to 48 hours.

COLLECTING SAMPLES: IN FLIGHT

After visible colonies develop, dip a steri le inoculating loop into a colony, and spread it across a microscopy sl ide—adding a drop of clean water as needed—to form a thin fi lm over the sl ide. The bacteria needs to be relatively uniformly distr ibuted over the sl ide.

Run the sl ide over a heat source to heat fi x the bacteria on the sl ide. I f using a Bunsen burner, hold the sl ide about several inches over the fl ame so that the sl ide is warm to the touch. Repeat this three times.

Add iodine crystal violet dropwise so that the bacteria on the sl ide is completely covered. Let stand for 30 seconds, then rinse with water.

Wash the stain off with water. Flood the sl ide with gram iodide, and let stand for 30 seconds. Rinse. Add ethanol. Let stand for 5 seconds before rinsing with water.

Flood the sl ide with saff ranin. Let stand for about thirty seconds. Rinse with ethanol.

Blot dry with bibulous paper ( i f the paper is avai lable). Place under microscope to identify whether or not the bacteria are

gram positive or negative. Focus at 10x magnifi cation, view detai ls at 40x magnifi cation. For more acute detai l , obtain mineral oi l , place a drop on the sl ide underneath the lens, and change the lens from 40x to 100x so that the lens is immersed.

Perform further biochemical tests based on identity, gram + or - , of the bacteria.

VIEWING SAMPLES

Gram + are violet Gram – are red

Gram Stain Procedure Video www.youtube.com/watch?v=4LVVJqD7LjM

1: Microbiome of the upper troposphere: species composition and prevalence, eff ects of tropical storms, and atmospheric implications, PNAS. DeLeon-Rodriguez et al, 2013 www.pnas.org/content/110/7/2575.full.pdf+html

2: Ubiquity of Biological Ice Nucleators in Snowfall. Christner et. al. Science, 2008. www.sciencemag.org/content/319/5867/1214.full

3: How do microorganisms reach the stratosphere ?. International Journal of Astrobiology. Wainwright et. al, 2006 eprints.whiterose.ac.uk/1556/1/wainrightm1.pdf

4: 5: Stratospheric superbugs off er new source of power.

ScienceDaily, Feb 2012. www.sciencedaily.com/releases/2012/02/120221212614.htm

6:Atmospheric Movement of Microorganisms in Clouds of Desert Dust and Implications for Human Health. Griffi n, Clinical Microbiology Reviews. 2007. cmr.asm.org/content/20/3/459.full.pdf+html

REFERENCES