BIOLOGICAL SAMPLING
DREAMS21/22
ATMOSPHERIC BACTERIA
 Recently, scientists have begun to analyze the biological
composition of the upper troposphere and lower
stratosphere 1
 Bacteria, fungal spores, and other microscopic biogenic
materials could play an integral role in climate, cloud
formation 2 , 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”
POSSIBLE APPLICATIONS
 In 2012, students at Whitecastle University cultured
stratospheric bacteria B. stratophericus to create a
biofilm that generates electricity 5
 A NASA scientist has noted that the adaptations of
stratospheric bacteria could be used as a model for
terraforming new planets 4
 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 issues 6
COLLECTING SAMPLES: ON THE GROUND
 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
sur face of the agar. Streak back and for th 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 hour s, checking
daily for growth.
COLLECTING SAMPLES: IN FLIGHT
 We a r i n g s te r i l e g l ove s, p r e p a re 9 n u t r i e n t a g a r p l a te s a c c o r di n g to d i r e cti on s
i n c l u de d w i t h a g a r.
 W i th a p e r m a n e n t m a rke r, l a b e l t h e c ove r a n d b ot tom o f e a c h a g a r p l a te . L a b e l
t h r e e “ fl i g h t ” , t h r e e “ c o n t rol ” , a n d t h r e e “ s i te ” . N u m b e r e a c h p l a te i n a s et 1 - 3 .
S e a l t h e m t i g h tl y w i t h t a p e . L e ave t h e c o n t rol p l a te s i n t h e l a b i n a r e f r i g e ra tor.
 B r i n g t h e s i x p l a te s l a b e l e d “ s i te ” a n d “ fl i g h t ” i n a n i c e - fi l l e d c o o l e r to t h e l a u n c h
s i te . A n h o u r b e fo r e l a u n c h , m o u n t to t h e to p t h e b oxe s w i t h Ve l c ro t h e 3 p l a te s
l a b e l e d “ fl i g h t ” .
 At s o m e p o i n t, r e m ove p l a te l a b e l e d “ s i te 1 ” f ro m t h e c o o l e r, a n d l et s i t fo r o n e
h o u r. U n s e a l a n d o p e n fo r 3 0 m i n u te s , n e a r t h e “ fl i g h t ” p l a te s , fo r 3 0 m i n u te s .
M i n i mi z e t h e a m o u n t o f c o n t a c t w i t h te a m m e m b e r s .
 At t h e l a s t p o s s i ble m o m e n t b e fo r e l a u n c h , u n s e a l a n d o p e n t h e p l a te s l a b e l e d
" fl i g h t ".
 I m m e d i a te l y a f te r t h e b a l l oon l a n d s o n t h e g ro u n d, s e a l t h e fl i g h t p l a te s .
 Fo r t h e fi r s t b a l l oon l a n di n g , p l a c e t h e p r e c o ol e d “ s i te 2 ” p l a te n e a r t h e l a n d i n g
p o i n t a n d l et s i t fo r s eve r a l m i n u te s . D o t h e s a m e w i t h t h e “ s i te 3 ” p l a te a t t h e
s e c o n d b a l l oon l a n d i n g .
 P l a c e a l l p l a te s b a c k i n c o o l e r o r s to ra g e .
 I n c u b a te a l l p l a te s a t 37 ° C ove r n i g h t. C h e c k t h e eve r y d ay fo r g row t h .
 I n c u b a te fo r u p to 4 8 h o u r s .
VIEWING SAMPLES
 A f te r v i s i ble c o l o n ie s d eve l op, d i p a s te r i l e i n o c ul a ti n g l o o p i n to a c o l ony, a n d
s p r e a d i t a c ro s s a m i c ros c opy s l i d e — a ddi n g a d ro p o f c l e a n wa te r a s n e e d e d — to
fo r m a t h i n fi l m ove r t h e s l i de . T h e b a c te r i a n e e d s to b e r e l a t i vel y u n i for ml y
d i s tri bu te d ove r t h e s l i de .
 Ru n t h e s l i de ove r a h e a t s o u rc e to h e a t fi x t h e b a c te r i a o n t h e s l i de . I f u s i n g a
B u n s e n b u r n e r, h o l d t h e s l i de a b o u t s eve r a l i n c h e s ove r t h e fl a m e s o t h a t t h e
s l i de i s wa r m to t h e to u c h . Re p e a t t h i s t h r e e t i m e s .
 Ad d i o di n e c r y s t a l v i o l et d ro pw i s e s o t h a t t h e b a c te r i a o n t h e s l i d e i s c o m p l ete l y
c ov e r e d. L et s t a n d fo r 3 0 s e c o n d s , t h e n r i n s e w i t h wa te r.
 Wa s h t h e s t a i n o f f w i t h wa te r. F l o o d t h e s l i de w i t h g r a m i o di de , a n d l et s t a n d fo r
3 0 s e c o n d s . R i n s e . Ad d et h a n o l . L et s t a n d fo r 5 s e c o n d s b e fo r e r i n si n g w i t h
wa te r.
 F l o o d t h e s l i d e w i t h s a f f r a n i n . L et s t a n d fo r a b o u t t h i r ty s e c o n d s . R i n s e w i t h
et h a n ol .
 B l ot d r y w i t h b i b u l ou s p a p e r ( i f t h e p a p e r i s ava i l a bl e ) .
 P l a c e u n d e r m i c ros c ope to i d e n ti f y w h et h e r o r n ot t h e b a c te r i a a r e g r a m p o s i tive
o r n e g a t i ve . Fo c u s a t 10 x m a g n i fi c a ti on , v i ew d et a i l s a t 4 0 x m a g n i fi c a ti on . Fo r
m o r e a c u te d et a i l , o b t a i n m i n e ra l o i l , p l a c e a d ro p o n t h e s l i de u n d e r n e a t h t h e
l e n s , a n d c h a n g e t h e l e n s f ro m 4 0 x to 10 0 x s o t h a t t h e l e n s i s i m m e r s e d .
 Pe r fo r m f u r t h e r b i oc h e mi c a l te s t s b a s e d o n i d e n tity, g r a m + o r - , o f t h e b a c te r i a .
Gram + are violet
Gram – are red
Gram Stain Procedure Video
www.youtube.com/watch?v=4LVVJqD7LjM
REFERENCES
 1: Microbiome of the upper troposphere: s pecies composition and
prevalence, ef fects 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 of fer new source of power. ScienceDaily,
Feb 201 2.
www.sciencedaily.com/releases/ 201 2/02/120221 21 2614 .htm
 6:Atmospheric Movement of Microorganisms in Clouds of Deser t Dust
and Implications for Human Health. Grif fin, Clinical Microbiology
Reviews. 2007. cmr.asm.org/content/ 20/3/459.full.pdf+html