Rocket and balloon borne exposure experiments on survival of microorganisms in space environmen

Hemenway, C. L.

Hotchin, J.

Lorenz, P.

Markusen, A.

English

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The Surv iva l  of  Mj-croorgaiiisms i n  Space 
Fur ther  Rocket and Balloon Borne Euyosure Experiments 
-foV 
John Hotchin, Pe te r  Lorenz, Aletha Markusen, and C u r t i s  Hemenway 
Dudley Observatory, 
Div is ion  of Laboratories and Research of the 
New York S t a t e  Department of Health, and t h e  
S t a t e  Universi ty  of N e w  York a t  Albany 
This r e p o r t  descr ibes  the r e s u l t s  o f  s u r v i v a l  s t u d i e s  of 
t e r r e s t r i a l  microorganisms exposed d i r e c t l y  t o  t h e  space environment on 
two bal loons and two rocket  f l i g h t s .  The work i s  a p a r t  o f  a program 
t o  develop technics  f o r  t h e  c o l l e c t i o n  of microorganisms i n  t h e  s i ze  
range of  micrometeorite p a r t i c l e s  i n  space or n o n - t e r r e s t r i a l  atmospheres, 
and t h e i r  r e t u r n  t o  e a r t h  i n  a v iab le  staie fr3r f i l r t h c r  s t s d y .  
Previous s u r v i v a l  s t u d i e s  were repor ted  (Hotchin, J. , Lorenz, P., 
Hemenway, C., Nature 206 No. 4983, 442, 1965) i n  which a f e w  r e l a t i v e l y  
l a r g e  a rea  samples o f  microorganisms were ex2osed on mi l l i po re  f i l t e r  
cemented t o  aluminium p l a t e s .  
newly developed t echn ics  have r e su l t ed  i n  a 25 f o l d  min ia tu r i za t ion  
r e s u l t i n g  i n  a corresponding increase i n  t h e  number of experiments 
Performed. This  has enabled a s t a t i s t i c a l  eva lua t ion  of t h e  r e s u l t s  
t o  be made. 
5 X 5 mm. i n  s i ze )  were flown i n  t h e  f o w  experiments, and  organisms 
used were coliphage T1, penic i l l ium r o q u e f o r t i  (THOM) mold spores,  
Po l iov i rus  type I (Pf izer  a t tenuated  Sabin vaccine s t r a i n ) ,  and b a c i l l u s  
s u b t i l i s  spores .  The organisms were deposi ted e i t h e r  by spraying d i r e c t l y  
upon t h e  v i n y l  coated metal un i t s ,  o r  by d r o p l e t  seeding i n t o  shallow 
depress ions  i n  t h e  mi l l i po re  f i l t e r  membrane coated u n i t s .  Groups of 
u n i t s  were prepared comprising f u l l y  exposed, inver ted  (screened by 2 m. 
of Al), and f i l t e r  p ro tec t ed  organisms. 
t h e  f l i g h t  se t ,  t h e  back up set ,  and a labora tory  con t ro l  s e t .  The 
a l t i t u d e  of t he  exposures var ied  from 35 km. i n  t h e  bal loon experiments 
t o  150 km. i n  t h e  rocke t  experiments. 
was approximately 6 hours f o r  t h e  bal loon f l i g h t s  and about 3 minutzs 
f o r  t h e  rocke t  experiments.  
-' 
I n  t h e  p re sen t  series of experiments, 
A t o t a l  o f  756 separa te  exposure u n i t s  (each approximately 
A l l  o f  these  were included i n  
Times of exposure a t  a l t i t u d e  
b 
RESULTS 
The r e s u l t s  of t h e  exposure experiments are too  voluminous t o  
be inc luded  here i n  to to ,  therefore  only t h e  main msults w i l l  be given 
and t h e  effects o f  t h e  var ious f ac to r s  s tud ied  w i l l  be r e l a t e d  t o  t h e m .  
It  w a s  found t h a t  severe l o s s  of v i a b i l i t y  occurred during t h e  drying 
of t h e  v i ruses  f o r  t h e  prepara t ion  of t h e  samples. In  t h e  colighage T1 
dry ing  l o s s  could be c u t  down t o  1 o r  2 loglo u n i t s  ( t i t e r  drops from 
approximately 1-08 t o  .IO6 pF!r*,+tt'a seeded) by careful c o n t r o l  of 
N67 11844 
0 
(THRUI 
L 
*PFU = Plaque Forming Units 
B 
(NASA CR OR TMX OR AD NUMBER) 
https://ntrs.nasa.gov/search.jsp?R=19670002515 2020-03-16T17:04:15+00:00Z
humidity during and a f t e r  the drying period The o v e r a l l  l o s ses  of the 
two con t ro l  groups ( inverted f l i g h t  s e t  ant? .I a?:;\oratary con t ro l  s e t )  a r e  
shown i n  Table 1. It was evident  t h a t  the drcp1e-L seeded labora tory  
cont ro l  coliphage showed a smaller  f r a c t i o n  recovered (0.00004) than 
the inver ted  f l i g h t  s e t  (0.004). 
seeded phage and was apparently prevented by evacuation and des icca t ion  , 
of t h e  sprayed v i rus  laboratory cont ro l .  
T h i s  d id  not occur w i t h  t he  spray 
Since the  su rv iva l  was b e t t e r  i n . t h e  inver ted  f l i g h t  s e t s  than 
i n  t h e  laboratory controls ,  the r e s u l t s  a re  expressed a s  surviving 
f r a c t i o n s  r e l a t i v e  t o  the  inverted f l i g h t  set .  Table 2 shows the  bas i c  
r e s u l t s  obtained w i t h  coliphage T1. Survival  of t h i s  b a c t e r i a l  v i rus  
w h i c h  was f u l l y  exsosed t o  space up t o  t h e  a l t i t u d e  of 150 km. f e l l  
by a f a c t o r  of 0.00007 i n  comparison t o  a comparable inver ted  con t ro l  
a t  the i d e n t i c a l  1ocation;assuming a s t r a i g h t  l i n e  inac t iva t ion  r a t e  
the  ha l f  l i t e  o r  'rl.phage wide2 these e e n d i t i m s  i s  nnly 20 seconds. 
The surviving f r a c t i o n  of the other  organisms used, under the same 
exposure conditions,  i s  shown i n  Table 3 .  It was evident  t h a t  the 
s t a b i l i t y  of -the coliphage was intermediate,  and both types of spores 
were very s t ab le ,  and a l so  the pol iovirus  showed some su rv iva l  i n  a l l  
f l i g h t  experiments a f t e r  d i r e c t  exposure. 
The e f f e c t s  of s eve ra l  other  condi t ions of exposure were 
s tudied,  and the  most s i g n i f i c a n t  r e s u l t s  a r e  expressed i n  Table 4 i n  
terms of t h e  su rv iva l  f a c t o r  of the d i f f e r e n t  condi t ions used. Seeding 
the  v i r u s  i n  two ha l f  volume separate  a l iquo t s  w i t h  complete drying i n  
between gave a 24-fold increase i n  su rv iva l ;  the reason f o r  this is not  
c l e a r .  I n  t h e  Dudley rocket flown on blovember 12, 1965, two days a f t e r  
t he  Luster  rocket,  shadowing o f  the specimens from d i r e c t  s o l a r  r ad ia t ion  
was very much higher owing t o  the  angle of f l i g h t  r e l a t i v e  t o  the sun 
of an average of  approximately C S o  from normal compared t o  approximately 
60° i n  the Luster experiment. 
s u r v i v a l  of the coliphage and ind ica tes  t h a t  the l e t h a l  e f f e c t s  o f  space, 
noted so  f a r  i n  b r i e f  exposures, a re  probably due t o  s o l a r  sources o f  
r a d i a t i o n .  The lower a l t i t u d e  (35 km.) of the balloons was l e s s  l e t h a l  
than the condi t ions of t h e  Luster rocket .  The broth-grown concentrated 
coliphage apparently contained a pro tec t ive  f a c t o r  which gave v i r t u a l l y  
complete p ro tec t ion  aga ins t  s o l a r  radiat ion. ,  The data  obtained from 
t h e  f i l t e r s  i nd ica t e  t h a t  approximately CCO A of gold o r  1400 8 
aluminum a l s o  gives  complete pro tec t ion  t o  t h e  coliphage. 
t h a t  study of t h e  s p e c t r a l  transmission of  these f i l t e r s  w i l l  throw 
f u r t h e r  l i g h t  on the wave lengths  responsible f o r  the l e t h a l  e f f e c t s .  
T h i s  r e su l t ed  i n  a 1400 fo ld  higher 
I t  is  hoped 
. . I  
I 1 .  3 .  
DISCTJSS ION 
These r e s u l t s  c lear ly  Indicc. -3 t h a t  the environment o f  space, 
s o  h o s t i l e  t o  more complex forms o f  l i f e ,  is by no means immediately 
l e t h a l  t o  var ious microorganisms. T h i s  might be p red ic t ed  from the 
known resistance of spores  and v i ruses  t o  vacuum and f r eeze  drying, 
y e t  experience w i t h  t h i s  branch of exobiology supp l i e s  d e f i n i t e  
answers unobtainable by theory alone. 
The choice of organisms used has not  centered upon t h e  v i ruses  
t o  be most l i k e l y  s t a b l e  i n  space, bu t  has  been determined b; many 
fac to r s ,  n o t  l e a s t  of which i s  the h e a l t h  hazard o f  us ing  some of the  
more s t a b l e  agents  such as sc rap ie  v i r u s  which are severe ly  pathogenic. 
I t  is  not  inconceivable t h a t  small  v i ruses  such a s  pol iovi rus ,  which  
have been shown t o  survive on t h e  f r i n g e  of  the atmosphere could be 
c a r r i e d  t o  great heights  during c e r t a i n  weather condi t ions  and thereby 
ob ta in  some of  the i r  unpredictable  epidemiological  behavior.  
The f a c t  t h a t  s o l a r  r a d i a t i o n  appears t o  be t h e  primary cause 
of i n a c t i v a t i o n  of  microorganisms i n  space, and t h a t  t h i n  lajjers of 
appropr ia te  f i l t e r i n g  material can offer  enormous pro tec t ion ,  suggests  
t h a t  t h i s  cause of death i s  n o t  a very g r e a t  b a r r i e r  t o  microbiological  
i n t e r p l a n e t a r y  t r a v e l .  
SUMMARY 
1. A human pathogen po l iov i rus  type 1 can  surv ive  a t  t h e  
L i m i t s  of  the atmosphere (balloon a l t i t u d e  c. 35 km.) f o r  long  periods,  
and a t  rocket a l t i t u d e s  ranging up t o  150 km f o r  b r i e f  per iods .  
2. L i t t l e  o r  no loss of v i a b i l i t y  could be de tec ted  i n  
b a c t e r i a l  o r  mold spores  (B. s u b t i l i s  and penici l l ium) a f t e r  exposure 
i n  space a t  a l t i t u d e s  up t o  150 km. for about 3 minutes. 
3 .  I n a c t i v a t i o n  of highly dispersed,  exposed b a c t e r i a l  v i rus  
(coliphage TI) was r ap id  a t  150 km 
showed a surv iv ing  f r a c t i o n  of lO-”after about 3 minutes. 
bu t  highly aggregated T1 phage 
4. The i n a c t i v a t i o n  appears t o  be due t o  so la r  r a d i a t i o n .  
0 5. The inBct iva t ing  rays were completely screened out  by 
880 A o f  Au o r  1400 A of Al-fi l ter  on t h i n  formvar membranes. 
6 .  If f u l l y  p ro tec t ed  from s o l a r  r ad ia t ion ,  d r i e d  organisms 
surv ived  b e t t e r  i n  space than  i n  s tandard  l abora to ry  condi t ions .  
7. Broth-grown coliphage T1 suspensions contained a P ro tec t ive  
f a c t o r .  
0 * . *  
i .  4. 
This work was slipportcd 3y tlie ihL- ona l  -1eronautics and 
space adminis t ra t ion under lJASA grant bEG-155 --!?l. 
The authors want to thank Dr. V. Tonlpkiils 1'02 h i s  support, 
Drs. R. Deibel, J. Barlow and J. Aronson f o r  t h e  v i r u s  mater ia l  and 
the  B. s u b t i l i s  s t r a i n s ,  M r s .  J. Gardner and Mr. A .  Loder f o r  
exce l l en t  t echn ica l  ass i s tance ,  M r .  D. Hallgren, M r .  T. Laudate and 
Mr. J. Highberg f o r  their cooperation I n  preparaing the  f l ig l i t  ware 
and Mr. R. Coon and Mr. J. Dugan for t h e i r  cooperation i n  the  
preparat ion of the f l i g h t  da ta .  
A' 
Table 1 
The survival G f  contrcl prcparation:: cLP TI ccli phage 
during drying and storage ehrcugh %e experimental period* 
Seeding Set PFU** PFU eluted Fraction Remarks 
technic seeded re covered 
Laboratory 3.9 4 . 5 2 0 . 4  0.00034 Helti in air at rt" and p*** 
Droplet control 
Inverted flight 8.9 6.5 f 0.3 0.004 Evacuated 
set 
Laboratory 5.2 2.8 i 1.0 0.004 Vacuum desiccatec! 
Inverted flight 5.2 2.3 f 0.3 0.001 Evacuated 
Spray cvn t rd  
set 
* Titers expressed throughout in loglo. 
** PFU = plaque forming units. 
***eo p = room temperature and pressure.  
Table 2 
The effect of rocket borne exposure to space at 150 km. for about 3 minutes 
(Luster experiment) 
No. of replicate PFU/seeded PFU/recovered Surviving frac tim 
units Exposed( a) Inverted (b) ( a/b) 
4 8 . 9  2.2k 0.1 6 . 5 5  0.3 0.00007 
Table 3 
The surviving fraction* of different microcryanisms during complete 
e x p s u r e  at 50 km. €or abour 3 minutes 
Microorganism Seeding me thcd k pproximate 
surviving fraction 
TI coli phage Droplet in 1 vclume Q .00007 
TI coli phage Zpray 0 
Penicillium spores Spray - 
Poliovirus type I Droplet + ** 
Poliovirus type I Spray 0 
B. subtilis spores 
strain M-1 Droplet 0.35 
B. subtilis spores Droplet 1 
s t ra in  M-4 
average number of viable orsanisms on exposed fligkt units 
* Expressed as -average number of viable organisms on inverted flipt-t units 
significantly different from the controls, and in all experiments only few 
plaques were found with this virus. 
** The survival of the poliovirus in most flight experiments was not 
The effect of different exposure conditims on rhe scrvival of ccli phage T1 
in space (150 lcm.) 
I_- -- 
Survival factcr** 9xperiment Exposure condition - 
Lua ter rocket Seeding 1/2 vol. 2 x 24 x 
h d l e y  rocket Shadowing f rom solar radiation* 1400 x 
Balloon I 35 km. altitude 3 x  
Balloon 11 35 km. altitude 230 x 
Luster rocket 3 x concentrated phage in  
broth medium 6300 x 
Luster rocket Filters *** 14000 x (no loss) 
-- 
*Due to relative angle of rocket and sunlight. 
* *  surviving fraction 
*** 877 > thick Au o r  1400 A thick A1 on formvar membranes. T h e  
difference between the survival under the Au filter compared with 
the A1 filter is not significant. Generally, ccrnplete survival of 
the droplet seeded materials was found in all fligbt experiments 
under these metal filters. 
Expressed as surviving fgaction of fully expcseci units in Luster rocket 


The Survival of Microorganisms in Space. Further Rocket and Balloon Borne Exposure Experiments

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