Castro, Victoria

Flint, Stephanie

Khodadad, Christina

Melendez, Orlando

Monsi, Roman

Ott, C. Mark

Oubre, Cherie

NASA Technical Reports Server

Investigation into an 
Alternative Method for 
Microbial Monitoring on ISS
CHRISTINA KHODADAD, ESC, KSC, FL
CHERIE OUBRE, KBR WYLE, JSC, TX
VICTORIA CASTRO, KBR WYLE, JSC, TX
STEPHANIE FLINT, NASA, JSC, TX
ORLANDO MELENDEZ, NASA, KSC, FL
MONSI ROMAN, NASA, MSFC, AL
C MARK OTT, NASA, JSC, TX
https://ntrs.nasa.gov/search.jsp?R=20170005910 2019-08-31T07:23:54+00:00Z
History
Microorganisms have been 
detected on the International Space 
Station (ISS) 
 Potential human pathogens
Introduce new microorganisms with 
every exchange of crew or addition 
of equipment or supplies 
Background
 2011 Workshop at JSC reviewed cutting 
edge technology (NASA/CP-2012-
217460, 2011)
 Environmental Microbiology
 Infectious Diseases/Pathogens
 Food and Water Safety
 Workshop determination
 Replace or supplement current 
culture based practices on ISS
 Selected molecular based 
quantitative Polymerase Chain 
reaction (qPCR)
Will add a picture of 
Aspergillus too
Background
 Molecular-based technology 
 Polymerase Chain Reaction (PCR)
 Real-time quantification and 
characterization
 Identifies specific targets
 Total heterotrophic growth
 Rapid assessment of the environment
 High reproducibility and accuracy
 Low detection limits on culturable and 
unculturable microbes
 Utilize COTS PCR units
 Operational in microgravity
 Meet ISS interface and safety conditions
Benefits of Molecular-based 
Methods
Current methods for microbial detection
 Labor and time intensive cultivation-based 
approach
Requires collection of samples on orbit and 
transportation back to ground
 Disadvantages to current detection methods
 Unable to perform quick, reliable detection on 
orbit
 Lengthy sampling intervals
 Minimal identification capabilities
 Can fail to detect or characterize all cells 
present
Purpose and Benefits
 Develop a rapid microbial identification system
 Reduce crew time & expedite operational decisions
 Provide an in-flight microbial identification system
 Increase monitoring of crew health
 Monitor air, water, food, & surfaces for potential 
pathogens
 Reduce or eliminate reliance on ground support
 Provide an independent system for long-term space 
flight
 Provide flexibility 
 development of customized species specific assays 
 Determine viability with RNA possible
Challenges
 Find a COTS, PCR instrument
 Effective in microgravity
 Compact 
 Low cost
 Identify to species level
 Live vs Dead cell via RNA 
 Quantitative
 Easy to use and interpret data
 Real-time information
 Short time from sample to answer
 Work with multiple sample types
 RAZOR EX, BioFire Defense, Inc, was tested & integrated as one of three 
instruments into the 2 x 2015 Water Monitoring Suite
 developed as an SBIR with DoD
 Completed engineering/safety evaluations and launched to ISS, SpaceX-9, July 2016
Goals of Tech Demonstration
 To perform in-flight and ground-based real-time quantitative 
Polymerase Chain Reaction (qPCR) validation using a 
commercial off-the-shelf system
 The proposed technology demonstration on ISS was aimed 
at determining:
1. If qPCR testing will work in the spaceflight environment
2. If qPCR testing during a flight operation yields results 
similar to those from a controlled experiment on Earth, 
thereby validating the instrument for potential use as 
a monitoring tool aboard the ISS.
3. Develop a CONOPS for water testing
4. Verify that qPCR is successful using ISS water samples
Methodology
 BioFire Defense, RAZOR EX
 Uses raw or prepared samples
 Pouch system contains customizable, optimized 
freeze-dried reagents
 Pseudomonas aeruginosa
 5 varying concentrations
 Sample to answer in approximately 1 hour
On-Orbit
Operations
Molecular 
Grade Water
Unfiltered 
PWD Water
Filtered 
PWD Water
September 20, 2016 X
September 22, 2016 X X
October 3, 2016 X
October 18, 2016 X
November 22, 2016 X
December 14, 2016 X
January 17, 2017 X
March 14, 2017 X
Pseudo LO
Pseudo HI
Results
0
5
10
15
20
25
30
35
40
45
0.02 0.20 2
A
VE
RA
G
E 
C
t V
A
LU
E
DNA CONCENTRATION (ng)
Average Ct Comparison 
Ground vs Flight - DI Water 
G-DI FLT-DI
0
5
10
15
20
25
30
35
40
45
0.002 0.02 0.2L 0.2H 2 20
A
VE
RA
G
E 
C
t V
A
LU
E 
DNA CONCENTRATION (ng)
Average Ct Comparison 
GROUND FLIGHT
Results–ISS Potable Water Samples
0
5
10
15
20
25
30
35
40
45
0.02 0.20 2
A
V
ER
A
G
E 
C
t V
A
LU
ES
DNA CONCENTRATION (ng)
Average Ct Comparison – Potable Water
Flight Samples
PWD-UF PWD-F
Results
0
5
10
15
20
25
30
35
40
45
0.02 0.2 2
A
V
ER
A
G
E 
C
t V
A
LU
E
DNA CONCENTRATION (ng)
Average Ct Comparison
Ground Samples
G-DI G-PWD-UF G-PWD-F
0
5
10
15
20
25
30
35
40
45
0.02 0.20 2
A
V
ER
A
G
E 
C
t V
A
LU
E
DNA CONCENTRATION (ng)
Average Ct Comparison
Flight Samples
F-DI F-PWD-UF F-PWD-F
Conclusions
 Microbial Monitor is the first device to successfully and accurately perform 
quantitative PCR using ISS water samples in the microgravity environment of 
space providing a fast sample to answer.
 The residual chemicals in the potable water had no effect on the PCR 
chemistry
 There was no difference between the molecular grade water and the ISS 
potable water
 There was no difference between the filtered and unfiltered ISS potable 
water samples
 The flight samples were not significantly different from the ground controls
What is proposed for future work 
with the Microbial Monitoring System
 Optimize additional assays for real-time detection
 Develop methods of sampling additional environments 
on ISS, i.e. Air, surfaces, food
 Equate genetic equivalents to colony forming units (CFU)
 Develop methodology to recover amplicons for further 
investigations
 Optimize Microbial Monitoring for future exploration
The team would like to thank:
Crissy Canerday
Michele Birmele
Jared Jones
Shawn Schumacher
Katy Larner
Logan Godfrey 
William Therrien
Susan Hanley
Joseph Benjamin
Ray Wheeler
I want to say how fantastic the 
procedures are and how easy this is 
to use. It is phenomenal that you 
have taken a piece of commercial 
hardware and … made it work in 
space the first time and is very user 
friendly. I think this is going to be a 
fantastic capability for the space 
station.
-Kate Rubins, Ph.D.
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Investigation into an Alternative Method for Microbial Monitoring on ISS

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