1. Biological Experiment Guidelines
Introduction
Life Sciences or biology is the study of living organisms and matter like plants, animals, and human
beings. For millions of years organisms have evolved on Earth in the presence of gravity. It is such
an ingrained aspect of all organisms that often times, scientists are not able to determine what
aspect of a physiological process is gravity dependent. For example, when a butterfly emerges
from her chrysalis, she typically “falls” out of the end of the chrysalis that points toward the ground.
What happens when gravity is removed and there is not falling? Gravity on the ISS is effectively
counterbalanced by the centripetal acceleration of the orbiting space station creating a weightless
environment. So, with the invention of space travel, living organisms can be studied minus the one
ever present variable on Earth – gravity – for the first time in human history!
Your Experiment
If you come up with a biological experiment and it wins the competition, it will be performed by
astronauts on board the International Space Station. Remember, you're not being asked to actually
do the experiment -- you're being asked to explain your experiment idea and how it would work
(although any prototypes or designs or diagrams that you show in your video might help people
understand your experiment better). The biological experiment will be conducted in a fully self-
contained piece of space flight certified hardware called a habitat (known as a Commercial Generic
Bioprocessing Apparatus or CGBA for short). Unlike many physics experiments that might use a
one-time, concerted effort by an astronaut, many biological experiments involve long periods of
growth and observation (from days to weeks). During this time, the habitat controls the experiment
determined environmental conditions (including temperature control, lighting, food, media, water,
growth environment, and humidity) and records stills and video of the experiment while it is being
conducted. The astronauts are only able to manipulate the experiment using the built-in interfaces
like plungers and valve handles (described for each habitat later). The winning entry will be adapted
for space flight and tested on the ground by experts to check it is safe for space flight. However
for an entrant or team to successfully design a biological experiment that is feasible and safe, it
must take into account the following guidelines and be appropriate for one of the four space flight-
certified habitats that are available to support biological experiments for this contest.
Guidelines
Microgravity
Remember that, because the ISS is far away from Earth and orbiting very fast, gravity is effectively
cancelled out on the space station. Things that seem “easy” on the ground are not necessarily easy

2. in space. There is no “up” or “down” and cultures will float freely within a container. Insects that
attempt to fly often tumble when they move their wings.
Life-Support
During transportation: The habitat will provide everything your experiment requires to thrive. From
the time your experiment is loaded into the space flight habitat, launched, transported to the ISS
and installed 10-14 days may have passed, so it may help to design a biological experiment that
can remain “dormant” during transportation to the ISS. Many biological experiments can be flown
in stasis or a slow-growth condition and activated by the introduction of food/media or both. If
investigating higher organisms (i.e. insects), it may be possible to keep the organism in a contained
space within the habitat until it is released so that the initial behavior or adaptation in microgravity
that is being studied can be seen on video. The astronaut, for example, can open “doors” to release
the organism into the primary portion of the habitat just before video and imaging begins.
Onboard the ISS: The habitats cannot be opened at any time once on board the ISS. Thus it is
important that you have some idea of how the needs of your proposed biological experiment could
be delivered or provided. Here are some questions to ask yourself when designing a biological
experiment: What is required to successfully support the organism, culture or sample I am
studying? How long does it need to live? Does it need to be fed, if so how often? Does it require
fresh media at different time intervals?
Temperature Control
During launch and transport to the ISS (which can take between 10-14 days) the experiment will
remain at ambient temperature between +16°C and +26°C depending upon location within the
launch vehicle. Once the experiment is installed on board the ISS, the temperature can be chosen
to be any temperature between 4°C to 37°C and may even be changed during the course of the
experiment. For example, it might be set to 25°C during the growth phase and then cooled to
refrigeration temperatures (4°C) to extend organism, culture or sample life.
Biosafety Levels
Many biological organisms are given what is called a Biosafety Level (BSL) rating. Only biological
substances and organisms with a rating of BSL 2 or lower may be flown. To be safe, a good rule to
follow is try to utilize non-hazardous cultures, organisms or samples for your experiment. Examples
of non-hazardous substances/organisms include: water, sugar water (nectar), Agar, Phytagel,
caterpillar food, fruit fly food, baby food, seeds, guar gum, plant cells, any invertebrate that is not
poisonous, non-toxic or cannot cause disease in humans or animals, tissue cultures, yeast, some
bacterial cultures. Some examples of low-hazard or low BSL materials are some bacteria and
viruses including Bacillus subtilis and Escherichia coli, as well as some cell cultures and non-
infectious bacteria.
Organism, samples and cultures
1. Anything considered poisonous, toxic or dangerous to human health is prohibited from use
on the space station.
2. The organisms, culture or sample being proposed for study must be available between
January and May when the testing will happen before the launch.
3. The organism needs to be able to be transported from the USA (where it will be tested) to
Japan (where the launch happens).
4. How long can the organism, culture or sample survive and will that meet the experiment
objectives? And when does the experiment end?
Length of experiment
The experiment can go on for a few days to a number of weeks depending on how long it needs to

3. collect results for. Where outside interaction is needed for longer experiments, the astronaut's time
should be considered - for example, 10 to 20 minutes a week would be reasonable.
Prototypes, Mock-Ups, Pictures & Diagrams
Many ideas shine as concepts until they are proven impossible or infeasible with prototypes.
For this reason, it is optional but encouraged that contestants build and demonstrate mock-ups
or prototypes of the devices needed to perform the experiment. This will make your entry more
clear to the judges and voting community and to the developer of the habitat if your experiment is
chosen. It is important for you to clearly understand the biological process or behavior of what you
are proposing to study. You should be explicit on how to keep the organism, culture, sample alive
for the necessary time. Experts will adapt the concept you demonstrate into hardware that meets
all the requirements for space flight. Explanatory pictures, diagrams, and schematics that help to
illustrate the experiment concept are also encouraged.
Data collection
Since the biological experiment will not be returned to Earth, high resolution digital video recordings
and still images will be used to analyze the experiment. Still images are automatically taken as
often as every five minutes, 24 hours a day, or can be taken more regularly for short periods. Video
imaging can also occur throughout the day for extended periods.
Be aware that some organisms may leave residue on the viewing windows of the experiment
habitats, which can make the experiment very hard to see. For instance, fruit fly larvae drag their
wet sticky food with them as they move around. On a previous experiment, fruit fly larvae were kept
in separate chambers when they were young and messy and only let out into the primary portion of
the habitat as adults for better viewing.
Habitats
Overview
The following is a description of the four space flight certified habitats that can be utilized for a
biological experiment. Your experiments do not need to be limited to the ways they have been
used before. Think of each habitat in terms of its capabilities and ability to support your proposed
biological experiment. For instance, do not think of habitat 1 as an insect habitat, rather it is a
vented box with lights and a plunger system for manual opening and closing of a smaller space
within the larger primary habitat space. As long as the box itself is kept sealed and intact, the
plunger and small container system within the primary habitat can be used for a variety of activities.
Just use your imagination!
Modifications
The habitats may be slightly modified to better support a proposed experiment but modifications
are not recommended because of the complexity they may add before the experiments are due to
go to space. Consider proposed changes carefully against how essential it is for the experiment to
succeed.
Habitat 1 – “Butterfly” Habitat
Summary
The “butterfly” habitat shown in figure 1 is a sealed box that has been used in previous biological

4. experiments to house different types of invertebrates such as caterpillars, spiders, fruit flies, and
butterflies. It is essentially just a lit box with a clear front, so it is the most versatile of the available
platforms. The clear front window allows video imaging of almost all of its contents. Up to four
plungers that are controlled manually can be used to open or close 4 smaller containers inside
the sealed habitat. The plunger system can also be used to inject fluid or start and stop another
mechanical system. In the past, this plunger system was used to expose food for butterfly larvae,
release fruit flies into the primary habitat for spiders, expose a water container for the primary
inhabitants of the habitat and/or confine the insect being studied until it was installed on the ISS.
Lighting for imaging is provided via white and infrared (IR) light-emitting diodes (LED). The white
lights can also be programmed to turn on and off to simulate a day/night cycle, and the infrared
LEDs enable imaging during the "night" phase. Six water-proof vents allow the box to “breath”
while sealing in liquids. The vents allow gas transfer to support aerobic metabolism and to equalize
pressure during changes in cabin pressure. The habitat body provides one level of containment.
Example Experiments
1. Butterflies
Three butterfly habitats containing two different species of butterfly larvae flew to the ISS. The
hardware setup was the same for both species with the only difference being the caterpillar’s food.
In each case, small caterpillars were loaded, launched and grown until they formed chrysalises and
later emerged as butterflies.
The caterpillar food tray (figure 2) was loaded with food in the exposed wells (on the left side) and
the covered wells (on the right side). The habitat was launched on a space shuttle and transferred
to ISS approximately four days after being handed over to NASA personnel. When the astronaut
installed the habitats into CGBA for temperature control and imaging, she also pulled up the food
plunger to expose the fresher food in the covered wells (on the right side of figure 2).
The nectar feeder (figure 3) was filled with sugar water for the butterflies to eat after they emerged
from their chrysalises. The feeder started in a “closed” position to keep it from evaporating. Shortly
before the butterflies emerged, the astronaut pulled the nectar feeder plunger up to align the hole
in the slider and expose the nectar for the butterflies to eat. Figure 4 shows the butterfly habitat
loaded for launch to the ISS with Monarch caterpillars.

5. 2. Spiders and Fruit Flies
The "Butterfly" habitats also flew orb weaving spiders and fruit flies to the ISS. When the butterfly
habitat flew in the configuration to hold spiders, four smaller units within the habitat (see figure
5) held fruit flies along with the appropriate fly food and water in a separate chamber. The water
chamber also provided a safe yet contained space to launch the spider so that she was unable to
spin webs until she was released into the primary chamber of the habitat box once on board the
ISS.
Alternate Configuration Ideas
While it is important that the plunger system mechanism remains the same on this habitat,
changes may be made to what the plunger movement does (the smaller containers inside the
butterfly habitat). For example, instead of the plunger system opening a smaller compartment to
expose fresh food within the primary habitat, it could open a small compartment to expose a clean
living space for the habitat inhabitants or it could “activate” a syringe pump. A biological experiment
could use 1, 2, 3 or 4 plungers.
Specifications
Dimensions: (3.5 x 5.0 x 7.0 inch)
Levels of Containment: One

6. Video/Imaging: Almost the entire habitat is imaged. The front half of the “top” is
omitted in order to avoid glare from the LEDs.
Materials: 5 sides are anodized aluminum. The front window is clear
polycarbonate. The mini hab and food and water trays are made from
several materials including: polycarbonate, Ultem, stainless steel,
PTFE
Types of experiments: Invertebrates, crystal growth, plant growth.
Habitat 2 – OptiCell Processing Module
Summary
The OptiCell Processing Module (figure 6) is a closed culturing system that is based on an off-the-
shelf culture chamber called an OptiCellTM. It is well suited for biological liquid cultures and can
grow things like bacteria, yeast, and very small organisms like nematodes. The syringe and valve
allow fluid to be drawn from one of the three OptiCells and injected into another. This feature can
be used to extend the period of growth by growing the culture in each chamber sequentially. The
OptiCellTM chamber consists of two durable, thin plastic films held 2mm apart by a rectangular
frame. They were designed to support cell cultures, but they have also been shown to work well for
bacteria, yeast, and nematodes. The clear plastic sides are good for imaging and are permeable to
oxygen and CO2. This gas exchange lets the culture “breathe” through the film.
Example Experiment
In preparation for a yeast experiment, all three OptiCellsTM chambers were loaded with yeast
media and the first was inoculated with yeast. It was immediately installed into a smart incubator
which cooled them to 4°C (refrigeration) in order to keep the yeast cold enough that it would not
grow much. A couple of days after arriving in space, an astronaut set the incubator to warm the
experiment up to 22°C (room temperature). At this temperature, the yeast started to grow actively.
After a few days, the yeast had filled chamber number 1 and used up most of the nutrients in the
chamber. At that point, the astronaut pulled a small amount of the culture from chamber 1 and
injected it into chamber 2. This inoculum started a new culture in chamber 2 which grew until it
saturated chamber 2 and the astronaut pulled some from it to start number 3. When the yeast had
grown to fill chamber 3, the incubator was set to cool again to preserve the culture for analysis.
Specifications
Dimensions: 3 x 11 x 12cm ( 1.2 x 4.5 x 5.0 inch )
Volumes: OptiCell – 10mL
Syringe – 3mL
Video: The two outside OptiCellsTM may be imaged.
Types of experiments: Cell and tissue cultures, microbiology, small organisms, micro-
organisms

7. Habitat 3 – Group Activation Pack (GAP) with 8 Fluid
Processing Apparatus (FPA)
Summary
This space flight hardware platform was designed to house many different types of biological
experiments and allow for fluid mixing while providing three levels of containment. This means
this hardware can house slightly more hazardous substances when compared to the previously
described space flight hardware platforms. Each GAP hardware (see figure 7) contains 8 glass
tubes (see figure 8) with moveable rubber septa and a gas permeable membrane that allows
modest gas exchange, if needed. The septa allow the tube to keep 2 - 4 fluids separate within the
tube and accomplish sequential mixing of these fluids at the appropriate times. Total liquid volume
within each of the 8 tubes is 6.5ml. All 8 tubes within one GAP are activated at the same time.
GAPs provide three levels of containment. A hand crank is used to manually move the septa to mix
the fluids.
An example of each fluid is:
1. A culture or organism in stasis.
2. An initiating media or other fluid that starts growth.
3. A fixative to terminate the experiment and preserve it for analysis upon return to Earth.
Example Experiments
Thousands of glass barrels have flown in hundreds of GAPs over the last two decades. Supporting
small plants/seed germination, small invertebrates, microorganisms, mammalian cells and tissues,
viruses, bacteria, protein crystal growth and biomaterials. GAPs may house organisms with a BSL
rating of 2 or less.
Alternate Configuration Ideas
There can be minor modifications to the septa configuration within each glass barrel.
Specifications
Dimensions: ( 3.5 x 4.0 x 5.0 inch )
Levels of Containment: Three. The tubes are the first level. The doubly-sealed cylinder
body and end caps are the second and third.
Video: The GAPs are not imaged while in CGBA. Two of the eight tubes
may be imaged at a time during experiment activation and/or
termination. No close-up video can be captured during actuation.
Materials: Glass tubes, silicone septa, anodized aluminum endcaps,
polycarbonate cylinder body.
Habitat 4 – Culture Flasks
Summary
Three standard culture flasks at a time are placed in a bracket that holds and illuminates them for
imaging (see figure 9). The flasks are typical off-the-shelf sterile polystyrene cell culture flasks.
Culture flasks are typically used with liquid media in a biological lab, but only non-hazardous gel-

8. like substances such as agar are acceptable for microgravity use. This is because the vented lid of
the flask is designed to allow gas exchange and it could become saturated and blocked if a liquid
culture were to come into contact with it in microgravity. The culture flasks, also called cell-culture
flasks, can be obtained on the Internet in almost any country. They are specified to provide 25cm2
of growth area when laid flat, and they hold approximately 50mL of fluid. White LEDs and infrared
LEDs are both available for this hardware platform.
Example Experiment
In September 2011, a seed-germination and directional root growth experiment is slated to occur
in this habitat. Its goal is to investigate the comparative effects of gravity, light, and touch on the
direction that roots grow. The flasks contain layers of an agar called PhytagelTM of a varying
densities which test the “touch” sensitivity of the roots (thigmatropism). The plants are also grown
with and without light to investigate the effect of light on the direction of root growth (phototropism).
Comparison of the flight experiment with its ground control counterpart distinguishes the directional
effect of gravity (gravitropism). This habitat may hold organisms with a rating of BSL 1 or less.
Alternate Configurations
Minor lighting changes could be accomplished.
Specifications
Dimensions: Flask (1.0x2.1x3.75 inch) Flasks in bracket (1.3 x 5.5 x 7.0 inch)
Levels of Containment: One
Video: All three flasks are imaged at once. White and/or infrared LEDs can
be independently switched to shine in through the angled “shoulder”
near the cap for illumination.