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 selfcontained 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 flightcertified 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

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 +16C and +26C 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 4C to 37C and may even be changed during the course of the experiment. For example, it might be set to 25C during the growth phase and then cooled to refrigeration temperatures (4C) 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 noninfectious 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

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

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 caterpillars 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.

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: Levels of Containment: (3.5 x 5.0 x 7.0 inch) One

Video/Imaging:

Almost the entire habitat is imaged. The front half of the top is omitted in order to avoid glare from the LEDs. 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 Invertebrates, crystal growth, plant growth.

Materials:

Types of experiments:

Habitat 2 OptiCell Processing Module

Summary
The OptiCell Processing Module (figure 6) is a closed culturing system that is based on an off-theshelf 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 4C (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 22C (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: Volumes: Syringe 3mL Video: Types of experiments: The two outside OptiCellsTM may be imaged. Cell and tissue cultures, microbiology, small organisms, microorganisms 3 x 11 x 12cm ( 1.2 x 4.5 x 5.0 inch ) OptiCell 10mL

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: Levels of Containment: ( 3.5 x 4.0 x 5.0 inch ) Three. The tubes are the first level. The doubly-sealed cylinder body and end caps are the second and third. 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. Glass tubes, silicone septa, anodized aluminum endcaps, polycarbonate cylinder body.

Video:

Materials:

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-

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: Levels of Containment: Video: Flask (1.0x2.1x3.75 inch) Flasks in bracket (1.3 x 5.5 x 7.0 inch) One 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.