Deployment of the MEED in deep space exposed the microbial systems to a potential thermal problem. A black and white thermal coating pattern was painted on the cover of the MEED assembly to provide the proper thermal control after deployment and before opening the MEED. The proportioning of black and white paint provided the proper internal temperature preconditioning inside the MEED. To meet the thermal balance requirement within all portions of the MEED, the device was designed to remain within the 20 degrees Celsius temperature range after deployment.
In most cases, the phenomena studied represent well-known model systems that can be correlated directly with disease or other medically important conditions that could affect the health of future astronauts. Each investigator selected a species of microorganism that was nonpathogenic to man, well characterized relative to the studied phenomenon, well suited to simple and rapid screening tests and compatible with the flight hardware environment. Nine species were chosen, including Aeromonas proteolytica, Bacillus subtilis, Bacillus thuringiensis var. thuringiensis, the T-7 bacteriophage of Escherichia coli, Nematospiroides dubius, Trichophyton terrestre, Chaetomium globosum, Rhodotorula rubra and Saccharomyces cerevisiae.
A. proteolytica was chosen because it produces an endopeptidase that elicits intracutaneous hemorrhaging and necrosis in laboratory animals, and a hemolysin which has the ability to hemolyse human erythrocytes. This microbe was retained in fluid suspensions and exposed to solar ultraviolet (UV) irradiation at peak wavelengths of 254 nm, 280 nm and 300 nm. In addition to survival evaluations, cells recovered from the flight hardware were quantitatively tested for alterations in toxin production.
Flight cuvettes were opened after flight, and contents were plated for surviving organisms. Dilutions of 100, 1000, 10,000 and 100,000 in duplicate were made from each cuvette. After 24 hours of incubation, counts were made and averages computed.
Twenty-four hours after plating, 50 clones were selected at random from each energy level at the three wavelengths. Each was transferred to heart infusion agar slants and inoculated into 50 ml of 2% casitone broth for cultivation. Following 28 hours of incubation at room temperature with constant aeration by shaking, the culture was centrifuged at 8000 rpm for 10 minutes at 4 degrees Celsius. The resulting supernatant was frozen until assayed. Clones not cultured were transferred to fresh slants after 14 days.
The 1:100 dilution made from each cuvette was retained and stored at 20 degrees Celsius. Eight weeks after opening the cuvettes, aliquots of the first dilutions were made, and 30 additional clones from each wavelength and energy level were selected for study and prepared for assay as described.
Endopeptidase activity was measured with an urea-denatured-hemoglobin substrate. For the assay, 1.0 ml of a 1:40 cultural filtrate dilution containing the enzyme was incubated in a water bath with 5.0 ml of the hemoglobin substrate for 5 minutes at 37 degrees Celsius. At the end of this period, 10 ml of 5% (weight/volume) trichloroacetic acid in water was added to terminate the enzyme action and to precipitate the undigested protein, which was removed by filtration. Filtrate absorbence and control activity were expressed in units. One unit of endopeptidase activity was defined as the amount of enzyme producing an absorbence of 1.0 in 5 minutes at 37 degrees Celsius under the assay conditions described.
The degree of hemolysis was determined on 2% type O-positive human erythrocytes, diluted in physiological saline. Doubling dilutions to 1:1024 were made of the filtrate in physiological saline with a final concentration of 0.2 % sodium azide added to prevent contaminating growth. Five drops of the culture filtrate and three drops of the 2% human-erythrocyte suspension was dispensed in culture plate wells. The hemolysis titer is defined as the reciprocal of the highest dilution that gives complete clearing in the well following 24 hours of incubation at 37 degrees Celsius.
B. subtilis was chosen for study by two groups because of the known stability of this species in extreme environments and because of the copious investigations that have been conducted regarding this microorganism. One group evaluated B. subtilis spore survival under space flight conditions and correlated these findings with those obtained for the same strain employed in the Apollo 16 Biostack flight experiment. The other group chose strains HA101 and HA101(59)F because both strains have three specific amino acid markers and because the HA101(59)F strain is defective in the ability to repair radiation damage. Therefore, it is highly susceptible to the damaging effects of UV irradiation. These cells were examined for genetic alteration, UV sensitivity and vacuum sensitivity.
Strain 168 Spores: Spore suspensions (2XE08 spores/ml) in cold, distilled water and cold phosphate-buffered saline (PBS) were prepared preflight and transported to NASA Lyndon B. Johnson Space Center (JSC) from Frankfurt, Germany. During flight, samples were kept in cuvettes with quartz windows. Wet samples were prepared at JSC by pipetting 50 microliter aliquots of the suspensions into type A (aqueous sample) cuvettes. Dry samples were exposed on cover glass squares, which had glass bars affixed on two opposing edges providing space for the bacteria. Each support was 0.8 mm high and charged with 0.001 ml of cold spore suspension in distilled water or PBS, yielding 200,000 spores/support. These samples were dried for 24 hours over silica gel at 4 degrees Celsius. After drying, samples were kept at room temperature until transported to JSC, where type B (dry sample, unvented) and type C (dry sample, vented) cuvettes were loaded with the predried samples.
Cuvettes were placed in the Microbial Ecology Evaluation Device (MEED). During flight, samples were exposed to solar UV irradiation at a peak wavelength of 254 nanometers (three different doses) and to space vacuum for 1.3 hours. Nonirradiated flight controls were also exposed to space vacuum. Ground controls and launch vibration controls were irradiated and exposed to vacuum at JSC. A simultaneous laboratory simulation experiment, performed at the University of Frankfurt, ran exactly parallel to the time schedule of the Apollo 16 flight.
The survival value, N/No, of bacterial spores (where N is the number of colony formers after exposure and No is the number of colony formers of the controls) was evaluated in terms of colony-forming ability. Results were compared with those of the simultaneous laboratory simulation experiment.
Strains HA101 and HA101(59)F: In ground-based experiments, 1.8 ml aliquots of spore suspensions (100 million spores/ml) were irradiated in Petri dishes. Ground control spore monolayers were prepared on 0.45 micrometer membrane filters by filtration of aqueous suspensions; approximately 100 million spores were attached to a 25 mm diameter filter. Monolayers were subsequently dried at 20 degrees Celsius for 1 hour. Spores were recovered after irradiation by suspending the filters in a 0.025% solution of Tween 80 and sonicating for 15 minutes.
For flight studies, purified spores were diluted to 50,000,000 spores/ml in distilled water. Wet cuvettes contained 2.5 million spores per 50 microliters of fluid. Dry cuvettes contained spore monolayers on filters prepared as follows. First, 3.8 ml of the diluted spore suspension were filtered through a 47 mm filter (0.45 micrometer pore diameter, 960-square millimeters). The filters were dried on filter paper for 1 hour in Petri dishes. Chips (25-square millimeters, 5,000,000 spores/chip) were punched from each filter under sterile conditions.
Spores from type A cuvettes were recovered by sonification of the cuvettes for 1 minute in a bath of 70% ethanol. After drying the outside of the cuvette, the contents were removed. The inside of the cuvette was rinsed five times with 1.95 ml of a 0.025% Tween 80 solution. Pooled contents were stored at 5 degrees Celsius and assayed for viable counts and morphological mutants on AK agar plates. Spores from types B and C cuvettes were recovered by transferring chips from cuvettes to empty vials. Cuvettes were rinsed with 2 ml of a 0.025% Tween 80 solution and added to the vials containing chips. Vials were sonicated for 15 minutes to remove the spores from the filters. Viable-count assays were made on AK agar plates.
T-7 Bacteriophage of Escherichia coli
Survival studies of the T-7 bacteriophage of E. coli were included to relate the present experiment to the space-flight-mediated effects reported by the Soviets for the E. coli phage flown on previous flights. Rather than the T-1 or K-12 (lambda) phage commonly employed by the Soviets, the simpler and more stable T-7 phage was chosen for this study in the hope that it would be more resistant to the rigors of space flight and, therefore, prove to be a better UV test subject.
Purified T-7 phage was stored in 20 millimolar (mM) Tris (pH 7.5), 0.5 M NaCl and 20 microgram/ml gelatin solution. Concentrated stocks were diluted to 100 million plaque-forming units/ml for ground-based and flight experiments. For flight experiments, each cuvette contained 5 million phage units. Phage assays were performed using E. coli B as the indicator host on TBAB agar.
Inflight samples were exposed to UV irradiation at wavelengths of 254, 280 and 300 nm. In ground-based experiments, UV irradiation was generated using either a germicidal lamp or a quartz/mercury-vapor lamp. The germicidal lamp emission was measured with a UV meter; most emissions were at a wavelength of 254 nm. Filters were used with the quartz/mercury vapor lamp to provide maximal emissions at wavelengths of 254, 280 and 300 nm. Survival rates and genetic mutations were studied.
B. thuringiensis var. thuringiensis, which has been used widely as a biological insecticide, was selected for this experiment because it produces three toxins which are reactive against biological systems, and because it lends itself well to both rapid screening and critical in vivo analyses. The toxins include a lipolytic alpha-exotoxin (a phospholipase), a deforming beta-exotoxin (a nucleotide that is heat stable and kills insects at time of molt or pupation) and a crystalline gamma-endotoxin (a proteinaceous factor that destroys midgut cells in many Lepidopterans, causing gut paralysis and eventual death). Several in vitro and in vivo systems, including reactions in the silkworm and housefly, were used to evaluate possible alterations in toxin production.
Spores were obtained by growing the microorganism on AK agar slants for 4 to 5 days at 30 degrees Celsius. The spores were washed from the slants with sterile, distilled water and heat shocked for 10 minutes at 60 degrees Celsius to kill remaining vegetative cells. After three successive washes, the spore pellet was suspended in distilled water at a final concentration of 640 million spores/ml. One milliliter aliquots of the spore suspension were then drawn through filter pads, resulting in a monolayer of spores on the filter. The spore-impregnated filter was cut into chips 0.5 X 0.5 square centimeters, and loaded into type B cuvettes. In addition to various inflight stresses (i.e., shock, vibration and increased g forces during launch and recovery, reduced gravity and cosmic irradiation), the organisms in 32 of the 41 inflight experiment cuvettes were exposed to various levels of solar irradiation for 10 minutes. Nine cuvettes were held as ground controls and nine as vibration controls (exposed to simulated launch vibrations).
Upon return of the experiment hardware, the spore-impregnated filter chips were unloaded from their cuvettes and placed in 1 ml of distilled water at pH 6.0 for 24 hours. This treatment was followed by mild sonification to facilitate removal of the spores from the filter pads. After serial dilution, 0.1 ml aliquots of each spore suspension were spread onto sheep blood agar plates and maltose agar plates in quadruplicate. All plates were incubated at 30 degrees Celsius for 24 hours.
Colonies growing on sheep blood agar were counted to determine survival and analyzed for abnormal morphology. In addition, colonies were examined for zones of beta-hemolysis that deviated from controls. All abnormal colonies and 10% of the normal colonies were transferred to maltose agar plates and Sarcina flava inhibition plates.
Also, colonies growing on maltose agar were observed for abnormal colony morphology and the ability to use maltose. All abnormal colonies and 10% of the normal colonies were transferred to sheep blood agar plates and S. flava inhibition plates.
The S. flava inhibition plates were observed for beta-exotoxin production. Zones of inhibition were compared with those of the control groups. Any colony surrounded by a zone that deviated from the control standard was subjected to further quantitative analysis by using the housefly assay system. The housefly assay system consisted of spotting 1 ml of the serially diluted toxin preparation onto circular filter pads fitted into 20 X 60 mm Petri dishes. Each dilution was seeded with 10 late third instar housefly larvae in duplicate and held at room temperature until all controls had emerged and died. Then, each dilution was scored for the number of deaths, emerging adults and mutants. Abnormal isolates were also tested for alpha-exotoxin and gamma-endotoxin production. The test for alpha-exotoxin measured phospholipase activity on egg yolk agar plates.
To encourage sporulation, organisms were cultured on tryptose phosphate agar with manganese sulfate added at the rate of 2 ng/ml. Duplicate plates were made for spore-crystal production. These plates were incubated at 32 degrees Celsius for 72 hours. Cultures were washed off the agar with sterile saline after a nigrosin smear was made of each. Then, suspended spores and crystals were adjusted in a colorimeter to give the same turbidity. Precise amounts of each culture suspension were injected into the midguts of 15 third instar silkworm larvae, and these larvae were held in Petri plates and fed ad libitum for 24 hours, at which time deaths were recorded. A total of 3250 insects were injected.
N. dubius was the only multicellular organism used in this study. It is pathogenic to laboratory mice, but not to humans, and is insensitive to the special holding conditions of the flight hardware. Specimens subjected to the space flight experiment conditions were evaluated for changes in survival, infectivity of mice, adult formation, egg production and egg development.
Type A cuvettes were filled 16 days before launch with approximately 200 larvae in suspension. These cuvettes were sealed and categorized as 32 flight experimental (16 UV1 and 16 UV2), 20 flight control, 20 vibration control and 20 ground control. Flight experiment cuvettes were positioned in the MEED trays under selected neutral-density filters. Flight control cuvettes were positioned in the trays to prevent solar UV irradiation. The MEED was transported to the NASA John F. Kennedy Space Center (KSC) and loaded on board the Apollo 16 command module. Ground and vibration control cuvettes were loaded into assemblies identical to the flight unit. The ground control unit remained in the proximity of the flight experiment package until launch. The vibration control unit was tested at the experimental dynamics laboratory at JSC simultaneously with the Apollo 16 launch, and the vibration control unit was returned to the JSC Lunar Receiving Laboratory for the remainder of the test period.
In 14 days of space flight, larvae were exposed to zero gravity, fluctuating magnetic fields, high mass ionizing radiation and 10 minutes of filtered solar irradiation at a 254 nanometer wavelength. Cuvettes were returned to the laboratory 5 days after splashdown. The wax seal of each cuvette was removed, and the larval suspension was transferred to the well of a spot plate containing 0.2 ml of distilled water. Each cuvette was aspirated with an additional 0.05 ml of distilled water to ensure the removal of all larvae. The suspension volume was divided equally into two individual wells of the plate yeilding 75 to 100 larvae per aliquot. Larvae in each aliquot were counted and inoculated into individual male C57 mice. All mice were housed in individual cages for an infection period of 28 days. On postinoculation day 22, fecal pellets were examined quantitatively for eggs and were subcultured for viability. On postinoculation day 28, all mice were killed by cervical dislocation. Ten centimeters of the proximal portion of the small intestine, including the pyloric sphincter, were removed from each mouse and examined for adult worms.
Four fungi species were selected. Trichophyton terrestre was included to study animal tissue invasion, because it attacks human hair under laboratory conditions. Chaetomium globosum was of special interest because of its cellulolytic activity on cloth fibers similar to those contained in crewmember flight garments. Two yeasts, Rhodotorula rubra and Saccharomyces cerevisiae, were well suited to drug sensitivity studies.
Fungi in the flight hardware were exposed to UV irradiation at wavelengths of 254, 280 and 300 nm, full light and dark. Cuvettes housed in the vented-exposed tray, unvented-exposed tray, flight control tray, ground control tray, and vibration control tray were unloaded systematically. The retrieved fungal cells were placed in a dilution series for viability, survival and phenotypic selection studies. Single-cell phenotype isolates were selected by colony development changes, which included variation in growth rates, sporulation, pigmentation, texture, density and perimeter.
Mature ascospores, yeast cells and conidia were harvested, and cell suspensions were made using sterile, distilled water. Wet cuvettes were filled with a diluted fungal cell suspension (10,000 or 100,000 cell counts/0.05ml cuvette volume, depending on the cell size). Suspensions were filtered, and filter paper squares with attached fungal cells were cut with a square punch and placed in dry cuvettes. To reduce cell loss from static electricity, filter paper squares were placed in the cuvettes before they were dry.
Tween 80 was used to wet the filter paper squares before removal and to wash the cuvette interiors. All procedures were conducted aseptically.
Two methods were employed to monitor the actual radiant energy penetrating selected optical components of the flight hardware. One of these methods involved Kodak High Resolution Film (Estar Thick Base) SO-343 which had been purged of oxygen and sensitized with dry nitrogen gas to decrease the latent-image fading rate. This system was reliable over a range of 40 to 520 ergs/square centimeter total energy with a 254 nm peak wavelength.
Solar irradiation within the range of 40,000 ergs/square centimeter to 400,000 ergs/square centimeter was monitored by an adaptation of the Potassium Ferrioxalate Actinometry System.
High Energy-Multicharged Particle Dosimetry
It was impossible, in the design of the flight hardware, to protect test systems from galactic irradiation. Therefore, this factor was measured to better understand any observed biological effects. Galactic irradiation measurements were conducted to evaluate the effect of high energy-multicharged (HZE) particles on biological systems. A 2.5x3.8 cm container was provided within the flight hardware and ground control units to house four types of dosimeters capable of recording HZE particles. Lexan dosimeters, identical to those employed in the crew personnel passive dosimeters, were used for direct correlation. Cellulose nitrate (CN) dosimeters were included in the MEED as well as in the Apollo Light Flash Moving Emulsion Detector (ALFMED) which was flown on Apollo 16, again allowing for direct comparisons. The other two detectors, Ilford G5 and silver chloride, were flown only in the MEED, but were of considerable value in establishing the HZE particle environment experienced by the flight hardware.
Lithium fluoride (LiF) thermoluminescent dosimeter (TLD) chips were used to provide an integrated dose from the broad spectrum of ionizing radiation present in the space environment. The 5 by 5 by 1 millimeter chips were annealed (1 hour at 400 degrees Celsius followed by 2 hours at 100 degrees Celsius) and loaded into cuvettes. Fifty cuvettes were loaded as follows: 19 cuvettes for mounting in the flight hardware; 21 cuvettes for vibrational testing and ground control; and 10 cuvettes for irradiation calibration. The TLD-loaded cuvettes were then placed at various sites within the hardware.
Postflight analysis indicated no significant difference between inflight and ground-based control viability or survival rates after exposure to UV irradiation. Results of endopeptidase and hemolysin production are unavailable.
Bacillus subtilis spores
The combined action of space vacuum and solar UV irradiation at a peak wavelength of 254 nm resulted in greater viability loss than in ground-based studies. Space vacuum alone did not cause a decrease in predried spore survival, indicating air-dried spores may survive exposure to space vacuum if shielded against solar irradiation. Additional space flight environmental factors did not measurably influence viability and irradiation responses of Strain 168 spores.
Strains HA101 and HA101(59)F were exposed to the space flight environment both in aqueous suspensions and dry layers. Lethal irradiation effects at peak wavelengths of 254 and 280 nm were greater for dried spores than for those exposed to distilled water. Additionally, the repair-defective strain was more sensitive at both UV irradiation wavelengths. As expected, survival rates for space flight-exposed spores did not differ significantly from analogous aliquots in the ground control units.
Mutation frequencies for morphological and asporogenous phenotypes are maximum at low doses of radiation. However, for the HA101 strain, the mutation frequencies are increased with higher energies at both wavelengths.
Bacillus thuringiensis var. thuringiensis
As with B. subtilis, no significant difference between the mean of B. thuringiensis survivors from ground, flight or vibration control units was observed. Also, there was no significant difference between the means of survivors for any of the groups exposed to solar UV light in space. There was a significant difference (p<0.01) in the survival rate of groups exposed to full sunlight in space when compared with the nonirradiated control groups. This indicates that space-flown spores of this species were resistant to levels of UV irradiation encountered in the test, but were sensitive to full sunlight. This follows previously established patterns obtained from ground-based studies and is not considered anomalous behavior.
T-7 Bacteriophage of Escherichia coli
Large losses in non-UV exposed flight subjects (as compared to ground controls) are not indicated during postflight survival evaluations. This supports the hypothesis that the T-7 phage is more resistant to the rigors of space flight and proves to be a better UV test subject than the T-1 or K-12 (lambda) phage. The lethal effect of inflight solar UV irradiation at a peak wavelength of 254 nm was considerably higher than ground-based controls exposed to the same irradiation levels; however, the characteristic shape of the dose response curve was similar to the ground control data curve.
A total of 200,000 ergs/square centimeter of solar inflight UV irradiation at a peak wavelength of 254 nm was sufficient to completely inhibit infection in the murine host and subsequent maturity to adult worms. Therefore, the survival of space flight irradiated larvae was too low for further comparative studies.
Comparison of nonirradiated flight and ground control subjects revealed no differences in survival, infectivity in mice, adult formation or subsequent egg production. A significant decrease in egg viability within the group of adults which descended from flight control larvae was noted. This was an important observation since this control group was not purposefully exposed to any experimental stresses and was simply a "passenger" on the space flight.
Postflight analysis indicated that dosimeters received as much energy as had been expected from calculations based on data from the NASA established Solar Spectral Irradiance Standard. The photographic film monitoring method proved to be a useful tool for measuring small amounts of UV irradiation in space.
Data indicate that neither the simulated launch vibration nor the total space flight exerted a detectable change in preirradiated control systems. The ferrioxalate monitoring system, therefore, was shown to have the stability required for successful measurements made within the flight hardware. Analysis of inflight irradiated actinometry systems verified that the optical filter components of the MEED allowed for critical evaluation of exposed biological test systems.
High Energy-Multicharged Particle Dosimetry
Both Lexan and CN detectors revealed track fluences (track/square centimeter) of HZE particles. Since the CN detector is more sensitive, it showed track fluences substantially higher than those found in Lexan. The CN records particles with a Z (atomic number) greater than six, while Lexan records particles with a Z greater than ten.
Comparison of Lexan and CN track fluences found in the MEED flight hardware were lower than those found in either ALFMED or the passive personnel dosimeters. These observations, along with depressed TLD values, imply that the MEED flight hardware had a greater average shielding as compared with either ALFMED or the personnel passive detectors. Likewise, data are slightly lower than those obtained from the TLD and CN detectors employed in the BIOSTACK flight hardware, which was stowed in the Command Module in an area of minimal shielding to ambient cosmic radiation.
Statistical analysis indicates that various areas within the MEED received uniform irradiation from ionizing irradiation components of the space environment. Therefore, it is valid to omit this factor as a variable when comparing inflight test systems. The mean dose of all MEED TLD was 0.48+0.02 rad with a range of 0.44 to 0.51 rad. Doses to crewmembers (from crew passive TLD measurements) ranged from 0.48 to 0.54 rad, with a mean of 0.51+0.02 rad. The dose of 0.48+0.02 rad represents a total absorption of 48 ergs of ionizing energy per gram of biological material within the MEED. This value was applicable to all samples within the flight hardware, including flight controls and UV irradiated samples.
In conclusion, no statistically valid differences could be detected in the survival of flight samples when compared to corresponding ground-based controls, although a reasonable variety of organisms (viruses, yeasts, filamentous fungi, bacteria and an invertebrate) were tested under several different conditions. In general, these evaluations were based on multiple observations of ten to thirty replicates of up to one million cells each. While the results of this experiment conflict with those of other space flight investigations, it must be observed that certain space flight conditions cannot be exactly duplicated, and therefore results from different flights are not directly comparable.
|Mission||Launch/Start Date||Landing/End Date||Duration|
|Apollo 16||04/16/1972||04/27/1972||11 days|