| Approved For Release 2000/08/10 : CIA-RDP96-00789R003800280001-0 [ | | (U) The heritable material of living organisms is contained in the DNA (RNA in some ™ viruses), a large molecule so constructed that it can replicate itself in a most exact fashion one cell generation after another. This is the basis of biological continuity and unity. It is also, = however, the basis for biological diversity, which occurs through mutations. Each mutation alters the action of a specific gene, which is a genetic entity with its own specific end product, imi or protein. Genes are very stable structures, but each has its own spontaneous mutation frequency. The probability that a spontaneous mutant cell will be obtained every time a cell oi divides is constant, provided the environmental conditions are unchanged. Changes in the environment are known to influence the mutation frequency. Such changes include the presence or absence of certain trace elements (e.g. selenium), plus the presence of physical ™ or chemical agents (mutagens). - ee _ ‘ Bacteria provide a convenient way to study mutations because millions of cells can be grown in a very short period of time. Over the past few years, several bacterial assays have been developed to screen chemicals for their ability to induce mutation. Because there “ is a close correlation between mutagenesis and carcinogenesis, such mutagenicity assays are very often used together with the in vitro tests that employ single microbial and/or mammalian = cells, as well as in vivo tests that employ multicell organisms from insects (fruit fly) to mammals (rodents). One of the best known bacterial mutagenesis assays is the Salmonella/ i | mammalian microsome histidine reverse mutation assay developed by Dr. Bruce Ames at the | University of California in Berkeley. The Microbial Genetics Department at SRI International | is using this assay system on a daily basis for Government agencies and commercial clients to ™ | determine the mutagenic potential of chemicals; they have performed such testing over a . period of more than 10 years. = | (U) The Salmonella assay employs several tester strains of Salmonella typhimurium, am: ‘each with a unique specificity for detecting chemical mutagens. The Salmonella strains, under | optimum conditions, have a generation time of less than 30 minutes. The bacterial strains are unable to grow in the absence of the essential amino acid histidine because ofa mutation in ™| - one of the genes that is needed for histidine synthesis. When these bacteria are plated on defined selective medium having litle or no histidine, little or no growth occurs except those =, few bacteria-that spontaneously mutate back to histidine independence (ability to grow in the | absence of histidine). In this case, a nonfunctional gene product is reverted back to a _ functional one. This event allows the mutant cells to grow and divide. Because a mutation is | stably inherited, all progeny of the mutated cells retain the ability to grow in the absence of Approved For Release 2000/08/10 : CIA-RDP96200789R0038 0001-0
I : : 7 7 7 7 7 : : 7 - * | —, 4 Approved For Release nL : CIA-RDP96-00789R003800280001-0 (U) histidine. Distinct individual colonies will appear on the solid selective growth medium, with each colony containing billions of progeny of the spontaneously mutated cells. Exposure of the bacteria to a chemical mutagen will result in an increased number of colonies appearing on the solid selective growth medium, due to an increase in mutation induction. (U) In the Ames Salmonella assay, a small amount of histidine is added to the growth medium to allow for a few cell divisions of all the plated histidine requiring mutants (~10*). Such growth is often necessary for chemical mutagenesis to occur. The results of the Salmonella assay are usually expressed in terms of the number of revertant colonies per amount of chemical added to the selective growth medium, which is usually delivered to the plate in 25-ml volumes. Because of the presence of limited histidine in the selective medium, the results of the Ames Salmonella are considered “semiquantitative,” since residual growth on all plates (control as well as chemical treated) does not allow for quantitative survival determination. A quantitative mutation frequency, however, can be determined. It is more labor intensive than the standard Ames assay, because survival determination requires diluting of the cell cultures, and a different growth medium is needed for determining (1) the mutant fraction and survivors for each of the controls, and (2) the different exposure concentrations of the test chemical. The mutation frequency is defined in terms of number of mutants per given number of surviving cells, usually per 108 cells. Because of its simplicity and the rapid response time of about two days, the Ames Salmonella assay can readily be adapted to study the effect of RA on the mutation frequency. Such an adaptation was established by SRI’s Microbial Genetics Department for use in this experiment; a detailed discussion of the specific biological procedures that were followed can be found in Section D, Protocols. C. (U) Experimental Design 1. (U) Conceptual Replication ; The experiment undertaken in this study represents a conceptual replication of the Nash experiment described in our Introduction chapter. The replication presented here is termed conceptual, because several of the experimental details of the Nash experiment have been changed and improved. First, two potential mechanisms have been postulated that could account for the acquisition of a statistically significant effect--that is, an IDS hypothesis has been advanced, in addition to the more established RA hypothesis. Second, Salmonella 10 Approved For Release 20 diosi10 : CIA-RDP96-00789R003800280001-0 | i i
—1 i pee Approved For Release 2000/98/10 : CIA-RDP96-00789R003800280001-0 typhimurium rather than Escherichia coli were used as the target bacterial cultures. Because this particular species of Salmonella is used most frequently by SRI’s Microbial Genetics Department in toxicity studies, its behavior is particularly well understood in terms of assay conditions and experimental protocols. Finally, the Nash analysis was extended to include multiple analyses of variance. As in the Nash experiment, nine test tubes filled with dilute bacterial culture were used per session. Mutation from histidine dependence to histidine independence was mentally promoted by the subject in three of the tubes, mentally inhibited in three, and the remaining three tubes served as controls. For the purposes of obtaining baseline data, two additional control test tubes (for a total of eleven altogether per session) were prepared in the same manner as the session test tubes, but were kept in the Microbial Genetics Laboratory. 2. (U) Model Testing Criteria a. (U) The IDS Model (U) As mentioned previously, there were two primary models under investigation in this experiment. A pivotal concept to the first, or IDS favorable model, is freedom of choice: namely, that by using some type of psi-mediated informational processes, subjects have the opportunity to select out locally-deviant subsequences from a larger random sequence. For example, in half of the sessions, the subjects were allowed to select the three test tubes in which they wished to promote mutation, and the three test tubes in which they wished to inhibit mutation. A statistically significant deviation from mean chance expectation (MCE) in this condition, therefore, could be interpreted theoretically in two ways: (1) the subjects somehow mentally “forced” genetic changes to occur in the bacteria in accordance with their desires to either promote or inhibit mutation rates (the RA hypothesis); or (2) given the natural spread of mutation rates in a biological system, the subject was able to psycho- energetically sort those test tubes containing bacteria with high mutation rates from those tubes containing bacteria with low mutation rates (a session—by-session IDS hypothesis). b. (U) The RA or IDSU Model (U) The second model under investigation has been termed the Remote Action (RA) or Intuitive Data Sorting Unfavorable (IDSU) model. In this condition, the conduits by which either the subject or experimenter are able to select test tubes are rendered 2 Approved For Release 20 0/08/10 : CIA-RDP96-00789R003800280001-0
Approved For Release poorer : CIA-RDP96-00789R003800280 01-0 (U) the seven had been participants in previous psychoenergetic experiments—-i.e., all four had to demonstrated some ability in remote viewing. One of these four subjects also scored significantly in an earlier SRI random number generator PK experiment, and another had demonstrated some ability previously in Computer—Assisted Search (CAS) tasks. All of the participants were SRI employees: one was a Statistician, two were secretaries, and the remainder were research professionals in either physics or computer science. b. (U) Experiment Site Locations ow j | For reasons stated in Section C.2.a, it was determined that the biological technician should be kept entirely blind as to all facets of the experiment, and that, in order to facilitate this situation, the psychoenergetic testing should occur in a location that was different from the one used for the biological preparations. The Microbial Genetics Laboratory, therefore, was used for all aspects of preparation of the biological cultures, and a room in another building at SRI was used for the psychoenergetic sessions. c. (U) Hardware Construction Once it had been determined that two separate facilities were necessary for conducting the experiment, a container had to be constructed that was suitable for transporting the biological samples from the Microbial Genetics Laboratory to the psychoenergetics facility. There were three primary factors that dictated the design of the container: (1) the biological samples had to be protected from extreme variations in temperature; (2) the samples had to be protected from sunlight; and (3) the container had to be lockable. \ » To control against extreme variations in temperature, which can greatly affect the mutagenicity of Salmonella, a Coleman® ice chest was chosen as the transport container. Triple-paned insulated glass windows were specially installed in the top and front side of the ice chest to allow an unobstructed view of the experiment test tubes. Because sunlight also affects the. mutagenicity of the bacteria, a tarpaulin was used to completely cover the cooler during transport between the biological laboratory and the psychoenergetics facility. A lock was installed; the key was retained exclusively by the biological technician, to preclude the possibility of tampering with the biological samples once they had been removed from the Microbial Genetics Laboratory. Approved For Release 2000/08/10 : CIA-RDP96-00789R003800280001-0