WEBVTT 00:00:00.000 --> 00:00:12.000 [NATIONAL LIBRARY OF MEDICINE FREDERICK FACILITY HF3412 16 mm COLOR PRINT OPTICAL SOUND] 00:00:12.000 --> 00:00:17.540 [THE P4 CONTAINMENT FACILITY FOR RECOMBINANT DNA RESEARCH] 00:00:17.540 --> 00:00:23.220 [Located at THE FREDERICK CANCER RESEARCH CENTER Frederick, Md.] 00:00:23.220 --> 00:00:30.500 [NATIONAL INSTITUTES OF HEALTH] 00:00:30.500 --> 00:00:36.600 [Narrator:] The first certified P4 containment facility in the United States for recombinant DNA research, 00:00:36.600 --> 00:00:44.110 a laboratory designed to meet the requirements set forth in the NIH guidelines governing recombinant DNA research, 00:00:44.110 --> 00:00:47.100 is now available to scientists. 00:00:47.100 --> 00:00:50.640 The laboratory itself is under negative air pressure. 00:00:50.640 --> 00:00:58.210 The experiments are carried out from beginning to end inside an elaborate system of enclosed gas-tight cabinets. 00:00:58.210 --> 00:01:02.800 This means that nothing, not even the tiniest particle of dust or air, 00:01:02.800 --> 00:01:04.750 or the smallest micro-organism, 00:01:04.750 --> 00:01:09.120 can penetrate the heavy stainless steel and glass cabinets. 00:01:09.120 --> 00:01:13.650 This system of cabinets measures about seventy feet in combined length. 00:01:13.650 --> 00:01:19.720 It is contained inside a room which has an area of about four hundred square feet. 00:01:19.720 --> 00:01:25.150 Using the shoulder-length rubber gloves, which are tightly attached to the sides of the cabinet, 00:01:25.150 --> 00:01:33.730 laboratory workers are able to move and control all the materials and tools used in the experiment without direct contact 00:01:33.730 --> 00:01:40.320 between their hands and anything inside the cabinet. 00:01:40.320 --> 00:01:46.470 Each experiment must be carefully planned well in advance, since all of the materials used in the experiment, 00:01:46.470 --> 00:01:50.630 including chemicals, cultures, and laboratory equipment, must be brought 00:01:50.630 --> 00:01:56.560 into the group of interconnected cabinets, or cabinet line, as it is called. 00:01:56.560 --> 00:02:03.440 One of the methods of bringing materials into the cabinet is through an autoclave, or steam sterilizer. 00:02:03.440 --> 00:02:12.220 Equipment and materials are loaded onto a shelf inside the autoclave for transfer to the interior of the cabinet lines. 00:02:12.220 --> 00:02:20.420 A heavy door is then swung into place and locked to provide a tight seal. 00:02:20.420 --> 00:02:25.230 A laboratory worker removes them from the cabinet side of the autoclave. 00:02:25.230 --> 00:02:28.590 Because the interior of the device has now been exposed 00:02:28.590 --> 00:02:30.530 to the inside of the cabinet, 00:02:30.530 --> 00:02:32.780 it will be necessary to run the autoclave 00:02:32.780 --> 00:02:41.840 through a sterilizing cycle before it is opened again on its outside end. 00:02:41.840 --> 00:02:47.340 The two-hundred and fifty degree Fahrenheit steam pressure cycle takes time. 00:02:47.340 --> 00:02:55.690 All large items have to enter through the autoclave, so planning its maximum use is essential. 00:02:55.690 --> 00:02:58.160 The cabinet lines are modular, that is, 00:02:58.160 --> 00:03:04.700 made up of individual connected units allowing the free movement of materials within the lines. 00:03:04.700 --> 00:03:10.340 At least two workers are required for most manipulations and movements of materials. 00:03:10.340 --> 00:03:15.130 A variety of equipment for experiments must be contained within the cabinets, 00:03:15.130 --> 00:03:26.930 and that includes a refrigerator which is used for both long- and short-term storage of materials. 00:03:26.930 --> 00:03:35.810 Here, supplies of growth media are being checked and readied for later use in preparing bacterial culture. 00:03:35.810 --> 00:03:41.120 Another designed feature of the refrigerator is movable shelves which slide in and out 00:03:41.120 --> 00:03:48.020 to allow the laboratory worker easier access to materials stored within. 00:03:48.020 --> 00:03:51.580 Note the tools used to extend the limited reach of the researcher 00:03:51.580 --> 00:03:59.090 as he works within the restricted space of the gas-tight cabinets. 00:03:59.090 --> 00:04:06.010 The cabinet system in this laboratory has two levels which are connected by a small elevator or dumbwaiter. 00:04:06.010 --> 00:04:08.540 Specially weakened strains of bacteria 00:04:08.540 --> 00:04:12.920 which cannot survive outside of the carefully controlled laboratory conditions, 00:04:12.920 --> 00:04:18.480 are grown in a waterbath located on the second tier of the system. 00:04:18.480 --> 00:04:22.500 Once the cultures have been grown and are ready for use in the experiment, 00:04:22.500 --> 00:04:25.230 they are transferred to the nearby elevator, 00:04:25.230 --> 00:04:30.650 which descends with them to the lower level. 00:04:30.650 --> 00:04:37.260 Here, most of the biochemical and microbiological steps in the experiment are actually carried out. 00:04:37.260 --> 00:04:42.010 The weakened strains of bacteria which are used in recombinant DNA research 00:04:42.010 --> 00:04:46.470 make up a second form of containment called biological containment. 00:04:46.470 --> 00:04:49.090 [simulated test] 00:04:49.090 --> 00:04:51.020 Although physical barriers or physical containment 00:04:51.020 --> 00:04:55.260 are highly effective in preventing the escape of micro-organisms, 00:04:55.260 --> 00:04:59.630 biological containment adds a built-in safeguard. 00:04:59.630 --> 00:05:05.650 The crippled bacteria are so weak that they self-destruct outside the laboratory. 00:05:05.650 --> 00:05:13.650 These two types of barriers, physical and biological, are always used in combination. 00:05:13.650 --> 00:05:18.750 This particular cabinet system has an area with glove ports on both sides. 00:05:18.750 --> 00:05:21.990 This arrangement allows two laboratory workers to team up 00:05:21.990 --> 00:05:24.710 for some of the more delicate operations, 00:05:24.710 --> 00:05:31.060 such as the transfer of cultures or reagents from one container to another. 00:05:31.060 --> 00:05:38.420 Continuing the inventory of research tools mounted within the cabinet line is a high speed centrifuge. 00:05:38.420 --> 00:05:42.110 This device, mounted flush with the bottom space of the cabinet, 00:05:42.110 --> 00:05:45.710 is using in purifying DNA. 00:05:45.710 --> 00:05:50.930 Spinning rapidly, DNA separates from protein and solvent. 00:05:50.930 --> 00:05:59.400 The control panel for this centrifuge is mounted outside the barrier of the gas-tight cabinet. 00:05:59.400 --> 00:06:07.790 Bacteria for experiments such as this are grown in single colonies which are cultivated in standard laboratory petri dishes. 00:06:07.790 --> 00:06:15.460 The petri dishes are kept in an incubator inside the cabinet line. 00:06:15.460 --> 00:06:20.210 The microscope is a vital tool in experiments involving micro-organisms. 00:06:20.210 --> 00:06:23.300 An instrument is mounted in this laboratory's line. 00:06:23.300 --> 00:06:27.520 Despite the restraints that are imposed by the heavy rubber gloves, 00:06:27.520 --> 00:06:32.940 skilled laboratory workers are able to make delicate adjustments on the microscope. 00:06:32.940 --> 00:06:44.690 A closed-circuit television system with the screen mounted just above the researcher's head provides the means for viewing. 00:06:44.690 --> 00:06:50.530 A second means of entering the enclosed cabinet line is through a device called a dunk tank. 00:06:50.530 --> 00:06:56.970 This consists of a tank filled with a strong liquid disinfectant and is an integral part of the barrier, 00:06:56.970 --> 00:07:03.410 and like the autoclave, prevents the passage of unwanted living organisms in either direction. 00:07:03.410 --> 00:07:12.880 A leakproof container is used to convey materials needed inside the line, safely through the dunk tank. 00:07:12.880 --> 00:07:17.270 Note how the attached gloves are pulled back inside the cabinet. 00:07:17.270 --> 00:07:23.350 This is due to the negative air pressure that is constantly maintained within the cabinets. 00:07:23.350 --> 00:07:30.140 This added safety feature ensures that no organism escapes, should the gloves be punctured. 00:07:30.140 --> 00:07:36.500 Although use of experimental animals is not a significant part of most recombinant DNA research, 00:07:36.500 --> 00:07:42.580 this laboratory is equipped to house animals on two tiers within the cabinets. 00:07:42.580 --> 00:07:49.550 Experimental mice like these can be inoculated and examined within the cabinets. 00:07:49.550 --> 00:07:55.710 The daily care and feeding of the animals is also carried out entirely within the cabinet system. 00:07:55.710 --> 00:08:02.040 Materials needed to work with and care for the animals are introduced through the dunk tank. 00:08:02.040 --> 00:08:08.490 When necessary, cages and waste are removed in the same fashion that all other material is taken out: 00:08:08.490 --> 00:08:10.390 through an autoclave. 00:08:10.390 --> 00:08:17.660 The material is placed inside and is then heated to a temperature sufficient to kill all organisms. 00:08:17.660 --> 00:08:20.090 At the end of a sterilization cycle, 00:08:20.090 --> 00:08:24.190 material is removed from the autoclave for further processing, 00:08:24.190 --> 00:08:27.760 either washing if it is reusable laboratory equipment, 00:08:27.760 --> 00:08:32.440 or disposal as trash if it is not reusable. 00:08:32.440 --> 00:08:37.520 As any material leaves the building, it is sterilized a second time. 00:08:37.520 --> 00:08:45.590 Material is loaded into the autoclave and again heated to achieve complete sterilization. 00:08:45.590 --> 00:08:47.970 [A Closer Look at DNA] 00:08:47.970 --> 00:08:53.020 We have seen the facilities, equipment, and techniques that will be used in this laboratory. 00:08:53.020 --> 00:08:55.570 Now let's take a close look at the substance 00:08:55.570 --> 00:09:01.980 which is the focus of the scientific activity which will be carried out here. 00:09:01.980 --> 00:09:06.380 In order to show more clearly some of the methods of DNA research, 00:09:06.380 --> 00:09:10.830 we are going through a non-recombinant DNA procedure. 00:09:10.830 --> 00:09:13.210 Close-up pictures, which you will see, 00:09:13.210 --> 00:09:20.700 of a bacterial DNA culture were made under P3 conditions. 00:09:20.700 --> 00:09:25.900 Reagents are added to the purified DNA to separate it from other materials 00:09:25.900 --> 00:09:32.210 such as protein and to prepare it for spinning. 00:09:32.210 --> 00:09:40.060 A glass rod can then be used to spin the purified DNA into a cohesive mass. 00:09:40.060 --> 00:09:44.370 Pressed against the side of the test tube to eliminate excess moisture, 00:09:44.370 --> 00:09:49.160 the long strands of DNA take on the appearance of transparent thread, 00:09:49.160 --> 00:09:54.620 wrapped around the end of the glass rod. 00:09:54.620 --> 00:09:59.350 The DNA molecules may be dissolved again in a salt solution 00:09:59.350 --> 00:10:09.280 for further study and analysis of the DNA's genetic or biochemical make-up if so desired. 00:10:09.280 --> 00:10:22.820 At this stage the DNA is ready for spinning or other processing as may be be called for in the experiment. 00:10:22.820 --> 00:11:29.585 [Film ends]