BRL eee DEPART My OF HEALTH, COUCATION, AND WELFA PUPLIC HEALTH SERVICE RE CRANT APPLICATION SECTION 1 D. A. Glaser Form Approved Budget Bureau No. CER LEAVE BLANK NUMBER TYPE |PROGIAM REVIEW GHOUP FORMERLY COUNCIL (Avoata, Weer} DATE RECEIVE TO CE COMFLETED OY MRIRCIPAL INVESTIGATOR fitems T through 7 and THA} yt. TITLE OF PROPOSAL (Do ast exceed $3 typawriver spaces) Scanner-Coapuver Irnvestigetions of Biological Svatems- 2. PRINCIF.AL INVESTIGATOR 3, DATES OF ENTIRE PROPOSED PROJECT PERIOD (This spplicsi OA, NAME (Last, First, Initiel) : FNOM THROUGH Glaser, Ponsld A. O11 June 1075 Fl May TORD DB. TITLE OF POSTION. 4, TOTAL DIRECT COSTS HE. [BT DIRECT COSTS REQUESTEL Professor of Physics and Molecular Biology QUESTED FOR PERIOD IN ITEM 3 6,124,938 | _i,f ’ FOR FIRST 12-MONTR PERI 1, 646,655 2C, MAILING AGORESS SHBG cay, Site, Zip Code} Denartment of Molecular Biology University of California Berkeley, California 94720 6. PERFORMANCE STTETEY (See instructions} University of California Berkeley, California 94720 — 20, DEGREE 2E, SOCIAL SECURITY NO. 2E.TELL Paces Coda TELEPHONE f o ENSION PHONE DATA LATS 6)2 14410 “a. BEAR IMEAY, SERVICE, LAEORATORY OR EQUIVALENT {See f; nstrustions) Virus Laboratory . + ZH, MAJOR SUEDIVISION (See frstructions} * Collerce cf? Letters and Science 7 Hesearen trveiving Wunisn Subjects (900 instructions) ABS HO §{C] YES Approved: C.(E] Ves - Pencing Review ete TO BE CONVLETED OY RESPONSIBLE ADMINISTRATIVE AUTHORITY (items & throuah 13 an 8 Inventions (keaava! Applicants Only + See fastryctions) ACIJNO 8.[(C) VES — Not previously reported C. CAIYVES — Previousty raported nt ThE) 9, APPLICANT GRGANIZATIGONIG) (Sce fasiructions) Vi. TYPE OF ORGANIZATION (Cncek anpicedls item!) The Regents of the University of California (FEDERAL CO] stare (CO tocalL COOTHEN (Specify) e/c Campus Research Office MLL Vireeler . 12, NAME, TILE, ADDRESS, AND VELEPHONE NUREER OF University of California OFFICIAL tN BUSINES 5 OFFICE WHO SHOULD ALSC gE NOTIFIED If AN AWARD IS MADE . Berkeley, California $4720 . . Mr. August G. Manga, Manager Carpus Research Office - MIL Wheeler, University of California TO RAGE, TITLE, ARD TELEMIONE NUIMSER OF OF FICIALE) Berkeley, California SIGWING FOR APPLICANT ORGANIZATIONS) Telephon umrct. PSIBERTTEY ORG i WICAVIGRAL 5 FOR INSTITUTIONAL GRANT PUSE Cees: ve fastructians} Bionedical Seiences Support Grant. . Telephone Sarcber (a: 14, ENTETY HUNBER (Formerly PHS Account Number) hei7o Poa 16, CENT FICAT iON AND ACCUPTANCE, knowledea ond ercont, 93 te any arent awarded, the obligation te comply ve We, the uncselgaad, certify that the ¢ tatements herein eru truo and comple to the best of our ith Public Health Service turms and conditions In effect ot the tive of ihe marr’, GIRS TOS BoA Snr ae nc (Si pnetuces be quad en . oN. orginal copy only. GB. SIGNAT Ricwlab bor SR NAMED IN TTEM 40 DATE - Lawl? ony Uen bok,” tpsepotpreat coma nor ho etn semngetite RedState shee vanes ee ee eee REMOVE AND USE FOR DRAFT COPY “Donald A. Glaser, ‘John Bercovitz, D. A. Glaser SECTION 1 DEPARTMENT OF HEALTH, EDUCATION, ANDO WELFARE LEAVE BLANK ' PUBLIG HEALTH SERVICE PROJECT NUMBER RESEARCH OBJECTIVES , NAME AND ADORESS OF APPLICANT ORGANIZATION University of California Bertteley, California 94720 NAME, SOCIAL SECURITY NUMBER, OFFICIAL TITLE, AND DEPARTMENT OF ALL PROFESSIONAL PERSONNEL ENGAGED ON PROJECT, BEGINNING WITH PRINCIPAL INVESTIGATOR » Professor of Physics and Molecular Biology, Virus Lab. Assoc. Development Engrg., Virus Laboratory , Acst. Development Engrg., Virus Laboratory Assoc. Development Mngrg., Virus Laboratory » Principal Programmer, Virus Laboratory Post-Grad. Research Biophysicist, Virus Laboratory Asst. Develop. Energ., Virus Laboratory » Sr. Development ingrg., Virus Laboratory » Principal Development Ingrg., Virus Laboratory (cont. on p. Ronald Baker, dames Berk, : Fraser Bonnel John Couch, Ted Fujita, Robert Henry, Leif Hansen, TITLE OF PROJECT Scanner-~Computer Investigations of Biological Systems USE THIS SPACE TO ABSTRACT YOUR PROPOSED RESEARCH, OUTLINE OBJECTIVES AND METHODS, UNDERSCORE THE KEY WORDE (NOT TO EXCEED 10) IN YOUR ABSTRACT, - Large scale genetic and physiological studies of bacteria, yeasts, and animal cells grown in tissue culture will be carried out using recently constructed automated eguapment and compyter-directed patvern recognition techniques. By automatic exami- nation of up to 10” colonies in'a batch, rare mutants will be isolated and partially characterized, mutagenic effects of chemical and physical agents will be measured , ever, at low dosés, and genctic recombination frequencies measured accurately for mapping purposes. Mutants for detailed studies of DNA synthesis in BE. coli and B. subtilic will be isolated and partially characterized. Mutants of E. coli, S. typhimuriun and Saccharomyces cerevisiac will be isolated vor study of biosynthetic and degra- dative pathways and for analysis of the mechanisms of genetic recombination. Genetic maps of E. coli, Saccharomyces cerevisiae, and some manmalian cells will be enlarged. Feasibility studies of automatic recognition of bacterial and fungal pathogens in medical and public health applications will be extended. Mutagenic effects of food additives and other enviromental chemicals will be tested in several bacterial, yeast, and animal cell systems. Carcinogenic effects of chemical and physical agents including ionizing rediation will be measured using animal cells. Interactions of hormoncs and other agents with tumor cells grown in tissue culture will be examined SO investigate the biochemical mechanism of the interactions and to test possible anti-neoplastic effects of a variesy of substances. Mutant tumor cells sensitive to some agents and resistant to others will be isolated for further study. Screening. programs may be undertaken when feasible for iwutagens, carcinogens, anti-necplastic agents, and effects oF low doses of mutagens and. ionizing radiation: Additional instrumentation will be constructed as needed. Pe bat wxe fil . : Donald A. Glaser Condsvatioa pou 2g ow ‘Privileged Communication... Professional Personnel (continued) Larry Johnson, é _— Assoc. Devel. Engrg., Virus Laboratory Alex Para, sst. Devel. Engrg., Virus Laboratory ve Ale Ur Laser ~ FROM THROUGH MONTH P a DETAILED BUDGET FOR FIRST 12-MONTH PERIOD June 1, 1975 May 31, 1980 ip yh ae DESCRIPTION (Iremizo) rime on AMOUNT REQUESTED (Omit cents} PERSONNEL EFFORT FRINGE AS, Y NAME TITLE OF POSITION we en BENEFITS TOTAL cca. [PRINCIPAL INVESTIGATOR (See details on sitttached p, 34) 505.872 CONSULTANT COSTS Occasional engineering consultents for special technology 1,500 EQUIPMENT (see details on atteched p. 4b) 828, 200 SUPPLIES ~ “(See details on attached p. 3c) 204 , 642 ._,. One major trip to Hast Coast for professional persons to DOMESTIC . ho attend a major corfermce (Gordon,CSH (52,000); attendance TRAVEL me SCORED by professionals and grads at local conference (Arrowhead, ~ Biophysics); confer with colleagues and equip.suppliers. 4,009 PATIENT COSTS (See instructions} . ($1 , 000 } ALTERATIONS AND RENOVATIONS THER EXPENSES (ftomize) (See details on attached pn. 3c) 103,451 TOTAL DIRECT COST {Enx. Page 1, Item 5) (Enter on Page 7 a 1,616,655 inbIRECT” saws DATE OF DHEW AGREEMENT: Cwaiveo } cost sha 5 F273 ‘ (C] UNDER NEGOTIATION WITH: (Sea Instructions) st ____% TOC = : FO THIS IS A SPECIAL RATE fee. off-site}, SO INDICATE, NIH 398 (FORMERLY PHS 398) . PAGE 3 Rev. 1/73 ThE ee yea ‘See §Steacgemdpea a bE. ee t | PERSONNEL “Privileged Communicatio — Za -. _ Donald A. Glaser... , , ~ Fringe 'Name Title of Position @Time Salary Benefits Total a Donald’ A. Glaser.. Principal Inves. 2@mos. 8,652 1,296 9,948 iMechanical Engineers — | Leif Hansen | Prin. Dev. Engrg 100 25,224 3,026 28,250 John Bercovitz Asst. Dev.Engrg-II 100 «14,748 1,770 . 16,518. { larry Johnson | ‘Assoc.Dev.Engrg.III 100 18,792 2,255 ~ 21,047 | Larry” Henderson Draftsman I 100 8,628 - 1,035 9,663... ;Shop and. Maintenance J _ * ; Walter Debold Prin. Lab.Mech.V 100 16,236 1,948 18,184 Lloyd Davis Prin.Lab.Mech.y. 100 16,236 1,948 18,184 James Munger lab.Asst.II » 100 8,196 983 9,179 | X Lab.Asst.I 100 7,560 907 8,467 xX (2) Lab.Helpers 100 «612,624 1,514 14,138 ‘Computer Programming - : ! Fraser Bonnell ‘Prin.Progr.V 100 «24,612 = 2,953 «27, 565 | x \ ‘Sen.Progr. 100 16,656 1,999 18,655 ; x . Programmer 100) 12, 444 1,493 13,937 !Computer Operations . - | Robert Henry Sr. Dev. Engrg.V 100 = 2k, 012 2,881 26,893 Xx(2) Computer Operator 100 19,906 2, 388 22,294 | tnetrumentation ; ; Ronald Baker Assoc.Dev.Rnerg.V 100 = 20, 748. 2,489 23,237 {| James Berk Assoe.Dev.Engre.I 100 17,052 2,046 19,098 | Ted Fujita Asst. Dev.Engrg.V -100 17,052 2,046 19,098 | Alex Pare . Asst. Dev. Engrg.I 100 14,040 1,685 15,725 “or. Pat Donahoo’ ‘Tab.Asst.I °° “100 -.7,560- 907 8,467 Biological Operations . . ~ ‘ i = John Couch Asst.Res.Biophysicist 100 15,290 2,294 ~17, 584 | Philip Spielman -Staff.Res.Assoc.II 100 = 14, O40 1,685 15,725 i Marilynn Brook. -Staff.Res.Assoc-II 100 ..13,0h4. 1,565... 14, 609 | Carol Greiner Staff.Res.Assoc.I 100 «12,144 1,457 13,601 i Eva Bennett Lab. Asst. II 100 8,208 985. 9,193 James Colby _ Lab.Asst.I 100 7,272 872: 8,144 Xxx (3) Post ~Doctorals 100 35,568 5,335 40,903 XXXX (4) Grad.Students 50% - 9 mo 23,292 2,796 26, 088 100% - 3 mo , ‘Administration and Procurement . o . '- Madeline Moore "Adm. Asst. II 100- 10,248 ~~ 1,230 (11,478 , ’ Total Salaries 450,084 55,788 505,872 Hi a ON Oey tee erm Rn “He GS Last { 8 ts" Sy lee ath Privileged Communteation 4 meet 9 a settee tame n= Caring sion oss « BD = EQUIPMENT PDP 10-I System -KI-10 _MF-10 RP-10 RP-03 DF-~10 “TM-108 TY-LO TD)~10 TU-56 DC-LOA. DC-10B LF-0F _ VB-10C PDP-11 Processor Memory (2)64K words Disk Control Disk Drives(3) Data Control for — Disk and MagTape(2) Mag TapeDrive Control Mag.Tape Drive (2) DEC Tape Control DEC Tape Drives(2) DataLine Scanner Data Line Group, 8 lines Line printer, 1250 lpm ~ Graphic display Controller for flying-spot scanner, (18 bits) “Unit Cost 240,000 - 80,000 ~ 26, 000 20,000 + ete 14,000 20,000 25,000 15, 300” 4.700 10,000 5,500 47,500 35, 000 30,000 ~ Donald A..Glaser . 4 Total 240,000 160, 000 26,000 60,000 -28, 000 20,000 50,000 15, 300 9,400 10,000 5,500 - 47,500 35,000 30, 000 Sub-total (PDP 10-1) 736, 700, PDP 10-I (Sortvare package/p. a.) Laser h watt tunable blue laser — (Spectrophysics Model 164) Flying-Spot Scanner--speed up PDP-11 ~ controller : New faster yoke and A/D converters PDP 10 to PDP 11 direct memory bus Total Equipuent "5,000 49,500 30, 000 10, 000 _ 37,000 828, 200 Donald: Aww -Glaser rm omens Var as wd se g2> FREE 7 tie oe we we od hPa tim arg ce : ag + ate Se Me TOTAL OTHER - Consineeten cege ea 3e eo - veneer _Priyileged Communication. = veneer SUPPLIES , : Software notebook updates (3300); softuare updates subscription (31,100/yr); teletype paper, printer . “paper, Calcomp plotter paper ($1,300); Mag tapes ($300); “DEC tapes (#00). . ~ 3,400 “Petri dishes at $25/case (500 dishes per case), oe 2 cases per week x 50 wks = 100 cases x $25. "2,500 Agar at $1. 30/liter; 0.030 liters per dish; 1000 dishes per week x 0.03 liters/dish x 50 weeks x i $1.30 1,950 ‘Agar for Cyclops trays~+20 trays /e x 1.5 Liters/ oo tray x 50 weeks x $1.30 | 1,950 Agar for Dunbwaiter--2 e xperiments/vk x 256 trays xo Lo 1.5 liters x 50 weeks x $l. 30 liter 49,920 . Miscellaneous arugs, chemicals, nutrients and glassware 7 5,000.” Film and Development $0.18/ft for 35 mm filn ; Cyclops--20 trays/wk x 50 wks x 32 sauares/tray x 6 photos/square x $0.18 ¢ 8 photos/ft. 4,320 Dumbwaiter--256 trays/expt. x 100 ‘oxpts/yr x 32 squares /tray x 6 photos/square, x $0.18 = 8 photo2/ft . ; Po . 110,592 , Miscellaneous small electronic, mechanical and , optical parts for constructing laser selector-inoculator electronic controls, - and new cell manipulation devices 25,000 TOTAL SUPPLIES $204, 632 OTHER EXPENSES a, _ Computer maintenance ($2, gl /no.--see De 3a) - 35,376 - EE Machine Shop (2,000 hrs. at $11/hr) - 22,000 LBL Machine Shop 20,000 Machine shops (special jobs on and off campus ) _ 10,250 LBL Supplies 9,600 Phones . 2,700 Xerox 1,800 . Page charges, 15 pp at-$75/page | 1,125 Publications - professional journals 500 Mail 100° $103,451 Donald A. Glaser Moscouttion vane = Dd = Privileged Comaunication Te ements eter oes ae net mn em emer a ae a ee ee ow wane Sree eee pr neeee ‘Corapuser Maintenance (continued from ps3) Maintensnce per mo KI-10 Processor ’ 550.00 MF-10 888 .00 RP-10 79.00 RP-03 510.00 ° ‘DF-10 134.00 TM-10B 43.00 TU-l.0 316.00° TD-10 _ 20.00 TU-56 . 68.00 DC-i0A 19.00 DC-~1OB 18.00 LP-10F 153.00 VB-LOC 150.00 20 tha NS nT ” ad “Total per mo 2,948.00 a we pe a me v f ret Fe Fy tee ys 4 at oom. GI a * - rid + m. aren a ~ + whe wot ™, fee ee i. a fo fs aoe o 4 @ tes fa CK : SESS vs Pg Te LS 20 ge in? a Y its ge> BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUSLIC HEALTH SERVICE DIRECY COSTS ONLY (Omit Cents) IST PERIOD | ADDITIONAL YEARS SUPPORT REQUESTE Dif Phis application only) DESCRIPTION GAME AS DS. . . TAILED BUCGETIT 2ND YEAR 3ROD YEAR 4TH YEAR 58TH YEAR GTH YEAR 7TH YEAR : “eee . * . ¥* . Penson 505,872 | 556,459 |612,104 | 673,214 {7h0,645 CONSULTANT COSTS 1, 500, 1,650 1,815 |. 1,997 é,197 Unctade fees, travel, etc.) ‘| eourpvent “ 828, 200 75,000 | 75,000 5,000 | 75,000 “| supPLIEs | 204,652 «| 225,095 27,604 | 272,364 299, 600 oe DOMESTIC 4,000 5,300 | 3,630 3,993 4.392 FOREIGN , : PATIENT COSTS LTERATICNS AND RENOVATIONS OTHEN EXPENSES 103,451 | 123,796 | 125,175 (237,692 [151,461 ’ TOTAL DINECT COSTS P6465 655 975,300 [1,065,328 |1, 16%, 36041, 273, 295 TOTAL FCR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1, Item 4) ————» | $ 6,124,938 — REMARKS: Justify aff costs for tha first yeer far which the reed may not be obviews. For future years, justify equipment costs, as well as any significant increases in any other category. If a recurring annual increase in personnel costs is requested, give percentage. (Use continustion page if necded.) . 4 Cost of living . figured at 10% By June 1975. - Hach consecutive year figured at 10% increase for cost-of-living increase and inflation . a oo . Employee benefits figured at 12% for non-academic; 15% for academic salaries. SECTION Il — PRIVILEGED COMMUNICATION -?- = , BIOGRAPHICAL SKETCH (Give tha following information for olf professional personnel fisted on pags 3, Seginning with the Principai investigator. Use continuation paces end follow the srme generel format for bach person. NAME TITLE BIRTHDATE (Ma., Day, Yr.) 7 ‘Professor of Physics and’ . =~ Donald ‘A. Glaser ; Molecular Biology 9/21/26 PLACE OF BIRTH (City, State, Country} PRESENT NATIONALITY f/f non-U,S citizen, SEX indicate kind of visa and expiration date) Cleveland, Ohio, USA USA (3 Mate (] Female EDUCATION (Begin wath bsecalaureste training and include postdoctoral) _ INSTITUTION AND LOCATION DEGREE CONFERRED sclentir iC Case Institute ofTechnolorgy . BS 1946 Mathematics ana Physi California Institute of Technology PhD 19h9 woo. " Case Institute of Technology Sc.D. 1959 " " HONORS Henry Russel Award, 1955; Charles Vernon Boys Prize, 1959; American Physical Society Prize, 1959; D. Sc., Case Institute of Technolosy, 1959; Nobel Prize (Physics), ‘1960; Ellio Cresson Model (Franklin Institute) 1961; Alumni Distinguished Service Avard (Cal.Tech.) 1967; Gold Medal Avard (Case Institute of Technolory) 1967. MAJOR RESEARCH INTEREST ROLE IN PROPOSED PROJECT Cell genetics and control mechanisms Principal Investigator RESEARCH SUPPORT (See instructions} NIH Grant GM 19439 bf/1/73 = 5/31/74 Genetic Control of Cell $e, 262 ‘ Physiology and Structure NIH Grant GM 1324 09) «= 6/1/73 - 5/31/74 Scanner-computer investi- $405,698 mo . gations of biological . systems RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Starting with present position, dist treining and experience relevant to area of project. List ali or most representative publications, Do not exceed 3 pages for each individual.) Visiting Professor of Biology MIT 1961-62 Miller Professorship UC Berkeley 1962-64 Consultant Brookhaven National Lanoratories, Argonne National Laboratory, and a varicty of other laboratories and agencies on scientific and instrumentation ~ problems in physics and biology. i. C. B. Ward, M. W. Hane, and D. A. Glaser, "Synchronous re-~initiation of chromo- rome replication in BE, coli B/r after nalidixic acid treatment," PNAS 66, 365 1970). 2. C. B. Ward and D. A. Glaser, “Control of initiation of DNA synthesis in BE. coli | B/r," PNAS 67, 255 (1970). ” 3. C. B. Ward and D. A. Glaser, "Correlation between rate of cell growth and rate of DNA synthesis in Escherichia coli B/r," PNAS 68, 1061 (1971). 4. oD. A. Glaser and C. BY Ward, “Computer identification of bacteria by colony morphology", Frontiers of Pattern Recognition, Acad. Press, N. ¥. (1972). D- Jd. Couch, J. Berk, D. A. Glaser, J. Raymond, and T. Wehr, "Automated recognition of bacterial strains by analysis of colony morphology", Proceedings of the 13th International Congress of Genetics, Berkeley, California, August 1973. 6. J. Raymond, J. Couch, D. A. Glaser, and C. T. Wehr, “Automatic selection of conditionally defective mutants of micrcorganiems," Proceedines of the 13th (continued) WIH 99¢ (FORVERLY PHS 392) Rey. 1/73 : ak veers ew al PR 2 NT “NM wy 7? afte ial OU ae me tae ee . OE WAeek ay a theese _Cortiewatios mye 1 6. Donald A.. Glaser Privileged Communication =» 7. C. T. Wehr, L. Waskell and D. A. Glaser, “Isolation and characterization of | ' cold-sensitive DNA mutants of Escherichia coli K12", Proceedings of the 13th International’ Congress: of Genetice, “Berkeley, California, August 1973 « 8. RM. Burger and D. A. Glaser, "Effect of nalidixic acid on DNA replication by toluene-treated Escherichia coli", Proc. Nat. Acad. Sci. 70, 1995 (1973). 9. D.-L. Parker and D. A. Glaser, "Chromosomal sites of DitA-membrane attachment . in Escherichia coli", submitted to J. Mol. Biol. September 1973. 10. D. L. Parker and D. A. Glaser, "Effect of growth conditions in DNA-membrane | attachment in Escherichia coli; in preparation. il. A. H. Dougan and - D. A. Glaser, "Rates of chain elongation of ribosom fal RUA molecules in Escherichia coli", submitted to J. Mol. Biol., 1973. 12. L. Waskell and D. A. Glaser, “The isolation and partial characterization of mutants of E. coli with cold-sensitive synthesis of DNA", in preparation. 13. D. A. Glaser, "Some effects of ionizing radiation on the format ion of bubbles in liquids", Phys. Rev. 87, 665 (1952). oS Us. D. A. Glaser, "Bubble chamber tracks of penctrating cosmic ray particles", Phys. Rev. 91, 762 (1953). ; of 15. D. A. ‘Glaser, "Progress report on the develépnent ‘of bubble chambers" 'y Nuovo Cimento 2, suppl. 2, 362 (1954). 16. D. A. Glaser and D. C. Rat, "Characteristics (of bubble chenbers", Phys. Rev. 2b hl (1955). | 17. OD. A. Glaser, “the Bubble Chamber", Scientific American 1955. . 18. J. L. Brown, D. A. Glaser, and M. L. Perl, "Liquid xenon bubble chamber", Phys. , Rev. 102, 586 (1957). | 19. OD. A. Glaser, D. C. Rahm, and ¢. Dodd, "Bubble counting for the determination . 4 ’ . of the velocitics of charged particles in bubble chambers", Phys. Rev. 102, 6, -1653 (1956). oe , So 20. OD. A. Glaser, Decays of strange particles, Kiev. Conference, 1950. 2l. D. A. Glaser, et al., "The neutral branching ratiog of K° particles", Phys. Rev. Tetters en . - 22. D. A. Glaser and L. on Roellig, "Elastic +p and p-p scattering at 1.23 Bev/e." . Phys. Rev. 116; 1001 (1959). - we 23. D. A. Glaser et al, "Direct proof of, 3} neutral decay", Phys. Rev. Letters 3 51 (1959). — 4 a , 2h. oD. A. Glaser and . I. Yettenbe Te, "ay automated syste for the growth end analysis of “arge mimbers of bacterial colonies using an environvental caamter Ca at poe 26. eit ae nee nite waienenin © 8 te esaey » 25. 2T. ; 28. 29. 30. 31. Cartleuation aga To + Donald.A..Glaser__...._... Privileged Communication ‘ —- a - . en eee and a computer-controlled Tying-spot scanner", Ann. N.Y. Acad. Sci. 139, 2h5 (1966). D. A. Glaser, "Biologic al objectives and stratery for the design of | a space vehicle to be landed on Mars." Chap. 18, Biolozy and the Exploration of Mars, Nat. Acad. Sei. Nat. Res. Counest publication, 1900. D. A. Glaser, Je MeCarthy and M. Minsky, "The automated “piological laboratory, B. Wolf, A. Newman, and D. A. Glaser, "On the origin and direction of. peplica« tion of the E. coli K12 chromosome", J. Mol. Biol. 32, 611 (1968). 2 M. L. Pato and D. A. Glaser, "The origin and direction of replication of the chromosome of Escherichia coli B/r. Proc. Nat. Acad. Sci.. 50, 1268 (1968). C. B. vard and D. A. Glaser, "The origin and direction of DNA synthesis in E. coli B/r.- Proc. Nat. Acad. Sci. 62, 681 (1969). c. B. Ward and D. A. Glaser, “Evidence for multiple growing points on the genome of rapidly growing E. coli B/r. Proc. Nat. Acad. Sci. 65, 800° (1909). . Cc. B. Ward and D. A. Glaser, "Analysis of the chloramphenicol sensitive and resistant steps in the initiation of DNA synthesis | in E. coli B/r. Proc, Nat. Acad. Sci. 6h, 905 (1969). (See beginning of list for more recent publications. > eared ae ee Se ee wm PR ge ee PeE OP LSE See wm 8 IT ma oi ¥ hy “AE se, BSE ‘ weg ad a te Soe nae a ee ate ey Bo. * ee FES Pas Ss * * a eH in.) ote te te etn aterm 6 me te me Nimo Om ; ee co eine a 2 ee ene Cogtvnuathe one 8 Donald A. Glaser eben Gam fe tapes - - BAT OEP aS TT __ Privileged Communication ' _ Eh LULULUCUCOCCtC‘S Ronalé Baler - Associate Development Ingineer. b. 2/16/29 in Fulhem, London, England. U. S. A. Ruislip Manor Secondary School, First Year Netional. Instrument Maker, Ingersole Ltd., England, 1951-54; Tool and Instrument Maker, G. E. ‘Research Labs., England, 1954-57; Mechanical Designer, Physics Department, U. of Michigan, 1957- 393 p Mechanica Designer. Lawrence Radiation Raboratorys.: 1959-65. John Bercovitz « Assistant Deve lopment maineer. b. 9/3 /k5 ; Baltinore, “Ma. Cal. Poly, Pomona, BSME, 1972. Design Engineer, Riverside 1969-1973. A a tern thes Mme ae OH James Berk - Associate Developzent Engineer, b. 9/17/42, Nev London, Wisconsin. UCLA, BA Physics, 1965; UCLA, MS Physics, 1967; UCLA, PhD Physics, 1969. ToC eT Biochemist, UcBerkeley, 1970-75. National Science Foundation Fellow 1966-57 . . UCLA, BA, 1957; UC Berkeley, MA, 1958; Teaching Assistant, Department of © Mathematics, UC Berkeley, 1939-613 Computer Programmer, Lawrence Radiation Laboratory, Livermore, 1961-65; Instructor, UC Extension Division, various semesters since 1961. _ John Couch - Research Associate. b. 5/6/h1, Hartford, Arkansas. MIT, SB Physics, 1963; Stanford, PhD Biophysics, 1970; Acting Instructor in Biophysics, St tanford, : 1971. {ss W. XR. Fair, J. L. Couch, N. Wehner, Biochemical Medicine 8 (329-339), Purifi- t cation and Assay o? the Prostatic Antibacterial Factor (PAP); Nakayama, H. and Couch, J. L., "Thymineless death in Escherichia coli in various assay ‘systems: viability determined in liquid medium", J. Bacteriol. 114, 228 (1975); Jd. I. Couch and P. C. Hanawalt, "DNA repair replication in temperature -sensitive DNA synthesis deficient bacteria", Biochem. Biophys. Res. Commun. 29, 779 (1967); J. L. Couch and P. C. Hanawalt, "Analysis of s-bromouracil distribution in | partially substituted deoxyribonucleic acids", Anal. Biochem. #1, 51 (1971); _ P. C. Hanavalt, D. F. Pettijohn, EB. C. Pauling, C. F. Brunk, D. W. Smith, L. C. Kanner, and J. L. Couch, "Repair replication of DNA in vivo," Cold Spring | Harbor Symposia, on Quantitative Biology, Vol. XXXIII (1968), Pp. 187. i | i | Fraser Bonnell - Principal Prograrmer. b. 7/28/35, Port Chester, N. Y., USA.. | j $ “Ted Fujita - As sistant Development Engineer. b. 9/19/43, Tovaz, Utah. Uc Berkeley, BS, 1964; UC Berkeley, MS, 1965. Project Engineer, Berkeley Selentific. Labs, , 1965-69. a ot — a ° ar we emma emo “Robert. ‘Henry. - Senior Develovinent Engineer. b. "6/8/36, Winfiela, ‘Kansas. U. Kansas, BS, 1959; UC Berkeley, MS, 1965; Boeing Airecraft-Electrical drafts man 1957-58 (summers) ; Western Electric Co-; Engineer, 1959-60; RCA, Engincer, 1960-64. | | Le Leif Hansen - Principal Development Rngineer. 5/29/27, Copenhagen, Denmart:. _ USA citizenship. ‘Technical University of Denmark MSME, 1954; Senior Design Engineer, General -Dynamics-Astr onautics,’ 1957-62; ‘Senior Engineer, Lawrence Radietion Laboratory, Berkeley, 1962-65. (vechanteak Eogineer, RDAP 1954-56). (cont inued), tote ‘Sp rom cht A vet os "s Bf 5 Po s af Poeeym RE Shwe BYE UN THIS “ = fs i ~~ 4 we, mop be . Coaatirantiog Tut oo” 9- Donald Ae ‘ Glaser. —— _ Privileged Communication ena “Larry Johnson - Associate Development Engineer. b. 2/28/37, Sioux Falls, South Dakota. §D School of Mines, BSME, 1959. Sperry, Project Engineer, 1959-65; Boeing, Design Engineer, 1965-66; FMC, Sr. Design Fngineer, 1966-70; - Thermidex, Sr. Project Engineer. Alex Para - Assistant Development Engineer. b. 2/22/50, Buenos Aires,. Argentine. Citizen of Argentina. Chabot College, AA, 19€8; UC Berkeley, BS, 1971; . ‘Engineer's Aid, UC Berkeley, 1969-70; Sr. Mngineers Aid, UC Berkeley, 1970-71. Sr. Developuent Ingineer, UC Berkeley 1971-72. co “oS i Wan os Donald.A.. Glaser. warner Sag2 ~ 9a = ~erivileged Communication... —_— _ Table of Contents Justification of First 12-Month Period Research Plan Specific Aims, Methods of Procedure and Significance Further Automation Instrumentation Development Significance Facilities Available , Collaborative Arrangements Principal Investigator Assurance 10 12. 2h 38 ho ho. Ta be -10- Glaser, Donald A. '. Privileged Communication Justification of first 12-month period Personnel During the first 12-month period we plan to maintain the engineering staffs at their present size, because we expect there to be extensive debugging, modification, and minor additions made to the machine as we gain experience in its use. Those who de- signed the machine will be the most effective at understanding its shortcomings and making necessary improvements. As time goes on the shop activities will shift from construction of new equipment to maintenance of the existing equipment at probably the same level of manpower as required during the construction phases of the project. The instrumentation and electrical engineering group will similarly be engaged in debugging, modifications, and minor additions to the equipment. , mo In order to operate the computer facility around the clock, we will need to have two full-time computer operators, but no other major expense is. contemplated. For biolo-~ gical operations, a Senior Biologist with considerable experience in computer program~- ming and instrumentation is being proposed and the budget also provides for the sala- ries of three postdoctoral researchers and four graduate students since training grants for these categories of people are no longer available. As the experimental program gains momentum, we will need to add two relatively junior programmers to help biologists formulate protocols and write programs to carry out the necessary operations. . Equipment a PDP1O-~I System to replace our PDP-6 System. By the time this proposed program begins in June 1975, we will have owned and operated: our present PDP-6 system for 1O years at an enormous saving in the cost of leasing the same equipment. Lease rates are usually computed to amortize the equipment in about 4O months and we will have operated the equipment for 120 months atthe same cost. Several years ago the PDP-6 computer became essentially obsolete when it was replaced by the PDP-10, and then by the PDPlO-I system. Probably by June 1975 there will be a yet newer replacement of the PDP1O-I system. At the present time’ (October 1973) there is only one operating PDP-6 computer left in the United States at the Rand Corpora- tion who are planning to get rid of it in the next few months. ‘There may also be another highly modified PDP-6 computer at M.I.T. not maintained by D.E.C. (Digi- tal Equipment Corporation) and perhaps used for special experimentation in compu-~ ter science. D.E.C. no longer maintains the software for the PDP-6 and it is costly and difficult for us to modify the constant improvements in PDP-10 soft- ware so they are useable on the PDP-6. New software, beginning to be issued by D.E.C., is not suitable at all for the PDP-6 computer and we will soon be unable to take advantage of the "community knowledge" and library of programs available for PDP-~1O applications. It is not practical for us to maintain the computer ourselves and D.E.C. maintains only one trained maintenance person who, in fact, can only be trained at our own computer by his immediate predecessor. We absolutely then depend on this one person because ours is the only computer of its kind still maintained by-D.E.C. The change to the new PDP 10 system most recently available in June 1975 is expected to give us a speed increase of at least a factor of h in analyzing photo- grephs from the Dusbwaiter and Cyclops. Since these instruments take photosranis at the rate of 1 per second and our precens rete of enalyzing pictures is about 1 psr 10 seconds to 1 pe» 20 seconds, we have an eutremely unfavorable ratio of analysis time to production time for these photographs. With this additional Glaser, Donald A. ~ll. a Se Privileged Commnication a Justification of first 12-month period (continued) factor of i or more available in the PDP-10 system, the ability of the computer to analyze. data will be nicely matched to the rate of production by the biological machines. For all these reasons, the switch to the new system is extremely desir- ab ie * . , . laser. This laser is needed to measure the light scattering of droplets of cell Culture formed in the high-speed dripper-inoculator in order to determine whether a droplet contains a cell and the kind of cell contained therein. By rejecting empty droplets and droplets containing multiple cells as described in the Biolo- gical Plans part of the proposal, we will increase the effective size of the Cyclops and Dumbwaiter by a factor of 3 and be able to carry out critical sorting operations for experiments on animal cells. Ss Flying-Snot Scanner--sp2ed un. To further increase the speed at which photographs can be analyzed, we propose to update the Flying Spot Scanner to current techno-. logy by the substitution of the PDP-ll computer to serve as a controller for the scanner in place of the home-made cireuit thet does the job now. In addition we will substitute new, improved versions of the deflection yoke system for the pre- cision cathode ray tube and faster A/D converters. Finally, we would add a PDP-~10 to PDP-11 direct memory access-dimp for bringing scanner information directly into the PDP-10 memory without going through the slower 1/0 Bus. Supplies--The cost of supplics is based on the assumption that the Cyclops will con- tinue to operate. for small-scale experiments and for "second-pass" experimental ‘material produced by lerge Duwsbwaitcr experiments. It will operate with petri dishes or with gloss treys at a modest level as described in the budget figures themselves. ' The budset for Dumbwaiter supplies is based on the expectation that we will be able to carry out 2 batches per veek for 50 weeks per year which seems at this time a reasonable average level of activity. . ~- Travel--On the average of one major trip to the East Coast for professional persons to attend a major conference such as the Gordon Conference and the Cold Spring Harbor Confermec, as well as attendance by professionals and graduate students at local conferences. Also conferences vith colleagues and equipment suppliers. Other--Computer maintenance contracts are based on present cost estimates by the manuiacturer who carrics out the maintenance. Machine-shop time and other campus shops is required from time to time vhen our own single machinist is overvorked or when special facilities and large machines are required for a particular job. The budget is baced on one man year of work for this purpose. ‘he Equipment Budget for subsequent years provides for new accessories bound to be required as the experimental program expands, including for instance, a television- scanner system for on-line real time analysis of growing colonies to eliminate the photography step and provide for the possibility of intervention in the experiment in real time and very rapid read-out necessary for particular applications. For study of animal cells it vill probably be necessary to design a camera that photographs a small area of agar at a time through a low-power microscope for studying very small clones of animal cells. Other requirements of these kinds are bound to arise. We will justify this budget item on a year-to-year basis. M emegmieatipes wed ae 1? ~ Donald A. Glaser we a . Privileged Communication 00 "Rescarch Plan AS insroduction | 1. Objectives. When this program-project began in July 1955, the overall goel wa: to automave many of the procedures of petri dish tecanicue on a large scale using computer-directed machinery and pattern recognition techniques in a flexible way so that a wide varicty of biomedical problems could be attacked. Now, in November | 1975, after successful operation of several prototypes, much of the equipment is in operation and all of the najor equipment will be in full operation at the end of the current grant period in June 1975. In its shorts period of operation the machinery has suecessfully aided in the ondole- tion of cold sensitive mutants of E. coli Kl2 unable to synthesize DNA at 20°C. Iz has also psrformed highly accurate automatic recognition of growing colonies of 10 species of bacterial pathogens important in medical diagnosis as.a demonstration of wr its abilities in many health-related applications. In the next few months we will 2 begin new experiments in genetic mapping, mutant isolation and- physiological charac- terization with H. coli, Saironclle typhimuriu:, Bacillus subtilis, Saccharomyces cerevisiae, and aninal cells grown in tissue culture. iiany os these projects will be Les done in collaboration with scientific investigators who have on-going projects in = these areas , . During ‘ che next five-year grant period beginning in June 1975, we propose to extend " these projects and add others involving the construction of genetic meps, the isola- i tion of important mutants, and the eharacterization of mutants and strains. Some , of these projects will be chosen to aid in critical steps of the productive work of i a& number of independent scientific investigators already working in these fields, rd and some will be important parts of our own biological programs. In addition, ve “fe propose to examine the feasibility of health related projects including screening x of environmental cheiicals, including food additives for their mutagenic effects on bacteria, ycast and animal cells, the potential carcinogenic and anti-neovlastic effects of various agents on animal cclls, and “the cffects of very low levels of Loniziny radiation on various cells. If large-scale screening projects appear feasi- ble and desirable, special funds will be sought to carry them out if necessary. Finally, a modest instruzentation program will be continued to add new capabilities to the machinery as they become necessery. Scientists from many laboratories are expected to take advantage of this facility. oo fee 2. Backsround. Since this progran includes a number of different biological es projects, the vwiological background, rationale, aims, and methods will be discussed a project by project in subsequent sections of this proposal. What brings them toge- ry: ther in this program is a similarity in the technical manipulations and the common Ss - requirement for large-scale experiments too tedious, slow and costly to carry cut by hand. In some cases quantitative measurements on proving colonies are extremely difficult without the automatic pattern recognition facility. Except for cnall labor-saving devices, techniques for growing colonies on solid media have changed little sine they were invented. Many projects in the contempor- ary biology of clonable cells are severely limited by the difficulty of isolating particular mutants, characterizing them, and locating them on the genetic hap of the organism. Numerous health-related programs including medical bacteriology; contami- nation monitoring; mass screening programs for mutagens, carcinogens, and anti- “neoplastic agents; and industrial strain-improvement programs utilize simile r teche~ nigues. It is hoprtd that this orocrom-projicet will be useful in all these fields ac wall as in vork in Vundenontel biolocy. fe mete Homme i a t 4 * Ree Se eR A GR MUR = em ne Gat | i t Donald A. Glaser rr Conteugntion rage - 13 - Privileged Comunication 7 nee 3. Rationale of this automation. To carry cut experiments requiring study of large numbers of colonics we are constructing a machine (the "Dumbwaiter") in which .256 hOcem x 80cm eagar-filled glass trays circulate in an.incubator past stations where | various operations can be performed. An inoculation device deposits single cells .carried in microdroplets in regular rovs and columns for maximum uniform packing and- easy subsequent manipulation. During incubation, time-lapse photographs of the colo- nies are made using up to 5 different colors of light. A flying-spot scanner. (similar to a television camera) under control of a computer (PDP-6) examines the photographs, finds all the colonics, and records their size, appearance, and growth rate. The. computer then computes the frequency of various clesses of colonies for measuring. muta~ tion rates, map distances, recombination rrequencies, and other required biological results. In addition the computer can direct a colony "picker" to retrieve part of a colony for replica plating, sus spension in liquid, restreaking, or delivery to a test tube or small petri‘dish for further manual work in the laboratory. Alternatively the computer can direct the spraying of some or all colonies with nutrients or drugs on some predetermined schedule or according to the actual performance of each parti-~ cular colony. Thus the conputer ean intervene in on-going experiments. Irradiation, genetic crossing on the agar, and similar operations can also be performed as the trays move through the Dumbwaiter. Design of the DW (Dunbwaiter) and associated equip. ment has been done to allow a wide variety of accessories to be added to carry out. epectal manipulations as they are required for particular experiments. If colonies ze placed 1 mm apart, the DW can hold almost 10% colonics per load of 256 trays. | Several loads can be processed each day for many types of non-~interfering experiments. * } What does this kind of large-scale automation have to contribute to -biomedical science? ; Solution of many biological problems depends’on the. ebility to isolate a particular ‘ kind of mutant, to measure the rate of a particular genetic recombinational event, or to measure responses of growing cells to specific chemical, biological and physical _ conditions. Automation allows highly reproducible experiments to be performed with large numbers of organisms so rare events can be observed and more common evénts meas- ured with high statistical accuracy. Computer-directed pattern recognition allows quantitative aspects of growth to be explored for regularities that would escape _ quest tative visual examination. , None of this increased statistical and quantitative power reduces the neéd for thought- ful study of the biological system in advance of large-scale experiments and of careful analycis of the results. Nor is this kind of automation likely to reduce the number or quality of people involved in a given research area. ther the same people will be able to accomplish tasks impossible without the machinery and to do many more- con=- ventional experinents with much reduced tedium. ! In medica 1, public health, and industrial applications, large scale sereening, con- | tamination surveys and diagnostic assays, and other similar tasks can be done with | : : nem ern Oe mi eC the unbiased reliability of automation and the cconomies of large-scale. It is expec- ted that these machines or adaptations of. them will be cost-effective and quality- leffective for a variety of immediate health-related applications. ~ —h. Comprehensive Progress Report. (a) Period covered by this report: June 1970 to November 1973. (b) Sumary. Equipment has been ‘built for inoculating up to 100 40 cm x 80 em agar-filled glass treys.with sins “le cells in regular rovs and columns of adjustable epacing, incubatins the treys under timttly controlled conditions, photographing them puriecdically, and analyzinh the photocraph: with e seamer-conouter system. Frecuen- cles of various colony types are recorded by the computer which can also direct the Donald A. Glaser Coatinuntios rode « Uh z ‘Privileged Communication ss automatic picking, replica plating, and restreaking. of colonies it is instructed to select. Nutrients, drugs, viruses, and other agents can be delivered to whole trays or selected colonies under computer control. Design and construction of a fully-automated system able to carry 10° colonies on 256 trays is near completion. . With the. presently operating system ve have isolated cold-sensitivé mutants of E. coli Kl2 unable to synthesize DNA at 20°C using 1/5 as much agar and much less labor than a parallel project using hand methods.. Nine bacterial species isolated from human urine and a laboretory strain have been studied with the automated system. Using newly developed programs, the computer can correctly identify unknown colonies of these ten types with accuracies better than 98%. m, A. 4(c) Detailed Report 1) Biological Projects. Although the goals and budget of this program-project were directed principally tovard development of the automation system, several biomedical projects have been carricd out to demonstrate the abilities of the system and to speed the work on biological projects in our laboratory supoorted by NIH as GM 19439 - (replacing GM 12524). The NIH preferred funding the instrumentation and biology pre- grams separately so that they would pe reviewed separately by appropriate panels. (a) Finding, Counting and Sizing Colonies. Computer programs have been written for scanning photographs of 1OO-mm-netri dishes prepared by hand for finding, ’ counting, and sizing the colonies correctly,in spite of overlaps of colonies ~ and wide variation in colony sizes. The counting algorithm has an accuracy of better than 99% on dishes containing up to about 400 colonies and’ requires about 10 seconds per dish. It is-thus greatly supericr to any commercial colony counter, but is not used currently because simpler and faster program’ are effec- tive with. regular array-inoculated dishes and trays. - (b) Isolation of Cold-Sensitive Mutants’ (by a method widely applicable to mutans hunting). Invectigationus of DNA synthesis in E. coli, its control, and its connection with cell division, require isolation and genetic mapping of conditionally lethal DNA mutants. Work on our laboratory and many others has uncovered 7 or 8 classes of heat sensitive mutants normal. at 50°C or 37°C but ‘unable to synthesize DNA at about h1°c. ‘These classes map at 7 or 8 distinct ~* sites, but probably DNA synthesis is even more complex and additional sites — defining more structural or control genes remain to be discovered. We are searching for new classes among coldesensitive mutants unable to synthesize DNA at 20°C by taking time lapse photographs of colonies grown from mutagenized cul- tures and shifted between 20°C (restrictive temperature)‘ and 37°C (permissive temperature). Imposing conditions on colony diameters and growth rates leads to efficient selection of cold-sensitive mutants by the scanner in a vay that saves much labor and materials when compared with a competitive hand experiment run in our laboratory. Cold sensitive mutants mapping at a known site (Cc class) have been found and 3 new mutants may represent a new class not yet precisely mapped. Since the colony picker is not yet in operation, the scanner aids in locating mutant colonies by displaying a map of each dish on the display scope. Holding the dish egainst the screen, mutant colonies are picked wherever the commer has drawn an kK. _systen semi-automatically on a reduced scale (about 100 trays maximum capacity). ‘ Photographs are examined by a flying-spot scanner (similar in operation toa tele- Copiaut tion octee 15 & | . Donald. A. Glaser ome og Privileged Communication _ _— This method (later -using tne picker Lo“eliminate hand labor) can ve used for any mutant selection based on colony size or appearance. we have tested it success- fully with knorm leucine auxotrophs by growing mixed prototrophic and auxotrophic . eultures in Limiting leucine and then spraying the agar with additional leucine, . taking photographs during the incubation intervals. 7 . (c) Automated Recopnition of Bacteriel Strains by Analysis of Colony Morvholory. To test the ability of the system to identity bacterial pathogens for medical and public\health applications, we photogrephed 24-hour colonies of nine species isolated from human urinary infections plus one Bacillus subtilis strain. Using methods of colony morphology analysis dcecribed below, tne system “learned” to -recognize the 10 test species by examining about 1000 colonies of each. Upon scanning an additional L000 colonies presented in mixcures or in pure cultures, the program makés tvo decisions: 1) whether to attempt an identification (ans- wered "no" if the "colony" is not round, is actually a piece of dirt, an imperfec- tion in the agar, etc.), and 2) to what species does the colony belong (if 1) is. ansvered "yes"). Results were as follows: . * . % Attempted _ % Correct Aerobacter aerogenes . 83 ~ * 200 Bacillus subtilis - 8B 100 Escherichia coli , 83 , 100 Herellca vaginicola TT 100 Klebsiella pncwnoniae 81 - Proteus morganii 86 - 00 Pseudomonas putida . 83 - 100 Salmonella typhimurium . 89 . ,100 Serratia marcescens 86 100 Staphylococcus aureus 89 > 100 2) Technical Prosress. When completed in January 1975, our automated system will prepare minimal agar medium in 400-liter batches, dispense it with a prograrmable variety of additives into 256 hOcm x 80cm prestcrilized glass trays, and circulate. “the trays inside a precision incubator past stations for inoculation, time-lapse photography, colony picking and replica plating or restreaking, and treatment with chemicals, radiation, viruses, etc. In January 1975, the prototype test version came into operation and is nov carrying out almost all of the operations of the final ty vision camera) connected to a medium-sized computer. The computer finds all the colonies, measures their diameter, characterizes their appearance (using up to about’ 100 parameters), and issues commands for colony picking, nutrient spray, mxtant ‘purification by colony restreaking and replica plating, according to a protocol written by a biologist. : . By June 1975, when the presently proposed program is due to begin, the system should be in full operation. Technical aspects of this vork have been reported in two published papers: D. A. Glaser and W. H. Wattenburg, An automated systemtor the growth and analysis of large numbers of bacterial colonies using an environmental chamber and.a computer-controlled flying-spot scanner, Ann. N. Y. Acad. Sci. 139, 243 (1966); D. A. Glaser and C. B. Ward, Computer identification of bacteria by colony rorpholosy, Frontiers of Pattern Recognition, Academic Press, N. Y. (1972), - : ~ ¢,;™ SEIT Fey 2 eat r : Se Be ye ae oa Centinestias sme 16 0... _ Donald A. Glaser. _ Privileged Comaunication a Nee ee oe ese vente eR oe ee me eee te me me OL Fa eel aR ett can no mmr Mtl ne anes trae ane tne mmm oe a larg re number of oral reports, and Progress Reports to the TICMS. Detailed publications will be: ‘prepared after the full system is completely operational. -We ea seen no reports of sinilar systems in operation elsewhere. — . (a) Oo jectives and General ‘Description. Many of the biological opdect ives of this program require the ability to examine about 10° fairly well isolated,colo- nies (about one colony per square centimeter). For other studies up to 108 colonies need to be examined but they may be crowded into a smaller space (about 100 colonies beg square centimeter). The machine must therefore have a capacity of about 10° ¢ em’ of solid grovth medium (agar, silica gel, or other medium) s To - provide the required area of agar in the smallest possible volume, the machine uses stacks of horigontal agar-covered trays spaced one inch apart. These trays are made of inespensive window glass with metal frames and can be washed and serilized very easily by reasonably standar techniques. They elso provide a very uniform growth surface of high optical quality so that good photograplis of growing colonies can be made. Ordinary plestic or glass pnetri dishes made by hand in the leboratory in small batches in the conventional way can be laid on the trays for incubation, photosraphy, and manipulation in the machine. Al- ternative ly, Large-scale e3 ~perinents ean be carried out by pouring a sheet of agar directly on the tray. A design has been chosen which makes it possible to intermix these two modes so that the petri dishes made by hand can be analyze at the samc time as large-scale experiments prepared automatically by the machines The entire machine is fully automated to perform large-scale microviological ex perinents in conjunction with o sophisticated data gathering and processing system. Because the stacks of trays are moved up and down by. mechanical devices, we have called the machine “A Dumbwaiter" The design concept of the Dumbwaiter is very simole. Glass trays carried in aluninun frames are stacked directly on top of each other in two stacks about 25* apart. Cross-ducts are“provided to transfer trays from the top of one stack to the top of the other, and from the bottom of one stack to the bottom of the other. The trays then circulate in a rectangular path moving up through one stack across to the top of the other, down through the second stack, and across from the bottom of the second stack back to the bottom of the first stack. This over-all design plan can be seen in the attached figure. On tl cross- ducts for moving the trays horizontally will be mounted cameras for photograph- ing the trays and special aceessores for inoculating the agar with organiems; administering drugs and nutrients, irradiating with ultra-violet light or other | radiation, picking, restreaking and replica plating colonies and other manipu- | lations. Trays are handled singly only in the cross-ducts. In every other part of the Dumbwaiter and auxiliary equipment, the trays will be handled in stacks of 64. The stacks and transfer paths are enclosed in housings in which a sterile grovth, environment is acntained. Mixing, sterilization, and pouring of agar is carried out outside of the Dumb- waiter. Accessory stations will also be provided for washing the trays for reeuse, for sterilizing them, and for special incubation and cold-storase of trays of colonies which do not need to be vhotosraphed very frequently. Four moveable magazines will be provided for storing stacks of trays and transporting them from the Dunbwaiter to and from various auxiliary stations where these special operations will be carried out. The separation of these necessary func- tions to a number of specialized stations was phehaia to be the best vay to provide : , . 2 renid, reliable ond econonica), oneration of the svetem. On the following pares ‘uve WLLL give the deteaile, status, and churachariesies oP tne Diesbusiter and its rd cg ae Sep gem an f . aunaliar; COUT Ge f A wee 4 & 3 OTS eo pad tel A Res SFT yey ae gta i we oon nae ele ¥ m ete 7 tat tat. RAL Oath (S OA bea VI a WOie Loanitnuctige C290 17 - Donald A. Glaser privileged Communication. ..._ €i ma (b) Operational prototype (cyclops) + As design. aill.testing of Dumbwaiter com- ponents procceds, we often need to construct temporary devices for testing de- sign principles and mechanical devices that will be used in the Dmbwaiter. At the same time, we were anxious to begin carrying out biological experiments before the Danbwaiter comes into full operation. We have, therefore, constructed a machine celled "Cyclops" consisting of a Dumbuaiter camera mounted on an x-y motion capable of handling one or tvo Dumbwaiter trays in the same way that vill be..done in one of the horizontal cross-ducts of the completed Dumbwaiter. Cyclops is capable of photographing agar-laden glass trays or trays carrying conventional plestic petri dishes, of inoculating sterile agar with organisms to be grown). of spraying drugs, nutrients and other substances, .of picking and restreaking colonies and carrying out most of the mechanical and optical operations of the Dumbvaiter.. It is not. able to incubate and circulate trays, however, end at the present time vequires the trays to be transported by hand. Nearly all of the. other ancillary facilities of the Dubwaiter are being used routinely for experi -, ments done on the Cyclops as will be described below. (c) “Moveable macazines. The moveable magazines serve many purposes. Their main function is to sranspors and protect the 6l-tray stacks. Each stack rests on a dolly on rails on the bottom of this magazine. When the moveable magazine is engaged to a fixed magazine for transferring a stack in or owt of the Dumbve iter, the rails in the moveable magazine mate with corresponding rails in the fixed magazine. The moveable megazine and the fixed magazine both have doors facing each other. ‘The space left between the doors after engagement is accomplished Will be sterilized by UV radiation: The doors vill then ve coupled together ‘and simultancously litted up into an enclosed UV irradiated container above the fixed magazine. The lifting of the doors is performed py an air cylinder. The ‘stack transfer can now be executed using a hand-driven transport serew located in the moveable magazine. - ‘Whenever actual stack transfer is not taking place, the dolly is locked in a fixed horizontal position and the stack is secured in vertical compression by hand-operated screws in the magazine top cover. This will prevent unwanted move- ment of trays in the stack during transport and handling of the magazine. The vertical compression will be especially important to keep all trays paraliel to cach other during the agar-pouring and annealing process. The agar-pouring will be done while the moveable magazine is reeting on levelling jacks in a combination sterilization, pouring, and annealing oven. ‘The stack dolly is > equipped with mercury levels (permitting 180°C dry sterilizat ion) to assure accurate Levelling of the stack before the agar pouring. ‘lhe agar Vi: rology 45, 366 (1970). 2. M. Hane, "Some effects of nalidixic acid on conjugation in Eecherichia coli K12", Je Bact. 105, 45-56 (1971). —_s- rs 3, C. Be Ward and D. A. Glaser, "Inhibit tion ‘of initiation of DNA synthes is by low - concentrations of penicillin" ; he RM. Burger, "Kinetics of labeling of fas b-renaturing WA in Bacillus subtilis", J. Mol. Biol. 55, 199- 201. “(a9n). ‘ 7 - RR. M. Burrer, Mmolucnesiented Reoherichta oo replicate only that DNA which iW. Aead. Set. 68, flo% (1971). ryt age oe + + yt — was aboub to ne renlicetcd inv ag al tain a a Cantippesss ae «Be Donald A, Glaser. _Privileg ged Communication oo . ne 6 R. HN. Burger and D. A. Glaser, "Effect of nalidixic acid on DNA replication by tolucne-treated Escherichia coli", Proc. Nat. Acad. Sci. 70, 1955 (1973). Te D. Le Parker and D. A. Glaser, "Chromosonal sites of DiA-nembrane attachment in Escherichia coli" ;» submitted to J. Mol. Biol. September 1973. -8. D. Le Parker and D. A. Glaser, "Effect of growth conditions in DNA-rembrane attachment in Escherichia coli," in preparation. -o 9. A. H Dougen and D. A. Glaser, "Rates of chain elongation of ribosomal’. ‘RNA molecules in Escherichia coli", submitted to J. Mol. Biol. 1973. 10. L. Waskell and D. A. Glaser, "me isolation end partial characterization of mutants of E. coli vith cold-sensitive synthesis of DNA", in preparation. wed ae vf ? a Wes vat A. he. Staffing Assistant Professor of Clinical Pathology 1970-71 W. Keith Hadley 7 and Laboratory Medicine, UC Medical Center. Postdoctoral Fellow 1967-69 oa a ~ Leif Hansen larry Johnson Alex Para Principal Development Engrg. Associate Development Imergc. Assistant Development Engrg. Calvin Ward - ‘ Assistant Research Biologist 1969-71 ant -Beverly Wolf Assistant Research Biologist 1965-72 a Ronald Baker Associate Development: Encre. 196h-present m. John Bercovitz Assistant Development Ingreg. 2/73-present te James Berk Associate: Development Iberg. 3/3 ~present ed Fraser Bonnell Principal Programmer - 1965=present ef John Couch Research Associate - 1971-present be fed Fujita Assistant Development Engrg. 1969-present aN Robert Henry Senior Development Engrg. 1964-present 1965-present 2/73-present 9/T2-present ~ oh . Glaser, Donald A. Privileged Communi cation 2a B) Specific Aims; C) Methods of Procedure; and D) Significance Since this program project is a collection of different biological projects, we will devote a section of this proposal to the aims, methods, and significance of each. project separately. These projects have in common the need to isolate and char- - acterize mutants or recombinants difficult to find by hand methods because they are rare and have no easy biological or chemical selection technique, but can be defined by growth rate or colonial morphology under particular growth conditions. In some cases the events are not rare but their frequency must be known with high accuracy so that large numbers of colonies must be examined. The isolation procedures involve | inoculation with single cells, incubation, time-lapse photography, replica plating, colony picking, colony restreaking, growth rate or morphology analysis, and other Operations -that our system is designed to carry out on a large seale. Some of these projects are already under way; some will be begun soon; others will require prelimi- nary feasibility studies; and still others will be added later. ‘They represent a sampling of projects proposed in conversations with a number of scientists and involve @ range of clonable cells from bacteria to mammalian cells. They include fundamental studies of molecular evolution and biochemical pathways as well as applied studies of mutagenic effects of environmental chemicals and efficacy of proposed antineo- plastic agents. With each project title is listed the scientific investigator(s) who propesed and will guide the work. In some cases a true collaboration with our laboratory is expected to develop; in others the effort will be to help provide mutants for independent and on-going research done in other laboratories; in still others a feasibility study or actual screening effort with direct health-related goals will be undertaken. 1) Isolate, map, and characterize temperature sensitive mutants of E, coli unable to synthesize DNA at 20°C or at 41°. oo Donald A. Glaser, Professor of Physics and Molecular Biology, University of California, Berkeley. “Method: . Automated replica plating and incubation at the permissive and restric-. tive temperatures followed by photography and computer matching of replicas is a straightforward method that will soon be possible. In current use is a series of time-lapse photographs taken of single primary colonies incubated at permis- sive, non-permissive, and permissive temperatures on a time schedule that allows the computer to impose limits on the colony size to define the mutant class. selected. Less agar and fewer manipulations are required for the time-lapse method, but some mutants may be killed at the restrictive temperature so different classes of mutants may be produced by the two methods. Mapping is done by inter- rupted mating or epvisomal complementation followed by measurement of co-transduc-~ tion frequency. Results are obtained by automated colony counting on selective media. Characterization of mutants will be done mainly by conventional methods. Significance: Knowing the number and location of genes involved in DNA synthesis and its initiation in E. coli is the first step in the genetic and biochemical dissection of this all-important cellular process. Mutants obtained in this study will be shared with other laboratories engaged in enzymological analysis to speed the overall progress in understanding DNA synthesis. (Dr. William Wickner in Professor Arthur Kornberg's laboratory, Biochemistry Department, Stanford University, is studying one of our cold-sensitive mutants that may repre- sent.a new DNA gene). An understanding of this most complex and central. process in bocteria is bound to be imnortams for understanding the analogous processes in cells of higucr organisins, including. proliferating animal ceils. Alternatively antibiotics that function by perturbing DNA synthesis may be understood or Glaser, Donald A. Privileged Communication 2a. 2) 3) rationally sought if vulnerable features of DNA syfithésis in pathogens is understood. feasure anomalous DNA synthesis events. for temperature-sensitive mutants, for UW sensitive and UV resistant mutants, and for recombination~deficient mutants, including gene duplications, deletions, point mutations, other chromosomal changes. D. A. Glaser Methods: Changes in proteins involved in DNA synthesis may produce detectable changes in the rates of occurrence of various mutational events including point mutations, deletions, and duplications. The rate of point mutations can be estimated from the rate of revertable auxotrophs. Deletions can be scored as non-reverting auxotrophs, and duplications can be ‘scored by assays for certain enzymes. In particular colonies are able to grow on lactobionate as sole carbon source only if there is a duplication in the lactose operon. Chlorate resistance is being used as a selective condition for deletion of chlorate genes whenever a nearby site for some other function is also affected. These and other assays will be used to study the roles of various DNA synthesis-related genes known to affect UW sensitivity, recombination, or any of the genetically-defined class of temperature sensitive DNA mutants, whether enzymatically characterized or not. Significance: In evolutionary changes to optimize survival, certain changes in the chromosome must be advantageous in pruning away unnecessary DNA, duplicating genes required to produce large amounts of product, providing surplus duplicate genes for future mutational experiments, and enlarging the chromosome to provide scope for greater complexity. The probability of these changes must be affected by the structure of DNA synthesis-related proteins. An understanding of these effects is critical for understanding evolution at the chromosome level and also necessary to understand diseases of higher animals that may result from slight perturbations of the DNA synthesizing machinery. Rational searches for anti- biotics against bacterial pathogens may be possible if this class of perturbations in their DNA synthesis can be understood. Intensive mapping of the E. coli chromosome and measurement of changes in size of the chromosome as frequencies of various mutational events are changed. D. A. Glaser Methods: i. Temperature sensitive lesions can be. readily introduced into the bacterial chromosome and mapped by Pl transduction using the already fairly densely placed well established markers in E. coli (or using P22 in Salmonella). Thus the map can be densely filled with temperature sensitive relatively well localized mutants. ; ii. Temperature sensitive mutants which densely cover small local regions of the map can be prepared by mutagenizing P22. transducing phage in Salmonella’ (or Pl transducing phaze in E. coli) and transducing in a wild type gene for a known lesion on the recipient strain. Transductants for this particular marker gene will then carry a number of lesions in the neighboring region (around 1% of the chromosome) corresponding to the size of the transducing phage (this method has been develoned very succes sfully in recent work (Hong, J., Smith, G., and Anes, B. W., FEAS 23, 0899 (1971))._