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VIFCS ST DIES Oi DRINKING FOUQTAIR AEEC AID ITS PCTEQTIAL REALTY VAZAQDS -& a1‘77 no-I‘v *' 2% "n m vi . L ".1. G '.L.~.‘ 0.3.7 A TUESIS S “”‘ttta to the Co11c*e of Setence and “rte ‘30“1 ‘ 85ft? 1“ve‘sit" of A"r70v1tvrc "3% A‘r“cfl Se‘c CG '* “Prticl fulftllmrnt of th“ rroa:rc rots for tbc i“?T€C of EASTER OF SCTEECE I ..~ '\ J‘ ‘ " I" ~ '. ‘ 3' . Q- ' 5 -‘_:‘Q1" " T- De_3rc eat 0; r-OEAQLClCT_ an: eoelto ' :ii‘f‘lt": }_.J V.) [1'1 \O q,/—;? 5,. 207$ ACLECnLED““T’hTS [-1 . 4.! ,_,-. .- v 4 - ‘ 1e F'I QOI‘.ALSFFT3 to ‘J.TPQSS rfijz serce‘m .. ’— + . . 4- I- ‘lr :' 1‘ ‘1- ' Y + 'A! -‘- ‘ : A r- eebrechclog co Dr. M. A. T-o , . t OUL isc -L . ~I- 4- 4- -— fl -— up 4- f .7" ~. 1—... l‘ n 1 :Mfllstant,“--c rc-t. tfi-trc1i 'nzcets ( 1 Lnu, . PC I , 4 x111*b“hess to helo, this investi rifle: would — ‘ “x 'L'fin got have ‘een .OSSLu_t. Artrcctetion is also ext n - Dr. L. C. Fer“ con Chi Dr. J. L. 13111: n for their helfif?1 sv~¢cutions sad 0: ticisrs of this thesis. Thcfi's are also 6”e to the Public Herltk n I. CC"itt€€ of the PEfier Cup 9nd Conta1_er Emotitute, dew York and fietional Aococizticn oi‘ 85:1;tcrflu 1:, .Dirl eT, C«:lorfx30 zfitosc- :iyftae desistanne eaehled the avthor to hold the rositio: of 9“Qc f_€1 fircd'w tc ragesrch Cns7sir FAEE IL\:TL.{OD'LJ:CTIC'..; . O O I I I I I O . . . . . . . . C O O O O O O O O C O O O U . C I I I O . 1 1. AS EXPERIfiixmAL AEROSOL STUDY OF A DRINAIAJ ?OUQTA-J Ifi THE LAT03‘TCR3 333E? CCgmflOLLED r5“ 7.! {'(1 L‘-) $.‘LICL! ococooooooooooooooooooo-coo-00...... 7 .. 3. : a: 4.1 r ter,L-C 18 5 L1{- l’Ae L114 Ode O O O O O O O C C I D O O O O O O O O O O O 4 D 4 ‘V L iLGSL‘l L13 0 O O O O O O O C I O O O O O O O ._. C C O O O O O D O O O O O O O O O 4 2. AITEKPTS TC ISOLATE A VIRUS FRO? DRIGKIEG FCJuTAIss USED 3" THE PUBLIC .................. ¢O :.nt€1fli€lg 311,5. ::rt‘ifi.OdS oooooooooooooooooooooo 2:" 5.9811146” ooooo0.0.0.000ooooooooouoooooooooooc :2; DISCUSSIOE ........................................ Lu ‘0 EIELIOE‘PquE-Itfoooooooooooooooooooooooooooo000.00.... b4 IAIRODUCTION In en attemot to deteriine the possible 1¢.lt risks Ln using public drinking fountains, an angle jet fountain was exp wxime -ntally contasinated with a virus and studies were made of the resulting water splash and aerosol created. Water samples were also collected from drinl hing foun- q tains used :3 the oublic and the sasples were tests for Q .L The design of drinking fountains has changed little since the first public health laws prohibited the comhon drinking cup in public filaces in 1909. Eerry (1) pointed .— A. n .3' {‘7 P: out that the bubbling fountain was as much a 1th menace as the comr on cup on the basis of his excerimental demonstra- tion. Dunlag, Hinman, and Maffit (2) reported the t both continuous and intermittent flow vertical—jet drinking foun- (I CW 1. r- p) '1) f‘ (D H (I) ’3 33 b1 CL m ry and recommended a slanting-jet foun- oain n: th a guard over the ori iice. ”he latter type foun- tain is widely used today. hitchehs and Ross (3) found that bacteria remained frog 30 ninutes to 2 hours on drinking fountain pav had been contaminated experimentally. They mentioned the ,csotctlicr of bacterial transfer by solassing water. Although the stady of Anderson, Ross and 3ross (4) was not connected directly with drinking fountains, they in- ves“aated the resultiig aero ol cont: .atcioo fron sac- Q —- (_'. teziolorical tecznioues and demons treted that, wieh drone of media containing bacteria fell to a hard surface, the C&Ct€?it were disseninated by aeros ls. C) Lzac cterial aerosol in corniec tiox with drinking foun— tains was first investigated by halluann and Fontes (5). The; introduced large numbers of bacterial cells into the 6’ ("F C) jet-stress of a drinking fountain. By exgosing agar gla at various distances from the test fountain, they were aole to collect viable cells. Mach (6) demonstrated that when snail amounts of virus were introduced into a jet-stream of a dr nking fountain, a viral aerosol was produced and could be recovered by aerosol sanpling methods. The study of viral o’ent has‘beeo enhanced Dulbecco and Vogt's (7) tissue culture methods. This 1 I I method as 'mproved oy ioun er ( ), provi ids d further as- C 5..» k4. stance with cultivation of HeLa cells (Scherer, Syverton {‘1‘ 1 pa a.d }ey ) (9) a dd other cell lines for viral, oiochenical and cytological pu poses. t was also found that certain tissue culture lines were more sensitive to some viruses than others (7, 8), maizin2: it necessary to use several cell lines for experixental virus work. With the new techniques for the detection of viruses, it was possible to test the water from drinking fountains usef by the public, in an attempt to demonstrate viral agents contaminating the fountains and surrounding area. ihe study was divided into two tarts: 1. An experi- mental aerosol study of a drinking fountain in the labora- tory under controlled conditions. 2. Attestts to isolate |\) a A x {4.117 Q o 1 en m fifi .h‘-‘ from: J. n "0/“ . o. pen o8 v r d l "L 1? V - 1. An Lnl'fILMHlAL AERQSOL STUD] OF A DRINKING FOUnlAIN In ZHE LM "Lt’ HOLE SABER CCHTRCLLED COSDITIOAS. Eaterials and lethods The drin Icing fountain used (Figure l) in the exyeri- q sental aerosol work was a jet—angle, star ude rd type, con- C l- (‘1) k.— ’3 .4 p) 1 n -'-, an orifice guard. This type of fountain is most co onlv-found in puolic buildings. The fountain has installed in the laooratory in a nanner simulating usual installation. The virus vas introduced into the jet-flow of the fountain by allowing the virus suspension to fall, drop by drop, to the apex of the stream of water in the fountain. No attempt was made to control the flow or treasure of the water and the fountain was permitted to flow throughout the experimental verlod. The-virus used in the experimental p;1ase of this study was the T-B bacteriophage. It was produced as lysates from infected Escherichia coli type B cells. The lysates were produced on the surfac of svnthetic "F" medium (10) bye 7H1 in: the virus to a heavy :rowtd of ;; coli cells. After incubation at 37 C for EA hours, the lysate and d:oris on the agar surface were flooded with Difco nutrient broth. The sultin" susteusion was (r. clearcd by slox--wecu ceitrifuxation and finally :iltered ‘h -1. , ... 1.-....» 7 '3, 4. M rm . .. 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I - .- F: 4' P r ‘ '\‘ -- ‘ \IL 4" .\J-L1..d315teu so , -DJ we” Ctlt. I ter'J. unrgr ens cl 4.11? -715—5238 Ltap 0* or‘ ‘Q (‘1 ‘/.‘r‘.' fir- J ' C‘ A '. h‘- (— I“ '. buy E‘s UA)OJ.1 t... 4 (_.. uda...k pk , L'-‘.. C-..'-L/L I'e \ ”3;”..- €10 ted ‘ “\f« I NI! k)‘. C .hL-L _ O t S . L O _ S 6 C .L t l s +u T Y T S l T C P C n u L .l t C e .Tu .... e C HM. Q... 0 HT. 60 ... . "V _.... g. 1.; 1A TL M...” 6 Val. A v A r . G ... m U . e t u r t. S A . H H R C .Q S ... _ O E. o. S ..-. .. l S n U .1 n .....n u... r: .L E ,.. S E u G P I l l e C 6 a h r. C l .....o d O C l t P .L S t .l e V n n .. we .1 ..l n. . . G. .l v. S .l . O L. t -. . E ......n. n t T l C T 6 C A l ..a. O C e C u 0 E S a r u a... . .... “W... mu H e 5.4“ by Cu ....c. ..,. a S H v u t l O ...U G . .3. l d r C h S S n a h P t 3 g G C G .n U C H, .. r e I + V .l n u n C t C t 3 r 6 ...U 8 C O .l a M .n .l l .. . t .C h T O n C i v _.-... mi ..r. E ..T . M ..Tu u 4.0 :1— 49 .3 fig ..HH 0 e .r... O C 1.4 d ....m C. .-.. 8 C C. . V L .,. A d .6. r T l s S O S C c t .. u w b t n, t r d .c D. n n .3. O 3,. t 1 e a e . e 1 O G -3. i i ...H. I G t S ......“ r . l C V d E "V 1 no W. :L ....w 6 LL .79 e 8 € 8 u s l W C S ...“ S O P S rl P U u... . Q .TV 8 ..ru 0 6 n... s O A. S 0. t , ,. W V S h e... .l S . J u 0 C d O C E t .1. v. .r. .. P T ..J P n 6 C 4. u PM“ .TL “I" l .....U. I. e 3:; u C.» n m .. . .. O V C l a n r e P . t 3 ..l l P l .1 >.._ t .l .l . r. .... d. l... .9. f O .1 m .1. 40 S P I S .. O C. _ O S 8. a n C C C C ....N T .3 m... C O O n. ....u n l ..I* “11““ 1;; AU t v .. r e mi db LU O T v v. ... t .Q. A ...l G J. ...u S O E . L m l S e - e u n .1 e .3. u S "J ”1. my. C O V. e 1 My“ .3. w. G ml 3 C .l n C O ..u S .n u .l . l .3 e C ...._ . m.. C C D. O --U V ..L .1 C V C O 2, on t C pl 5 .l T T T. C 4; r. .a O C O t l a .1 C. d J. u l u U. 6 0 e u S t E e l ,. .C A. -0. u _ _.J O .1. C ...c .L 9 u“ 3 x0 “U T .Q . l u C .....h l 46 1Q C d 7. ”U ...... v...“ r L0 ”J. ... + AU Lu 9 ...fi PU l V T . ._ S e S 1. .... r n . :1 H . G . .u T m... G u... .L . ..f n u l 1 n .l c e u H O C e G .l D C .. . J. O . . N... t .1 3 . 3 L1 TABLE IV ELCCV‘RY CF VIABLh AIR—BORnE VIRUS PLEIICLRS JITE LEL SLIT LAEFLLR go. of plaques counted in each 1704 liters_of air tested 0 H, :r- m ’ t (7 ’D H st hour F1 - wi h virus I? t l 3 0H t " “1‘ 7 4: ! r". d ' V , 4i; 0 3 __ r n 3 l 2 o r O 4 76 25 5 l ( ‘\ H ( ‘1 C) ~43 *W v.-.-‘ Afl.—.——-J— .-w' ‘ .r‘ r T v i: Q Em C ‘ Y i V S ivC 137 13 52 8 Average 72 Eltquce, too numerous to count. 1: by- 707' fi‘; w.—-.,. . 7T7" Ir; ’J. L‘LLIU X A C. .... r.‘— - us 2 ‘C [Jka ...) n ,I *J y ”sed Founteiie [\ ... lJ O 1008 LLnS - J- r 4-; UC .!‘ . 40. “‘0 .L13. Jo! ‘ .. t ' f‘ a.‘ K4 L.‘ 9 ty litrcr one univerel Ct LIV cuts ’- ‘:‘ ‘ 1 r- t. £4ka - 'A Q . = . . u ‘ 0) One foun- b . ¢~ ‘ o v‘ )1} C 1‘0- 3 ... U v-- ‘l\ 5.4L. g wro -. a L . .L u‘.‘ ~ U \fel‘fl 7 ufi'fl. -. Lie ‘-.L1.;. 4. L; ‘vx C k; r... .f\ . 3‘ ap— .. (I ‘ o V‘5— f". .I". L, \ .CIC 6‘ I A least 0 collec '— D a. QCil 4 @176 ‘v .’ lr h u ‘ \“.-J I; S ...L. 1 1" ount Lflo ~ 0 n3 dr: F‘s " l' C.) .... . ULLC’ ‘- - f‘ 1- : ;‘ . e.-.t.c.-.c be... .1 r'o.-: 6176 v — r. II liquid fir '. ‘1'; J'- ...—_.. 'JI‘C "\ v ...L"; I OHTS. ests 4. t. r to 3.0 *5") let ‘1 .G- Ad 7 L Cy S S .1 +b xtle of no ’ .-- V u;;€i‘. J. ‘w ‘ v I tectiou. (.6 Q l ._ .L L---J. ,- gr, r1 . ... i c :‘N O .1. -. Up. "me' ' * ‘ .L ' .--r 3 .~ ~ n we (.., CCf‘gL‘ll q of AJ .’ NS =-. q .f‘ -Av ~ ”ed, a. -ucounte we ~ to O n --.L i r L, n ' ‘f (I UOJXL.) V- 6 UL; — . vo~.-' - (' . v- ..— v o’.‘ LL 0 ( Z.‘ U n I" I", “ I ‘ l ‘41:». 1-- .02." ‘ a - (x 3... a. -..r~ ,. ' k" " ‘ ; " u u " VA. ' ‘_\—<-_4. (L. v C .l ‘1 C.‘ 11“; J- LL av ‘- ”0.2. ,f l .L— Jere , 't r. 4...: cc? '1 T7 rd J"‘.+ (J bM\U\‘, (1‘: fl.) DJ, T.. . a _.'J. 6 G n C V IV ... “1‘. ..J ‘. F'QQ roam .. ‘—J e a; '.‘-..' 6 -. 1°C :7. ‘v .3. . LI L1. :1 of 0 LI]. ) U f!" _L. r...°..J--.,.,.-.. .. .-.c«.1..L 92.44..-. C L‘-J.Cl"C ‘ql fro _.- ;.scd XAl \2' v -r._. . .4.‘ ,._ u_ - .-‘1 .7 -.. . '. ..,, ‘i....~. ~- r.‘\ . .° .' ."\ . x.“ T‘: __: . . -l Cl routs JLQluJ was regleced u. g c.; ll Cl le-ure f" g -— q ‘ .Vv m; .0 .'\ I " ‘ 5.'\‘ J‘ . ‘ ‘ ‘. “ J“4 ‘V r l,;, CCVClOgCC LJ so-v&s ‘t E - \l~,, Cvflvaiflln. LJC .91 - .L - ,1 ,..-.,'1 _ .o -,...‘ r‘ ‘ , --1. ., ~,' . . Ctnb ;.Lc-lv.uec cull SElu . .he JOHACJ sldney cells 1" '-‘ \ i‘r“ I: \ p Y. —I I”? Q 'P ‘ ‘. I‘. _.. 'fl V't ’r‘ " 4‘ "o -e.e LU u ;e gours at ,; C oelore inoculet-o; filt” ”rte; “ll mciifi contained ;eniclllln, 130 units per ml, '_"‘P Q". v\1 w“ J. ..il Keck of three tubes of HeLa cells was inoculated with ..l ml of the {rocessed water sgec: en. The cells were o 'err;d ever; ot?cr Ca; for slgn of cytoye'hogenic effect (CJE), over :l dC‘ gerlcd. the water senile: wile; showed evldcnce of Chi c; Kch cells were inoculated i*'o each of three tubes of 1‘. -onhey kidney cells ix use same renter as LcLe cells. ‘ . 1 "- 1‘ rs. -. .. .~. ..~— - - , ' '2 -, .-'- 'm. ‘-, 4-! ~ Ale: were 5 so oeserued .or cl QC'S alter lsoc.letloa. 1; q ‘I 1 J. T. c. (w . . r' a ‘v L. kJ. ... U L- 7' r - s J- - r ' ~ We c nu':c* oi :ter s: glee te(c«u _rc CCC“ d°-uu— .1 .. " . . . J- ’ - ., ... e I' ‘ ln_ -o;.te-- .30 fr ‘0 S Llldvn3s is s ex“ .“ 3” l: V. V . .1. , ,— J. 4. W "' -4- ' .. 4. -l3 b' .-J r- les were tested, A; scr5les ire- the library, 22 Ire: tLe grade school, i each from the men's 33;nesium and t-e student union and 3 Iron the university auditorium. Of the 22 ea m5les tested from the grade school, 6 scm5les (27.2%) 5roduced sane CPE in the ori3inal IeLa tissue culture and additional sample was {u tion'b e. A subculture, into tr e sane cell line, with nutrient fluid from the ori Qnal culture pro tdu ccd some CPE vith 5 o: the ...I 7 original samp lees. With an additional sub culture (secczzr ‘ passage) only 2 of the samples continued to show CPE. There was a total or 43 samples of water tested iron the university library and 7 samples (16%) showed som— tyge of OPE and 3 were questionable. On subculture, a ain 7 srecincns -iv;L ed some chen es in the tissue cultLre cells to warrant an additional subculture. However, on this third culture (second 5assage) only one sample affect- ed the cells of the tissue cultures. From the next two locations (Table V) 18 sam:les ‘5roduced only 6 showing CPE of HeLa cells. Addi ional sibcultures of these 5 san5les failed to show any effect u5cn the tissue cultures. The selples from the auditorium building showed 2 sau5les with CPR :ut u on pess a3e lost [‘0 K)! ...».~ r‘r -...L—JUL, rt~u p Lil. CLLJ. will; ..- -J A I ' ggLII—KF cell (:3 Monkey kidney cells samples tests . so. of sam;lles :40. of ’30. of i shouin5 CPLi ,3sam5les sar5les i “ri3. lst 2nd 3rd ‘testcd s owing . geis. nass pess pass i CELt "_‘_ . I: _.-". a II 221‘: ‘\-.V (fif‘ / \ e: .1; E E r— /‘ school as o (l)- , 2 O i ( a 4 x ljjzreiti i g — ’ \ r ' r- lst Flo El 3 \l) ' 3 s Q I A U i 5 Litre f I : é Cree» it 22 , 4 (2) 5 t l c y e l g I . ; Sauerlt ? Uni on 51' E g O O O :5. Q i : iler's . E 3 ncsiuv C 4 (l) ! C C G 5 . O ' I F . i : Ludlicrlus 7 E ' 2 1 Q 2 O . J 3 l ' i - ? Total :6 21 (5) g 14 4 0 so 3 * CPE — Cvt 5C.thic effect ** he” :11 ( ) - Se Jles testeei'fiti ouesi ionatle ess.t£w L fl (.3 *- 3- -. ~ .- Tz * , ~. 4 ,— 1-. ._ s cvln5 enJ ClE Gnu en: gueetloncLle -'$’ 9_ '\ \ ~ 4- ‘0 . n . -‘ -L . ‘~ -.-~. ‘ 'R; ... — - sat .-,,les were t: ensicrred to £2 bill-CL tissue ..E.s:_.t...)e .LL HeLa cells. none 5=educed any definite effect ugon the Jie CPL, cs desecibed in detail Felon, was not unlike that described by End ere (15) on the isolation oi }. f \L C? (7‘ .irus in bu en kidney tissue cu ture. If the agent in ‘ 4" ‘ V . Vv“v‘~ . -‘ » ’ '1' .’ ‘. ‘ -\ a“ q «- 4» .. r. mater seL5les was measles, it hOulQ be Lovcn e sous to ‘ F 1- A, .,.L . , .1. J. 1- -. 5 ' _ L . . o - ”sen Le cl eel, es Lhab p'ocuced CPL and the 5 question— ‘ f us --‘ V‘ ’ ’- ’ . ‘ 1 0’ v —- A v» -- o. r \— e. :: sel~l;s a=uez tested.3xl:uonLed -Lhim3. e5 tweliil , Orly .1 f-.. V 1- ..-— '— u - "n- ‘ .v ,. '_ .‘ ..-.“ 1 litrarv QESGMCHL 5rooucoc soLe CPL. Inc tnree sLL5les _roducing GEE in the ori5inal monkey kidney tissue cultures die notzflxw: c L... r‘r- ~r (4".flfi (‘ '- ’_' [ 014.36, ‘Vl..b.;K/-$ _ find. rs? ’) (.- a . \I.‘ I... .‘I‘I’ l D", 1.11‘ .I I .l‘llllll ‘1’). 4 Figure Outgrowth of normal HeLa cells in an uninoculated tube (600x) Control for Fig. 5 and 6. culture. 28 Appearance of giant cells in culture, 5 days after inoculation. (810x) 29 Figure 6 Cell aggregation and granulation, 8 days after inoculation. (810x) 30 Figure 7 Outgrowth of normal monkey kidney cells. Control for Fig. 8, 9 and 10. (600x) 31 ’0‘ Giant cell formation with multinuclei, 7 days after inoculation. (600x) 32 Giant cells with small cytoplasmic vacuoles, after inoculation. (600x) 33 ; 'gure 10 ‘ . ‘ t ' o " ‘ \\. .-,? . f ‘0 ' I ' . . . '3‘.“ '2’ . ‘ a} D 0 a «8. d' ‘3“ . a. 5 Vacuoles showing lace-like network at cell sheet 3!) margin, 10 days after inoculation. (600x) 34 Figure 11 Outgrowth of normal monkey kidney cells stained with H. d E. Control for Fig. 12, l} and 14. (1200x) 35 Giant cell formation and nuclear changes, 7 days after inoculation, H. & E. stain. (lZOOX) 36 Figure 13 Giant cell formation and nuclear changes, 7 days after inoculation, I. & E. stain. (l200x) 37 14 Figure II, .. _.IK.‘ ‘Il I’ll. I‘II ll. [OI I'. I'll It“. | 0‘ ' I Nuclear changes of the cells around the vacuoles, lO (1200x) days after inoculation, H. 8 E. stain. 38 "C'- .,‘ o ..— Y" c ~n". _ . l 2. L- f ka-.. ‘1' ‘x ' f .' ‘ ICN ‘.‘\1‘_Cj Li... C L, “a I - LL" 7- V i D .:> u t O L.) COLI -. r.-_.‘\ .L',‘ '_J . C -.L h 5 _ .l i. ... lb .1 A. i . d V r . . -. n E ..C . .. .... .U C C .J C t r.” .. S .l r a t i .l O r. s n . l r F t _ .... l 1. r S _ . . m. 6 l ...e l V -.. E C n C t C. _..c O . l E C C R u... G t .1“ C r). C .. S L. t S r. C ..H T .l .L .... G -.-. j E . . C C L T L .1 C ..wh C nL. u... «l .. , vn . ...i mi H U r E .l m i C S S -1 a F . c J. o O u 1 C G .l C t is U .C n-“ u -1. t t t T. is .C u. - u u u m." G ..i ... 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C v . ,u £11013 3V6 Ci ”1/ \ (I I v \_; cezluni ' 0‘ —.&A V 31' (1‘ -L). 'I'I" - I u ‘7‘ foll- .F I .5...A-- V. 'Y I" C\ Lu. IL- v ." V ,3 Tint ln~ '; :1}: Ler~ '. v'\ r? 1 . '51.: t '9‘ II 1" tissue cultures 4.r~ °.».J..- C8 1“ be. in DJ. ‘6 ‘. '1‘3 4-- a 'n‘.‘ _--Lp; 95.. b ,- ~- J . iiO LIL.-- 331 GCOVGI‘ . ('v . ‘ LL- UL: f. '\ ‘r. .- LC .4. _ 'u ..L g 91" la". ‘ "tOjlz. v lnly C (A gent cert , L he mm; S r . 4. . ‘ . I‘v.r nL‘ crlc :0 c;‘ u G» «L a ~$ C“ c K“ C" h _L . L4... ‘1. r" L1 ~- La ,.‘ 4. lb“ - J-l \I 366; A ,H -Vc 2' CL». .1’ Lugs. S 7-1 '! c (7‘ -_ J. b-5—I ---~ '- J I 1|". , 0 11711 1 rlturcs re“ ' 'OQLIC 6' uh l~o ‘ . J.‘ I I; u. cell CT 8 cc .., could not en “'56”? 1.}! fl" ‘ L Y‘ K.’l l to cells ' i J. 5:! t cells HeLa '\ L.) E I A .. q bl -cculated e - I1 .1. n k. I". ‘ . ... s ‘3- rc 11 e n of W seen in ..— ... 'on. 41 ti ‘UJ react ‘I. . -.,-.. f. V _...L ‘,"\‘,'.J Y ’ rid. 4L4..i\.-- -‘ 'fi ’ “e L-ll'lCC. .L c J. Ht". “kl-ls ‘8165 'I l ‘ r. s... similar to that ble X" P L-(.a tic cell cultures 0 n.) d 1U. e C t he -2. i (3. q 4-11 ZIN'SCITL I16 ‘- 0 w I -llOI' I.“ C 1 lncte‘ 10? was a. c2 ‘ 'u.l§ .; (-3 kn wig, or pv, z- I .- hours. CA :or ” I X" ‘ O ~-"o A ULu‘.‘ Cu: 18 an .L 1 V 4. L .a - b; J.‘ .L ‘L V” ’ \ -\/ \ J-‘ss L-Cn’lrélfz. cure , LL Lee- e . -. , ~ ..5‘ 1 fl .2. cl VI? ‘0 e ound -.—. r‘ - - l C-G ‘ '-. I- L; ‘fi ...L W 1'1 O I v .L- V A“ L o J- \m‘ __ Us. x ‘ o,‘ , I T l A.-- .'.-.- 7- , o$\-'-A.'L "C J 1. U L1 110 " rc _.K O k; :1 l i l \U. C ." 7 v 8 ——~ .., .1,- 1.x. ‘ (f;“lA) a. l u ‘ “Cl; ! ..-.- 7) .‘ $ t:- ccle ' ,J 5“,- _. ....a.~ L1,- - --¢ 3 9 o . L :k 1.— V W ’\ ‘_I cL-L“*-I‘- ‘,. .\ mu A14 I I \I ;.. f I.‘ V a.--- ‘ t -—L‘. ‘ 0“» \ f f! " " ~lL-gl. ‘Jl\- g} .d L (‘- ‘VI 0' (:< -LG r _ J ‘. fl J \IL-lb (N lGCtl A Col .,1 h mck to >16 3 "O E 31". C' u. l . .‘L- Jul. 1 rlv ’3 ( “a q l 6 June 0 c VJ». .'1 15.0 i O C C#;Ln1n° b I to be 11 “'. Y. -3. - 1 in: ..- \4 f'.‘ L. J . L C 9 P :1. v I gfl - .3: .g \~ ‘- n.’ .1, r“; 7,1 - ‘- (v 5.1 4.1. - p.11 L ‘0 is J ': ofid \J A P r’ A a.) ~_ - A five fi K 1n— q, m1! "_ _-.C: ~~sa 1.; O J. CL! ,—. \ -p-A .‘ ‘-\_l— .1. ‘ I J AC V‘,‘ f‘. .1 u l.‘ “0“ u '1‘ "UL U k 'c. ’- ,L ‘Lcrl0“ .‘r ,, yak, -5 1. C. h 1. u. .l‘l-I (,. K.‘ Jatex 1 8 L n Dc... .- rcgs of e fc1y :‘W v..'. 4.1 C. Q q ¢ roan". a C I ~.— ..u'. .31". a to 1.. U ‘— l 111'- \:J '1 -ter - fl‘Y O «in v .... l L; A .- u c. t .A. the 53:13. J V 1" 318. C .1. J. U b 11 r-v 5“ fl, J" I q 60. 4. La «L r LE) 1. .ncre avllr .n p .L 01.1.11; 1 ‘V i h "r . -. 145 ‘ ..-- A 7) ”SS A.— \' to he 1 (‘7; VC.\ "'1‘": '4‘ -‘ _- ‘J ~J . . m f r‘ r. “ ‘\ 161' r * - -- .-. - v 0 {,I J. ;.LJ‘ , ,c ..l n 1 L" ..L '— J. U "en lC-C: n C-. glue ,4 cultures. '16 } ..-- n A O 3 C H 116. ( ) 44 u /(r 4.4 C. 21 (' .l U .1. r“ --P ‘ CLISLEfrC . 6 tr r ‘ v.16 J. €I‘ ‘ I“ 946‘“ ‘ t r1 (.4, 4.: b‘. elieve fl U ‘ I 0 V1 1"]le ’\ LA I SIC ”ms- ulqtl A ‘ 4. L2 [I Cf Alt 0 an: 1 _ y _ "' ., O ---— was-L ‘. S u ,- ,V v. r. \. -—‘ l a. 1;COLulte A J. n LS t "c J r (-2. L153 V g -v ...; .. c 2 .4 4 1.4 k)! H] BIBLIOGRAPHY Berr", J. L. Bubble fountain te free the -urea; of Lecoretor a: l?5, 1914-1915. Hi‘.:flen, end D‘L1l-187‘-’ J. -J o 3 J. J. Pro~rees recort of Drinkin: Fcantei c. Jour. ”8300., Z: 33 920. , l C. A. 30:3 Jour. Amer. fl? V 2 or common : I: r. :octcrio sts k L O O 18 S D Amer. W;;“lin“ Ass Collecte , City of L. Mfffltt. er‘ Jorfl:e S . SS I '18 1. LI 6r “actrr‘clo Kellmenn, w. L., and A. A. Fonte study of drinking fountains. Jogr. Amer. Works Aesoc., A7: SEE-2&2, 955. Neck, w. H. Aerosol coitcvinction b? drinleinj tains. The Scniteri a.n, 99: 173-173, 195 {lift-'71.", 18.57. "s, fl. ‘ V , '. . . I 3 ti stWR ev Yo the Committee on Sanitary Vr.ter horks :ztlzizirr 00., 3Q: Pre- ions of Proc. Dulbecco, 9., end H. VOjt. Plaque formation isoletion of pure lines with poliomveli Jour. Exctl. ikfii., 99: 167-132, 1:F12 ounfer, J. S. Ecnolever tissue culture . I. ceration ane stenderdizetiou of sus e2' trvosin dis:crscd n.nkev kidne" cells.8 see. Evotl. ficl. & ch., 9:: 209-23?, semer‘E/r, I]. F. , J. T. SV'VJ'? --;tO_-‘, €1-65 3. C‘ 0 SC‘ 01 the froe.1€tion in vitro of rolicmf viruses. IV. Viflel :sltifiliceti n in strein of bums: mrli nant eiit eliel ce Belg) iC"ived from c iderncid crrcinomr cervix. Jo r Ev t ch., 9": Ad.1s, 1. T. Leticia in mi 81 Rese cerc., er: J. H. Cc.roe, Jr., gze l- -73, 1950. ,— 1-“ 'V r“ N I; ‘ VI" D-?ntr, J. 9., enc a. I. ailson. collect nf sir-borne siczoo v: . -2 , a F nrn - 101,018.}.010 1 , C: 1-6 _ILI 44 A r .3 .4' 1.: 11 1 _J 'I.}\ ’3". , .' x P.) M 7:. vv : 4-° ~ .1 4- -1.0.- £ . acorr, s oe hkkP01101 of serctr,soid c€.rr er: in J- v.‘ 7 4'3 vv __..,~ “, -- . -.. - if ‘: ‘.' LCHQS “1 merit 01 se are elem nstion. no :tx. 12.. 1» 1., TI 1 .v, r- r‘, if, Q 4..-.11 o - _.-11‘1 o .LLG-lt.fi‘, 7: dill—L. i(.l, 191;u o «(“70, y. -“tF‘thl needs of iELUCllf cells In tissue ~1tnwe. sc11nce,122: fil-T?A, 1355- . J. Morton, and R. G. Parker. f e.n miiel cells in tissue CthU :. .4 I U I . 1di s on a synthetic medium. Proc. bet. E: tl. Biol. 8 Ked., T§z 1—1, 1959. Enisrs, J. F., on: T. C. Peebles. Protrfietion in tissue cultures of crtope hofenic ements from netients wi1h recs es. Proc. Soc. Etntl. 2101. f Eed., ii; €7”-296, lvf—"lL CJ IfictVlcr, I. 3., E. L. Fincher,end fl. . Gown. Ire effect of sernlinf mete d unon the enforcnt resnonre of sir-borne Esoteric to tennerature 5nd relative humidity. Jour. Infect. Diserr:es, £22: g7- 00 10:7 \Jl... , -,' ./ | 0 el CC1“”lICu ion. Fort Detrick Contrsct I1-li-3Cl:-CkL—162, licnifen State "n_ve“' tr. . E. kinner, rnd J. H. Smith. nts recovrred from t ssue cultures of center kidney cells. Am er. Jour. nffiiene, 63: 23A-215, 1956. Bull, R. N., J viral e~s Buckle, 9. tudies with the wonTev-11tre-chleen- Inclusion-Aficnt (MINIA) and -oeiirj-€;rnt derived fro: snontaneouslv defeneretin_; monker kidney cultures. Archiv iur die Gcs erte Virus— forschnng, VIII: 139—166, 1953. C") m H. b ’ :3 9.4 _ 0 L *3 le liic1., J. L. Poliovvclitis in ur:en sew eni661c and in nonepidemic tines. Ar. 7? ions, 3;: 243-253, 1947- Kcllv, . I. Det.eo:7 ion Mid occurrence of Coxsesxie viruses in sewe‘e. Em =.e:. Jour. Purlic Healtr, 1529—15 " 19:3. --.1/ 9 Tech, fl. ., N. L. Kcll~a;n, E. H. 5100:, end P. J. Krueéer. E‘ntc: ic viruses 1‘1 '1 Selig: 1ell€c~ isolction. I. Colifcr: Chi :nterococc1 i;cide;ce to isolation of viris s. SCJE e an? Inius‘rie fies :s fig: 9FC—“od, lf's. a w- -. ., ...- Y ' Y“ r. . > 5?; r FJL, J. fl., 5. E Ps‘ne, Ffld . 4. - 4-1 _. .. Concepinnf‘j the Q" ”_‘F‘.L.:‘:O' 611;-0 6- J. I ~1 Ian. ‘-