-‘r.— “— Hll I l 1 ’l I I I II‘ II I I 1 I t I II I I OBSERVATIONS ON SOME HAZARDS OF WORK IN THE TUBERCULOSIS DIAGNOSTIC LABORATORY Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY Walter A. MiIIer I955 .THESIS This is to certify that the \ thesis entitled Observations on Some I-{azards of work in the Diagnostic Tuberculosis Laboratory. I presented by Walter A. I-i'lller has been accepted towards fulfilltgent of the requirements for MS __ , , degree in____BTiCt"_TiOJ-OFZV " ,4 J‘ -- : I."- Hr‘IH-w I .L L \ Majoft: professor Date Mill” 90, 1955 0-169 1‘“"’.‘"-"fl'| A: ~ (3 ‘rp 7T!”7'fi" (" ‘7. TT’T'WT 01D ,JQ‘.‘»'_I‘..LIIA5.TS or"! L) .'._J .d-LuleD'fl 0}. 3011.5. IN TTE TUE UCUIOSIS DIAGNOSTIC LiPORITORY By HALTER A . 111.1113 A THTSIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of BESTER OF SCIUICE Department of Bacteriology and Public Health 1955 THESIS The writer wishes to express his appreciation to Professor U. L. Hallmann, Departnent of Microbiology and Public Health, Michigan State University of Agriculture and Applied Science, for the sugges- tions and assistance given him on this study. Appreciation is also expressed to Samuel R. Damon, Ph.D., Director, Bureau of Laboratories of the Indiana State Board of Health for making available the lahoratory facilities to conduct this study. OBSERVATIONS ON BORE HAZARDS OF WORK IN THE TUEELCULOSIS DIAGHOSTIC LABORATORY By Walter A. Miller AI ABSTRACT Submitted to the School of Graduate Studies of Mich'gan State University of Agriculture and Apolied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1955 Walter A. Killer AN ABSTRACT The occurrence of viable tubercle bacilli on the outside surface of specimen bottl 9 used to submit sputum to a laboratory for tuberculosis exarinatisn were determined. The outside surface of 297 sputum bottles was w shed with four percent sodium hydroxide. The sodium hydroxide was then neut"alized with 2 N hydrochloric acid and a porti n of this neutralized solution was cultured on Lowenstein-Jensen's tuberculosis medium. Twenty percent of all specimens containing tubercle bacilli in the sputum were found also to have virulent tubercle bacilli on the outside surface of the specimen bottle. The ability of tubercle bacilli to survive two different arid-fast staining procedures was investigated. Duplicate slide preparatiens were made from twenty five cultures of fifgobagteriug f' tuberculosis. One set was stained with the Aiehl-Keelson technique and a second set was stained by a method in which a wetting a was added to tea stain to aid penetration. Cultures 01 organisms removed from slides staincc with the Ziehl-Neelson technique failed to produce gr wth. Cultures of organisms removed from slides stained by the method using the wetting agent produced eleven isolations. OPSZEVATIOUS ON SOTT IAZARDS OF WORK IN TIT-3 TTE'IRCUIOSIS DL’LGIIOSTIC -»‘~.TI-T‘;1.TORY Introduction Review of the Literature The Hazard of'work in Tuberculosis Hazards in Diagnostic and Research Laboratories One Possible Source of‘Worker Infection A Tuberculosis Diagnostic Laboratory Purpose of this Study Experimental I II The Examination of the Outer Surface of Specimen Bottles Used for the Collection of Sputum A. Procedure for Determining Susceptibility of Myco- bacteria to'Uashing Solution 1. Cultures Transferred Immediately Following Exposure to Washing Solution 2. Cultures Transferred After One Hour Exposure tolfashing Solution 3. Cultures Transferred After Two Hours Exposure to Washing Solution B. Procedure for Washing the Outer Surface of UnOpened Pottles Containing Patients Sputum as Received in the Laboratory .P The Viability of Tubercle Dacilli After ’cidAFast Staining O- A. Ziehl—Neelson Vethod B. Modified Kinyoun Method III Summary OBSEPVJEIONS on sow tnzusns or won't. IN THE TUBERCUL SIS DIAGNOSTIC L'LPOF.:‘.TORY INTRODIETION Improved techniques in diagnostic procedures, for the laboratory diagnosis of infectious diseases, have been consistent with the progress achieved by esearch in allied scientific fields. These improved tech- niques have raised the quality of diagnostic tests, which in turn has in- creased the demand for these tests, by practicing physicians. To meet these demands it has sometimes been necessary to increase the work load of he laboratory beyond the limits dictated by the availability of ade- quately trained personnel. Adoption of these new and improved methods too often occurs without due consideration being given to the increased hazards of infection which are sometimes introduced by these newer tech- niques. Mhny laboratory workers are trained to perform diagnostic tests on the job, without adequate training in protecting themselves against the hazards involved in handling diagnostic specimens. Protection of those who come in contact with infectiousagents in the laboratory is of utmost importance. Anderson (1) states that "We must not take it for granted that scientists will invariably be in- fected by the materials with which we work. If the probability exists, greater efforts should be made to remove it". REV TEL-I OF THE LITDPAT UF‘E A comprehensive survey has been made by Sulkin and Pike (10) of infection in this country among laboratory workers. A total of 1,334 infections in the United States was tabulated. The infections presumably were acquired as a result of laboratory exposure. Thirty-nine ases re- sulted in death, a fatality rate of 3.0 percent. Approz’nately one-third of the infections had been reported in the literature. The remainder was detected through questionnaires mailed to approximately 5,000 labora- tories including those associated with state and local health departments, accredited hospitals, private clinics, schools of veterinary science, un- deregraduate tea hing institutions, biologic manufacturers, and various governmental agencies. The majority of the cases reported occurred dur- ing the last 25 years. f the laboratory-acquired infections, 775 were bacterial, 261 viral, 200 rickettsial, 29 parasitic and 61 fungi. The performance of diagnostic tests and research accounted for at least 668 of the 775 bac- tcrial infections while the remaining 107 occurred in classwork, pro- duction of biolopics or during the performance of a combination of those activities. HATLRUS IN DIAPIOSTIC A?) RflVELTJF LABORA SKIES During the past few years considerable attention has been directed D niection to which microbiologists are exposed during H. to the hazards of the course of routine work in diagnostic and research laboratories. Stue dies conducted at Camp'Detrick, Maryland (9) showed that bacterial aerosols are produced by some of the common laboratory operations. Sieve type air samplers, placed at approuriate distances around laboratory work— ing areas were used to demonstrate bacterial aerosols produced by the removal of stoppers from dilution bottles and removal of inocula from vac- cine bottles with a hypodermic syringe as well as various pipetting and mixing procedures. A combination of hijh speed photosraphy and the sieve type air sampler was used to demonstrate bacterial aerosols produced by the high speed blendor. Sulkin and Pike (10) indicated that infections in laboratory personnel have occurred with much greater frequency in scientifically trained workers than in animal caretal {ers and lessor trained laboratory assistants. WWWMPOwWWKFTWmmmB The incidence of laboratory acquired tuberculosis wcs second among bacterial infections with 153 cases reported. Trained scientific workers were involved in 136 cases. The remaining 17 cases involved stup dents, animal caretakers, dishwashers, and janitors. Most of the tuberculos1mfections, with the exception of autopsy infections, were contracted from clinical specimens. Other sources of infe wtio included laboratory accidents, aerosols, and handling dis- carded glassware wherea some sources were not indicated and were considered as undetermined. The iniections are more often pulmonary than non—pulmon- ary. Long (i) pointed out that since most infections appear to be the re- sults of diagnostic procedures they are perhaps to be explained on the grounds that greater danger exists when working wit h materials of uncnown character than with those of known cha-acter. Although the monetary loss involved in laboratory infections with tuberculosis is not the mos t important los s it cannot be overlooked. Plunxett (7) estimated that when one considers not only the actual cost of a case of tuberculosis, but also allied expenditures, the total cost is approximately “10,000 per case. During he past fe ew yee.rs, tuterculosis laboratories have adopted the procedure of culturing specimens to recover tufercle bacilli. This procedure h.s een shown to be superior to the simple acid-fast stained film examination. However, he adoption of the culture procedure has introduced hazards to the worker which were not experienced in the exam- ination of stained film preparations. Failure to recognize these haze ds may well be the reason for the high incidence of labore1tory im fections with tuherculos is cited by Sulkin and Pi] :e (10), Long (3), Fish and S pendlove (2 ) . OLE POSSIPLT new“? ,3 hq“v““ IVF"CTI“}? IN THE TUBJPCULOS 8 D13 "‘STIC LHPTZ-TO’Y A common opinion shared by workers in the tuberculosis labora- tory is that a possible source of infection is in handling contaminated specimen conto1iners. Although this opinion is rarely denied it is not emphasized to the extent th:rt adequate precautionary measures are usm1lly dzen d.urinq the processing of the specimens. The specimen bottles generally used by health departments for the collection and subm WlS sion of sputum specir1ens to the laloratory con- .3 sist of a glass bottle 65 mm. high with a diameter of 35 mm. fitted with a cork or rubher lined metal screw cap. This bottle is of convenient size,e nd,wnen ew1clos d in a double container mailing outfi it, complies with the regulations of the United St tes Postal Service. Cpecimen collection outfits are usually furnished to physicians by the healt h depm_rt1ent. The physicians then ri ve the cont.1iners to the patient and instructs him to deposit the sputum in the bottle and mail it to the l1boratory. The patient often holds the bottle before him as he ‘ 5 coprhs to produce tae specimen. Lerosols of infectious material may be expelled by this action. Tiny droplets may dry on the outer surface of the bottle. Furthermore, a bottle is often contaminated directly by the patient as he deposits the succinen but, regs 1rdless of the manner in which the bott le becomes contaninated, tubercle b1cilli dried in mucus rer1in Viable for long periods of time. Tiese contaminated bottles becoue a he zerd to those who come in direct contact with the: 1 in the lehoretory. Refore culturel teCVniqucs gained wide spread acce tance as the 0-5" more eiiicient and exacting examination, sputum was examined by simply spreading a small amount of the material on a glass slide. The film was stained by one of several methods for acid—fast bacilli and examined microscopically. A specimen to be examined by this method could be sterilizeC by steam under pressure before it was opened. De-contanination of diagnostic specimens by steam under pressure is impossible when cultural procedures are to be employed ans, thfs, a contaminated speci— men bottle becomes a hazard. memfitfi‘WESSTBY The following study was undertaken to demonstrate the possible occurrence of viable tUlchlO bacilli on the outside surface of specimen bottles which are used to submit sputum specimens to the laboratory for tuberculosis examination. - .Specimen containers used for this study were those which were furnished to physicians throughout the state of Indiana by the State Board of Health for the collection of sputum for tuberculosis culture examination. The collection bottle has been described above. Infectious ornanisms present on the outer surface of these bottles may be kept viable by the dried mucus. The bottle thus becomes a hazard to the worker unless some measure is taken to decontaminate it in the laboratory. EXPTRITEHTl When contamination does occur the number of tubercle bacilli present may be few. It was therefore necessary to devise a method to wash the surface of the bottle with a small amount of liquid which would be capable of removing the mucus but would not be toxic to the tubercle bacilli. The washing solution must also be bactericidal for non-pathogenic organisms, present on the bottles. The effect of several wa M1 g solutiors on several types of so aprOpgytic mycobacteria for V0 rious times 0: exposure WES 5. first studied. Test organisms used: Eycobacterium phlei ’icobe cterium smegmatis Theobacterium designated as white saprophyte Mycobacterium designated as radish bacillus 1 .fe.sning solutions studied: Physiological saline Tri- so odi um phosphate (Na3P04.12 H20) 23 percent Five percent solution of non-ionic detergent (Triton X-100) in 23 per- cent tri-sodium phosphate Two percent solution of non-ionic detergent (Triton X—100) in 23 per- cent tri—sodium phosphate Fi 1ve percent solution of non-ionic detergent (Sharples 2181) in 23 per- cent tri-sodium phosphate wo percent solution of non-ionic detergent (Sharples 2181) in 23 per- cent tri—sodium phosphate Four percent solution of sodium hydroxide (without neutralizc.tion) Four percent solution of sodium hydroxide (with neutralization) I PTOC DUTT F0 D‘TTT“ITIHJ of ‘TTIPILITY Or IJCOP CT TIA TO u-oTI‘G SOII TITTB Test organisms were transferred from original culture slants (Lo- .en- stein-Jensen) to tufes containing 10 ml. of liquid Dubos medium and incubated at 37 C. for 72 hours. The 72 hour cultures were placed in a mechanical shaker for 15 minutes to disperse organis IS evenly thrOIr hout the liquid medium. 5. This procedure was repeated twice. One-tenth ml. of the suspension of the third sub—culture of each test organism was transferred to a tube containing 9.9 ml. of each of the washing solutions listed above. Immediately following mixing of the organisms with the first seven washing solutions listed above, 0.1 ml. was transferred to two tubes of Lowenstein-Jensen's tuberculosis medium slants. This procedure was following with all solutions except the 4 percent sodium hydroxide (see page 6). This sodium hydroxide solution was neutralized with 2 M hydrochloric acid with phenol red added as an indiCator. One—tenth ml. of the neutralized sodium hydroxide was then transferred to two tubes each of Lowenstein-Jensen's medium. One—tenth ml. of each washing solution containing the test organisms was withdrawn after exposure to each solution for l, 2, 3, 4, 24, and 48 hours and placed in two tubes of Lowenstein-Jensen's medium. The Lowenstein-Jensen slants were incubated and any colonial growth observed on the slants was recorded as 4+, indicating very heavy growth with no evidence of inhibition, 3+ indicating a detectable amount of inhibition, 2+ with 15-20 colonies, l+ with l—lS colonies and negative indicating no growth. Physiological saline was used for a control. The growth obtained on slants from inocula transferred from saline for the various exposure times was used to compare the inhibition of the various washing solur tions for the same exposure times. The results are reported in Tables 1, 2, and 3. TAPLE I A‘. ROSUltS l. GDGUTH Q§_CUTTUVVS TEXTSFEIFTD IIZCDIXTYLY FTLLTTIHG EXPOSUTE IQ W 5181? IIJG SOI.-UTI7'II. White V adish 73.53531: Solution _I‘-_I.E}31.§_'_ I_~I. smefmatis Saprophyte Bacillus Saline (control) 4+ 4+ 4+ 3+ TSP* - 23'") (Na3P04o12 H20) 4+ 4+ 4+ 3+ TSP +-Triton X-lOO (5%) 3+ 3+ 4+ 3+ TSP + Triton X-100 (21) 2+ 2+ 4+ 1+ TSP + Sharples 21551 (51) 1+ 2+ 3+ - SP +-Sharples 2131 (2%) 1+ - 3+ - 4% NaOH (not neutralized) - - 4+ - 4% NaOH (neutralized) 4+ 4+ 4+ 4+ *TSP Tri—sodium phosphate 4+; very heavy growth with no evidence of inhibition 3+ a detectable amount of inhibition 2+ 15 to 20 colonies 1+ 1 to 15 colonies - no growth TAB-IE 2 2. SOI_.TTI‘I"N Tashing Solution EL £333; Saline (Control) 4+ TSP’~‘ - 23”. (1333120442 .1120) 3+ TSP + Triton X-100 (5-3) - TSP + Triton X—100 (2fi) 1+ TSP + Sharples 2181 (5%) 1+ TSP + Sharples 21.91 (263) - 43 NaOH (not neutralized) - :3 N's-.03 (neutralized) 4+ TSP* Tri-sodium phosphate White Saprophyte Bacillus GRV‘UTH 93 CUT-TIRES TZ‘STFTPR'CU TT"? 1 HOUR EXPOSURE TO M "531313 q" (7“. 4"} ,L ,-$—L(. 1 4+ 4+ 4+ 3+ 3+ 3+ 3+ 4+ very heavy growth with no evidence of inhibition a detectable tmount of inhibition 15 to 20 colonies l to 15 colonies no growth 4+ 1+ 1+ 2+ 4+ 10 TAPLE 3 3. C333TW‘93'CUITUW73 TiXTTFTPRTD AFT?" 3 HOURS EXPOSU33 E9 WISHING S 0L UTI "l N White Radish War in: Solution kgzglgi ‘3.smegmatis Saprophyte Bacillus Saline (Control) 4+ 4+ 4+ 4+ TSP* - 23:3 (131904.12 H20) 2+ 1+ 4+ 2+ TSP + Triton 32-100 (523) - - 2+ — TSP + Triton :{-100 (2:1) - 1+ 3+ - TSP +-Sharples 2131 (5%) - - 2+ - TSP +-Sharp1es 2181 (2% - - — - 43 NaOH (not neutralized) - - 3+ - 453 351011 (neutralized) 4+ 4+ 4+ 4+ * TSP Tri—sodium phosphate 4+ 3+ 1+ very heavy growth with no evidence of inhibition a detectable amount of inhibition 15 to 20 colonies 1 to 15 colonies no growth Althourh transfers were exposure to he \;sIfng solutions control and in the neutralized 4% NaOH 1 “P CLLL‘ selefited as the solution of choice made also after 3,4,24, srowth was obs tubes. was 11 and 4? hours erved only in the saline Therefore, 4% NaOH was neutralized immediately fol- lowing the washiné of each specimen bottle. IT PRmEDITPF FOP I-I‘rSHIpG TIFF). CO ITT.11_II TIIIG P T LITE? The glass placed in a glass ointment jar. OUTER SUPFlCE OF UNOPWL {TWHHETAS P CHIVTD IN THE LiPOTXTORY Ten ml. of 4$ ‘WD POTTIE sputum bottle w s removed from the mailing ores and NaOH with phenol red in- dicator was added to the oin oment jar. a metal screw cap was placed on the jar containing the sputum bottle a'17d the 4f? Ila OH. This bottle was then rotated manna. 11y so that the 471' 10H was allowed to wash the entire surfs ce of the specimen bot le to remove via11e tubercle bacilli which might be present. The wishing solut 2 N UCl and a portion of the neutrali slants of Lowenstein-Jensen tuberculosis medium. bottle with the diagnostic specimen was laboratory and. the sputum w.as cultured in the routine manner. from these wa of tubercle bacilli. A. Observations Th -e outer surface of 297 sputum see in the manner described above. were obtained examination. yielded in 12 cultures of tubercle ba those specimens found positive in the routine exalination I 1011 WES cilli. immediately neutralized with 1 zed w-shing was then placed on The washed specimen then returned to the txberculosis Cultures shings were observed weekly for six weeks for colonial growth cimen bottles was washed Si 1xty isolations of tubercle bac 111i from these 297 specirens in the course of routine culture The washings from the outer surface of the 297 specimen bottles Therefore, 20 percent of were shown to 12 have viable tubercle bacilli on the outer surface of the bottle. THE VIIFIIITY OF TUDIRCI ‘BICILLI LFTUH iCID-F1“T STAIHING Several nodificati: ns of the origi- nal Ziehl—Neelso n method for steini n3 smear pre_ rations of tubercle Iacilli have appe ea.red in the literature (8). Although no claim is made for sterilization of bacterial cells stained by these methods, some bacteriologists depend upon the alcohol and phenol us ed in th e staining so ution to destroy the hm 111i The heat employed by the original technique is also relied upon to "kill" tubercle bacilli. Iflddlebrook and his co—worlcers (5) h vs shown the t II. tubercu - losis has a tendency to grow in a corded or serpentine configuration and when such suspensions, consisting of clumps of bacilli are stained, it is possible that organisms in the center of tLese clumps are protected from the bactericidal substances in the sto mining solution. The preparation of slides for microscopic examination of "raw" diagnostic spoolnens and of "live" cultures 18 common practice in many .L tuberculosis laboratories. Slides with the infectious material may be allowed to "air dry" before staining. The process of drying is sometimes hastened by placing the unprotected slide over a hot plate and convection currents produced by the hert may result in the flaking of particles from the slide and the dissemination of infectious material. The ability of virulent tubercle bacilli to withstand two different methods of acid-fast staining was studied. The orifinal acid-fast U) tsin procedure described by Ziehl-Neelson in 1882 (8) was compared with no afic. tien of the Kinyoun carbolfuch . sin ac 1 e-fast stain reported by Q) [—10 Muller and Chermock (6). Duplicate slides were prepared from 25 cultures of virulent tubercle 13 bacilli grown on Lowenstein—Jensen medium. The slides were placed in coyered Petri dishes and dried in an incubator at 37 C. for 24 hours. One slide from each culture was placed on a staining rack and stained by the Ziehl-Neelson technique. Procedure 1. Each slide was flooded with a solution made of 10 ml. (10 per— cent alcoholic solution) of basic fuchsin and 100 ml. of a 5 percent aqueous solution of phenol. N c (Q ‘lides were steamed gently over a flame for 5 minutes. (The slides were not boiled). Stain was added to the slides as it evaporated. 3. The slides were then washed with water and decolorized with 3 percent HCl in acid alcohol until no color flowed from the slides. 4. The preparations were then counterstained with a solution of methylene blue. Tr ' The duplicate slides were stained by the modified procedure of nlnycun ,s described by MUller and Chermock (6). E' 1 The carbolfuchsin for this stain was prepares as follows: Basic fuchsin 4 gm. Phenol 8 ml. Alcohol (95 percent) 20 ml. Distilled water 100 ml. The basic fuchsin was dissolved in alcohol and added to the water while shaking. The phenol was melted in a 56 C. water bath and 8 ml. was added to the stain with a pipette. Turgitol number 7, a wetting agent, was added to the Kinyoun carhol- fuchsin stain in the ratio of 1 drop to 30-40 ml. of stain. The slides were flooded with this solution for 5 minutes (the oricinal instructions specify 1 minute), no heat whs applied to the slides in this series. The slides were then washed with water and decolorized with 3 percent HCl in acid alcohol until no color flowed from the slides. The preparations were then counterstained with a solution of methylene blue. After the film preparations we-e stained each slide was placed in an ointment jar to which had been added 5 ml. of 4 percent NaOH with phenol red added as an indicator. The jars w‘re vlaced in a wire basket and placed on a Kahn shaker for 10 minutes to remove as much of the stained film as was possible. The NaOH from each jar was titrated to the neutral point with 2 N 901. A portion from each of the neutralized washings was placed on two tubes each of Lowenstein—Jensen's tuberculosis medium. Cultures inoculated from washings of the 25 slides stained by the orihinal Ziehl-Neelson failed to produce colonies of tubercle bacilli. O Uashinbs from slides stained with the modilied techniques described by Muller and Chermock produced ll positive cultures. No detrimental effect was observed after the test organisms had been in contact with p ysiolop ical saline for as long as forty—eight hours. &li sht toy ic effects were noted from the use of Tri-sodium phos- phate for one hour. A sh rp increase in to: {icity was noted at two hours. No growth was obtained in the subcultures after twenty—four and forty— eight hour exposure periods. The toxicity of his solution is of particu- lar interes since this chemical is commonly used as an ag ent for de- contaminating and d1"est1n" sputum in many laboratories. Combinations of non-ionic detergents and tri-s odium pb 05 he te proved to be even more toxic than triésodium phosphate used alone Test organisms kept in four percent sodium hydroxide for approximately ten minutes before neutralization with 2 N hydrochloric acid were not affected. Twelve isolations of E. tuhqrgulgsis obtained from the outer surface of contaminated specimen bot p188 cle- arly supports Long's (3) sug— gestions that greater dangers may exist when working with materials of unknown character. A change of procedure was introduced in the tuberculosis labora- tory of the Indiar na State Board of Health as a res.ult of the findings of this study. All tuberculosis specimens submitted to the laboratory for diagnosis are assumed to be contaminated and are transferred to clean sterile bottles before they are processed. The recovery of viable tubercle bacilli from "cold" stained film prep care tions indicate a possible hazard not only to laboratory workers but to wash room workers as well. This hazard can be eliminated completely by'sterilizing all film preparations witlm steam under pres sure before staining. f the 1ahoretory worker. .10 J :3 (1) Anderson, Robert J., Editorial, Protects“ Purlic Health ieport, 65: 463, 465, 1959, (2) Fish, Charles H. and Spendlove, George A., Safetv ne.sures in E tuberculosis laboratory. Public Health Reports, 65: 466, 467, April, 1950. (3) Long, Csmond 1., The hazards of aeguirin: tuberoulosis in the laboratory. Anerican J. of Public Health, 41: July, 1951. (4) tickle, Friend Lee, Growth of tuhcrculosis literatory work and .L cuities it has presented. C nnecticut Health Tulletin, 65: ’W 1‘ .- «an- a t . la, JeCedter, 1,; . ~f. " [v ‘ 4- s n ‘ '1. ~ ~'\'\“1 ."L' . .1"- 34”! 9 av ‘ (5) .1dd1ehr001, Carener, Pacte11a1 ¢,u ¥LCOth Iaiections or an. J. P. Iippencott, i Co., Page 299, 1948. L) O 1.1. Q.» I (6) Muller, H. E., and Chernock, R.: 1 rapid stainin" tecnni.us for fast organism. J. of Lab. and Clin. I-fed., 30: 169, 1945, (7) Plunkett, R. 3.: Tuherculosis as an econonic and social proilem. Connecticut Ted. J., 3: 9, 1944. *v 1 O 1 (V (3) Schaub and Foley. Diagnosuic Bacteriology. C. i. .osey U0., 4th Ed., 1952. (9) Stein, Leon, Anderson, Raymond E., and Gross, Hoel 3.: (Piolo:ical Dept. Chem. Corps., Camp Detrick, Frederick, Ed.) Potential in- fectious hazaris of conmon bacteriological technigues. Paper presented at 49th General fieeting of the Society of American Pacterlolocists, Way, 1949, Cincinnati, Ohio. (10) Sulkin, Edward 8., Pike, Pobert H., Survey of laboratory-acguired infections. Amer. J. of Public Health, 41: 769, 781, 1951. MIC H!IGAN STATE UNIVERSITY LIBRARIES IIII IIIIIIIIIIIIIIII