J. clin. Path. (1954), 7, 290.

TEMPERATURE LEVELS IN HOT-AIR OVENSBY

E. M. DARMADY AND R. BARRINGTON BROCKFrom the Portsmouth and Isle of Wight Area Pathological Service

(RECEIVED FOR PUBLICATION OCTOBER 14, 1954)

During the past few years the popularity of hot-air ovens for sterilizing has been increasing. Thisis partly due to the ease with which they sterilizesmall objects such as syringes, and partly becausethey are inexpensive to run. At the same timeautoclaves, which appear to be popular in America,proved to be difficult to obtain in this country afterthe war, and were often large and cumbersome;they also required skilled handling. The intro-duction of syringe services has led to increasingnumbers of hot-air ovens being used for steriliza-tion, and it was primarily because of the formationof a syringe service in this area that the presentinvestigations were undertaken.

Aim of the InvestigationOur preliminary investigations were made to

assess:(1) Whether the heat actually penetrated to the

syringes. (2) Whether the type of packing em-

ployed was satisfactory. (3) At what position inthe oven should the thermocouple of an automaticrecording apparatus be placed.Normally it has been the practice to test an

oven with thermocouples placed in glass tubeswhile the oven is empty. The efficiency of theoven is gauged by the degree of correlation of thetemperature levels recorded throughout the oven.In our investigations the ovens were tested fullyloaded just as in a syringe service, since it is onlywhen heat penetrates the centre of the syringesthat sterilization is effective. In the literatureopinion varies considerably as to what the tem-perature of the oven should be and the timerequired to ensure adequate sterilization. Tem-peratures varying from approximately 1400 C. to1800 C. from half an hour to two hours are men-

tioned. However, it is probable that in thiscountry 1600 C. for one hour, as advocated by theMedical Research Council Memorandum No. 15(1945) on the sterilization of syringes, would beaccepted in a court of law. This memorandumfurther recommends that ovens should be loaded

cold and allowed to heat up to 1600 C. and heldat this temperature for one hour. In our investiga-tion this temperature has been used in all the testsundertaken.

Recommendations in the LiteratureAlthough experience with hot-air sterilization

dates back as far as 1881, Koch and Wolfhugel(1881) found that dry heat was not as effective aswet heat in destroying bacteria and spores, andthat, although vegetative bacteria could be des-troyed at a temperature of just over 100° C. for oneand a half hours, spores required a temperature of1400 C. for three hours. The type of hot-air ovenis, however, not mentioned, and it is probable thatthese were of the gas type.

Since then a number of sterilizing temperaturelevels have been advocated, and, in view of the

TABLE ITEMPERATURE LEVELS FOR HOT-AIR OVENS

Name Hot-air Sterilizer

Bigger, J. W. (1949) .. .. 1600 C. for I hour1400 C. for 3 hours

British Pharmaceutical Codex 1500 C. for I hour(1949)

British Veterinary Codex (1953) 1500 C. for 1 hourBurrows, W. (1949) .. .. 1700 C. for 2 hoursAmerican Public Health Asso- 1600 C.-180° C. for 2 hours

ciation (1950)Dubos, R. J. (1948) .. .. 1600 C. for 90 minutesFairbrother, R. W. (1953) .. 1600 C. for -1 hourGerhards, G. A. (1952) .. 140° C. for 1 hour (electric fan oven)Gradwohl, R. B. H. (1943) 1700 C. for 2 hours (gas oven)M.R.C. Memorandum No. 15 1600 C. for 1 hour from cold

(1951)Mackie, T. J., and McCartney, 1600 C. for I hour

J. E. (1953) 1800 C. for + hourMcCulloch, E. C. (1945) .. 1600 C. for I hourSmith, D. T., and Martin, D. S. 1500 C.-160° C. for I hour

(1948)Stitt, E. R., Clough, P. W., and 1600 C. for 90 minutes (syringesBradenham, S. E. (1948) must be separated)

Walter, C. W. (1948) 160° C. for 1 hour if instrumentsare clean

1600 C. for 4 hours if oily or greasyWhitby, L. E., and Hynes, M. 160° C. for 1 hour

(1951)Wilson, G. S., and Miles, A. A. 1400 C.-1600 C. for 1-1+ hours

(1946) 4000 C. for 20-30 secondsUnderwood Weedon, B. (1941) 3200 F. for 1 hour (electric oven

without-fan) (1600 C.)

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variety of recommendations, have been set out inTable I. In the majority the type of oven has notbeen specified, but Gradwohl (1948) quotes 1700 C.for two hours in a gas oven as being satisfactory.Weedon Underwood (1941) advocates 160' C. forone hour, and illustrates a satisfactory electricoven without a fan. More recently, however,Gerhards (1952) using a fan oven has shown thatspores can be satisfactorily destroyed at 140' C.for one hour. Ewald and Schmid (1953) alsodraw attention to the importance of testing elec-tric ovens with maximum thermometers, and foundlarge variations from shelf to shelf. Walter (1948)in his monograph The Aseptic Treatment ofWounds draws attention to the limitation of thehot-air oven, and points out that most ovens heatuip slowly and unevenly and that sterilization istherefore uncertain. He advocates that the wholeload should reach 160' C. and be held at this tem-perature for one hour, provided that the instru-ments are clean and free from oil or grease. Ifthe instruments are oily or greasy he considersfour hours after 160' C. satisfactory, but alsoadvocates that " air blowers " in the ovens hastenheating and assure uniform conditions.

Method of InvestigationIn our investigations " chance " all-glass metal-

tipped syringes were used, and were packed in eitherthick-walled glass tubes or metal containers. It canbe stated now that it was found by experiment thatthe type of containers used made no appreciable differ-ence to heat penetration or the temperature reached.In the preliminary experiments needles were alsoused, and these were packed in glass test-tubes withcot on-wool plugs. Throughout the experimentsattempts were made to simulate the exact conditionsof a normal syringe service. Normally syringes wereloaded into wire baskets 9 in. x 9 in. x 9 in. of atype commonly used in bacteriological practice. Thebaskets were either (1) loosely packed, when syringesin their containers were held apart by metal skewers,leaving a gap of approximately I in. through whichair could flow freely, or (2) tightly packed, when asmany syringes in their containers as possible werepacked in the baskets.The number of baskets loaded into each oven de-

pended upon the overall capacity. In each case theload was considered as average for the size of theoven. No attempt was made to overload the oven.In two ovens metal trays were provided which didnot allow the use of baskets. In these ovens thesvringes were laid two deep on their sides to allowthe free passage of hot air as indicated in Fig. 1. Inselecting ovens care was taken to choose those whichwere, or might be, in normal use in the laboratory,and were of the standard pattern produced by well-known laboratory furnishers. A multipoint copper

5

'6

.4

/ NK2J2 1 21

FIG. 1.-The distribution of syringes in electric oven B, packedloosely, 40 syringes to a shelf. The figures indicate the positionand number of the thermocouple used.

constant thermocouple temperature indicator wasused throughout the experiments. The indication wasobtained by a null point method using a potentio-meter circuit of high precision, i.e., back E.M.F. wasapplied against the E.M.F. generated by the thermo-couple until a sensitive galvanometer registered zero,and the readings of the potentiometer were calibratedagainst actual temperatures applied to the thermo-couples, which were measured with sensitive thermo-meters immersed in hot oil. This method cancels outany internal electrical errors in the system.

Specially calibrated mercury and glass thermometersgraduated from 100' to 200' C. in half degrees wereplaced in position as indicated by the manufacturers.The readings from these thermometers were consid-ered to indicate the air temperature of the oven, sinceit is these readings which guide the operator and areused as a reference point in all experiments except inone electric oven in which the manufacturers hadprovided a circular-dial type of thermometer.

Special borings were made in the glass piston ofsome syringes and thermocouples and leads passedinto the cavity so that the point rested as close tothe base as possible. The syringe was then placed ina container and the lead so arranged that normalpacking was not disturbed. The wire leads wereintroduced into the oven through the hole used forthe thermometer. Normally 10 or more leads wereused in each experiment, and were distributed in

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baskets throughout the oven. Usually two tightlypacked baskets and two loosely packed baskets wereplaced on the top and bottom shelves respectively,and leads placed in the centre of each. Other leadswere also placed in a syringe on the outside, or inother baskets. The type of distribution of the basketsand position of thermocouples are shown in Fig. 2.This distribution was varied sligh.ly from oven tooven according to its capacity and the experiencegained in previous experiments.

FIG. 2.-The distribution of the thermocouples and baskets used inmost of the experiments. The number indicates the number ofthermocouple used. Thermocouple: 11 and 16, outsideloosely packed syringes. 12 and 17, centre loosely packedsyringes. 14 and 19, outside tightly packed syringes. 13 and18, centre of tightly packed syringes. 15 and 20, centre of looselypacked metal syringes.

In this manner we have tested two gas ovens andfour electric ovens without fans and two with fans.With the exception of one gas and one electric oven,all ovens tested conformed to the ±30 C. when testedempty. In the experiments the ovens were loadedcold and allowed to heat to 1600 C. and held forone hour at that temperature during which time thetemperature recordings were taken at 10 to 20-minuteintervals.

ResultsGas Ovens.-In the two gas ovens wide varia-

tions of temperature were apparent from the startand generally the temperatures were considerablylower in the bottom shelf than in the top. It wasalso found that syringes tested near to the back

wall were consistently overheated. In the first oven(gas oven A) the reference thermometer reached1600 C. after 50 minutes. At that time the majorityof the syringes had not reached 1300 C., anddifferences of approximately 60° C. were noted, iftightly packed syringes are excluded (leads 6 and 9).Most of the syringes on the top shelf seriouslyovershot the air temperature of the oven, whereasat the end of one hour most of the syringes on thelower shelf, except those placed at the back, had notreached 1600 C. Even after one hour there was avariation of 400 C. The recordings of thermo-couples are shown in Fig. 3. Similar findings werefound in the second gas oven (gas oven B), whichalso showed some interesting features. This ovenhad been claimed by its owner as being satisfactorybecause of the time taken for it to reach 1600 C.It was thought that this slow rise would ensurean even distribution of heat. Experiments showedthat this oven took 136 minutes to reach 1600 C.At this time six out of the 10 thermocouples werebelow 1550 C., the lowest recording being 1460 C.The remaining four were at temperatures varyingfrom 1660 C. to 182° C. However, during the next15 minutes the oven air temperature had risen to1630 C. and the syringe temperature varied from1520 C. to 1920 C. At the end of one hour sixof the syringes were between 1930 C. and 1690 C.,while one syringe still had not reached 1600 C.,giving an overall variation of 340 C.

Electric Ovens.-The four electric ovens allshowed improved performance over the gas ovens.In one electric oven there was only 90 C. differencein the recorded temperature of the syringes at theend of one sterilizing period. In the other threethe overall variations of the temperature at theconclusion of the sterilizing period were 210 C.,280 C., and 170 C. if the tightlypacked syringes areexcluded. This in itself might prevent certainsyringes from reaching a temperature necessaryto ensure bacterial death. Perhaps the greatestdisadvantage of the electric oven without fan isthe time taken for the syringes to take up heat.Even with electric oven C, in which the finaloverall variation was less than 90 C., heat wasslowly taken up. For example, although the tem-perature of the oven reached 1600 C. in 63 minutes(and no tight packing was used), the syringes atthis time varied from 1220 C. to 1500 C. (320 C.),and even after 15 minutes there was a variation of250 C., the temperature of one syringe being only1260 C. It was not until three-quarters of thesterilization time had been completed that thetemperature of the syringes became stabilized.These recordings are shown on Fig 4. This oven

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FIG. 3.-Serial temperature recordingsof syringes in gas oven A startedfrom cold. Syringes Nos. 1 and 8 wereplaced in the back of the oven onthe bottom and top shelf respec-tively. 0 denotes those syringesplaced on the bottom shelf. Xdenotes those placed in the centreof tightly packed baskets. Note thewide variation of temperature andthe length of time taken for syringesto attain the temperature of theoven.

t70 161

160

150_

140

o 120-

Li 110Ib-

6I

TIME MINS.

60 80

TIME MINS.

Fio. 4.-Records of thermocouplesplaced in syringes in gas oven C.0 denotes those placed in the bottomshelf, and A denotes those in thecentre shelf.

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FIG. 5.-Thermocouple reading fromelectric oven D. Note the slowrise of temperature and the widedistribution of temperature at theend of one hour's sterilization.

60 80

TIME MINS

FIG. 6.-Thermocouple readings insyringes, the distribution of whichis shown in Fig. 2. Note the promptrise in temperature but that those inthe centre of tightly packed syringesfail to take up heat satisfactorily.X denotes centre of tightly packedsyringes. 0 denotes centre oftightly packed syringes on thebottom shelf.

Lu.I

: I

a:

I--

TIME MINS

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was fitted with four shelves and a built-in circular-,dial type thermometer. It is probable that the dialin this case was faulty and by its correction thetemperature of the syringes could have been main-tained in the neighbourhood of 1600 C. It wouldnot, however, have altered the length of timenecessary to bring the syringes up to a stabilizingtemperature. It also points out the necessity ofchecking the recording temperature with thatattained by the syringes. Fig. 5 shows the record-ing in electric oven D, which again shows thedelay in the time taken for the syringes to take upheat, and suggests that there is a danger that someof the syringes may not receive sufficient heat toensure sterilization. Electric ovens A and Bshowed exactly similar trends.

Fan Ovens.-Two different patterns of electricoven with fans were tested, and it was interestingto note that, although the final overall variation oftemperature in the syringes was less than 80 C.and 100 C., the tightly packed syringes again showeda serious delay in taking up heat. In these ovensheat was promptly taken up as shown in electricoven B with fan (Fig. 6), and the overall variationof syringe temperature was less than 120 C. and130 C. from the start of the sterilizing period, thelowest temperature of any syringe recorded, ex-cluding tightly packed syringes, being 1440 C.This ensures that all syringes have similar heattreatment.

Overloaded Ovens.-In all experiments so farquoted, there was a delay in the time taken forthe packed syringes to take up the oven tem-perature. It was therefore decided to test specifi-cally the effect of tightly packed syringes and atthe same time using syringes of varying sizes. Forthis experiment electric oven A, with fan, was used,since it appeared to be the most efficient oven(Fig. 7) tested at that time. The recorded tempera-tures of thermocouples are shown in Fig. 8 andshould be compared with Figs. 3. 7, and 9. It isseen that the syringes in the centre of the tightlypacked baskets take a considerable time to reach therequired temperature, and further that the overload-ing of the oven interferes with the normal air flowwith the result that many of the syringes fail toreach oven temperature. In general the bottomshelf shows a lower temperature, confirming theinterference with air flow which is normally higherin ovens with fans.

Heat-loaded Ovens.-The Medical ResearchCouncil Memorandum No. 15 (1945) specificallymakes the point that ovens should be loaded coldand the oven allowed to attain the temperature

of 1600 C., when it should then be held at thistemperature for one hour. However, in a busysyringe service considerable time may be wastedto allow the oven to cool to room temperaturebetween batches. It was therefore decided to testspecifically the effect of loading the oven when stillhot. For this purpose the ovens were allowed toheat to 1600 C., and as soon as that temperaturewas reached the ovens were opened and while theywere still hot were loaded with syringes alreadyplaced in baskets. The ovens were then allowed toregain the temperature of 1600 C. and held forone hour. Two ovens were tested in this way,gas oven A and electric oven A with fan. Thedistribution of syringes and thermocouples wasexactly similar. The temperature recordings areshown in Figs. 8 and 9 and should be comparedwith Figs. 3 and 6. No appreciable difference isseen in gas oven A, while in electric oven A withfan the uptake of heat is more even and complete,presumably because heat has already been ab-sorbed into the fabric of the oven.

DiscusonIt must be clearly understood that the placing

of thermocouples enclosed in syringes and shieldedby containers are all variables which contributeto the variations in temperature shown earlier. Thearrangement of the basket also plays an importantpart in interfering with hot air currents. Theexperiments must therefore be considered as in-dicating trends of temperature changes rather thandefinitive alterations. Nevertheless, in construc-ting any piece of sterilizing apparatus it is essen-tial to ensure that all articles receive similar heattreatment, and that the temperature reached cor-responds to that of the recording thermometer,reaching this temperature with a minimum ofdelay. With the exception of fan ovens themajority of ovens do not meet these criteria. Inaddition, our experiments again stress the impor-tance of avoiding tight packing, since it is clearthat by tight packing it is possible that somesyringes may receive insufficient heat treatmentand may not reach the thermal death point ofbacteria. There must be a free flow of air aroundall articles to be sterilized, and enclosing syringesin metal boxes or similar sealed containers mayprevent the heat penetrating to those syringes nearthe centre. It is also clear that overloading ovensmay seriously interfere with heat distribution, apoint which has been frequently stressed in theAmerican literature but which does not seem tohave had sufficient emphasis in Britain. MaxwellSavage (1936) has also shown that tight packing

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FiG. 7.-Reading of electric oven A withfan. X denotes those placed insyringes in the centre of tightlypacked syringes. 0 denotes thoseplaced in lower shelf. In the finalstages the oven was allowed tooverheat, and the rapid take-up ofheat should be noted.

170 -

160

ISO _

140

V 130

120/a:

Id-11

U0Id0

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I--

TIME MlNS.

FIG. 8.-Effect of overloading an ovenin which all the baskets containtightly packed syringes. X denotescentre of the basket. 0 denotesbottom shelf. A denotes centre oftightly packed 20 ml. syringes.

TIME MINS.

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FIG. 9.-This should be compared withFig. 3 (gas oven A). The same ovenposition of syringes and baskets,number of thermocouples, andsymbols as those used in Fig. 1. theonly difference being that the ovenwas first heated to 160° C. andloaded hot and allowed to regain thetemperature of 160° C. before thesterilization procedure was restarted.Note the time saved by this method,and that the uptake of heat andvariation of temperatures recordedare similar to that loaded cold.

20 40 60 80TIME MINS.

10O I20

FIG. 10.-The effect of loading electricoven A with fan while it is still hotNote that the take-up of heat isslightly more rapid and that theoverall variation of temperaturetends to be less.

Li

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TIME MINS.

OVEN TEMP. DEG. C., K~~~~~~~~~~1s 1 1 I- :

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IAs.

170

160

150

140

V1306w40

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is equally dangerous in autoclaves and it is essen-tial that steam should penetrate to all articles tobe sterilized.The uneven distribution of temperatures recorded

in gas ovens suggests that this type of oven is un-suitable and unreliable for syringe services. Notonly is there a danger that heat may not penetrate,but also there is a possibility that excess of heatmay cause the solder to disintegrate if glass andmetal syringes are used. A further danger, shownby Gerhards (1952), is that the point of the needlemay be seriously damaged by extremes of tem-perature.

In the electric oven without fan the overallvariation of temperature was not so great as witha gas oven. However, without fans the transferof heat is slow, and variations even after stabiliza-tion may be extreme and suggest that the heattreatment may be of such short duration and ofsuch low temperature that the thermal death pointof bacteria may never be reached. It is thereforeimportant that in the electric oven the time ofsterilization must be so adjusted that the instru-ment showing the lowest temperature receivesadequate heat treatment. This may mean thatsome articles receive more heat and for a longerperiod than is necessary. In a busy syringe servicewhen ovens are in constant use the time may be ofimportance. The value of the fan oven is thatthe load takes up the heat rapidly and equallyfrom the time the oven reaches the desired tem-perature and that the variation of temperaturefrom one syringe to another is less than 100 C. Thisindicates that in a fan oven the temperature neednot be so high nor the heat treatment so prolonged,both valuable assets in a busy syringe service.The belief that a slow rise in temperature is

necessary to ensure that heat is taken up evenlyis discounted by experiences in gas oven B, for inthis experiment 136 minutes elapsed before theoven reached 1600 C., and even then a wide varia-tion of temperature was recorded. The experi-ments of loading the oven while hot indicate thatthe heat is taken up more evenly and more quickly.This is probably due to the fact that the fabricof the oven has already absorbed heat and allowsa greater and more even transfer to the articles tobe sterilized. The fact that so many of the ovensshowed a satisfactory distribution of heat whenempty suggests that perhaps more rigorous testsshould be employed by the manufacturers beforeissuing ovens for distribution. It is suggested thatprobably a standard load when testing might benecessary to ensure an adequate margin of safety.

No attempt has been made in this investigationto determine the thermal death point of bacteriausually encountered. Experiments with spores withthe oven at 160° C. for one hour seemed satis-factory. However, no record of temperaturesafter loading seems to have been carried out, andit is probable, in accordance with Gerhards' paper(1952) on the thermal death point of bacteria infan ovens which stated that 1400 C. for one hourwas sufficient, that this temperature is in fact effec-tive, and it is assumed that the reason why so manyauthoritative manuals have specified a higher tem-perature is because in the past the wide variationof temperature within gas and electric ovens neces-sitated this precaution.The organization of syringe services demands

simplified cleansing and sterilizing procedures.The fact that in autoclaves syringes should besterilized unassembled to ensure adequate steampenetration makes this method clumsy, since itrequires two further steps. The syringes must bereassembled after sterilization and the entranceholes to the containers closed. Hot-air steriliza-tion is therefore preferable, since it avoids boththese extra operations. Preliminary experimentswith thermocouples with syringes during the pro-cess of boiling show that even after three minutesthe syringe is not yet at the temperature of 1000 C.and that by wrapping in lint heat penetration isprevented for five minutes or longer, and provesthat little reliance can be placed on this methodof sterilization. The work of Bigger, Blacklock,and Parrish (1940) proves conclusively that evenafter 20 minutes not all bacteria may be destroyed,and this is accentuated if any biological materialadheres to the syringe. Removing adherent bio-logical fluids such as pus or blood together withpossible spores is one of the most important pre-liminary steps in the sterilizing process. Oncethis has been done, a standard period of dry heattreatment will ensure adequate sterilization.

Spores are known to be most resistant of allorganisms to dry heat. They therefore suggestthemselves as suitable test organisms, but they toovary in their resistance to heat. Unfortunatelymost authors do not indicate the precise organismsused to test the reliability of their ovens, and itis possible that this omission accounts for someof the discrepancies in the literature. For thisreason it is essential to carry out further work onthe thermal death-point of organisms in thepresence of biological fluids with special referenceto the length of time and the temperature neces-sary to ensure sterilization. An authoritative bodyshould define the organisms to be used for testingdry heat ovens.

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SummaryAn investigation with multipoint thermocouples

into loaded ovens has been carried out. Twogas ovens showed a wide variation of tem-perature, which makes these instruments unsatis-factory for syringe services. Experiments withfour electric ovens without fans showed that inthe main heat is taken up slowly and that, eventhough close correlation of temperature is obtainedwhen the oven is empty, wide variations of tem-perature may occur. In the fan oven heat ispromptly absorbed and the variation of tempera-ture is reduced. Loading ovens while still hotdoes not interfere with the transfer of heat to thearticles to be treated, and in fact in the majorityof instances it is to be preferred, but tight packingof syringes and needles is condemned as unreliable.

Our thanks are due to the directors of Messrs.Townson & Mercer for the loan of apparatus, to Mr.W. H. R. Saunders for assistance in the technical work,and to Mrs. W. Tuke, S.R.N., for general assistance.

REFERENCESAmerican Public Health Association (1950). Diagnostic Procedures

and Reagents, 3rd ed. New York.Bigger, J. W. (1949). Handbook of Bacteriology, 6th ed. Balliere,

Tindall and Cox, London.-- Blacklock, J. W. S., and Parish, H. J. (1940). Brit. med. J., 1,

582.British Pharmaceutical Codex (1949). Pharmaceutical Press, London.British Veterinary Codex (1953). Pharmaceutical Press, London.Burrows, W. (1949). Jordan-Burrows Textbook of Bacteriology,

15th ed. Saunders, Philadelphia.Dubos, R. J. (1948). Bacterial and Mycotic Infections of Man.

Lippincott, Philadelphia.Ewald, H., and Schmid, W. (1953). Off. GesundhDienst., 15, 159.Fairbrother, R. W. (1953). A Text-Book of Bacteriology, 7th ed.

Heinemann, London.Gerhards, G. A. (1952). Arch. Hvg., Berl., 136, 547.Gradwohi, R. B. H. (1948). Clinical Laboratory Methods and

Diagnosis, 4th ed. Mosby, St. Louis.Koch, R., and Wolffhugel, G. (1881). Mitt. GesundhAmte. 1, 301.Mackie, T. J., and McCartney, J. E. (1953). Handbook of Practical

Bacteriology, 9th ed. Livingstone, Edinburgh.McCulloch, E. C. (1945). Disinfection and Sterilization, 2nd ed.

Lea and Febiger, Philadelphia.Medical Research Council (1945). War Memorandum No. 15.

H.M. S.ationery Office, London.Savage, R. Maxwell (1936). Quart. J. Pharm., 9, 366.Smith, D. T., and Martin, D. S. (1948). Zinsser's Text Book o

Bacteriology, 9th ed. Appleton Century Crofts, New York.Stitt, E. R., Clough, P. W., and Branham, S. E. (1948). Practical

Bacteriology, Hematology, and Parasitology, 10th ed. Blakiston,Philadelphia.

Underwood, W. B. (1941). Text Book of Sterilisation, 2nd ed.American Sterilizer Co., Erie, Pa.

Walter, C. W. (1948). The Aseptic Treatment of Wounds. Mac-millan, London.

Whitby, L., and Hynes, M. (1951). Mfedical Bacteriology, 5th ed.Churchill, London.

Wilson, G. S., and Miles, A. A. (1946). Topley and Wilson'sPrinciples ofBacteriology and Immunity, 3rd zd. Arnold, London.

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