APPLIED MICROBIOLOGY, July 1968, p. 991-997Copyright © 1968 American Society for Microbiology

Vol. 16, No. 7Printed in U.S.A.

Instrument for Study of MicrobialThermal Inactivation

R. W. DICKERSON, JR., AND R. B. READ, JR.

Food Protection Research, National Center for Urban and Industrial Health, Public Health Service, Cincinnati,Ohio 45202

Received for publication 27 March 1968

An instrument was designed for the study of thermal inactivation of microorga-nisms using heating times of less than 1 sec. The instrument operates on the prin-ciple of rapid automatic displacement of the microorganism to and from a saturatedsteam atmosphere, and the operating temperature range is 50 to 90 C. At a tempera-ture of 70 C, thermometric lag (time required to respond to 63.2% of a step change)of the fluid sample containing microorganisms was 0.12 sec. Heating time requiredto heat the sample to within 0.1 C of the exposure temperature was less than 1 sec,permitting exposure periods as brief as 1 sec, provided the proper corrections aremade for the lethal effect of heating. The instrument is most useful for heat exposureperiods of less than 5 min, and, typically, more than 500 samples can be processedfor microbial inactivation determinations within an 8-hr period.

Few data are available on thermal inactivationof vegetative bacterial cells for inactivation timesfrom 1 to 25 sec-a range of times of primaryinterest for pasteurization of milk and milk prod-ucts and for the heat-processing of other foods.This lack seems to be due primarily to the diffi-culty of developing techniques and instrumenta-tion that will yield valid data for brief heat expo-sure times. Stumbo (6) developed a device for thestudy of thermal inactivation of bacterial sporeswith heating times as brief as 1 sec and exposuretemperatures above 100 C. This device and modi-fications of it (3) have been used extensively toobtain thermal inactivation data on bacterialspores. For temperatures available with steamunder pressure (> 100 C), inactivation occurs inconsiderably less than 1 sec for most bacterialvegetative cells. Accordingly, to be useful forvegetative cells, the steam chamber must operateunder a vacuum to obtain a saturated steamatmosphere at operating temperatures of less than100 C. The change from pressure to vacuumoperation required extensive modification of theinstrument; this paper describes these modifica-tions and gives operating characteristics of themodified instrument.

DESIGN

Basically, the instrument consists of a steamchamber with provision for six pistons as shown inFig. 1. A 7.6-cm deep reservoir of distilled waterin the bottom of the chamber was heated by im-

mersed electrical resistance heaters, and tempera-ture control was accomplished by regulatingpower to these heaters. A saturated steam atmos-phere of below 100 C was obtained in the chamberby applying a continuous vacuum by use of acondensate pump. The rate of steam removalfrom the chamber by the condensate pump wascontrolled by a manually operated needle valve inthe vacuum line. A float admitted make-up waterto the steam chamber as required, and the waterwas heated to chamber temperature in the en-trance pipe as shown in Fig. 1. Each of the sixpistons (Fig. 2) transported a sample cup (con-taining the microbial suspension) to and from thesteam atmosphere. Movement of the six pistonswas simultaneous and was effected by a pneu-matic cylinder. Piston diameter was 2.51 cm anddiameter of the piston cavity was 1.4 cm. Specialconsideration was required in designing the sealsfor vacuum operation, because leakage of roomair into the instrument might affect sample tem-perature. End caps and shoulders on the pistonwere used as mechanical stops, and the rubberrings sealed the chamber at each end of pistonstroke. Leakage during piston movement wasminimized by the rubber rings mounted on eachside of the chamber walls.One of the most significant changes from the

designs of Stumbo (6) and Pflug and Esselen (3)was the use of a sleeve to surround the piston andforce all the steam generated in the chamber toflow through the piston and sleeve assembly. Th;e

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DICKERSON AND READ

FIG. 1. Instrument for thermal inactivation research on microorganisms.

was done to remove condensate from the samplecup and improve heat transfer. Steam flow wasvertically downward over the cup, which gavehigher heat transfer rates than upward flow be-cause gravity accentuates downward flow of con-densate (2). Direct impingement of steam on thefluid sample was prevented by placing the samplecups in the pistons with open ends down. Surfacetension caused the sample fluid to adhere to thebottom of the metal cup. Upon entrance to thechamber, the metal cup was heated rapidly incondensing steam; however, the rate of heatingthe microorganisms was governed by the size andgeometry of the fluid sample. Pflug and Esselen(4) measured heating rates in open metal cups andconcluded that heating times were insignificantfor 0.01-ml samples spread over the bottom of thecup.Sample cups were 1.09 cm in diameter and 0.8

cm deep, and were drawn from 0.5-lb tinplate(0.02 cm thick). A thin cylinder of Whatman no.42 filter paper (0.76 cm in diameter) was used tospread a 0.01-ml bacterial test suspension over thebottom of the cup to a depth of about 0.018 cm.

Calculated performance parameters are shownin Table 1 for an operating temperature of 65 C.Steam flow, water flow, instrument heat loss, and

power for steam generation, were obtained bymass and energy balance calculations. Steam tem-perature perturbation during piston operationwas caused by the explosion of room air from thepiston cavities into the steam chamber, and wascalculated by use of the analysis of Gillespie andCoe (1) for matter crossing the boundary of athermodynamic system. The volume of the steamchamber was made large enough so that the tem-perature perturbation resulting from piston opera-tion was less than 0.03 C. The calculation of ther-mometric lag was based on the assumptions thattemperature of the environment in the pistoncavity and the temperature of the metal samplecup were brought to exposure temperature in-stantaneously. Schneider's graph (5) was used tocalculate thermometric lag of the 0.01-ml sample.The value of 0.1 sec is the theoretical minimum fora sample 0.018 cm thick.

INSTRUMENT OPERATIONThe assembled instrument is shown in Fig. 3,

and hardware for inoculating, loading, and re-covering sample cups is shown in Fig. 4. Cupswith precut filter-paper discs (for spreading thefluid sample over the bottom of the cup) were dry-sterilized prior to use, and a 0.01-ml droplet of the

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INSTRUMENT FOR STUDYING THERMAL INACTIVATION

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FIG. 2. Piston assembled in steam chamber.

TABLE 1. Calculated performance parameters

Operating temperature .......... 65 CChamber pressure.............. 19.3 cm of Hg

(absolute)Steam flow through instrument 140 liters/minWater flow through instrument.. 1.5 liters/hrHeat loss from instrument ...... 500 wPower for steam generation ... .1,000 wMaximal steam temperature per-

turbation resulting from pis-ton operation ................ 0.03 C

Thermometric laga of a 0.01-mlsample (0.76 cm in diameterand 0.018 cm thick) ........... 0.1 sec

a Time required to respond to 63.2% of a stepchange in temperature.

bacterial test suspension was placed in each with amicrometer syringe. The cups were then placedupside down in the spacing bar shown in Fig. 4.Six cups were picked up simultaneously by use ofa magnetic bar and were loaded into the pistonsof the instrument. After the sample-containingcups were in the pistons, an automatically pro-grammed sequence of operations was activated bypushing a button. The pneumatic cylinder drovethe six pistons rapidly into the steam chamber,and the samples were held in the chamber in thepath of the flowing steam. After the preset timeon the program control timer had elapsed, thepistons were expelled from the steam chamberas the pneumatic cylinder returned to its originalposition. Upon emergence from the chamber, asolenoid on each piston pulled the support rodfrom under the cup, which dropped the sampleinto a medium that supported growth of thesurvivors of the heat process. The cup dropped 15cm (0.18-sec free fall) before immersion in re-covery liquid. Tubes containing the recoverymedium were held under each piston by means ofthe tube holder shown in Fig. 4.During preliminary runs, we found that leakage

of room air into the instrument decreased tem-

perature in the piston cavity as much as 8 C. Con-sequently, detection and prevention of leakagewere imperative. Thermocouples were installed inthe steam flow, above and below the samplecup on each piston, and the 24-point recordershown in Fig. 3 was used for continuous monitor-ing of these temperatures.

MEASURED PERFORMANCE

Stumbo (6) has calculated temperature at thecenter of a sample similar to those used in thisstudy, and the calculations indicate that it washeated to the exposure temperature in less than 1sec. Using a 5% bentonite solution to suppressconvection currents, Pflug and Esselen (4) meas-ured heating rates in metal cups filled to depths of1.2 to 0.1 cm. Extrapolation of these data to ob-tain heating rates for a sample of a depth of 0.018cm yielded an estimated thermometric lag of 0.08sec and, correspondingly, a heating time of lessthan 1 sec. To evaluate the performance of theinstrument used in the present study, we measuredtemperatures in the piston cavity and in the 0.01-ml sample.

Temperature in piston cavity. A copper-con-stantan thermocouple was fabricated from 0.0079-cm diameter wire (No. 40 American Wire Gauge)and mounted in the piston cavity as shown in Fig.5. The lead wires were enclosed in Teflon tubingand brought through a small longitudinal hole inthe piston, terminating in a standard thermo-couple plug connection. An adjacent piston with ametal cup in the piston cavity is also shown inFig. 5. A light-beam oscillograph and d-c ampli-fier were used to record the signal from the ther-mocouple. The slowest component in the record-ing system was the oscillograph galvanometerwith a natural frequency of 200 cycles/sec. Ther-mometric lag of the bare wire thermocouple wasmeasured by exposing the thermocouple to a stepchange of 40 C. Thermometric lag was less than0.005 sec.A metal cup was placed in the piston cavity,

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DICKERSON AND READ

FIG. 3. Assembled instrument with temperature recorder anid automatic controls.

FIG. 5. Thermocouple mounted in pistoni cavity.

FIG. 4. Hardware for inoculating, loading, and re-covering sample cups.

under the bare wire thermocouple, and tempera-ture of the piston cavity was recorded as theinstrument was operated through an automaticprogrammed sequence of events (Fig. 6). Heatingof the piston cavity began when the piston reachedthe fully inserted position, and temperature in thepiston cavity responded to 63.2% of the stepchange in less than 0.05 sec. When the pistonsreached the fully inserted position, a roller-leafswitch opened a solenoid-operated steam valve,

bypassing the needle valve, and produced a largeflow of steam through the piston cavity, whichimproved heating rates. An autommatic timer heldthe steam bypass valve open for about 0.8 sec,and this time was recorded by an oscillographgalvanometer. Except for a slight temperaturedepression (< 0.5 C) caused by the steam bypassvalve, temperature was constant during the expo-sure. The effect of room air exploding into thesteam chamber was evidenced by the temperaturereduction as the piston cavity entered the steamchamber. The implosion effect that must occur asthe piston cavity emerges from the steam chamberwas not evidenced as a temperature increase (Fig.6). Instead, the implosion of room air in the

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VOL. 16, 1968 INSTRUMENT FOR STUDYING THERMAL INACTIVATION

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FIG. 6. Temperature in piston cavity with an exposure temperature of65 C.

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FIG. 7. Special cup-thermocouple assembly showing IFIG. 8. Thermocouple made from 0.0079-cm diam-

lead wires, protective tubing, and metal cups. eter wire installed in metal cup. Normal cup shown forcomparison.

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FIG. 9. Temperature at midpoint ofo.01-ml sample with an exposure temperature of65 C.

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996 DICKERSON AND READ

cavity cooled the thermocouple rapidly (5 C dropin 0.05 sec).Temperatures of flowing steam above and be-

low the piston cavity were also measured with theoscillograph and were constant at the exposuretemperature. Stroking time of the pistons wasmeasured with roller-leaf switches at each end ofstroke. Position of the roller-leaf switches wasrecorded with the oscillograph, and stroking timeof the pistons was 0.09 sec when the pneumatic

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FIG. 10. Temperature at the center of 0.01-, 0.02-,0.03-, and 0.04-ml samples with an exposure temperatureof5O C.

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APPL. MICROBIOL.

cylinder was operated with an air pressure of 1.4kg/cm2 (gage).A precision timer was operated by a roller-leaf

switch mounted at midstroke and automaticallyindicated exposure time. Voltage to the precisiontimer was recorded with an oscillograph galva-nometer simultaneously with temperature andpiston movement. True exposure time (time at65 C) was 0.25 sec shorter than the time indicatedby the precision timer.

Temperature in 0.01-ml sample. A copper-con-stantan thermocouple was fabricated from 0.0079-cm diameter wire (No. 40 American Wire Gauge)and mounted in a sample cup. Each wire wasenclosed in Teflon tubing and brought through thewall, near the bottom of the cup, as shown in Fig.7. The Teflon tubing was terminated just insidethe cup, and the bare wires were stretched dia-metrically across the bottom ofthe cup and joinedat the center for the thermocouple junction.Figure 8 is a view looking into the cup. A piece offilter paper was placed under the thermocouple forcontrast, and the arrow on the filter paper locatesthe thermocouple junction. A normal cup withfilter paper is shown in Fig. 8 for comparison.The thermocouple junction was sandwiched be-

tween halves of filter paper, and the cup-thermo-couple assembly was mounted in the piston cavity.An oscillograph recording system was used torecord temperature at the center of the 0.01-mlsample.When 0.01 ml of distilled water was pipetted

into the filter paper-thermocouple sandwich, itwas apparent that the filter paper disc was notcompletely filled with fluid. It appeared that thethermocouple wires and 0.018-cm thick filterpaper (Whatman no. 42) made a sandwich toothick to absorb 0.01 ml; therefore, a filter paper-thermocouple sandwich was made of 0.01-cm

50 60 70 80

EXPOSURE TEMPERATURE, °C90

FIG. 11. Thermometric lag of temperature in the piston cavity and at the midpoint ofa 0.01-mi sample at five

exposure temperatures.

FLUID SAMPLE

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VOL. 16, 1968 INSTRUMENT FOR STUDYING THERMAL INACTIVATION

thick (Whatman no. 50) filter paper. This gavean assembly 0.018-cm thick (the same as a no. 42filter paper without thermocouple), and a visualinspection indicated that the 0.01-ml sample filledthe filter paper-thermocouple sandwich. Ac-cordingly, thinner filter papers were used for alltests with the thermocouple.A 0.01-ml sample of distilled water was pipetted

into the cup-thermocouple assembly, and an auto-matically programmed run was made with anexposure time of 6 sec. Thermometric lag of tem-perature at the center of the 0.01-ml sample was0.15 sec (Fig. 9).The explosion effect (temperature decrease)

occurring as the piston entered the steam chamberwas in evidence; however, no rarefaction couldhave occurred if the thermocouple had been com-pletely immersed in water. Consequently, it isexpected that some air existed at the thermocouplejunction even after the 0.01-ml sample had beenpipetted into the filter paper-thermocouple sand-wich. When the pistons emerged from the steamchamber, an implosion effect (temperature in-crease) was in evidence, further suggesting thepresence of air bubbles at the thermocouple. Sincethe liquid sample is incompressible, the tempera-ture changes due to explosion and implosion canoccur only on the air bubbles trapped at thethermocouple and on the air at the exposed sur-face of the liquid sample; the microorganisms inthe main body of the sample would be unaffected.However, because of the importance of tempera-ture in microbial thermal inactivations, it wasnecessary to determine whether the temperaturechanges were, in fact, the result of pressurechanges imposed on the sample as it was trans-ported to and from the vacuum steam chamber.A test was performed wherein the implosion

effect was not operative during the return strokeof the pistons. This was accomplished by startinga normal test with a thermocouple in a 0.01-mlsample, and, after the sample reached exposuretemperature, the automatic sequence was inter-rupted and chamber pressure was raised to roompressure by slowly admitting room air to thechamber. With equal pressures inside and outsidethe chamber, the automatic sequence of opera-tions was resumed and temperature in the 0.01-mlsample remained constant at the exposure tem-perature during the return stroke of the piston. Inthe absence of a pressure change, the temperatureincrease did not occur, and it is therefore con-cluded that the temperature changes recordedduring piston movement were the result of rare-faction and compression of air bubbles at thethermocouple.By running tests with samples of 0.01, 0.02,

0.03, and 0.04 ml, it was found that the thermo-couple was never completely free from air (Fig.10). The instrument was operated at an exposuretemperature of 50 C because the implosion effectis maximal at the lowest operating temperatureand correspondingly highest vacuum. The effect isless than 0.5 C for fluid samples of 0.02 ml butnever vanished-not even for the 0.04-ml sample.The effect of sample size on thermometric lag isalso shown in Fig. 10. Thermometric lag wasprogressively larger for each larger sample, withthe 0.04-ml sample having a thermometric lag of0.95 sec.To show the effect of operating temperature on

thermometric lag, heating rates of the 0.01-mlsample were measured at five different exposuretemperatures and are shown in Fig. 11. Above70 C, thermometric lag of temperature in thepiston cavity was constant at 0.02 sec. Thermo-metric lag of temperature at midpoint of the fluidsample decreased with increasing exposure tem-perature up to 70 C and then slowly approachedthe theoretical minimum of 0.1 sec for tempera-tures up to 90 C. These values compare favorablywith Pflug and Esselen's (4) prediction of 0.08sec.By use of the instrument described herein, a

fluid sample can be heated to the exposure tem-perature in less than 1 sec. From values of ther-mometric lag (Fig. 11), heating rates can be deter-mined for any set of conditions. With an exposuretemperature of 70 C and an ambient temperatureof 25 C, midpoint temperature of a 0.01-ml samplewill reach 69.5 C in 0.54 sec. For exposure periodsas brief as 10 sec, corrections for heating and cool-ing may be neglected.

ACKNOWLEDGMENTS

The instrument was fabricated by J. A. Castelli andF. Reis, and the automatic control system was de-signed by A. B. Arnold. We are also grateful for thetechnical assistance of R. W. Parker and C. H. Bern-hardt.

LrrIERATURE CITED1. Keenan, J. H. 1941. Thermodynamics. John

Wiley & Sons Inc., New York.2. McAdams, W. H. 1954. Heat transmission, 3rd ed.

McGraw-Hill Book Co., Inc., New York.3. Pflug, I. J., and W. B. Esselen. 1953. Development

of an apparatus for study of thermal resistanceof bacterial spores and Thiamine at temperaturesabove 250 F. Food Technol. 7:237-241.

4. Pflug, I. J., and W. B. Esselen. 1955. Heat transferin open metal thermo-resistometer cups. FoodRes. 20:237-246.

5. Schneider, P. J. 1955. Conduction heat transfer.Addison-Wesley, Reading, Mass.

6. Stumbo, C. R. 1948. A technique for studyingresistance of bacterial spores to temperatures inthe higher range. Food Technol. 2:228-240.

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