APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, p. 705-709 0099-2240/78/0036-0705$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 36, No. 5

Printed in U.S.A.

Dry-Heat Destruction of Lipopolysaccharide: Design and Construction of Dry-Heat Destruction Apparatus JOHN H. ROBERTSON, DAVE GLEASON, AND KIYOSHI TSUJI* Control Laboratories and Instrumentation, The Upjohn Company, Kalamazoo, Michigan 49001 Received for publication 21 August 1978

A dry-heat oven with automatic, multiple-sample introduction and withdrawal has been constructed to achieve instantaneous heating and cooling of samples. The oven temperature fluctuation at set points of 170 to 2500C was ±0.10C, with temperature variation between the replicate samples of ±0.20C. Correction required for a sample come-up time was miniimal, i.e., less than 0.25 min of the dryheat destruction time. been constructed by modifying a Hewlett-Packard model 810 gas chromatograph (Fig. 1). The unique feature of the dry heating oven is the use of gravity to achieve rapid entry and exit of samples in aluminum cups (19- by 8-mm ID, 0.5-mm wall thickness) into and from the oven. This is accomplished by simultaneous introduction of six aluminum cups into the aluminum heating block (20 by 10 by 6 cm) through six 1.43-cm-outer diameter stainless-steel tubings. The aluminum heating block acts as a heat sink. Six aluminum cups are introduced into and exit out of the heating block by two inlet and two heating coin slot mechanisms, each constructed of a stainless-steel plate (16.5 by 3 by 2.5 cm). The inlet coin slot acts as a cover for the sample entry tubes to prevent heat loss. The coin slot plate is attached to an air piston cylinder for automatic operation. The two three-sample-capacity coin slot plates can be timed and operated independently. The aluminum cup is heated in a hole in the coin slot plate while resting on the aluminum heating block. The bottom of the cup was made flat to increase the contact surface with the heating block for rapid heating. The mode of the heating was changed from convection of the gas chromatograph to conduction for uniform, even heating. The chromatographic heating-rod element and a fan in the gas cfiromatographic oven were removed, and four 250-W contact strip heaters (model S-802, E. L. Weigand Co., Pittsburgh, Pa.) were bolted directly onto two sides of the aluminum heating block. The entire cavity of the oven was filled with calcium silicone insulation. Fiber glass insulation was also placed around the two shafts connecting the heating coin slot plates to the air piston cylinders. Thus, temperature variation between replicate samples approximates ±0.20C. The silicone control rectifier heater control circuit of the gas chromatograph was modified to handle increased power demand of the four contact strip heaters. The temperature feedback thermocouple to control the oven temperature was mounted adjacent MATERLALS AND METHODS to the heating block. To monitor the oven and the Design and construction. The laboratory-scale, heating block temperature, one thermocouple was dry heating oven and its temperature controller have mounted in the center of the heating block and the

The presence of bacterial lipopolysaccharide (LPS) above threshold levels in parenteral products may result in pyrogenic or febrile response in patients receiving the products. To assure that parenteral products are nonpyrogenic, a dry-heat process, either continuous or static, has traditionally been used as the means to depyrogenate and sterilize glassware and heat-stable compounds. Dry heat has also been the principal process for the sterilization of spacecraft (9). To determine the dry-heat resistance of bacterial spores, several apparatus have been described and used. They include: (i) the thermal death time tube-silicone bath method (1, 2); (ii) the thermal death time can-retort method (6, 12); (iii) an air oven with manual sample introduction and withdrawal technique (3-5, 7, 10); and (iv) various elaborate apparatus including a thyrister controlled infrared unit (8), a dry-heat thermoresistometer (11), and a laminar hot-air flow apparatus (14). None of these reports, however, has provided data on the critical parameter of sample "heat-up" time. Since heat transfer in dry conditions is slower than in steam, instantaneous sample heat-up may not be assumed. Therefore, a laboratory-scale, dry-heat oven has been constructed to meet the following specifications: rapid sample heat-up and cooling, uniform temperature between replicate samples during processing, precise control of heating temperature, capability to achieve and operate up to 4000C, automatic operation of multiple (at least six) samples for simultaneous introduction and withdrawal, relatively inexpensive, and trouble-free operation. This paper describes construction and operation of such a dry-heat apparatus.

705

706

ROBERTSON, GLEASON, AND TSUJI

APPL. ENVIRON. MICROBIOL.

FIG. 1. Automated dry-heat destruction oven showing coin slot mechanisms for multiple-sample introduction and withdrawal. (A) Coin slot mechanism; (B) heating block; (C) air piston; (D) stainless-steel tubings; (E) sample inlet; (F) sample outlet. other was mounted adjacent to the heating block. These two thermocouples were connected to a digital thermometer (model 3175A, J. Fluke Mfg. Co., Mountlake Terrace, Wash.) and to a single-pen strip chart recorder (model SR-205, Heath Co., Benton Harbor, Mich.). The range of the recorder was adjusted to record 200C full scale around the oven temperature. The sample heating time was controlled by an automatic timer (model 325A347A1OPX, Automatic Timing and Control Co., King of Prussia, Pa.), which is activated by opening an inlet coin slot mechanism. Either a minute or a second timer may be selected. At the end of the preset timed interval, the timer actuates an air piston which opens a heating coin slot mechanism for the sample cups to drop into receiving test tubes containing diluent. The test tubes are kept in an ice-water bath for instantaneous cooling of the samples. Figure 2 is a schematic of the timer sequences. The intricate timer sequence was required to prevent interaction of events and to accurately time and control each event. Upon activation of the oven temperature controller, temperature was monitored with the digital thermometer. Approximately 3 h were allowed for the oven to

equilibrate at the desired set temperature. After temperature equilibration a strip chart recorder was used to continuously monitor the oven temperature. The oven temperature did not deviate more than ±0.30C from the set temperature during the dry-heat destruction kinetic study. The desired number of aluminum cups containing dried LPS was placed into the inlet coin slot mechanism. The desired length of heating time was set, and the coin slot mechanism was actuated. This will actuate the air piston to allow the cups to fall onto the aluminum heating block. The event was marked on the strip chart recorder. Depyrogenated disposable test tubes containing endotoxin-free diluent water were placed in an ice bath under the stainless-steel sample discharge tubings for instantaneous cooling of the sample cup. At the end of the heating time the timer actuated the air piston to open the heating coin slot mechanism. The aluminum cups then dropped into the receiving test tubes. The event was marked on the strip chart recorder, and the length of the heating time was confirmed. The operation was repeated by changing the timer setting to vary the heating time. The relative humidity

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(RH) and temperature of the room were recorded with a certified hygrometer and temperature indicator (model HTAB-176, Abbeon Cal. Inc., Santa Barbara, Calif.).

RESULTS AND DISCUSSION Determination of aluminum cup heating time. To determine the heating characteristics of the aluminum cup at each set temperature, a thermocouple (part no. ICSS-116U-24, Omega Engineering, Stamford, Conn.) was placed in the bottom of a cup and dropped into the heating block through the inlet coin slot mechanism. The procedure was repeated at least twice at each of the six inlet positions. Since the thermocouple has its own heating lag time, the thermocouple by itself was placed into the heating block through the inlet coin slot mechanism to calculate the true cup heating time. Typical heating curves for a cup with a thermocouple (TTc) and the thermocouple alone (TT) at 170°C (TB) are shown in Fig. 3. These heating curves may be linearized by the following linear equations with a correlation coefficient of >0.9999 (Fig. 4). YTC = ATCX + bTc (1) + YT= ATX bT (2) where YTC = log [-( TTC - TB)]; YT = log [-( TT - TB)]; TTC = cup temperature with a thermocouple (degrees Celsius); TT = thermocouple temperature (degrees Celsius); TB = oven temperature (degrees Celsius); ATC = slope of the 170

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cup with the thermocouple heating curve; AT = slope of the thermocouple heating curve; bTc = Y intercept of the cup-thermocouple heating curve at x = 0; bT = Y intercept of the thermocouple heating curve at x = 0; and x = heating time (minutes). The time in minutes required for the heating curve to traverse one log cycle (fA), can be calculated by fh = 1/A. The slope of the true cup heating curve may then be expressed by the following equation:

OC = OTC - 90 - OT (3) where Oc = tan-' Ac; Ac = slope of the true cup heating curve; GTC = tan-' ATC; and OT = tan-1

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AT. For example, the slope of the true cup heating curve (Ac) at 170°C (TB) may be calculated as follows: Since YTC = (-1.675)x + 2.17 fh = 0.597 and YT = (-1.024)x + 2.18 fh = 0.976 C= tan-1 (-1.67450) - 90 - tan-' (-1.02357) = -103.490 Ac = -4.17 or fh = 0.240 bC = (bTc + bT)/2

2.17 Therefore, the true cup heating curve may =

DRY-HEAT DESTRUCTION OF LPS

VOL. 36, 1978

709

then be written as Yc = (-4.17)x + 2.17 and is tems. Control of the RH of a given sample during plotted in Fig. 4. The true heating temperature the dry heating cycle may not be practical. Howof the cup at various times, x, may be calculated ever, the effects of RH on the rate of bacterial spore resistance to dry heat have been amply by using the equation: (2, 4, 5, 7, 12). Therefore, this drydemonstrated (4) heat oven may be modified in the future to study TC = -{[log-'(ax + b)] - TB) Although it took approximately 0.5 min for the the effect of RH on the dry-heat destruction aluminum cup to reach 169°C, TB - 1 (Fig. 4), kinetics of bacterial LPS. some degree of destruction or lethality to LPS ACKNOWLEDGMENTS is expected during this time. Therefore, the dryis made to A. R. Lewis and S. J. Harrison Acknowledgment heat destruction time must be corrected to com- for technical assistance. pensate for the lethality generated during the LITERATURE CITED cup come-up time. The lethality accumulated during the come- 1. Alderton, G., and N. Snell. 1969. Chemical states of bacterial spores: dry-heat resistance. Appl. Microbiol. up time was calculated by integrating the le17:745-749. thality for the first 1 min of heating at each 2. Angelotti, R., J. H. Maryanski, T. F. Butler, J. T. heating temperature. Peeler, and J. E. Campbell. 1968. Influence of spore

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where CETB = correction time required to assume instantaneous come-up to the oven temperature (TB) when the rate of LPS destruction is z; L = lethality, log-[( Ti - TB)/z]; t = heating time (minutes); z = 46.6°C (zl, reference 13); TB = oven temperature; and Ti = true cup heat-up temperature calculated from equation 4. The C values thus calculated for oven temperatures of 170, 190, 210, 230, and 250°C were 0.22, 0.10, 0.15, 0.10, and 0.25 min, respectively. These values were subtracted from the heating time to correct for lethality accumulated during cup come-up time. Since the cup is dropped into an ice-watercooled diluent at the end of the heating period, the cup cooling has been assumed instantaneous, and no effort to correct for lethality was made. Heating system. The dry-heat oven can be classified into two categories: open and closed (6). An open system has been referred to as a system where the sample can gain or lose moisture without limit during heating. In an infinite time, therefore, the sample will be in equilibrium with the moisture level (RH) in the environment. The system also would pose no restriction to the rate of moisture transfer. A closed system, on the other hand, is a system in which moisture movement or availability is restricted. For practical purposes, an open system has been chosen for the design of the dry-heat oven. Dry-heat ovens, either static or continuous, used in the pharmaceutical industry are open sys-

moisture content on the dry-heat resistance of Bacillus subtilis var. niger. Appl. Microbiol. 16:735-745. 3. Bond, W. W., M. S. Favero, N. J. Petersen, and J. H. Marshall. 1970. Dry-heat inactivation kinetics of naturally occurring spore populations. Appl. Microbiol. 20:573-578. 4. Brannen, J. P., and D. M. Garst. 1972. Dry heat inactivation of Bacillus subtilis var. niger spores as a function of relative humidity. Appl. Microbiol. 23: 1125-1130. 5. Drummond, D. W., and I. J. Pflug. 1970. Dry-heat destruction of Bacillus subtilis spores on surfaces: effect of humidity in an open system. Appl. Microbiol. 20:805-809. 6. Fox, K., and B. D. Eder. 1969. Comparison of survivor curves of Bacillus subtilis spores subjected to wet and dry heat. J. Food Sci. 34:518-521. 7. Fox, K., and I. J. Pflug. 1968. Effect of temperature and gas velocity on the dry-heat destruction rate of bacterial spores. Appl. Microbiol. 16:343-348. 8. Molin, G., and K. Ostlund. 1975. Dry-heat inactivation of Bacillus subtilis spores by means of infra-red heating. Antonie van Leeuwenhoek J. Microbiol. Serol. 41:329-335. 9. National Aeronautics and Space Administration. 1969. Planetary quarantine provisions for unmanned planetary missions. National Aeronautics and Space Administration Document no. NHB 8020.12. Government Printing Office, Washington, D.C. 10. Oag, R. K. 1940. The resistance of bacterial spores to dry heat. J. Pathol. Bacteriol. 51:137-141. 11. Pflug, I. J. 1960. Thermal resistance of microorganisms to dry heat: design of apparatus, operational problems and preliminary results. Food Technol. 14:483-487. 12. Pheil, C. G., I. J. Pflug, R. C. Nicholas, and J. A. L. Augustin. 1967. Effect of various gas atmospheres on destruction of microorganisms in dry heat. Appl. Microbiol. 15:120-124. 13. Tsuji, K., and S. J. Harrison. 1978. Dry-heat destruction of lipopolysaccharide: dry-heat destruction kinetics. Appl. Environ. Microbiol. 36:710-714. 14. Wegel, S. 1974. Short time sterilization of glass materials under ultraclean conditions. Bull. Parenter. Drug Assoc. 28:122-135.

Dry-heat destruction of lipopolysaccharide: design and construction of dry-heat destruction apparatus.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, p. 705-709 0099-2240/78/0036-0705$02.00/0 Copyright i 1978 American Society for Microbiology Vol...
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