PLACE N RETURN 30X to move this chockommm your mood. TO AVOID FINES Mum on o: baton duo duo. DATE DUE DATE DUE DATE DUE MSU loAn Afflrmdlvo AalaVEqml Oppommly Inflation DIS-M EFFECT OF POUCH FILM PERMEABILITY ON THE GROWTH OF PSEUDOMONAS AERUGINOSA By Mark Gammage A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE School of Packaging 1984 ABSTRACT EFFECT OF POUCH FILM PERMEABILTTY ON THE GROWTH OF PSEUDOMONAS AERUCINOSA By Mark Cammage ‘Hany food products experience end of shelf-life, due to problems associated with aerobic bacterial contamination. Meat products are a prime example. Growth of aerobic contaminants has been controlled in the past by using high barrier films with some success. However, the mechanism of inhibition is not well understood. This research concentrates on the dynamic relationship of headspace gases and bacterial growth and metabolism, with respect to film permeability. Experimentation was confined to a single aerobic organism in a sealed pouch, which contains a liquid growth medium. It was found that inhibition of growth increased with an increasing barrier film. Headspace 02 concentration decreased in the pouches, where CO2 concentration increased, with a concurrent decrease in the bacterial population. It appeared that the organisms altered their metabolism when stressed by 02 depletion and C02 increases. Once these dynamic relationships are further quantified, computer shelf-life predictions will be possible. To my wife, Debbie, who made it all possible, my friends for their support and interest, and to Whiskey, who waited so patiently. ACKNOWLEDGEMENTS I would like to thank Theron W. Downes, Ph.d., my major professor, for his guidance and for making this thesis a truly educational experience. Also, James Pestka, Ph.d., for his generous donation of laboratory space and time, without which none of this could have been possible, and Bruce Harte, Ph.d., for being a committee member and donating his time. Special thanks to Donna Owens for wrestling with the word processor. List of Tables.. List of Figures. Key to Symbols.. Introduction.... Literature Review... Materials and Methods Results......... Conclusions..... Recommendations. Appendix........ Bibliography.... TABLE OF CONTENTS ..20 ..36 37 54 Table Table Table Table Table Table Table Table Table Table Table Table 8. Q9 10, 11, 12, LIST OF TABLES Film Permeabilities........................ Carle 8700 Gas Chromatograph Conditions.... Sealing Conditions......................... Super-Vac Sealing Conditions............... Growth Inhibition.......................... 02 Consumption............................. C02 Production............................. 02 Availability and Consumption............ Respiratory Ouotients (Stationary Phase)... Total 02 Consumed......................... Total C02 Produced........................ Respiratory Ouotients (Exponential Phase). 00.0.09 .....10 .....ll .....11 .....24 .....26 .....27 .....29 .....31 .....32 .....32 00.0.33 Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Nitrogen Iniection Apparatus...................12 MoCon Oxtran 100...............................lS MoCon Permatran-C..............................l9 Polyethylene (2 mil) Growth Curve..............21 Saran Coated Nylon (0.75) Growth Curve.........22 Saran (2 mil) Growth Curve.....................23 POUCh Gas DynamiCSOOOOOI.....OOOOOOOOOOOOOOOOOO31 vi LIST OF SYMBOLS CAL - Calibration PE - Polyethylene (2 mil) 8 - Saran (2 mil) SN - Saran Coated Nylon (0.75 mil) Std. Dev. - Standard Deviation Average XI I vii INTRODUCTION It is well known that aerobic psychotrophic bacteria are common contaminants of prepackaged meat products (Ayres, 1959). Even under the most sanitary conditions these microorganisms cause a premature end of shelf-life due to organoleptic changes and potential health risks. It was soon realized that packaging could be instrumental in controlling the growth of these obligately aerobic microbes, by controlling the atmosphere inside the package. The easiest factor to control in this sense is the packaging material itself, since it's permeability to oxygen will mediate the oxygen available for bacterial metabolism. In the early days of research it was thought that oxygen permeability was the prime factor determining the amount and rapidity of growth of contaminants. However, later research showed that other factors played a larger role in the growth of aerobes than first anticipated. Notably, the concentration of carbon dioxide in the headspace of the package, generated through bacterial metabolism or tissue respiration, has shown significant inhibition of Pseudomonas species. The permeable pouch is a very interdependent dynamic system, in which the organism itself plays an integral role in the change of its environment. Initially, 02 permeates in, because the 02 concentration inside the pouch is lower than outside, due to vacuum packaging. A short time later, the organisms inside the package start the exponential phase 1 2 of growth, which increases it's respiratory rate, thus using more 02. As the 02 is metabolized, CO2 is being produced in the package and as the CO2 level builds up and the 02 level decreases, this places a strain on the organisms and inhibits their growth. The gas permeability of the film affects the amount of 02 permeating in and the C02 permeating out, due to an increase in C02 concentration in the pouch as compared to outside the pouch. Theoretically, a high barrier film would inhibit the bacteria more by letting in less 02 and trapping 002, which is inhibitory. A low barrier film would not have as great of an effect. This study will examine the effects of oxygen and carbon dioxide concentration on the growth of a single microorganism, Pseudomonas aeruginosa, a common aerobic contaminant of prepackaged meat products. Three different materials will be used, representing high and low permeabilities to oxygen and carbon dioxide. A commercial bacteriological substrate, commonly used to grow Pseudomonads, was used to negate any effect that tissue respiration might have on headspace gas concentration. Obviously, it is no easy task to untangle all the factors affecting microbial growth in permeable packages. However, this study will examine the effects of gas concentrations on the growth of a single organism under specific conditions. LITERATURE REVIEW Effects of O2 and CO2 Concentration On The Growth of Aerobic Organisms To understand the effects of a film's permeability to gases on the microbial growth within a package, one must first have a basic understanding of the effect of the gas itself on microorganisms. This discussion will be confined to the genus Pseudomonas and other aerobic gram negative rods, which may act similarly to Pseudomonas. The gases under scrutiny are carbon dioxide and oxygen. Coyne (1933) observed that bacterial growth on fresh fish was inhibited by a 002 enriched atmosphere. Optimal inhibition of bacterial growth seemed to occur at a C02 concentration of 40-60%. Visual observations of the fish carcasses were made to evaluate the amount of microbial growth. Haines (1933) conducted a more in depth study on the effect of carbon dioxide on Pseudomonas and other organisms. Several conclusions were drawn as to cause and effect. He found that at a COZ concentration of 10-202 the lag period of Pseudomonas is increased and the generation time was cut to almost one-half at 20°C. Another interesting observation was that generation time was almost doubled by 10% 002 when the temperature was lowered to 0°C, indicating that C02 inhibition is more effective at lower temperatures. ‘It was noted that the maximum number of organisms reached was the same, independent of CO2 3. 4 concentration, it would just take longer with an increasing CO2 concentration or lower temperature. It was previously thought (Valley and Rettger, 1927) that pH change to a more acidic condition was responsible for inhibition of bacterial growth, but Haines (1933) suggested that it was due to the action of the gas on dehydrogenases in the cells. Since the research of Coyne (1933) and Haines (1933) several studies have been conducted, which basically support their contentions on COZ inhibition of microbial growth. Dual studies by Gill and Tan (1979) showed that all concentrations (0-450 mmHg) were inhibitory to Pseudomonas at 30° in a complex medium and maximum inhibition occurred at 250 mmHg. They also found, as did Haines (1933), that with a constant CO2 concentration inhibition increased with decreasing temperature. King and Nagel (1975) came to similar conclusions in their research. It has been suggested that a linear relationship exists between CO2 concentration and generation time under controlled conditions (King and Nagel, 1967). However, it was found that the inhibitory effect of 002 is not permanent and cultures once exposed will return to normal growth upon return to atmospheric concentrations of CO2 (Enfors, 1979). The question now becomes, where does the C02 necessary for inhibition come from in a package? In meat packaging the initial increase in C02 concentration is a direct result of tissue respiration. This occurs in the first 3-5 hours after packaging. C02 concentration increases after that 5 point are primarily due to microbial growth (Gardner et a1, 1976, Seideman et a1, 1976 a, b). Seideman et a1, (1976 a, b) also suggested that vacuum packages fit tighter and thus increase the partial pressure of carbon dioxide in the package and that any residual oxygen in the package would be available for conversion to carbon dioxide. Clearly, from the evidence presented, 002 plays a very important role in the inhibition of bacterial growth. "However, the previous discussion has not addressed the effect of 02 concentration on the growth of bacteria. Boneless beef roasts were packaged in various atmospheric concentrations of oxygen and it was found that Pseudomonas species were present in high concentrations of oxygen, but not in the low concentrations (Christopher et al 1979). The results of this experiment coincide with what one would expect with respect to 02 concentration and aerobic bacterial growth. Other studies temper this statement to a degree and show that oxygen dependency has its limits. King and Nagel (1967) found that by depleting oxygen in the atmosphere a limitation in the growth of Pseudomonas aeruginosa was not observed until more than 752 (volume/volume) of air was replaced with nitrogen. Clark and Burki (1972) endeavored to find the concentration levels of oxygen necessary to inhibit Pseudomonas. They found that the oxygen concentration could be lowered to 22 without significantly inhibiting growth. At 0.5-ZZ oxygen, the effect was to increase the lag time of Pseudomonas and at 6 concentration lower than 0.51 cell generation time was increased and cell yield at stationary phase was decreased. This research was done while maintaining a constant atmosphere, with respect to oxygen and carbon dioxide concentrations. In reviewing the literature it becomes obvious that carbon dioxide concentration is just as important, if not more so, than oxygen concentration. Possibly the aerobic organisms can utilize oxygen that is dissolved in the substrate and do not rely on headspace oxygen until the dissolved oxygen is used up. Carbon dioxide, on the other hand, is not used in metabolism as oxygen is. It's effect is purely inhibitory. Therefore, it may be as important to look at carbon dioxide levels in dynamic systems, such as gas permeable packages. Effect of Film Permeability With a better understanding of how oxygen and carbon dioxide affect aerobic bacteria, it now becomes possible to discuss how that bacteria might act in a dynamic system, such as a gas permeable package. Given there are no holes or bad seals in a package, its permeability depends on the permeability of the film itself (Eustace 1981). Ingram (1962) did an extensive study on the effect of film permeability on bacterial growth and came to some interesting conclusions. He found that Pseudomonas species were predominant on beef carcasses that were loosely wrapped with highly permeable film, while carcasses wrapped in a better barrier selected for more anaerobic organisms. In his studies he noted that aerobic organisms could grow in atmospheres that only had 12 oxygen, again suggesting that carbon dioxide is limiting growth. Films that allow carbon dioxide to escape would maintain aerobic conditions where more.impermeable films would trap the carbon dioxide in the package. Thereby, he concluded that the concentration of gases in the package depended on gas diffusion through the film and metabolic activity.inside the package. Also, as oxygen is depleted in the package more will permeate through the film and prolong the aerobic phase. Ingram observed, as others had, that a vacuum packaged product has an increased partial pressure of carbon dioxide due to the decreased headspace, but this tight fit can be eliminated as more carbon dioxide is produced, either by tissue respiration or bacteria growth. Hess (1980) found that obligately aerobic spoilage flora depended chiefly upon the oxygen permeability of the film and the effectiveness of the vacuum applied. He concluded that oxygen permeation into the bags caused the growth of aerobic bacteria and he found a reduction in the growth of Pseudomonas with films of oxygen permeability less 9 than 10 cc mil/m“ day atm. He also found that aerobic organisms were considerably inhibited by vacuums of 0.001 - .02 atm and the growth of Pseudomonads was almost entirely suppressed with a high vacuum. 8 Another study conducted by Lund (1968) with sulphite treated peeled potatoes found that, when wrapped in saran and stored at 23°C for 3 days and at 6°C for 7 days, there was a decrease in oxygen concentration and an increase in carbon dioxide concentration. She also observed that they were lower bacterial counts of aerobes in the saran bags than in perforated films, which would allow gases to transfer easily in and out of the bag. An observation made by Hannan (1962) also serves to shed more light on the dynamic relationship of permeability and biological activity. Hannan's research led him to the conclusion that the temperature coefficient of permeability is considerably less than that of biological activity, particularly respiration. In other words, permeability is not affected as greatly per degree of temperature change as bacterial respiration. Other articles have been written as summaries of the type of research previously discussed (Dallyn et a1 1973, Apple et a1 1983, Finne 1982). Similar conclusions were drawn in these papers, providing supporting evidence to the previous research. Finally, it is of interest to note that Pseudomonas aeruginosa produces one mole of CO2 for every mole of O2 utilized in metabolism (Nester et a1, 1973). MATERIALS AND METHODS Materials Three materials were chosen for this study, one with a high gas permeability constant, and the other two with a low permeability to oxygen and carbon dioxide. The materials selected were saran (2 mil), saran coated nylon (0.75 mil), and polyethylene (2 mil). These films represent samples of low and high gas permeabilities (Table 1) and all are heat sealable. Table 1 Film Permeabilities Permeability (cc/m2/24 hrs.) Film ' Oxygen Carbon Dioxide Saran (2 mil) 13.0 54.8 Saran coated nylon (0.75 mil) 17.0 67.8 Polyethylene (2 mil) A,967 21,333 A silicone sealant was attached to the bags and functioned as a septum to aseptically remove or introduce materials into the bags without altering the headspace of the bag or contaminating the interior of the package. Brain-heart infusion broth (BBL) was used as a medium inside the bags to grow the microorganisms and was chosen because of its known capability to grow cultures of Pseudomonas aeruginosa (Difco Labs). This broth was also used as a medium for starter cultures and bag innoculations. Plate-count agar (BBL) was selected as a medium for growth in enumerating microbes, utilizing a pour-plate technique 9 10 (Housler 1972). A 0.5 molar solution of phosphate buffer was used in the 99 m1 dilution blanks used in the colony count procedure. (Formula in Appendix E). Pseudomonas aeruginosa ATCC #27853 was selected as the organism for the study, because of its aerobic requirements for adequate growth (Buchanan). A Super-Vac machine (Smith Equip. Co., Clifton, N.J.) provided the ability to draw a vacuum on the bags and make the final seal of the package after the broth was added._ For headspace measurements of oxygen and carbon dioxide 3 Carle 8700 gas chromatograph was utilized for quantification (see Table 2 for conditions) and BBL Gas-Pak jars were used to keep an anaerobic atmosphere for the control culture. Table 2 Carle 8700 gas chromatograph conditions Oven temp. - 60°C Attenuation — 16 Chart Speed - l inch/minute mV response - lOmV Columns - Pora-Pak and Molecular Sieve Methods All film samples were formed into 8"x6" pouches using an impulse sealer to heat seal three sides of the package. All pouch fabrication was performed in a laminar flowhood to avoid contamination of the interior of the bags. For sealing conditions see Table 3. 11 Table 3 Sealing Conditions Film Heat Gas Vacuum Satan (2 mil) ' 6 O 3 0 Saran coated nylon (.75 mil) 4.0 0 1.5 Polyethylene (2 mil) 6 0 1 5 Following fabrication, brain-heart infusion broth was aseptically added to the pouches and they were vacuum packaged using the Super-Vac machine. See Table A for settings. Table 4 Super-Vac Sealing Conditions Film Heating Cooling Pressure (sec.) (sec.) (p.s.i.) Saran (2 mil) 0.35 1.5 30 Saran coated nylon (.75 mil) 0.35 1.0 30 Polyethylene (2 mil) 0.25 1.0 30 This reduced the amount of oxygen in the headspace, requiring the microorganisms to rely partially on oxygen permeating through the film for growth. After this was completed 20 cc of nitrogen was injected into the bags through the silicone septum for headspace, as shown in Figure 1. Once the nitrogen was added to the headspace the bags were incubated at 32°C for 24 hours and visually inspected for any contamination that may have occurred during pouch fabrication. Contaminated bags were rejected and only sterile ones used. 12 Plastic tubing Glass wool in ampule N drawn off and injected into pouch Apparatus was sterilized before use. Figure 1 Nitrogen Iniection Apparatus A starter culture of Pseudomonas aeruginosa ATCC #27853 was prepared by inoculating 20 ml of brain-heart infusion broth with a mature culture 16 hours before use to ensure that the starter culture would be in exponential phase when needed. Before the culture was used it was serially diluted using a phosphate buffer of pH 6.5 to obtain an inoculum 4 cells/ml. At this point the that was approximately 103-10 inoculum could be injected into each pouch for the start of the test. Plate counts were also done to determine the exact initial count of organisms to be inoculated. Each material was tested in triplicate, making a total of nine pouches tested for all the materials. An inoculum of 1 cc of starter culture was injected into each of the 13 test bags. Once injected with inoculum the bags were immediately tested for headspace gasses on the Carle 8700 gas chromatograph. The pouches were then incubated at 32°C in a horizontal position in order to maximize the headspace/media interface. Colony counts and headspace measurements were taken at 0, 6 and 12 hours and thereafter as indicated by the growth rate of the organism which was a function of the permeability of the material being used. Colony counts were performed by aseptically removing a 1 cc sample from the pouch using a syringe and then serially diluting the sample in 99 ml phosphate buffer dilution blanks. Either a 1 ml or 0.1 ml aliquot was removed from each bottle (depending on dilution required) and aseptically transferred to a petri dish, which was then filled with molten plate count agar and gently swirled to mix before hardening. Duplicate aliquots were taken for each dilution and the average of the two used for the colony count. Only plates with 30-300 colonies were counted after 24 hours of incubation at 32°C. Colony morphology of the plates was also noted, so that any contaminating organism might be detected. Headspace measurements were taken on the Carle 8700 gas chromatograph using a 1 cc sample, removed aseptically from the pouch with a syringe. Due to the number of measurements taken and the limited amount of headspace available, only one sample was taken per pouch and no gas replaced since the pouch was flexible and would conform to the missing volume. 14 This ensured that alteration of the individual gas partial pressures would be minimized. Aerobic and anaerobic control runs were synchronized with the beginning of the bag runs and inoculum for the controls were taken from the same starter culture that was used for the pouches. The aerobic control consisted of placing 20 ml of brain-heart infusion broth in a sterile petri dish and adding 1 cc of starter culture, prediluted as was done for the pouches. The petri dish was then incubated 32°C. The anaerobic control was similarly treated, except that incubation at 32°C was done in a BBL Gas-Pak jar, which provided the anaerobic atmosphere necessary. This device has a generator and catalyst which produces hydrogen and converts the oxygen in the atmosphere to water. The controls were sampled for cell concentration at the same time intervals as the pouches, using the same pour plate technique. Permeability Measurements of Films A MoCon Oxtran 100 was used to measure the oxygen transmission rates of the films (Modern Controls, Elk River, MN). This device utilizes the isostatic test method, which means that a constant total pressure is maintained on both sides of the film. A schematic representation of the Oxtran 100 is shown in Fig. 2. A film sample is clamped into the diffusion cell, exposing a 100 CW2 area of material. An oxygen-free carrier 15 W ,\\\\_\\\ t . — I :—"‘ Na. . M L\K\\\\\\\ paves/aw 1 a“ an; 3157‘ % MAST/77" JMJOR 54/193447? sat/:02?" #2 03m: 7:: 1—1 a 9 a [ rm £531ch «AA/w» ”2 LLLLL nae L~M l [1 L «.0 Figure 2. MoCon Oxtran 100 16 gas is continually flushed over both sides of the film to remove residual oxygen. Once an oxygen-free baseline has been established, oxygen is exposed to one side of the film, while carrier gas flows over the other side and into the coulometric detector. The coulometric detector is a constant current generator, the output of which is a linear function of the mass flow rate of oxygen entering the detector. At the surface of a graphite cathode each oxygen molecule reacts to capture four electrons. 02 + 2 H20 + 4e- -- 40H- These hydroxide ions react at the cadmium anode to form cadmium hydroxide and release the four electrons. 20d + aou' - 4e- -- ca2 Each molecule of oxygen causes the transfer of four electrons, therefore one mole of oxygen equals four Faradays. l Faraday - 96,500 ampere-seconds therefore, one mole oxygen - 4 x 96,500 - 3.86 x 10 ampere-szconds, at S.T.P. 1 cc of oxygen/24hr. - 1.99 x 10 amperes. As oxygen starts to permeate through the film the mV response increases and is displayed on the chart recorder. This increase will continue until a steady state is reached, which represents the equilibrium oxygen transmission rate of the sample. The oxygen transmission rate depends on the resistor used. 5.3 Ohm: ImV - lOOCC/day/atm 17 53 Ohm: 1mV - lOcc/day/atm The films were tested at 32°C and OZ RH. A MoCon Permatran-C was used to measure carbon dioxide transmission rates of the films, utilizing the isostatic test method. The Permatran-C is similar to the Oxtran 100, except the detector system consists of a pressure modulated infrared (PMIR) concept. Instead of using a mechanical chopper to modulate the light beam, it modulates the gas pressure at a fixed frequency. Pressure is achieved by means of a metal bellows, which compresses the gas at a cyclic rate of 25-30 Hz. Since pressurized gas absorbs more infrared radiation than non-pressurized gas, the pressure waves act to modulate the infrared beam. An optical band-pass filter is used to limit the light passing through to 4.3 um, which is the wavelength associated with stretching and bending of C-0 bonds found in carbon dioxide. The amplitude of the modulated signal is an indication of how much carbon dioxide is present. The signal from the detector is amplified and fed into the chart recorder. One side of the film is exposed to 100% carbon dioxide, while the other side is purged with a stream of air passed through a bed of ascarite. The sample is allowed to condition for a period of time, during which time a uniform diffusion rate is established. Once equilibrium is reached, a valve allows the air stream to bypass the ascarite, circulate in a closed loop, past one side of the sample, into the sensing chamber and back to the sample again. As 18 the permeant diffuses through the sample, it accumulates in the capture volume, which is recorded on the strip chart recorder. The slope of the line made on the chart paper represents the change in volume per change in time, which is proportional to the diffusion rate through the film. To determine what the recorder deflection means in terms of cc's of carbon dioxide, a known volume of carbon dioxide is introduced into the calibration loop and the deflection is observed. Several injections from the calibrating loop allows for the construction of a calibration curve of volts versus volume. A schematic of the Permatran-C is shown in Figure 3. 19 MANIFOLD Hm; INLET FLOHMETERS 0 "o (’0\, TEST ' \\\‘ \\\‘ \\\‘ " J; 33% METAL BELLO‘JS PUMP 'll l___: .. . PURGC-TFST VALVE - % I J SENSING CHAMBER Figure 3. MoCon Permatran-C RESULTS Headspace measurements and colony counts are presented in Appendices A and B, respectively. The results of which are summarized in Tables A4-6 and B4-6 and are used to construct the graphs for each material tested (Figures 4, 5 and 6). Standard deviations and statements of precision should be noted and considered when the Results section of this text is read. In Figure 4 it is evident that the 02 concentration initially starts to rise, due to the partial pressure difference between the inside and outside of the package. During this time the bacterial culture is progressing into exponential phase, which causes a drop in 02 concentration in the headspace immediately thereafter, along with an increase in CO2 concentration. The O2 and CO2 concentrations seem to decrease and increase respectively, until they reach a similar value. Compared to the aerobic and anaerobic controls the culture appears to be only' slightly inhibited, with respect to total all yield, by the O2 and CO2 permeability of the film. The saran and saran coated nylon films (Figures 5 and 6) show similar results, most likely due to their similar gas permeabilities. Saran and saran coated nylon also exhibit a concurrent decrease in headspace 02 with an increase in bacterial growth. Also, the headspace gases approach a value, which starts when the culture approaches stationary phase. The bacterial culture, was more 20 'Ifl/ S1130 :IO 90'! 21 12 A C02 . L O 02 . BO . I AEROBIC CONTROL d a ANAEROBIC CONTROL . 11 L O POUCH COUNT .18 1b 10Er 6 9 .. 34 8 r' J12 7 . JIO 6 . .. 8 5 b J 6 » 4 _ .. 4 3 . 1 2 4 8 12 16 20 24 - TIME [HRS] FIGURE 4 - POLYETHYLENE GROWTH CURVE % GAS 'IW/S'I'IHO :IO lOO'I 22 FIGURE 5 - SARAN COATED NYLON GROWTH CURVE 1 A C02 .10 O 02 a AEROBIC CONTROL l A ANAEROBIC CONTROL 11 o POUCH COUNT . 9 1 . 8 9. . 7 8 _ . 6 'I 7 I. O In 5 $ J " 5 _ .. 4 5 _. . 3 4 "'3’ —a 4 2 3 _ .. 1 12 ’ 24 36 48 so 72 TIME IHRSI % GAS 'IW/S'HEO :IO 901 12 11.. ODIOD 23 C02 02 AEROBIC CONTROL ANAEROBIC CONTROL POUCH COUNT 12 24 36 48 6O 72 TIME I HRSI FIGURE 6 ' SARAN GROWTH CURVE 1O 99 GAS 24 inhibited, with respect to total cell yield, in these films than in the polyethylene. To better quantify the degree of inhibition the culture is experiencing, calculations are made to determine what percentage the bag's colony count is of the aerobic control's colony count. The aerobic control's colony count is used as 100% and is calculated at the same time point for each film. 2 of Aerobic Control - Bag colony count x 1002 Aerobic Control Colony Count The higher the percentage of Aerobic control, the less the inhibition and the lower the percentage, the greater the inhibition. Table 5 shows the results of these films calculated at 24 hours. Table 5 Growth Inhibition Bag Colony Count Aerobic Control Z of Aerobic Film (cells/m1) Colony Count Control (cells/ml) pa 2.00x109 8.91x109 22.4 sn 1.9Ox108 1.55x109 12.2 s 1.26x107 2.51:103 5.0 The percent of Aerobic Control figure in Table 5 support the contention that inhibition increases with decreasing gas permeability of the film used. It should be noted that it appears that this inhibition may increase even further later in the growth cycle, although this cannot be calculated, due to a lack of colony counts for polyethylene past 28 hours. 25 It is not clear whether the inhibition is due to O2 depletion or CO2 increase. It is most likely a combination of both. Apparently, since the 02 level in the headspace drops, some 02 in the atmosphere is being used up by the organism. More specifically, the bacteria is utilizing the O2 in solution and more is taking it's place from the headspace. The fact that all samples have a similar growth rate up to 12 hours is probably due to the culture utilizing 02 left in solution. If one mole of CO2 is produced for every mole of O2 in the metabolism of Pseudomonas, through the amount of O2 consumed should equal the amount of CO2 produced. Oxygen consumption can be described by the following equation. Total 02 Consumed - Initial + Amount + Amount in Amount Permeated Solution (headspace) In Initially - Amount In - Final Concentration Solution (headspace) (Final) Initial Amount - Z 02 initially x 20 cc headspace volume Amount Permeated In - Permeability Rate x Time x Driving Force x Surface Area Amount In Solution - 0.02541 cc/ml x 20 ml (broth) Initially x Z 02 initial Amount in Solution - 0.02541 cc/ml‘x 20 m1 (broth) Final x Z 02 final Final Concentration - Z 02 final x 20 cc headspace volume Note: The driving force used in the amount permeated in calculation is taken from the point where the 02 concentration starts to level off for the saran and saran coated nylon samples and half way between the highest and lowest 02 26 concentration for polyethylene. The calculation of the O solubility constants are given in Appendix D. CO2 production is described in the following equation. Total C02 - Amount In + Amount In + Amount Produced Headspace Solution Permeated Out Final (highest level) - Atmospheric Level of C02 Amount in Headspace - 2 C02 final x 20 cc headspace Final volume Amount in Solution - 0.650 cc/m1 x 20 .1 (broth) x 2 CO2 (highest level) Amount Permeated Out - Permeability x Time x Driving Rate (days) Force x Surface Area Atmospheric CO2 - 0.0332 x 20 cc headspace volume Level Note: The driving force used for the amount permeated out calculation is the average of the lowest and highest CO2 levels. The 2 surface area is, 8 in. x 6 in. - 48 in. (one side) 9 2 2 48 in. 5 0.000645 m“/in . 0.03 m I 0.06 m A130, the value for the atmospheric level of CO was obtained from the "Handbook of Chemistry and Physics," 53rd edition. x 2 sides Total 02 consumption and C02 production calculation results are shown in Tables 6 and 7, respectively. Table 6 22 Consumption Film +A +B +C -D -E Total 0 Consumption (cc) PE 1.8 39.7 0.04 0.03 1.1 40.4 SN 1.1 _O.5 0.03 0.01 0.5 1.1 S 0.7 0.5 0.02 0.01 0.4 0.8 Note: A - Initial Amount (cc) B = Amount Permeated In (cc) 27 C - Amount In Solution Initially (cc) D - Amount In Solution Final (cc) E - Final concentration (cc) Table 7 222 Production Film +F +G +H -I Total C0 Production (cc) PE 0.8 0.49 55.8 0.01 57.1 SN 0.4 0.35 0.3 0.01 1.0 S 0.3 0.20 0.2 0.01 0.7 Note: Amount In Headspace Final (cc) Amount In Solution (cc) Amount Permeated Out (cc) F G H I Atmospheric CO2 Level (cc) The values calculated for O2 consumption and 602 production match quite well, except for polyethylene. In that sample 02 consumption is considerably lower than CO2 production. This may be due to an error in the amount permeated in calculation. If the equilibrium 02 concentration is used in calculating the driving force, B now equals 54.5 cc and.the Total 02 Consumption equals 55.2 cc, which is much closer to the 002 production value. The fact that 02 consumption and CO2 production are approximately equal indicate that the change in the concentration of headspace gases are mostly or entirely due to the metabolism of Pseudomonas aeruginosa and permeability rates of the films. If this was not true the CO2 production would not equal the amount of O2 consumed. The results in Tables 6 and 7 suggest that the organisms are consuming approximately 50 times as much 02 in the PE than in the higher barrier film. This conclusion is 28 not confirmed by the colony count measurements, since they do not show a fifty-fold increase in population. It must be remembered that respiratory rates of bacteria are not constant throughout it's growth. Also, cell concentrations and 02 headspace concentrations were not exactly the same at the outset of the run. Combine this with the fact that the respiratory quotients change, through the growth cycle of the microbe, one would doubt there is a linear relationship between 02 consumption and cell concentration over the length of the run. Earlier the statemenc was made that inhibition of total cell yield was evident in the stationary phase. For this to be true, and to prove that the permeability of the film governs total cell yield, and analysis of the quantity of 02 permeating in versus the amount of 02 being consumed by bacterial metabolism must be made. Research on Pseudomonas fluorescens showed that it's respiratory rate increased during exponential phase and decreased, tending toward a value of 3 cc 02/g dry wt./min. during stationary phase (Sadoff et a1, 1956). To be able to use the value in conjunction with the data collected in this research one must know the weight of an individual cell and it's percent water by weight. According to (Nester, 1973) one bacterium weighs 5.0 x 10-12 g wet wt./cell and is 70% water by weight. Therefore, 1 bacterium - 5.0 x 10'12 g wet wt./ce11 x 0.30 - = 1.5 x 10'12 g dry wt./cell. 29 With this information the analysis can be made. First, the grams in dry weight of cells at time t must be determined using the following set of equations. (cell conc. at time t (cells/ml)) x 20 ml (broth) . no. of cells (No. of cells) x (1.5 X 10-12 g dry wt./cell) = dry cells (g) From this the amount of 02 being consumed by bacterial metabolism at time t can be calculated. (3 cc 0 /g dry wt./min) x (grams of dry cells) - Amount 2 being consumed at time t (cc/min) This value can then be compared with the amount of O2 permeating into the package, which is calculated in the following manner. Permeability Rate x 1 day x Driving x Surface of Pi m 1440 min ‘Force at Area (ccOZ/m day atm) Time t = Amount 02 permeating in at time t (cc/min.) The results of these calculations are shown in Table 8. Table 8 22 Availability and Consumption Amount 0 Amount 0 Film A B C D Consumed Permeate (cc/min) In (cc/min) PE 24 2x109 4x1010 0.060 0.180 0.02800 SN 60 8.9x106 1.8x108 0.00027 0.00081 0.00013 5 6O 3.55x107 7.1x103 0.00105 40.0032 0.0001 Time (hrs.) Cell Concentration at Time t (cells/m1) No. of Cells Dry Cells (g) U0w> IIIII For each film the amount of O2 permeating in is less 30- than the amount required by the culture. Therefore, the 02 permeability of the film must be exerting, to some degree, an inhibitory effect on the growth of the bacteria. In the graphs (Figures 4, 5 and 6) this inhibitory effect appears to be manifested in a decrease in total cell yield at time t and it's magnitude is related to the O2 permeability of the film. Also, since 002 has been shown to be inhibitory to Pseudomonas, it may be more correct to speak of 02 being a limiting factor to bacterial growth at a certain CO2 concentration. Both the O2 and CO2 concentrations are in part determined by the permeability of the film being used and in part by bacterial metabolism. Although the values calculated in Tables 6 and 7 for O2 consumption and 002 production are approximately equal, there is a slight discrepancy. This might be explained by looking at an isolated section of the graph; the area where the headspace gases come to equilibrium. At this phase the respiratory rate starts to level off and becomes more constant (Sadoff, 1956), causing the gases to have a more constant concentration. Now, with a constant respiratory rate, the amount of dissolved 02 and C02 in solution becomes constant and the amount of 02 and C02 in the headspace reaches equilibrium. With those assumptions made, the respiratory quotient can be described by the amount of 02 permeating in the pouch and the amount of C02 permeating out. Figure 7 graphically illustrates this concept. 31 BACTERIAL i METABOLI SM T 02 CONSUMED C02 PRODUCED C02 Figure 7 Pouch Gas Dynamics By calculating the permeability rates of the films to O2 and C02, the respiratory quotient can be determined. 02 Permeability - Permeability x Surface x Driving Rate at Rate at Ar a Force Equilibrium 1 atm (m ) (atm) (cc/day) (cc/m2 day atm) CO Permeability - Permeability x Surface x Driving Rate at Rate at Area Force Equilibrium 1 atm (m2) (atm) (cc/day) (cc/m2 day atm) The results of these calculations are summarized in Table 9. Table 9 Respiratory Ouotients (Stationary Phgse) O Permeability CO Permeability Respiratory Film at Equilibrium at Equilibrium Quotient (cc/day) (cc/day) C02:02 PE 49.20 57.18 1:1.16 SN 0.19 0.09 ' 1:2.11 S 0.14 0.06 1:2.33 The respiratory quotients tend to increase, with respect to O2 demand, in the higher barrier film, but not significantly in the lower barrier film. Therefore, the change in respiratory quotients in the high barrier films is most likely due to the organism being stressed by the combination of 02 reduction and C02 increase in the 32 headspace. The respiratory quotient of the bacteria in the polyethylene bag did not change significantly, because the organisms were not as stressed as the ones in the high barrier films. This is evident by recalling that the colony counts for the polyethylene bags were only slightly less than the aerobic control, which shows a lesser degree of stress on the cells. To confirm these observations it is necessary to go back and calculate the respiratory quotients of the bacteria during it's exponential growth phase. The 0-12 hour interval was chosen for these calculations, because it best represents the exponential phase of growth. The same equations are used to determine O2 consumption and CO2 production during this time interval, as was used previously in Tables 6 and 7. The results of these calculations are displayed in Tables 10 and 11. Table 10 Total 02 Consumed Film A + B + C - D - E I Total 0 Consumed (cc) PE 1.78 10.7 0.04 0.07 2.76 9.69 SN 1.11 0.32 0.03 0.03 1.05 0.38 S 0.75 0.29 0.02 0.02 0.71 0.33 Table 11 Total 002 Produced Film F + G + H - I = Total CO Produced (cc) PE 0.14 0.09 6.20 0.01 6.42 SN 0.25 0.16 0.02 0.01 0.42 S 0.20_ 0.13 0.02 0.01 0.34 33 The respiratory quotients can now be calculated for each film over this time period, by comparing the amount of 02 consumed to this amount of C02 produced. These comparisions are shown in Table 12. Table 12 Respiratory Ouotients (Exponential Phase) Film Respiratory Ouotients (C02:02) PE 0.66:1 SN 1.10:1 S 1.03:1 From the table it is evident that the saran and saran coated nylon pouches adhere very well to the predicted 1:1 respiratory quotient. The polyethylene sample seemed to have strayed more from the 1:1 quotient.h This is most likely due to the amount of accuracy in the CO2 headspace measurement, since a small amount of error would cause a large change in the respiratory quotient. Therefore, it has been observed that Pseudomonas aeruginosa follows typical metabolic processes at first, until the majority of the O2 dissolved in the media is used up. Once this is gone, and providing the bacteria is enclosed in a barrier material it's metabolic processes appear to change, apparently from the stresses caused by a increased 002 concentration. This concept was not evident in the low gas barrier material, which indicates that permeability plays an important role in determining the metabolic pathways used by the organism. This stress is also evidenced by the reduction in cell yield, as previously discussed. 34 SUMMARY AND CONCLUSIONS The permeable pouch is a very dynamic system with respect to microbiological growth and headspace gases. Several observations were made with respect to this dynamic process. It was found that the number of colony forming units decreases with decreasing permeability of the film used. There was a reduction in 02 concentration in the headspace and an increase in C02 concentration as the number of organisms increased. Also, the headspace gases tended to an equilibrium, once the bacteria reached stationary phase, where the O2 and CO2 concentrations are equal. The 02 and C02 concentrations are determined by the permeability of the film and the bacterial metabolism. 02 consumption and CO2 production by the bacteria can be calculated by considering the permeability of the film, dissolved gases in solution and gas concentrations in the headspace. The respiratory quotient of the organisms can be calculated by comparing O2 and CO2 headspace concentrations at the equilibrium phase. Placing bacteria in a barrier film causes stresses on the microbes, by reducing the concentration of O2 and increasing the concentration of CO2 in the headspace. This stress appears to cause a change in the organism's metabolism, thus changing it's respiratory quotient. 35 RECOMMENDATIONS The research done here is enough to show that a pattern exists between microbial growth, film permeability and headspace gases. This opens up some intriguing possibilities, such as computer prediction of cell yield given the permeability of the film, initial headspace has concentrations and initial cell concentration. A concept such as this could greatly aid shelf-life predictions of many food products that are packaged in gas permeable packages. Before computerized shelf-life predictions can become a. reality much work needs to be done. Better precision in the quantification of data is necessary. More precise measurements of headspace gases and a quicker method of cell counts must be devised, since time is an important consideration. A method of equilibrating the amount of dissolved 02 in the media before inoculation of the organisms is needed. A great quantity of data is needed for a data base. Finally, this experiment does not consider the mixed flora effects. Many contaminated food products contain more than one organism, which could lead to neighbor-neighbor inhibition and microbial succession, which would probably alter the results obtained here. 36 APPENDIX Appendix A Headspace Measurements Appendix A Headspace Measurements Table A1 Polyethylene (2 mil) Time Peak Peak Peak Gas (hrs) Pouch Gas Height Width Area AU/Z Z v/v 0 CAL 02 7.500 0.1250. 0.938 0.062 - 0 CAL CO2 2.700 0.100 0.270 0.054 - 0 PE 1 02 5.350 0.100 0.540 - 8.56 0 PE 1 C02 0 0 0 - 0 0 PE 2 02 6.100 0.100 0.610 -- 9.76 0 PE 2 CO2 0 o o - o 0 PE 3 02 5.400 0.100 0.540 - 8.64 0 PE 3 C02 0 O 0 -- 0 6 CAL 02 7.500 0.100 0.750 0.050 -- 6 CAL CO2 2.500 0.120 0.300 0.062 -- 6 PE 1 02 6.350 0.100 0.635 -- 12.7 6 PE 1 C02 0.075 0.075 . 0.006 - 0.018 6 PE 2 02 6.500 0.100 0.650 -- 13.0 6 PE 2 C02 0 0 0 -- 0 6 PE 3 02 6.300 0.100 0.630 -- 12.6 6 PE 3 C02 0 0 0 - O 12 CAL 02 7.450 0.100 0.745 0.050 - 12 CAL C02 2.700 0.125 0.338 0.068 ~- 12 PE 1 02 7.150 0.100 0.715 -- 14.39 12 PE 1 C02 0.600 0.100 0.060 -- 0.89 12 PE 2 02 6.800 0.100 0.680 -- 13.68 12 PE 2 C02 0.300 0.100 0.030 - 0.44 12 PE 3 02 6.650 0.100 0.665 -- 13.38 12 PE 3 CO2 0.400 0.100 0.040 - 0.59 28 CAL 02 7.550 0.100 0.755 0.050 -— 28 CAL CO2 2.500 0.100 0.250 0.050 - 28 PE 1 02 2.850 0.100 0.285 - 5.67 28 PE 1 C02 1.900 0.100 0.190 -- 3.80 28 PE 2 02 3.550 0.100 0.355 -- 7.06 28 PE 2 C02 1.600 0.100 0.160 -- 3.20 28 PE 3 02 1.650 0.100 0.165 -- 3.28 28 PE 3 CO2 2.150 0.100 0.215 -- 4.30 37 38 Table A? Saran Coated Nylon (.75 mil) Time Peak Peak Peak Gas (hrs) Pouch Gas Height Width Area AU/Z Z v/v 0 CAL 02 6.100 0.125 0.762 0.051 -- 0 CAL C02 2.650 0.150 0.398 0.080 -- 0 SN 1 02 3.000 0.100 0.300 -- 5.88 0 SN 1 C02 0 0 0 -- 0 0 SN 2 02 2.750 0.100 0.275 -- 5.39 0 SN 2 C02 0 0 0 -- 0 0 SN 3 02 2.800 0.100 0.280 -- 5.49 0 SN 3 C02 0 0 0 - 0 6 CAL 02 6.000 0.100 0.600 0.040 -- 6 CAL C02 2.600 0.150 0.390 0.078 - 6 SN 1 02 2.100 0.100 0.210 -- 5.25 6 SN 1 C02 0.100 0.100 0.010 -- 0.13 6 SN 2 02 2.000 0.100 0.200 -- 5.00 6 SN 2 C02 0.150 0.100 0.015 -- 0.19 6 SN 3 02 1.900 0.100 0.190 -- 4.75 6 SN 3 002 0.150 0.100 0.015 - 0.19 12 CAL 02 6.000 0.100 0.600 0.040 -- 12 CAL C02 2.600 0.125 0.325 0.065 - 12 SN 1 02 2.100 0.100 0.210 - 5.25 12 SN 1 C02 0.600 0.125 0.075 - 1.15 12 SN 2 02 2.000 0.100 '0.200 -- 5.00 12 SN 2 C07 0.600 0.125 0.075 -- 1.15 12 SN 3 0; 2.200 0.100 0.220 -- 5.50 12 SN 3 C05 0.800 0.125 0.100 -- 1.54 24 CAL 02 8.000 0.100 0.600 0.040 -- 24 CAL 002 2.600 0.125 0.325 0.065 -- 24 SN 1 09 0.900 0.100 0.090 -- 2.25 24 SN 1 C05 1.090 0.125 0.136 -- 2.10 24 SN 2 02 1.100 0.100 0.110 -- 2.75 24 SN 2 002 1.140 0.125 0.143 -- 2.20 24 SN 3 02 1.000 0.100 0.100 - 2.50 24 SN 3 CO2 1.040 0.125 0.130 - 2.00 48 CAL 02 5.700 0.100 0.570 0.038 - 48 CAL C02 2.600 0.125 0.325 0.065 - 48 SN 1 02 0.850 0.100 0.085 -- 2.24 48 SN 1 002 1.09 0.125 0.136 -- 2.10 48 SN 2 02 0.800 0.100 0.080 -- 2.10 48 SN 2 C02 1.170 0.125 0.146 -- 2.25 48 SN 3 02 0.750 0.100 0.075 -- 1.07 48 SN 3 C02 1.270 0.125 0.159 -- 2.45 Table A2 (cont'd.) Time Peak Peak Peak Gas (hrs) Pouch Gas Height Width Area AU/Z 2 v/v 72 CAL 02 5.400 0.100 0.540 0.036 -- 72 CAL C02 2.500 0.125 0.312 0.062 - 72 SN 1 02 1.200 0.100 0.120 -- 3.33 72 SN 1 002 0.975 0.125 0.121 -- 1.95 72 SN 2 02 0.800 0.100 0.080 -- 2.22 72 SN 2 C02 1.090 0.125 0.136 -- 2.20 72 SN 3 02 0.900 0.100 0.090 - 2.50 72 SN 3 CO 1.120 0.125 0.140 - 2.25 40 Table A3 Saran (2 mil) Time Peak Peak Peak Gas (hrs) Pouch Gas Height Width Area AU/Z 2 v/v 0 CAL 02 7.200 0.100 0.720 0.048 - 0 CAL CO2 3.600 0.100 0.36 0.072 -- 0 S 1 02 2.500 0.075 0.188 - 3.92 0 S 1 C02 0 0 0 -- 0 0 S 2 02 1.750 0.100 0.175 -- 3.64 0 S 2 002 O 0 0 -- 0 0 S 3 02 1.750 0.100 0.175 - 3.64 0 S 3 C02 0 0 0 - 0 6 CAL 02 7.600 0.100 0.760 0.051 - 6 CAL 002 2.800 0.100 0.280 0.056 -- 6 S 1 02 2.600 0.100 0.260 - 5.10 6 S 1 C02 0 0 0 -- 0 6 S 2 02 2.150 0.100 0.215 - 4.22 6 S 2 002 0 0 0 -- 0 6 S 3 0 1.950 0.100 0.195 -- 3.82 6 S 3 C02 0 0 0 - 0 12 CAL 02 7.600 0.100 0.760 0.051 -- 12 CAL CO2 2.800 0.100 0.280 0.056 - 12 S 1 02 1.950 0.100 0.195 - 3.82 12 S l 002 0.750 0.075 0.056 -- 1.00 12 S 2 02 1.850 0.100 0.185 -- 3.63 12 S 2 CO? 0.750 0.075 0.056 -- 1.00 12 S 3 Oi 1.650 0.100 0.165 -- 3.24 12 S 3 C02 0.750 0.075 0.56 -- 1.00 36 CAL 02 7.300 0.100 0.730 0.049 -- 36 CAL CO2 2.200 0.100 0.220 0.044 -- 36 S 1 02 1.200 0.100 0.120 - 2.44 36 S 1 C02 0.750 0.100 0.075 -- 1.70 36 S 2 02 0.950 0.100 0.95 -- 1.94 36 S 2 CO2 0.700 0.100 0.070 -- 1.43 36 S 3 02 1.000 0.100 0.100 -- 2.04 36 S'3 C02 0.550 0.125 0.069 -- 1.57 78 CAL 02 7.700 0.100 0.770 0.051 - 78 CAL C07 2.550 0.100 0.255 0.51 '- 78 S 1 0i 1.100 0.100 0.110 -- 2.16 78 S 1 C02 0.750 0.100 0.75 -- 1.47 78 S 2 02 1.000 0.100 0.100 -- 1.96 78 S 2 CO2 0.800 0.100 0.080 -- 1.57 78 S 3 02 0.900 0.100 0.090 -- 1.76 78 S 3 CO2 0.700 0.100 0.070 -- 1.37 41 Table A4 Polyethylene (2 mil) Time Gas m Std . Dev . 0 02 8.99 0.67 0 C02 0 0 6 02 12.77 0.21 6 C02 0.01 0.01 12 02 13.82 0.52 12 C02 0.64 0.23 28 09 5.34 1.91 28 C05 3.77 0.55 Table A5 Saran Coated Nylon (.75 mil) Time Gas 2 v/v Std. Dev. 0 02 5.59 0.26 0 002 O 0 6 02 5.00 0.25 6 C07 0.17 0.03 12 05 5.25 0.25 12 C02 1.28 0.22 24 07 2.50 0.25 24 COé 2.10 0.10 48 O, 2.10 0.14 48 C05 2.27 0.18 72 02 2.68 ' 0.58 72 C02 2.13 0.16 42 , Table A6 Saran (2 mil) Time Gas 2_;7V Std. Dev. 0 02 3.73 0.16 0 C02 0 0 6 02 4.38 0.65 6 C02 0 0 12 02 3.56 0.30 12 C02 1.00 0 36 02 2.14 0.26 36 002 1.57 0.14 78 02 1.96 0.20 78 C02 1.47 0.10 All peak widths were measured at 1/2 the peak height. Peak Area - Peak Height x Peak Width The gas chromatograph was calibrated for oxygen and carbon dioxide at each time interval. The peak area calculated represents the response for a sample of 15% O2 and 5% C02. These values must be converted to Area units/Z gas, so they may be used to calculate headspace gas concentrations. Therefore, 02 calibration - Peak Area - Area Units 15% Z gas and C02 calibration - Peak Area = Area Units 52 2 gas To calculate the concentration of gases in the headspace the Peak Area for the respective gas is divided by the calibration factor for that gas. 2 gas (volume/volume) = Peak Area Area Units/7 Appendix B Colony Counts 43 Appendix B Colony Counts Table Bl Polyethylene (2 mil) Time Log of m (hrs.) Pouch Colony Count Colony Count Colony Count Std. Dev. 0 A11 5.65x103 3.75 - - 0 All 4.75x103 3.68 3.71 0.05 6 AER 4.10x105 5.61 - - 6 AER 2.50x105 5.40 5.50 0.15 6 ANA 4.90x104 4.69 - - 6 ANA 4.10x10“ 4.61 4.65 0.06 6 pa 1 1.55x105 5.19 - - 6 PE 1 1.29x10S 5.11 5.15 0.06 6 PE 2 1.46x10S 5.16 -- -- 6 pa 2 1.28x105 5.11 5.14 0.04- 6 PE 3 1.52x105 5.18 -- .. 6 PE 3 1.46x10S 5.16 5.17 0.01 12 AER 2.57x108 8.41 - - 12 AER 2.49x108 8.40 8.40 0.01 12 ANA 5.50x104 4.74 -- - 12 ANA 5.10x10‘ 4.71 -- -- 12 PE 1 1.30x108 8.11 -- -- 12 PE 1 1.24x108 8.09 8.10 0.01 12 PE 2 1.37x108 8.14 -- -- 12 PE 2 1.21x108 3.08 8.11 0.04 12 PE 3 1.74x108 8.24 - - 12 PE 3 1.66x108 8.22 8.23 0.01 28 AER 2.86x1010 10.45 -- — 28 AER 2.64x1010 10.42 10.44 0.02 28 ANA 5.8x104 4.76 -- - 28 ANA 5.2x10‘ 4.72 4.74 0.03 28 PE 1 4.8x109 9.68 - - 28 PE 1 3.6x109 9.56 9.62 0.08 28 PE 2 5.9x109 9.77 - -- 28 PE 2 4.71.109 9.67 9.72 0.07 28 pa 3 4.7x10q 9.67 -- ~- 28 PE 3 4.5x10° 9.65 9.66 0.01 44 Table B2 Saran (2 mil) Time Log of m (hrs) Pouch Colony Count Colony Count Colony Count Std. Dev. 0 All 1.76x103 3.24 - - 0 All 1.54x103 3.19 3.22 0.04 6 AER 1.09x107 7.04 -- -— 6 AER 1.01x107 7.00 7.02 0.03 6 ANA 4.00x10‘ 4.60 -- -- 6 ANA 2.80:10“ 4.45 4.52 0.11 6 s 1 2.80x106 6.45 - -- 6 s 1 2.68x106 6.43 6.44 0.01 6 s 2 2.86x106 6.46 - - 6 s 2 2.80x106 6.45 6.46 0.01 6 s 3 2.98x106 6.47 - - 6 s 3 2.84x106 6.45 6.46 0.01 12 AER 4.90x107 7.69 -- - 12 AER 4.10x107 7.61 7.65 0.06 12 ANA 1.68x10S 5.22 - -- 12 ANA 1.54x105 5.19 5.20 0.02 12 s 1 1.55x107 7.19 - —- 12 s 1 1.51x107 7.18 7.18 0.01 12 9 2 1.13x107 7.05 - - 12 s 2 1.03x107 7.01 7.03 0.03 12 s 3 1.35x107 7.13 -- - 12 s 3 1.27x107 7.10 7.12 0.02 36 AER 1.37x109 9.14 -- -- 36 AER 1.27x10 9.10 9 12 0.03 36 ANA 2.56x105 5.41 -- -- 36 ANA 2.50x105 5.40 5.40 0.01 36 s 1 9.90.1106 7.00 -- -- 36 s 1 9.50x106 6.98 6.99 0.01 36 s 2 1.39x107 7.14 -- - 36 s 2 1.31x107 7.12 7.13 0.01 36 s 3 1.03x107 7.01 -- - 36 s 3 9.10x106 6.96 6.98 0.04 78 AER 4.50.1109 9.65 - - 78 AER 4.10:109 9.61 9.63 0.03 78 ANA 2.56x105 5.41 -- - 78 ANA 2.50x10S 5.40 5.40 0.01 78 s 1 1.00x108 8.00 -- - 78 s 1 8.80x107 7.94 7.97 0.04 78 s 2 9.00x107 7.95 -- -- 78 s 2 8.20x107 7.91 7.93 0.03 78 s 3 8.60x107 7.93 -- - 78 s 3 7.60x107 7.88 7.90 0.04 45 Table 33 Saran coated nylon (0.75 mil) Time Log of ‘EEE-gf (hrs) Pouch Colony Count Colony Count Colony Count Std. Dev. 0 A11 9.48x103 3.98 -- -- 0 A11 9.4211103 3.97 3.98 0.01 6 AER 3.70x105 5.57 -- - 6 AER 2.70x10S 5.43 5.50 0.10 6 ANA 3.3011105 5.52 -- - 6 ANA 2.9011105 5.46 5.49 0.04 6 SN 1 5.5051105 5.74 -- - 6 SN 1 4.30x10S 5.63 5.68 0.08 6 SN 2 4.10x10S 5.61 - - 6 SN 2 3.30x10S 5.52 5.56 0.06 6 SN 3 4.60x105 5.66 - - 6 SN 3 4.00x105 5.60 5.63 0.04 12 AER 1.11x108 8.04 -- - 12 AER 9.70x107 7.99 8.01 0.04 12 ANA 2.94x106 6.47 - -- 12 ANA 2.78x106 6.44 6.46 0.02 12 SN 1 3.04x107 7.48 -- - 12 SN 1 2.86x107 7.46 7.47 0.01 12 SN 2 2.9654107 7.47 - - 12 SN 2 2.70x107 7.43 7.45 0.03 12 SN 3 2.86x107 7.46 -— - 12 SN 3 2.66x107 7.42 7.44 0.03 Table BB (cont'd.) 46 Time - Log of m (hrs.) Pouch Colony Count Colony Count Colony Count Std. Dev. 24 AER 1.61x109 9.21 -- - 24 AER 1.49x10 9.17 9.19 0.03 24 ANA 3.00x106 6.48 - - 24 ANA 2.88x106 6.46 6.47 0.01 24 SN 1 2.0751108 8.32 -- - 24 SN 1 1.9121108 8.28 8.30 0.03 24 SN 2 1.9011108 8.28 - - 24 SN 2 1.6611108 8.22 8.25 0.04 24 SN 3 2.05x108 8.31 -- - 24 SN 3 2.01x108 8.30 8.30 0.08 48 AER 1.005110lo 10.00 - - 48 AER 9.2011109 9.96 9.98 0.03 48 ANA 3.06x106 6.48 - - 48 ANA 2.90x106 6.46 6.47 0.01 48 SN 1 4.6011108 8.66 -- -e 48 SN 1 2.40x108 8.38 8.52 0.20 48 SN 2 4.70x108 8.67 - -- 48 SN 2 3.7Ox108 8.57 8.62 0.07 48 SN 3 4.9011108 8.69 -- - 48 SN 3 4.1031108 8.61 8.65 0.06 72 AER 6.30x109 9.80 -- - 72 AER 4.90x10q 9.69 9.74 0.08 72 ANA 3.08x106 6.49 -- - 72 ANA 2.80x106 6.45 6.47 0.03 72 SN 1 1.8211109 9.26 -- -- 72 SN 1 1.66x109 9.22 9.24 0.03 72 SN 2 1.92x10° 9.28 -- -- 72 SN 2 1.80x109 9.26 9.27 0.01 72 SN 3 1.76x109 9.24 -- -- 72 SN 3 1.62x109 9.21 9.22 0.02 47 Table B4 Polyethylene (2 mil) Log of Colony Time Count of Pouches Std' Dev. 6 5.15 0.02 12 8.15 0.07 28 9.67 0.05 Table BS Saran (2 mil) Log of Colony Time Count of Pouches Std' Dev. 6 6.45 0.01 12 7.11 0.08 24 7.03 0.08 78 7.93 0.04 Table 86 Saran coated nylon (0.75 mil) Log of Colony Time Count of Pouches Std' Dev. 6 5.62 0.06 12 7.45 0.02 24 8.28 0.03 48 8.60 0.07 72 9.24 0.02 Appendix C Film Permeability Measurements Appendix C Film Permeability Measurements Oxygen (Oxtran 100) The oxygen permeability of the films was calculated by multiplying the calibration factor (determined by the resistor used) by the mV response observed on the chart recorder. (X) cc/m2/24 hrs. 0 permeability - mV response x 2 1 mV Polyethylene (2 mil) sensitivity - 100 mv full scale 2 5.3 Ohm resistor: 129.2219 l24 hrs. mV Runs made at 32°C and OZ RH Table C1 Polyethylene mV Response Run No. mV Response 1 50.5 2 49.5 3 49.0 i - 49.7 Std. Dev. 8 0.80 2 02 permeability - 49.7 mV x.199_EELEHLZi_EE§; - 4,970 cc/mzlday 1 mV Saran (2 mil) sensitivity a 10 mV full scale 10 cc/m2/24 hrs. mV 53 Ohm resistor: Runs made at 32°C and 02 RH. 48 49 Table C2 Saran mV Response Run No. mV Response 1 1.3 2 1.3 3 1.4 §'- 1.3 Std. Dev. I 0.10 2 O2 permeability - 1.3 mV x 10.SE£E.L£3_EEE;.- l3 cc/m2/day mV Saran coated nylon (0.75) sensitivity - 10 mV full scale 10 cc/m2/24 hrs. mV 53 Ohm resistor: Runs made at 32°C and 02 RH. Table C3 Saran coated nylon mV Response Run No. mV Response l t-DD-IO— wuss 2 3 §'- 1.7 Std. Dev. - 0.10 10 cc/m2/24 hrs. 02 permeability - 1.7 mV x mV Carbon Dioxide (Permatran-C) The methods used to analyze the film samples for carbon dioxide, were identical to those suggested by Modern Controls Co.. The saran and saran coated nylon samples were tested using the good barrier method, where the polyethylene sample was tested with the poor barrier 50 procedure. The CO2 transmission rate can be calculated using the following equation. C02 Transmission - capture volume (cc) x 1440 min. x 196152312? Rate time to reach day surface area (cmz) calibration point (minutes) Polyethylene (2 mil) 5 V full scale chart speed - 4 incBes/hour surface area - 5 cm capture volume - 0.02 cc calibration response - 1.8 units Table C4 Calibration Response Time (Polyethylene) Trial # Calibration Response Time (min.) 1 2.7 2 2.7 3 2.7 C02 Transmission Rate It ..._.—— x —— x — __ 0.02 cc 1440 min. 1 4 662/62 2.7 min. day 5 cm2 - 21,333 cc/mz/day Saran coated nylon (0.75 mil) 500 mV full scale chart speed - 4 inchss/hour surface area - 50 cm capture volume - 0.002 cc calibration response - 1.8 units Table C5 Calibration Response Time (Saran coated nylon) Trial # Calibration Response Time (min.) 1 8.5 8.5 3 8.5 4 2 2 CO:2 Transmission Rate - M x M x .32 2'1 /_m 8.5 min. day 50 cm2 . 67.8 cc/mzlday 51 Saran (2 mil) 500 mV full scale chart speed - 4 inches/hour surface area - 50 cm capture volume - 0.002 cc calibration response - 1.8 units Table C6. Calibration.Response Time (Saran) Trial # Calibration Response Time (min.) 1 10.5 2 10.5 3 10.5 4 2 2 CO2 Transmission Rate - 93M 1: M x .12 £2 .62 10.5 min. day 50 C1112 = 54.8 cc/mz/day Appendix D Calculation of Solubility Constants for 02 and C02 Appendix D Calculation of solubility constants for O2 and CO2 A linear interpolation of the solubility constants for O2 and CO2 at 32°C was obtained by using the values at 30°C and 35°C. 02 solubility - 0.02608 - [(0.02608 - 0.02440) x 2/5] coefficient - 0.02541 cc/ml at 32°C and 1 atm of gas pressure c02 solubility - 0.665 - [(0.665 - 0.592) x 2/51 coefficient - 0.650 cc/ml at 32°C and 1 atm of gas pressure Note: Solubility constants obtained from the text (CRC, 1936) are given on a volume gas/volume water basis at their respective temperatures and at a pure gas pressure of 1 atm. .52 Appendix E Buffer Formula Appendix E Buffer Formula 3.631 g KB P04 7.125 g K26P04(3H20) 1 liter ' 820 0.05 Molar 53 BIBLIOGRAPHY BIBLIOGRAPHY Apple, J.M., Terlizzi, F.M. 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