oumaowm 05 TYPE E QLQSAAEUA AOTULEAug-A As AFFECTEB av VAAQAA smut-‘55 EN TA: MEQEUM AND av :AmBATEQ-A TEAAEAAMA The“: for {'{19 Degree of M. S. MECHEGAN STATE UNIVERSWY Aiexander Emodi 1967' THESIS " $35333? ESE (KY ABSTRACT OUTGROWTH OF TYPE E CLOSTRIDIUM BOTULINUM.AS AFFECTED BY VARIOUS SOLUTES IN THE MEDIUMHAND BY INCUBATION TEMPERATURE by Alexander Emodi The objective of this study was to investigate the effects of the addition of various concentrations of solutes to one basic medium formulation upon the outgrowth of the spores of Type E Clostridium botulinum at different incubation temperatures. Four strains, K, VH. SF, and A6247 were studied. An inoculum of 1.0 x 106/m1 was inoculated into trypticase— peptone-sucrose-yeast extract (TPSY) medium and additions of sucrose, sodium chloride (NaC1), potassium chloride (KCl). glycerol and sodium formate were used. Equilibrium relative humidity (ERH) determinations were performed to determine the water activity (aw) of the media. The possibility of an osmotic effect was also investigated. The molar concentrations and osmotic pressures of the different solutes were calcu- lated and related to the approximate inhibitory concentrations of 38.5% sucrose (1.12 m), 5.0% Nale (0.86M). 6.0% KCl (0.80 M), and 5.5% sodium formate (0.80 M). No inhibition of growth was observed with the 15% (1.63 M) glycerol con- centration. The use of 15% glycerol lowered the aw but Alexander Emodi did not inhibit outgrowth as observed with the other solutes. Samples were incubated at 30, 21.1, 15.6, 10 and 7.2 C temperatures. Growth was observed by production of turbidity and gas. Toxicity was checked with the mouse toxin assay. Only those tubes which showed gas and gave a positive toxicity test were considered positive for Type E outgrowth. The inhibitory aw level in these solutions appears to be between 0.9708 and 0.9755. The effects of heat and the size of inoculum on out- growth were also studied. Viable spore inocula of 1.0 x lOZ/ml, 1.0 x 104/m1 and 1.0 x 106/m1 of the K and VB strains were tested with and without heat shock in the various media. Spore suspensions were heat shocked for 15 minutes at 60 C. The size of the inoculum affected both lag and outgrowth times. Increased lag times and some growth inhibition were observed using 1.0 x 102 spores/ml at concentrations not inhibitory for the larger inoculum sizes. Heat shock in most cases delayed or inhibited outgrowth at concentrations where spores without heat shock grew well. This adverse effect of the heat treatment was best demon- strated with the smaller 1.0 x 102/m1 and 1.0 x 104/m1 inoculum sizes. However, there was some indication with the VH strain that heat shock may permit outgrowth at concentra- tions previously found inhibitory for growth, and that the beneficial or adverse effect of the heat treatment depends very much on the strain involved. Incubation temperature and growth response were in- versely related as measured by increased lag times and in Alexander Emodi decreased amounts of cellular material produced by each of the four Type E strains tested. OUTGROWTH OF TYPE E CLOSTRIDIUM BOTULINUM.AS AFFECTED BY VARIOUS SOLUTES IN THE MEDIUM AND BY INCUBATION TEMPERATURE BY Alexander Emodi A THESIS Submitted to Nuchigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1967 \ x \V. \ ACKNOWLEDGME NTS The author wishes to express his appreciation to Dr. R. V. Lechowich for his thoughtful guidance throughout this study and for his aid in preparing the manuscript. To his wife, Magda, the author is especially grateful for her encouragement during the course of his study. The author is deeply appreciative of the funds and facilities provided by Michigan State university and the Bureau of Commercial Fisheries to make this study possible. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . Definition of Water Activity Description of Water Activity Limits for Growth of Microorganisms General References on Water Activity of Microbial Growth Factors that Affect the Water Relationships of Microorganisms Definition of Osmotic Pressure REVIEW OF METHODS FOR DETERMINING ERH . . . . . . Manometric Methods Electric Hygrometer Methods Weight Equilibrium Methods Other Methods METHODS AND MATERIALS Methods Used to Determine Equilibrium Relative Humidity Humidity Sensor Method Salt-Paper Strip Method Graphical Interpolation Method Microorganisms Viable Count Procedure Toxicity Assays Water Activity Media Solute-containing Media RESULTS AND DISCUSSION . Water Activity Ionic Effect Osmotic Effect Temperature Effect The Effect of Preheating and the Size of the Inoculum on Outgrowth in Water Activity Media iii 10 ll 12 l3 l6 l6 l6 19 21 22 25 26 26 26 27 27 35 38 40 42 Page Effect of the Size of the Inoculum 43 The Effect of Preheating 46 The Temperature Effect 52 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 54 LITERATURE CITED . . . . . . . . . . . 58 iv Table 10. 10a. 11. LIST OF TABLES Percent ERH obtained with sensors using 10% NaCl as medium . . . . . . . . . . . . . Graphical interpolation data for TPSY medium with KCl to control a . . . . . . . . . w Percent ERH values of NaCl standards Equilibrium relative humidity values of TPSY media containing various amounts of NaCl Percent ERH values of TPSY medium with NaCl, KCl, NaCOOH, sucrose, and glycerol . . Outgrowth of four Type E Clostridium botulinum strains in TPSY medium with sucrose to con- trol aw . . . . . . . . . . . . . . . . Outgrowth of SF and VH Type E Clostridium botulinum strains in TPSY medium with KCl used to control aw . . . . . . . . . Outgrowth of K and VH Type E Clostridium botulinum strains in TPSY medium with sodium formate to control aw . . . . . . Calculated molar concentration and osmotic pressure values for glycerol, sucrose, NaCl, KCl and sodium formate solutions . The effect of inoculum size and preheating upon outgrowth of the VH Type E Clostridium Page 19 22 24 28 3O 32 34 36 39 botulinum strain in TPSY medium with sucrose to control aw . . . . . . . . The effect of inoculum size and preheating upon outgrowth of the K Type E Clostridium botulinum strain in TPSY medium with sucrose to control aw . . . The effect of inoculum size and preheating upon outgrowth of the VH Type E Clostridium botulinum strain in TPSY medium with sodium formate to control aw . . . . . . . . V 44 45 47 Table 11a. 12. 12a. The The The Page effect of inoculum size and preheating upon outgrowth of the K Type E Clostridium botulinum strain in TPSY medium with sodium formate to control a . . . . . . . . . . . . . . . . . . . . 48 w effect of inoculum size and preheating upon outgrowth of the VH Type E Clostridium botulinum strain in TPSY medium with KCl to control a . 49 w . . . . . effect of inoculum size and preheating upon outgrowth of the K Type E Clostridium botulinum strain in TPSY medium with KCl to control a . . . . . . . . . . . . . 50 w 0 vi LIST OF FIGURES Figure Page 1. Graphical interpolation for TPSY medium with KCl to control aw . . . . . . . . . . 23 vii INTRODUCTION Microorganisms, as living cells, require water for metabolism and growth. The water requirement of bacteria may be described with the water activity concept since water activity is a useful measure of the ”availability" of water for microbial growth. When substances are dissolved in water or bacterio- logical media there is a substantial interaction between solute and solvent. The extent to which the water activity is lowered depends primarily on the total concentration of all dissolved molecules and ions. Potassium chloride, sodium formate, sodium chloride and sucrose are very effective solutes to both lower water activity and change the osmotic pressure when added to bacteriological media. In this study, the effects of water activity and osmotic phenomena on Type E Clostridium botulinum strains were examined using the variables of different solutes, incubation temperature, size of inoculum, and effect of preheating of the inoculum. LITERATURE REVIEW It is now widely accepted that the water requirements for growth of many microorganisms can be defined in terms of the water activity of their environment. In the present <:ontext the measurement of the equilibrium relative humidity (ERH), the control of ERH, and some of its effects upon the growth of Clostridium botulinum Type E strains will be discussed. The effects of certain solutes upon possible osmotic phenomena were also examined. Definition ofeWater Activity The availability of water has been expressed in dif- ferent ways. Scott (1957, 1961) defined water activity or aw as a fundamental property of aqueous solutions that is equal to: where p is the vapor pressure of the solution and po is the vapor pressure of the solvent, and n1 and n2 refer to the number of moles of solute and solvent in the solution. Since water contains 55.51 moles/kg, for a 1.0 molal solution of an ideal solute the right—hand side of this equation will equal 55.51/56.51 = 0.9823. The aw also is numerically equal to the corresponding relative humidity expressed as percentage divided by 100. Mossel and van Kuijk (1955) expressed 2 availability of water in terms of equilibrium relative humidity (h) which is numerically equal to aw. Description of awfiLimits for Growth of Microorganisms Microbial growth has been observed by Frazier (1958), at aw levels ranging from very close to 1.00 down to about 0.62. Christian (1963) discussed the concept of aw in relation to the water activities of foods and the aw limits of the microorganisms involved in food spoilage and food poisoning. Molds differ considerably among themselves as to and range of a for germination of the asexual optimum a w w spores. The minimum aw for spore germination has been found to be as low as 0.62 for some molds, and as high as 0.93 for others. Yeasts require more moisture than molds and on the basis of aw may be classified as ordinary if they do not grow in high concentrations of solutes, that is, a low aw, and as osmophilic if they do. Lower limits of a range from w 0.88 to 0.94 for ordinary yeaSts and from 0.62 to 0.75 for osmophilic yeasts. Most bacteria grow well in the 0.995 to 0.998 aw range. The lowest reported limit for growth is about 0.55. General References on water Activity and Microbial Growth Walter (1924) found that none of the bacteria he tested could grow at relative humidities of less than 96%, whereas several molds grew at much lower humidity. Scott (1936, 1937) working with thin slices of beef muscle equilibrated with sulphuric acid solutions, found two strains of Achromobacter to have a lower limit for growth of approximately 96%, and two strains of Pseudomonas to have a lower limit greater than 98%m NUmerous workers (Tomkins, 1929, Heintzeler, 1939 and Stille, 1948) working with molds agree that the greatest tolerance to low aw is near the optimum growth temperature and that at lower and higher temperatures the range of a permitting germination and W growth is reduced. Changes of 0.01 to 0.05 in the minimum aw for a change of 10 C in incubation temperature have been noted. Similar data for bacteria have not been found. Von Schelhorn (1950) noted that the greatest tolerance of Aspergillus glaucus to low aw was manifest at the optimum pH, and that at lower and higher pH values the range of aw permitting germination and growth was greatly reduced. Similar data for bacteria have not been reported. Observation of staphylococci have shown that they generally tolerate somewhat higher solute concentrations than most other bacteria, and this has formed the basis of media used for enrichment and isolation of staphylococci as pro- posed by Chapman (1946). In experiments with dried meat adjusted to various water contents, Segalove and Back (1951) reported that a strain of Salmonella enteriditis required a greater amount of water for growth than Staphylococcus aureus or an alpha hemolytic-type Streptococcus. Scott (1953) discussed the importance of the aw in the relation to the growth of fourteen food poisoning strains of S, aureus. Aerobic growth was observed at aw levels between 0.999 and 0.96. The rate of growth and the yields of cells were both reduced substantially when the a was W less than 0.94. Anaerobic growth proceeded at slightly higher a than aerobic growth. He also showed that the w reduction of the aw below 0.99 led to an increase in the lag period. Christian and Scott (1953) grew sixteen strains of Salmonella in various media of known water activities at 30 C. The reactions of fifteen motile strains were very similar, whereas the single non-motile strain grew mare slowly and over a smaller range of water activities. Aerobic growth of the motile strains occurred in liquid media at water activities between 0.999 and 0.945. In food, the lower limit for growth was slightly less. Anaerobic rates of growth were only slightly less than the aerobic rate at all water activities. A large percentage of the cells could form colonies on agar media with water activities as low as 0.96. There is some evidence for the belief that the water requirements of most coliform bacteria may be similar to those of the Salmonellae. Foda and vaughn (1950) found that 5 to 8% sodium chloride (NaCl) was needed to inhibit the growth of Escherichia coli. The 5 to 8% concentrations 4 correspond to water activities of about 0.97 and 0.95 respective— ly. They also pointed out that most strains of Aerobacter aeroqenes did not tolerate more than 6.5% NaCl (w/v), corresponding to 0.96 aw. Factors that Affect the Water Relationships of Microorganisms Severens and Tanner (1945) studied the inhibition of growth by solutes such as NaC1, and reported that 3% NaC1 was the highest concentration permitting growth of unadapted strains of Salmonella pullorum and Salmonella schottmuelleri (S, paratyphi B). After adaptation for about 10 weeks, growth was observed in media with 8% NaCl. Christian and Scott (1953) found that the range of aw over which growth occurred was substantially the same when sugar, salts or miscellaneous nutrients were the predominant solutes controlling the aw. Mbssel and van Kuijk (1955) stated that the develop- ment of microbes in foods may be inhibited by appropriate desiccation or by addition of solutes such as sugar or salt in sufficient quantities. A study of the growth of the Salmonella oranienburg in dried meat, dried milk and dried soup by Christian and Scott (1953) showed that at the limiting water activities of 0.93 to 0.94, the water content of the soup was some three to four times as great as those of the milk and mutton. It is clear therefore that it is aw rather than the water content which determines the availability of water for growth of these bacteria. The water content of food gives no explicit information on ERH, as noted by Overbeek and Mossel (1951), since relatively small changes in the non-aqueous phase of biological material (e.g., partial replacement of cellulose by sugars or starch) may strongly influence its water binding capacity and hence its h value at a given water content. Scott (1957) reported that the biological response to a particular water activity was, at least for some organisms, largely independent of the type of solute present and the total water content of the substrate. The use of aw as a measure of total solute concen- tration was employed by Wodzinski and Frazier (1960). The aw of the medium was adjusted by the use of potassium chloride (KCl). Since sucrose alone and a salt mixture of NaCl, KCl, and sodium sulfate (NaZSO4) were just as effective in adjusting the a as KCl, it was evident that the effects w observed were not due to either the concentration of the K ion or of the Cl ion. Both of these ions had a much lower concentration in the salts mixture than in KCl alone. These observations further insure that the responses observed were a function of aw and not of the concentration of a particular solute or solutes. Further support that the minimum aw permitting growth changes with environmental conditions is found in the report of Bowling and Ramsey (1958). They showed that the combined effects of salts and chloramphenicol on Staphylococcus pyggenes were additive with most concentrations tested and frequently were synergistic. Christian and Scott (1953) showed that various basal media supported the growth of several strains of salmonellae at water activities down to about 0.945. When observations were extended (1955) to a simple medium of inorganic salts and glucose, the expected decrease in growth rate at the optimum aw was accompanied by a considerable reduction in the range of water activities permitting growth. This result indicated that growth of water activities of the order of 0.95 to 0.96 could only be realized when certain nutrients, absent from the simple medium, were provided. It is generally agreed that most of the microorganisms tolerate low aw at temperatures and pH levels most suitable for them as indicated by Ingram (1957) and Scott (1957). This is further supported in studies by Wodzinski and Frazier (1960, 1961). They investigated the water requirement for growth of A. aerogenes, Lactobacillus viridescens and Pseudomonas fluorescens in relation to temperature, pH, oxygen, carbon dioxide and some nutritional factors. When the temperature was lowered from 30 to 15 C, the minimum aw increased and the same effect was observed when pH was changed from pH 7.0 to pH 5.4 or pH 8.8. There are very little data published on aw limits of l. botulinum. Williams and Purnell (1953) reported that growth of Cl. botulinum Type A is inhibited in the range of 0.94 to 0.96 aw. Scott (1955) suggested that the minimum aw for these organisms would be 0.95. He also discussed the growth of these organisms in canned ham and showed that the aw levels of the samples examined were not low enough to prevent growth. Kadavy and Back (1951) conducted a study of the growth of Cl. botulinum in canned bread. They inoculated the bread and found growth in regions where spoilage by other organisms had occurred. In these regions the measured water content was higher, about 9 to 14%, than in the other regions. Water activity was not determined. Since biologists sometimes consider water avail- ability in terms of the osmotic pressure it is of interest to show the relation between these quantities, thus Osmotic pressure = :%2 1n E- o where V is the partial molal volume of water, R is the gas constant, T is the absolute temperature, p0 and p are the vapor pressures of water and the solution respectively. Ingram (1957) showed that the fraction p/po represents the "activity” of the water (aw) for aqueous solutions. REVIEW OF METHODS FOR DETERMINING ERH The measurement of the ERH of a substance—-that is the relative humidity at which the substance will neither gain nor lose moisture under given temperature conditions-- is an important determination that may be used to predict the storage characteristics of food products. Until recently the ERH of food was not easily measured and food processing practices have been based on the water content of commodities. The methods used to determine ERH can be classified in the following basic groups: A. Manometric methods. B. Electric hygrometer methods. C. Weight equilibrium methods. D. Other methods. Manometric Methods These methods consist of direct measurements of water-vapor pressure of a closed chamber with a manometer at controlled temperature. Makover and Myers (1943) described a relatively simple vapor pressure apparatus, consisting of an oil manometer, a sample bottle and a trap. The apparatus of Vincent and Bristol (1945) is based on the measurement of pressure exerted before and after 10 ll freezing of moisture vapor. Adams and Merz (1929) used an isoteniscope to measure the vapor pressure of saturated salt solutions. Legault, kaover and Talburt (1948) used a Dubrovin manometer having a sensitivity about seven times that of an ordinary U tube mercury manometer. Taylor (1961) determined moisture equilibria in dehydrated foods by an improvement of Makover and Myers's (1943) method. The improvement allowed a whole range of observations to be carried out on one sample, whereas the original method required a different sample for each moisture level. Electric Hygrometer Methods These methods consist of direct measurement of ERH by the use of an electric hygrometer. An electric hygrometer is an instrument made of two electrodes connected with a hygroscopic salt bridge. As the salt absorbs or loses moisture in equilibrium with the atmosphere to which it is exposed, the electrical resistance of the bridge changes. The resistance can be measured with the appropriate measuring instrument. Graebner (1944) described a method using an electric hygrometer to measure the relative humidity of the atmOSphere after the desired equilibrium, controlled by sulphuric acid, was reached. Yes (1944) used an electric hygrometer to measure ERH in fertilizers. Brockington (1949) measured the ERH of whole kernel 12 corn placing the hygrometer in direct contact with the corn. Menger (1954) suggested that the ERH in foods might be measured with a hair hygrometer. Generally, however, such methods have a low reliability. Weight Equilibrium Methods These methods use the principle that the gain or loss in the moisture content under any given atmOSpheric relative humidity increases with increasing difference be- tween atmOSpheric and product vapor pressures. The samples are exposed to controlled atmospheres of different relative humidities and the differences in weights are recorded. Dittmar (1935) used sulfuric acid of different con— centrations to control the relative humidity in humidity chambers. Makover and Dehority (1943) placed the sulfuric acid of known concentration in vacuum desiccators, which were evacuated to 5 mm of mercury. Yee and Davis (1944) designed a Special humidity chamber where the atmOSphere was constantly circulated. Humidity was controlled by supersaturated salt solutions. Cleland and Fetzer (1944) controlled the humidity by a stream of air of desired relative humidity. The relative humidity of air was adjusted by passing it through saturated salt solutions first and then through sulfuric acid. Winkfs (1947) method differed from the others in that he used a specially designed platform to replace the 13 left pan of the balance accommodating the crystallizing dishes. This way the samples could be weighed without changing the test conditions. Hellman and Melving (1948) used a vacuum pump to evacuate the air and reduce the vapor pressure to that pro— vided by saturated salt solutions. After equilibrium was reached the vapor pressure of the atmosphere was measured with a closed-end mercury manometer. Landrock and Proctor (1951) developed the graphical interpolation method. The method is based on the principle that the rate of gain or loss in the moisture of a product at any given initial moisture content and under any given atmospheric relative humidity increases with increasing difference between atmospheric and product vapor pressures. This method is further discussed under Methods and Materials. Other Methods Filter paper strips impregnated with certain salts were used to determine ERH above 90% relative humidity (RH) by Kvaale and Dalhoff (1963). The state——whether dry or wet-- of the test papers can be used as an indication of the ERH of a sample when they have been left together in a closed space. This method is further described under Methods and Materials. When a sensitive and reliable lithium chloride cell for the measurement of the deWpoint of air was announced by Hickes (1947) an attempt was made to adapt this cell for l4 measuring the ERH of foods. Values for ERH obtained in this way for NaCl solutions correlated satisfactorily with the data derived from measurements according to classical techniques. Determinations with the new method required considerably less time, one hour at the most, versus at least one day in other electric hygrometers as indicated by Waldman and Halvorson (1954) or several days by the older static methods Mossel (1951). Gur-Arieh, Nelson, Steinberg, and Wei (1965) developed a method to determine the aw of flour of high moisture content. The flour sample was allowed to equilibrate with water contained in a porous membrane, while under a constant mechanical pressure. After equilibrium was achieved the sample was analyzed for moisture content. A thermodynamic relationship enabled calculation of the a corresponding to w the pressure used in the cell. Results obtained with this method were in agreement with those obtained with the constant relative humidity desiccator technique in the over- lapping range of water activities. The two methods were also in agreement that the aw of flour was not affected by the particle size distribution. Monteith and Owen (1958) developed a method using thermo—couples for measuring relative humidity in the range of 95-100%. At high relative humidities a current of 30 milliamperes through a 38 single wire gauge chromel—p/ constantan thermocouple produces sufficient Peltier cooling to form a film of water on the junction which may then be used as the wet—bulb of a psychrometer. The method has a 15 sensitivity of.: 0.01% relative humidity when measuring the vapor pressure of water in liquid and porous material. METHODS AND MATERIALS Methods Used to Determine Equilibrium Relative Humidity To determine ERH in foods, three different methods were evaluated: a) The method using humidity sensing elements consisting of lithium chloride sensors and a potentiometer to measure changes in the electrical resistance of the cells due to changes in adsorbed moisture. b) The salt paper strip method of Kvaale and Dalhoff (1963). c) The graphical interpolation method of Landrock and Proctor (1951). Humidity Sensor Method The first method is a relatively new one which utilizes a Minneapolis-Honeywell W611A relative humidity precision indicator and sensing elements mounted in slotted metal cases which protect the elements without interfering with air circulation over them. The W6llA Portable RH Readout instrument reads relative humidity directly from the atmOSphere of almost any area. There are seven different lithium chloride sensors for the different humidity ranges which are accurate over 16 17 almost the entire humidity range from 0% to 100%. The instrument is temperature compensated for normal use from 25 to 110 F with special compensation possible below 20 F. This method appeared the most promising of the three. It provides a precise (i 1.0%), but greatly simplified method of determining the relative humidity in most atmospheres where the temperature is between 20 and 140 F, and measurement can be made in a matter of seconds. The portable W611 RH readout instrument is easy to read by using expanded scale plates, each calibrated for a particular sensor range. Each scale plate has three concentric scales: compensation, temperature in degrees F, and RH. The interesting feature of this instrument is its ability to take a reading from a sealed area without the user having to open the vapor seal. The sensor is mounted inside the area and brought through the seal with a vapor—tight connection. For our purpose, one hundred m1 freeze drying flasks were redesigned so that the sensors could be mounted in the upper portion of the flask, leaving the lower portion for the samples. The sensors had their copper leads brought through teflon plugs and were then connected with the meter to read ERH. Each flask could accommodate three sensors or two sensors and a thermometer. The sensors were furnished by the company with 1 1% RH tolerance and with calibration data. Two sensors were obtained in the 80 to 100% and two in the 65 to 90% RH range. To reach equilibrium more quickly a mercury seal glass stirrer was brought through the standard l8 taper joint normally used to connect the flask to the freeze drying apparatus. It is essential that the sample, the container, and the sensors be at the same temperature within : 0.01 C at all times. A thermostatically controlled bath filled with distilled H20 adjusted to 30 C was used for this purpose. Five and ten percent standard sodium chloride (NaCl) solutions giving calculated ERH values of 98.42% and 96.66% were prepared and used to check the sensors. Ten ml were pipetted into the test chamber and with the sensor mounted in the upper part of the flask, the flask was sealed and immersed in the 30 C water bath leaving only the sensor leads and stirrer port exposed. Relative humidity readings were made three times a day. An ERH was reached within 24 to 48 hours. For the first few weeks the sensors seemed to be working properly giving readings close to the calculated values, but the two sensors were in disagreement with one always sensing lower RH values than the other. Also, relatively minor changes in the water level of the bath lowered the RH value considerably, which could not be explained by temperature difference alone. The air temper- ature above the water was the same as the water temperature since the bath was covered. When an incubator with constant temperature was used to replace the water bath, fairly constant RH values were obtained but they were much lower than the calculated values for the NaCl solutions used. 19 The experimentally determined RH values presented in Table l were obtained when the actual RH was 96.66%. The table also revealed that one of the sensors was registering 2% less RH than the other. This should not be the case since the calibration curve provided by the manufacturer showed very good agreement between these two sensors. The low readings obtained could be an indication that the sensors require recalibration since with time the cali- bration of these devices change and for very precise work they may have to be returned to the manufacturer for re- calibration, possibly after each experiment. Table 1. Percent ERH obtained with sensors using 10% NaCl as medium. Time in hours Sensor No. 0 24 48 72 246-2 88.3 89.8 90.2 89.8 246—41 90.2 91.2 91.5 91.3 Salt-paper Strip Method To ensure that the sensors were registering lower RH values in the chambers than the actual percent RH, we turned to our second method, the salt-paper strip method. This method uses strips of filter paper impregnated with certain salts. The state (whether dry or wet) of such test papers can be used as a direct indication of the ERH of a sample when the papers and the sample have been left together for some time in a closed Space. This method was developed 20 primarily for the measurement of humidities above 90% RH for use in studies of prepacked meat products. Filter paper strips were impregnated with salts covering the range of 90 to 100% RH and were placed in the test containers together with the sensors and 5 ml of 10% NaCl was added to control aw. An ERH was reached usually in 48 to 72 hours. Salt strips showed RH values in agreement with the calculated ones; 96 to 97% RH for 10%»NaC1, while one of the sensors registered 87.8% and the other one registered 86.4% RH. Salt paper strips were also tested in plastic petri dishes and the results obtained were in good agreement with those obtained previously when the strips were placed to- gether with the sensors. A 97.7 to 100% RH‘was obtained for a 5% NaCl solution (calculated percent RH is 98.42). The salt paper strip method was less sensitive to temperature changes than were the sensors. They could be placed at room temperature without any difficulties caused by minor temper- ature changes. Smaller containers are more advantageous since the smaller the air Space, the sooner an ERH is reached. It is well known that ERH determinations are rather uncertain at high humidity levels. Some of the salts gave uncertain results. In some instances, the strips got wet when the percent RH indicated by the other salts was lower than indicated by the first salt. Most of the uncertainties occurred above 96% RH which is the range in which we are 21 most interested. This discrepancy in the salt paper method and the limited accuracy with the first method focused attention to a third method: namely, the ”graphical inter- polation method." Graphical Interpolation Method This method requires that samples of the product under test be brought to weight equilibrium when exposed to a series of different RH. It is generally used for food products, gives satisfactory results with simple equipment, but requires numerous weighings and is time—consuming. Five m1 of liquid media or 5 g of solid media were put into petri dishes and weighed to the nearest 0.1 mg and then placed in a Stender dish containing various sulfuric acid solutions to produce atmospheres of known RHS. The samples were exposed to these controlled atmospheres for 48 hours at room temperature. After the eXposure period, the gain or loss in weight of each of the samples was determined (Table 2). These values were then plotted with reference to the RHS with the gains in weight above and the losses in weight below a horizontally drawn ”zero baseline" representing no change in weight. Interpolation of the ERH was made at that point where the smooth curve intersected the zero baseline (Figure 1). To check the accuracy of the method, 4.0, 4.5, 5,0 and 10.0% (w/w) NaCl solutions were used as standards having calculated ERH values of 98.95, 98.62, 98.48 and 96.91%, 22 Table 2. Graphical interpolation data for TPSY medium with KCl to control aw. .4 TPSY with Change (mg) in weight per hour at relative % KCl humidity (%) of (w/v) ‘ 96.8 97.4 97.8 98.2 98.7 99.3 4.5 — -1.00 -o.30 +0.46 +1.55 +2.88 - -1.30 —0.38 +0.50 +1.34 +2.58 5.0 - —0.66 +0.06 +0.82 - - - -0.72 +0.19 +0.92 - - 5.5 -1.10 —0.24 +0.30 - - — -l.60 —0.60 +0.38 - - - 6.0 -1.01 -0.11 +0.90 - - - -0.70 - +0.94 - - — 6.5 —1.07 0.00 - - — — —0.94 +0.07 +1.01 - - - respectively. The ERH values obtained with the method are tabulated in Table 3 and show very good agreement with the calculated values and were readily reproducible. Since this method exhibited the best accuracy, it was thus the method of choice for our further studies. Microorganisms: The microorganisms studied in the experiments were four strains of g1. botulinum type E. The strains and their sources were: 23 5.0% l.0~ 6.5% 6.0° .9— Mg. gain in weight per hour m 1 loss in weight per hour m L .6‘ .7~ .84 o '9‘ m S l 0... 9‘6 9‘7 978 9‘9 % Relative Humidity Figure l. Graphical interpolation isotherm for TPSY medium with KCl to control water activity. 24 Table 3. Percent ERH values of NaCl standards. Percent ‘ Percent Percent NaCl Std. (w/W) RH determined RH calculated 4.0 98.99 98.95 4.5 98.91 98.62 5.0 98.82 98.48 10.0 95.42 96.91 25 Name of strain Source Kalamazoo (K) Mr. R. W. Johnston, Food and Drug Administration, Detroit, Mich. Vancouver Herring (VH) Dr. C. F. Schmidt, Continental Can Co., Chicago, Ill. Seattle Forks (SF) Dr. J. T. Graikowski, Bureau of Commercial Fisheries, Ann Arbor, Mich. A 6247 Mr. R. W. Johnston, Food and Drug Administration, Detroit, Mich. Viable count procedure: Viable counts of Spore suspensions were obtained using Beef Infusion medium of the following formula: Beef infusion 1 liter Bacto peptone 1% Disodium phosphate 0.2% Glucose 0.2% Sodium chloride 0.5% Sodium thioglycollate 0.1% pH 7.2 Agar 1.5% Appropriate dilutions were prepared so that aliquots of the dilutions would produce 10 to 50 colonies per 16 x 150 mm screw cap test tube. Colonies were counted after 24 to 36 hours incubation at 30 C using triplicate tubes per dilution. 26 Toxicity assays: Toxicity was tested using intra- peritoneal injections into 15-20 9 Swiss Webster white mice. One ml of a sample was digested with 1 ml of 1% trypsin solution for 60 minutes at 37 C. A volume of 0.2 m1 of this digest was injected into a mouse. A second mouse used as control also received 0.2 ml of digest but was protected with 0.1 m1 of type E antitoxin (1:200 dilution of the Porton antitoxin.) Death of the unprotected mouse within 96 hours was considered a positive test. Solute-containing Media: All the water activity and osmotic pressure studies were conducted in Trypticase Peptone Sucrose Yeast extract broth medium (TPSY) to which various solutes were added. The TPSY formula used was: Trypticase 5% Peptone 0.5% Yeast extract 1.0% Sucrose 0.2% Na thioglycollate .0.02% pH 7.2 RESULTS AND DISCUSSION In our previous unpublished studies, it was established that 4.87% NaCl in TPSY medium inhibited the growth of Cl. botulinum Type E strains at 60 and 50 F incubation tempera- tures. At lower temperatures such as 45 F, 4.17 to 4.6%‘was the inhibitory NaCl range and at 41 F, 2.5 to 3.0% were inhibitory depending on the strains. To determine if the inhibition of outgrowth was a result of lowered aw of the media due to the added NaCl, ERH relationships were examined at various temperatures using NaC1 and other solutes. Water Activity The ERH values of 4.0, 4.5, 5.0 and 10.0% NaC1 solutions were determined and are presented in Table 3. After satisfactory results were obtained with the standard NaCl solutions, the ERH of TPSY media containing 2.0-5.0% NaCl were determined (Table 4). The ERH values obtained with the TPSY plus NaCl media are somewhat lower than those obtained for pure NaCl solutions, but this is quite under- standable since this medium is composed of several different ingredients, all of which would tend to depress the ERH. From the data in Table 4, it seems that the inhibitory value of ERH for Type E 91, botulinum in TPSY media with NaCl 27 28 Table 4. Equilibrium relative humidity values of TPSY media containing various amounts of NaCl. Percent NaCl (w/v) ERH 5.0 97.54 4.5 97.83 4.0 98.05 3.5 98.34 3.0 98.50 2.5 98.61 2.0 98.90 29 lies between 97.83 and 97.54%. Additional salts such as sodium formate (M.W. 68.01) and KCl (MAW. 74.55) were used to determine if this inhibition was due to a small amount of available water in the media or if NaCl was acting in higher concentration solely as a chemical preservative, or was an ionic effect due to high concentration of certain ions or an ion imbalance. Some nonelectrolytes such as sucrose, galactose, and glycerol were also evaluated. TPSY media with these electrolytes and non- electrolytes were prepared and concentrations were adjusted so that the inhibitory range previously obtained with NaCl would be covered. The ERH of these media containing the different concentrations of salts and carbohydrates was then determined (Table 5). The obtained ERH data agreed very well with the calculated data minus the ERH value of the TPSY medium. The ERH determinations showed that 35% sucrose decreased the aw of the TPSY medium to 0.9766 and 12.5% glycerol decreased the aw to 0.9648, while 5.5%,KC1 reduced the aw of the medium to 0.9762 and 5.0% sodium formate decreased it to 0.9761. According to the NaCl data these aw values may not permit growth. To investigate this, the different concentrations of these solutions were inoculated with Type E strains of Q1, botulinum. Four strains; K, A6247, SF, and VH were used for the sucrose studies, while the SF and VH strains were used for the KCl test and the VH 30 Table 5. Percent ERH values of TPSY medium with NaCl, KCl, NaCOOH, Sucrose and Glycerol. Percent a Solute (w/v) Percent ERH w NaCl 2.0 98.90 0.9890 2.5 98.61 0.9861 3.0 98.50 0.9850 3.5 98.34 0.9834 4.0 98.05 0.9805 4.5 97.83 0.9783 5.0 97.54 0.9754 KCl 4.5 97.97 0.9797 5.0 97.76 0.9776 5.5 97.62 0.9762 6.0 97.44 0.9744 6.5 97.31 0.9731 NaCOOH 3.5 98.50 0.9850 4.0 98.03 0.9803 4.5 97.91 0.9791 5.0 97.61 0.9761 5.5 97.08 0.9708 6.0 96.94 0.9694 6.5 96.41 0.9641 Sucrose 18.5 99.03 0.9903 22.8 98.76 0.9876 26.7 98.50 0.9850 31.0 97.97 0.9797 35.0 97.66 0.9766 38.5 97.55 0.9755 Glycerol 2.5 99.20 0.9920 5.0 98.57 0.9857 7.5 98.13 0.9813 10.0 97.83 0.9783 12.5 96.48 0.9648 15.0 95.40~ 0.9540 31 and K strains were used for the studies with sodium formate. Three screw cap test tubes of each concentration containing 15 ml of solution were inoculated with suspensions con- taining 0.1 m1 of 1.5 x 107 spores/m1. The tubes were then sealed with vaspar (mixture of 25% wax and 75% petroleum jelly) and incubated at 30, 21.2, 15.6, 10, and 7.2 C. Growth was observed by turbidity and gas production and toxi- city was checked by mouse toxin assay. Only those tubes were considered having type E outgrowth which showed gas and gave a positive toxicity test. Experiments using galactose could not be carried out since the amount required to depress the ERH to the 97.66% value was above the level of solubility of this carbohydrate. Sucrose was very soluble in the TPSY medium and dissolved easily up to 38.5%.concentration (w/v). The four strains tested produced good growth at all five temperatures in the triplicate tubes of TPSY medium with 31% sucrose concentration at aw 0.9797 (Table 6). However, none of them was able to grow in 35% sucrose con— centration at aw 0.9766, except that in one instance one tube inoculated with the SF strain showed outgrowth at the 21.1 C incubation temperature after 29 days. This could be an indication of strain variation, in that this strain was capable of growth at a somewhat lower aw than the other strains. The tubes inoculated with the K, A6247 and SF strains showed growth after one or two days incubation in the 31% sucrose concentration at 30 C, while the VH strain .©m>ummno mm3 £u3oum oz I .Umms mmB mamamm 02 A 32 I I mm I I m> I I hvmmfl I I & mmhm.o m.mm I I mm I I m> I I hvmofi I I I M mohm.o o.mm mm.mm.mm mm.mm.mm h A m.m.m N.N.N mm mm.©m.®m no.h©.h® m H NH.NH.NH ¢.¢.¢ m> mm.mm.mm hm.hm.hm b h h.m.m N.N.N hvmofi mm.mm.mm .mm.mm.mm m m m.m.m ¢.¢.H & hmhm.o o.Hm hm.hm.bm HN.HN.HN m .m N.N.N N.N.N mm I I I mm.mm.mm m .m ¢.¢.v H.H.H m> mm.mm.mm HN.HN.HN @ .0 N.N.N N.N.N h¢~©< mm.mm.mm hm.hm.hm w A N.N.N H.H.a & ommm.o b.0m HN.HN.HN HN.HN.HN A A A mm m¢.m¢.m¢ I I I A A A m> HN.HN.HN HN.HN.HN A A A hwmom ma.ma.ma ma.ma.ma A A A M mhmm.o m.NN HN.HN.HN A A A A hm oe.o¢.ov A A A A m> HN.HN.HN A A A A hfimo< momm.o m.mH ma.mH.oa A A A A M NS 3 om 3 3}; cflmupm m X .OGOO ADV musgmummawu coaqusOsH mmouosm m Houucoo OD Umms mmouosm nufl3 EsHme Mmma CH mmmp oma pmumflsocfl mcflmuum ESQHHDDOQ 2AM m mama Asom mo m>mm SH mEHu £D30Hmuso .o magma 33 required a lag period of 4 days. At lower temperatures this difference in the lag time became more pronounced. At 21.1 C, the lag time of the VH strain increased to 12 days, at 15.6 C to 15 to 20 days, and at 10 C to 67 days. The comparable lag times of the other three strains were 5 to 7, 5 to 7 and 29 to 35 days, respectively. Microscopic observation of the cultures in sucrose solution showed normal cell structure with some chain forma- tion when incubated at 15.6 C or higher. Sporulation was also readily observed. Control of aw by glycerol did not produce the expected results. A concentration of 12.5% glycerol (aw 0.9648) increased the lag time but did not inhibit growth, although at this aw level an inhibition of growth was observed with the other compounds. Even when the glycerol concentration of the medium was increased to 15% (aw 0.9540) there was no inhibition of growth at 30 and 21.1 C but an increased lag time occurred. The ERH values determined for these two glycerol concentrations were much.lower than found by calcu- lation (12.5% measured ERH = 96.48%, calculated = 97.60%; 15% measured ERH = 95.40%, calculated = 97.14%). Why glycerol, the alcohol form of a three carbon sugar, does not inhibit growth at the same aw ranges as the other inves- tigated compounds is not as yet known. There is no clear cut answer to this problem yet, and until further evidence is obtained we have to settle with a negative answer. When the aw was reduced with KCl (Table 7), Good 34 A A I I I mm ammo.o 0.0 A I I I I m> A I I I I mm AAAm.o 0.0 I I I I I ma m> I I 1.0N.oa I I mm N0hm.o m.m I I NN.¢H.¢H mm.HH.HH m.m.m m> I I 0.0.0 0.0.0 I I 0 mm 0bhm.o 0.0 I I 0.0.0 m.m.m m.m.m E I I 0.0.0 0.0.0 0.N.N mm 5050.0 m.¢ N.» 3 0.3 HAN om 3 3}: cannum m .x .0200 ADV wusumummfiwu coHquSUSH HUM 3 . m Houucoo ou 00m: HUM nufl3 E5H0®E NmmB SH m>m0 oma pmumnsocfl mcflmuum ESSHHDUOQ .Am.m mama m> 0cm mm mo m>m0 SH mEHu QHBOHmuSO .h mHQmB 35 growth was observed up to 5.0% KCl concentration (aw 0.9776), while only some sporadic growth was observed at the 5.5% (aW 0.9762), concentration. One tube inoculated with the VH strain showed growth after 18 days at 30 C, and two tubes inoculated with the SF strain produced growth after 10 and 20 days at 15.6 C. In the medium where aw was controlled with sodium formate (Table 8), outgrowth occurred at the 5.0% level (aw 0.9761), and at lower concentrations, but not at the 5.5% concentration (aw 0.9708). Microscopic observation of the cultures grown in the KCl and sodium formate controlled media Showed filamentous structure of the vegetative cell and long chains. Sporu- lation was never observed. The inhibition of growth with the tested solutions could be inhibition due to aw. Since no growth has been observed with concentrations of 38.5% sucrose (aw 0.9755), 6.0%.KC1 (aw 0.9744), 5.5% sodium formate (aw 0.9708), and 5.0% NaCl (aw 0.9754) thus far, the inhibitory aw level of growth for the tested Type E strains in these solutions appears to be between 0.9708 and 0.9755, if inhibition is due to aw. The slight difference between the expressed inhibition by the salts and the sucrose may be an indication of a dif- ference in ionic concentration or it could be merely an experimental error. Thus, the possibility of ionic effects were examined further. Ionic Effect: Sucrose in the media inhibited out- growth at the 0.9755 aw level while NaCl, Na formate, KCl 36 H H H u H mm mem.o m.m H H H H H “Aw «mom.o 0.0 H H H H H mm monm.o m.m H N AA AAA AAA A... H AAA AAA AAA mm.~w.wM NA.NW.NA A A A mm momm.o o.v mewmumm A A A A Aw ommm.o m.m «.5 OH o.mH H.Hm om aflmuum 3m x_mwme AOV musumummfimu coaquDUGH mumfinom mz .3m Houucoo 0p 00m: mumEHom m2 EDHS ESHUGE MmmB SH m>m0 oma magmasocfl mcflmuum 8523309 .40. m 0&3. E 6cm x no ammo ca 0.5» nusoumuso .m magma 37 inhibited at aw levels 0.9744 to 0.9708. The difference in aw values was not appreciable. Although the concentration of ions was high in the electrolyte solutions and practically no ions were present in the non-electrolyte solution, the aw levels inhibiting growth were very close. The following calculations show the percentage composition of the salts: NaCl (M.W. 58.4§)_ NaCOOH (M.W. 68.01) KCl (M.W. 74.55) Na = 39.2% Na 33.6% K 52.5% c1 60.6% c 17.6% C1 47.5% 02 46.9% H 1.4% The above calculations reveal that the percentage of sodium is different in the two sodium salts. NaCl contains 6.4% more Na ion than sodium formate. The KCl salt contains 52.5%1K ion. The anions are also in a dif- ferent ratio, 60.0% C1 ion in NaCl and 47.5% in KCl, while in sodium formate there is 65.9% formate: however, all three salts inhibited outgrowth within close aw levels, and at the same approximate a levels the non-electrolyte w sucrose was inhibitory. The lag times at 30 C for the VH and K strains in 4.5% sodium formate concentration (aw 0.9791) were 3 to 4 days, 2 to 6 days in 4.5% KCl for the SF strain and was 5 days for the VH strain. At the same incubation temperature in 35% sucrose (aw 0.9797), the SF and A6247 strains re- quired only 2 days and the VH and K strains required 4 days 38 to Show growth. At almost identical aw levels the ability of the strains to germinate and start to grow was only slightly different. They all started to grow in 2 to 3 days except the VH strain. This strain seems to require somewhat more time to Show growth in the sucrose medium and even more time at lower temperatures. Also, the minor differences existing among the other strains could indicate either a strain difference or an ionic effect. The possibility of osmotic effect was also investigated. Table 9 Shows that the 38.5 sucrose solution, which is approxi- mately 1.12 molar (M) has an osmotic pressure of about 25.08 atmospheres. A 6.0%.solution of KCl is 0.80 M‘with respect to both K+ and Cl- ions, hence its osmotic pressure is equal 2 x 0.80 x 22.4 or about 36.0 atmOSpheres. A 5.5% sodium formate solution which is 0.80 M produces an osmotic pressure of about 35.8 atmOSpheres, while a 5.0% NaCl solution is 0.86 M and produces an osmotic pressure of about 38.5 atmospheres. The osmotic pressure of a 15%, 1.63 M glycerol solution is about 36.5 atmospheres and at this molarity or osmotic pressure, glycerol still permitted outgrowth. The molar concentrations of the inhibiting salt con- centrations are similar (0.80 to 0.86) and thus so are the osmotic pressures. The molar concentration and the calcu- lated osmotic pressure of the inhibiting sucrose Solution (1.12 M) is different from that of the salts. Therefore, the growth inhibiting characteristics of the solutes tested thus 39 Table 9. Calculated molar concentration and osmotic pressure values of glycerol, sucrose, NaCl, KCl and sodium formate solutions at 0 C. —— —V_—— Percent solute (W/v) Molarity Osmotic pressure in atmospheres Glycerol . 10°C 1'08 24.20 15'0 1.63 36.50 20°C 2.17 48.61 Sucrose 10.0 0'29 6.54 22.8 0.66 14.78 26'7 0.78 17.47 31°C 0.90 20.16 35°C 1.02 22.84 38.5 1.12 25.08 NaCl 4'5 0°77 34.40 4'7 0.81 36.28 5'0 0°86 38.52 10'0 1.72 77.04 KC 4.5 0.60 27.01 5'0 0'67 30.02 5.5 0.73 33.00 6'0 0.80 36.02 Sodium Formate 4.0 0.58 25.98 4.5 0.66 29.56 5'0 0.73 32.70 5.5 0.80 35.80 40 might not be explained using the molar concentrations of solutes that were added to the TPSY medium. However, the gas law equation is valid only at low solute concentrations and osmotic pressure generally increases with higher sucrose concentrations much more rapidly than predicted by the simple equation used to calculate osmotic pressure. The inhibiting sucrose concentration could have an osmotic pressure similar to those calculated for the salts. Therefore, the growth inhibiting characteristics of the solutes tested thus may also be explained with the osmotic phenomenon, if selective permeability is involved. The glycerol data are not readily explained by either the aW theory or osmotic pressure phenomenon and should be the subject of further study. Microscopic examination of the cultures showed that with sucrose in the medium the bacteria grew normally and only an increase of lag time was noticed. Also the rate of growth was not affected and Sporulation followed growth. In the salt-controlled medium the cultures showed long filamentous vegetative cells, chains, some curved rods, and a few straight rods. Sporulation was never observed. These differences in the electrolyte and non-electrolyte controlled media indicate that the salts beside lowering the aw may also affect the normal growth of the bacteria. Temperature effect: Generally.temperature had an effect on the lag period, the rate of growth, and the number of cells produced. The lag time increased with lower _ 41 incubation temperatures, and the rate of growth and the amount of cellular material produced decreased. The optical density (OD) of the cultures dropped from an absorbancy of 0.7 to 0.1 at 625 mu as the temperature decreased from 30 to 7.2 C. When 31% sucrose concentration (aw 0.9766), was used to control the aw at 30 C all four strains started to grow within 1 to 4 days, while at 21.1 C the lag times were Slightly increased and with the exception of the VH strain they showed growth in 3 to 7 days. The VH strain required 12 days to initiate growth. At 15.6 C, the lag times were again increased a few days, while at 10 C the increase was very substantial. The K, SF, and A6247 strains started to grow only after 21 to 37 days incubation. The VH strain had the longest lag time of 67 days. With less sucrose in the medium, the aw level is increased and the microorganisms can better tolerate the adverse effect of lower incubation temperatures which results in reduced lag times, increased growth rates, and an in- creased production of cells. This is well demonstrated with the 26.7% sucrose concentration in the medium. Four per- cent less sucrose in the medium increased the aw from 0.9797 to 0.9850, and at 10 C the lag time was decreased by 8 days for the K strain, 16 days for the A6247 strain, 12 days for the VB strain, and 8 days for the SF strain. In the media containing KCl and sodium formate, the observed lag times at 30, 21.1 and 15.6 C incubation tempera- tures were very Similar to those obtained from the experiment 42 with sucrose. Hewever, at the lower temperatures (10 and 7.2 C) no growth was observed with the SF and VH strains in the 4.5% (aw 0.9797), and 5.0% (aw 0.9776) KCl media, while the 4.5% (aw 0.9791) sodium formate concentration inhibited the outgrowth of the K strain but not the VH strain. This strain showed growth after 42 and 53 days incubation at 7.2 C. If these findings are compared with the data obtained with the sucrose controlled media the data Show that at aw 0.9797 all four strains grew after a lengthy incubation time. This may indicate that at low temperatures such as 10 and 7.2 C the electrolytes are a little more effective in inhibiting growth, or that the actual aw is slightly different from the recorded one. The inhibitory aw levels of the K, A6247, VH and SF strains were measured as 0.9755, 0.9708 and 0.9744 when sucrose, sodium formate, and KCl, respectively, were used to control the aw of the TPSY medium. Strain variation may produce growth at slightly lower aw levels, but Since no growth has been observed at aw 0.9755 (sucrose), aw 0.9708 (sodium formate) and aw 0.9744 (KCl) thus far, the inhibi- tory level of growth for the investigated Type E strains in these solutions appears to be between the 0.9708 and 0.9755 aw levels, if inhibition is due to aw. The Effect of Preheating and the Size of the Inoculum on Outgrowth in Water Activity Media The aw range of the K, A6247, VH, and SF strains was established in the previous experiment, using a 1.0 x 106 43 spores/ml inoculum. To determine how the size of inoculum and preheating spores before inoculation affects outgrowth the following sizes of inocula were used: 1 x 106/ml, l x 104/ml and l x 102/ml. All three inocula were tested with and without heat shock. The Spore suspensions were heat treated for 15 minutes at 60 C. Since, at this temper- ature a number of Spores would be injured or killed, the suSpensions were readjusted based upon viable counts to compensate for this loss. Spores were inoculated and incu- bated in the aw media at five different temperatures as in the previous experiment, but only the K and VH strains were used in this experiment. Effect of the size of the inoculum: In the sucrose medium (Tables 10, 10a) both strains produced outgrowth in the 31% concentration (aw 0.9797) but not in the 35% concentration (aw 0.9766). The size of the inoculum affected lag times, but not outgrowth of the VH strain. The lag time increased significantly as the size of the inoculum decreased in the 15.6-21.1 C temperature range. At higher or lower temperatures only the K strains Showed appreciable differences in the lag times due to inoculum size. This strain Showed poor outgrowth even at 30 C when the 1.0 x 102/ml inoculum was used, and at the 7.2 to 10 C temperature range no outgrowth at all was observed. The VH strain showed outgrowth at 30 C in 2 to 3 days in all tubes regardless of the inoculum size, while in the 7.2 to 10 C temperature range outgrowth was observed only after 32 days of incubation. 44 ..pmummuu ummn mm3 cofimcmmmsm whommIm .00>Hmmno mm3 £u3oum 02 I I I I I I moa x o.H I I I I I mmoa x o.H I I I I I AVOH x o.H mmhm.o 0.0m I I I I I mwoa x o.H I I I I I 00H x o.H I I I I I N0OH x o.H I I I I I NOH x o.a I I I I I mmoa x o.H I I I I I 00H x o.H 00mm.o 0.0m I I I I I EACH x o.H I I I I I 00H x o.H I I I I I 300H x o.a Nm.mm.mm mm.mm.mm 0N.¢N.vm OH.OH.OH I.N.N «OH x o.H I I.mm I.0v.0¢ I.HN.HN I.MH.HH m.m.m mmoa x o.a mm.mm.mm mm.mm.~m mH.mH.mH HH.HH.HH N.N.N «OH x o.H Amnm.o o.Hm I.0v.mm mm.mm.mm 0w.mH.mH NM.HH.HH m.m.N mvoa x o.H mm.mm.mm hm.hm.hm SKK h.m.m N.N.N 00H x 04 mm.mm.mm om.om.om NH.NH.NH 0.0.0 N.N.N m0oa x o.H m4. 3 o .3 H. S om 535005 3 3}: 00 m X .ocoo ADV musumnmeMp coflumflsocH wNHm wmouosm .30 Houusoo on mmonosm £0H3 ESH00E MmmB GA mwmp om pmumnsocfl cflmnum EDSHHDHOQ aflw m 0Q>B m> 050 MO mhmm SH 05H“ SUBOHmuso com: msflpmmsmum 0cm mNflm Egasoocfi mo uommmm 0:8 .oa manma 45 I I I I I I I I I I I I I I I H.HNOH x o.H I I I I I I I I I I I I I I I mNoH x o.H I I I I I I I I I I I I I I I H.HAVOH x o.H 0050.0, 0.0m I I I I I I I I I I I I I I I M¢OH x o.H I I I I I I I I I I I I I I I m0OH x o.H I I I I I I I I I I I I I I I mNOH x o.H I I I I I I I I I I I I I I I mNOH x o.H I I I I I I I I I I I I I I I EvOH x o.H 00mm.o 0.00 I I I I I I I I I I I I I I I MVOH x o.H I I I I I I I I I I I I I I I m0OH x o.H I I I I I I I.ov.o¢ 0N. I.0N I .mH mNOH x o.H I I I I I I I I.0N I I I ENOH x o.H I I I 00.0v.ov 0N.0N.0N 0H .0H .0H 0.0.0 mfioH x o.H hmhm.o o.Hm I I.H¢ I I.H¢ hN.hN.hN I.H¢. AN I.m.0 mvoH x o.H 00.00.00 00.00.00 0.0.0 0.0.0 ¢.v.H H.H0oH x o.H 00.H¢.Hv H¢.H¢.H¢ I.H¢.ON I.¢H.¢H m.m.m m00H x o.H N.h 0H 0.0H H.HN om EDHSUOSH 3 A>\3v mo mNHw m x..ucoo HOV musumummfimu soHumnsocH mmonusm .3m Honusoo on mmonosm nuH3 EsHpmfi Nmms CH mmmp om pmmeSUSH CHmHuw.deHH5uon .Aw.m mm>B M 03“ mo m>00 CH wEHp LHBOHmuso coma mcHummzmum paw muflw ESHDUOSH no #00000 038 .MOH 0HQMB 46 When sodium formate (Tables 11, 11a) was used to control aw, outgrowth was observed in the 4.5% concentration (aw 0.9791), while in the 5% concentration (aw 0.9761) only the 1.0 x 106 Spores/ml inoculum produced outgrowth in the 15.6 to 30 C temperature range. Again, in this medium the outgrowth of the K strain was affected more than the outgrowth of the VH strain in the lower (1.0 x 102/m1 and 1.0 x 104/ml) inoculum ranges. These inocula were inhibited at the lower (7.2 and 10 C) incubation temperatures. The K strain exhibited poor outgrowth at the higher temperatures as well. Only two tubes out of the three were positive with the 1.0 x 102 spores/ml inoculum at 21.1 c and no outgrowth was observed at 15.6 C. In the KCl medium (Tables 12, 12a) the two strains showed widely different outgrowth patterns. The VH strain produced outgrowth in the 5.5% concentration (aw 0.9762) while the K strain was completely inhibited at this concen— tration. Both strains were inhibited in the 7.2 to 10 C temperature range in the 4.5%.KC1 concentration (aw 0.9797). The 1.0 x 102 Spores/ml dilution of the K strain was inhibited even in the 4.5% concentration (aw 0.9797) except for one tube, which was positive after 10 days of incubation at 30 C. The VH strain showed erratic response in the higher dilutions. The effect of_preheating: No beneficial effect of the heat shock was observed with the K strain. 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N0 I.o~. 00 kOKOI a; \ .00 LOKDI I.m .0 .0 m 00 00 £00 00 $00 00 O r-lv-ir-lr-II-lv—l 000000 000000 00000 00000 xxxxxx \OWKOQ‘Q'NN hmbm.o m.¢ N.h 00 ®.m0 0.0m om AUV 00000000500 000000000H 00000000 00 0000 3}: 0 X. . 0000 I 00% 3 . 0 0000000 00 00% 0003 E00008 M009 00 0%00 om 000000000 000000 000000000 aflw m 0Q>B K 000 00 0>00 00 0800 00300m000 0000 0000000000 000 0000 00000000 00 000000 008 .0m0 0000B 51 7.2 C, but in most cases Spores with heat treatment required longer times to germinate and to produce outgrowth. With smaller inoculum sizes, irregular growth response and some- times complete inhibition were observed. especially with the K strain. The heat shocked inoculum containing 1.0 x 102 spores/ml of this strain did not produce any growth in the 35% sucrose medium (aw 0.9797), except for one tube which was positive after 28 days of incubation at 15.6 C (Table 10a). The spores without heat treatment produced outgrowth at 30 C in one tube in 13 days, at 21.1 C in two tubes in 26 days, and at 15.6 C in two tubes in 4Q days of incubation. The inhibition and irregular response were more evident at the lower temperatures. Outgrowth of the K strain was inhibited except for one tube when the 4.5% (aw 0.9797) and 5.0% concentrations (aw 0.9776) of KCl media were inoculated with 1.0 x 104 heat shocked spores/ml (Tables 12,12a). The heat shocked VH spores produced poorer outgrowth in this medium also. The 1.0 x 102/01 inoculum size was inhibited at all three concentrations tested, and prolonged lag times could also be observed. This prolongation of the lag times or even inhibition was observed in the sodium formate medium, too, (Tables 11, 11a), especially with the K strain. Tubes receiving inocula containing 1.0 x 102 heat shocked spores/ml were negative even at 30 C, while the non heat shocked inocula produced positive tubes. 52 The temperature effect: Temperature definitely had an effect on the lag period as temperature became limiting for the bacteria. Lag time increased as the temperature cbcreased. As shown in Tables 10a and 11a, the 7.2 to 10 C incubation temperatures inhibited the growth of the K strain in the 4.5% KCl and Na formate concentrations (aw 0.9797 and 0.9791), except for two positive tubes at 7.2 C in the sodium formate medium. The VH strain was also inhi- bited at these temperatures in the 4.5% KCl medium (aw 0.9797, Table 12). The inhibitory effects of decreasing incubation temperatures produced erratic outgrowth when the size of the inoculum decreased. The K strain was more sensitive to the adverse effect of lower temperature than the VH strain, although in the KCl media both strains were inhibited at 10 and 7.2 C. In the sodium formate medium the VH strain produced outgrowth at these temperatures and the sucrose medium yielded more regular outgrowth. These observations point out again that functional differences between the strains do exist. Generally, a decrease in the size of the inoculum resulted in irregular outgrowth and inhibition at higher concentrations in all of the three media tested. When a decreased number of Spores was inoculated into the media, inhibition was found at aw levels previously found uninhi- bitory for the larger inoculum sizes. Heat shock generally had an adverse rather than a beneficial effect on the spores of the strains tested. In 53 most cases the heat treatment delayed or even inhibited outgrowth at concentrations where spores without heat shock grew well. This adverse effect of heat was best demonstrated with the smaller (1.0 x 102 spores/ml and 1.0 x 104 Spores/ml) inocula. There was some indication that heat may initiate outgrowth at concentrations otherwise inhibitory for growth and that the beneficial or adverse effect of the heat treat- ment depends on the strain involved. The temperature effect became more defined at the lower incubation temperatures. At these temperatures the lagtjnmawas greatly increased and the rate of growth and the amount of cellular material was decreased. SUMMARY AND CONCLUSIONS The outgrowth of the K, A6247, VH and SF Type E Clostridium botulinum strains was studied in trypticase- peptone-sucrose-yeast extract (TPSY) medium with the addition of various concentrations of solutes. An inoculum size of 1.0 x 106/01 was inoculated into the TPSY medium and sucrose. sodium chloride (NaCl)0 potassium chloride (KCl), sodium formate and glycerol were added. The equilibrium relative humidity (ERH) of these media was determined with the graphical interpolation method and expressed as water activity (aw). The molar concentrations and osmotic pressures of the various concentrations of solutes were calculated and related to the observed inhibitions. The inoculated samples were incubated at 30, 21.1, 15.6, 10 and 7.2 C. Outgrowth was observed in the concentrations of 35% sucrose (aw 0.9766), 5.5% KCl (aw 0.9762), 4.8% NaCl (aw 0.9766) and 5.0%.sodium formate (aw 0.9761) and at lower concentrations but not in the 38.5% sucrose (aw 0.9755), 6.0%IKC1 (aw 0.9744), 5.0% NaCl (aw 0.9754) and 5.5%Isodium formate concentrations (aw 0.9708}. The approximate osmotic pressure of the 38.5%, 1.12 molar (M) sucrose solution is 25.08 atmOSpheres. The 6.0%.KC1 and 5.5% sodium formate solutions are approximately 0.80 M and produce osmotic pressures of about 36.0 atmOSpheres, 54 55 while a 5.0% NaCl Solution is 0.86-M and has an osmotic pressure of about 38.52 atmOSpheres. No inhibition of growth was observed with the 15% glycerol concentration. The use of 15% glycerol lowered the aw of the media to 0.9540, but did not inhibit outgrowth as observed with the other solutes. The 15% glycerol solution is approximately L.63 M and has an osmotic pressure of about 36.5 atmospheres. The existing difference in the inhibitory molar con- centrations between the sucrose and salts solutions suggest that inhibition of outgrowth may not be explained by the molarity concept. However, the osmotic pressures of the inhibitory concentrations of sucrose and NaCl solutes were 25.08 and 38.5 atmospheres respectively, and the gas law equation is valid only at low solute concentrations. Osmotic pressure generally increases with higher sucrose concentrations much more rapidly than predicted by the simple equation used to calculate osmotic pressure. The inhibiting sucrose concentrations could have an osmotic pressure similar to those calculated for the salts. Therefore, the growth inhibiting characteristics of the solutes tested thus may be explained with the osmotic phenomenon if selective permeability is involved. Since all the inhibitory salts and sucrose con- centrations exhibited similar aw values, the inhibition could also be explained by the aw phenomenon and if inhibition was due to low aw of the media tested then the inhibitory 56 level of growth for Type E strains in these solutions appears to be between 0.9708 and 0.9755 aw. Microscopic examination of the cultures showed that with sucrose in the medium the bacteria grew normally and only an increase of lag time was noticed. The rate of growth also was not affected and Sporulation followed growth. In the salt-controlled media the cultures showed long fila- mentous vegetative cells, chains, some curved and a few straight rods. Sporulation was never observed. These differences in the electrolyte and non-electrolyte con- trolled media may indicate that the salts also affected the normal growth of the bacteria. The effect of heat and the size of inoculum on out— growth was also determined with the K and VH strains. Three spore inoculum sizes, 1.0 x 102/01, 1.0 x 104/01 and 1.0 x 106/01 were inoculated into the aw media with and without a heat treatment. Incubation temperatures were the same as in the previous experiment. Heat shocking was done for 15 minutes at 60 C. An increased lag time, irregular outgrowth and in some concentrations complete inhibition was observed with the smaller inoculum sizes at concentrations found not inhibitory for the larger inoculum sizes. In the 5% concentration sodium formate medium (aw 0.9761) no out— growth was observed with the l.O x 102/01 and 1.0 x 104/01 spore inoculum sizes, while the 1.0 x 106/01 produced good growth. In the KCl medium, the VH strain grew in the 5.5% concentration (aw 0.9762) but the small 1.0 x 102/m1 size inoculum produced irregular outgrowth. The outgrowth of 57 the K strain occurred only with the larger inoculum (1.0 x 106/ml) in the 5% concentration (aw 0.9776). Inhi- bition was complete in the 5.5% concentration. Both strains showed more regular outgrowth in the sucrose media with the higher inoculum, while with the smaller inoculum erratic response occurred. When spores were heat shocked, an increase in lag time occurred especially with the smaller (1.0 x lOZ/ml) inoculum. A change in the inhibitory concentration was also observed: inhibition thus occurred at concentrations where spores without heat shock produced outgrowth. Incubation temperature affected the lag time, the rate of growth and the amount of cellular material produced. 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