An Investigation Into Water Relations Of Micro-Organisms Isolated From Intermediate Moisture Substrates

The influence of water activity (aw) on growth of a coccal isolate in media supplemented with three different a, controlling solutes is presented. Literature is reviewed concerning minimum ay for growth of bacteria, yeasts and molds with particular emphasis on the microbiology of Intermediate Moisture (IM) foods. The influence of ay on Staphylococcus aureus is also reviewed together with the effect different solutes have and the role and function of compatible solutes in microbial water relations. Preservation of IM foods using ay and other factors such as pH and preservatives is discussed. The concept of water activity and the structure of water in foods is presented and methods available for measuring ay are reviewed. A NovaSina HPL instrument was used to measure ay at 25°C. Statistical analysis of ay measurements showed that accuracy in the range 0.80 to 0.92 ay was within +1% of expected results. Precision (coefficient of variation) was in the range 0.07 to 0.70% for all sensors tested and between 0.12 to 0.18% for the sensors used in the study. Duplicate ay measurements gave mean values with a confidence interval of approximately 0.01 ay units. Details of the isolation of three cocci from a spoilt IM food at 0.82 ay and their identification are given. Growth studies were completed using isolate M10 with characteristics shown to be Similar to Staphylococcus epidermidis. Methods to maintain cultures including use of a "passage medium" to help preserve low ay tolerance and details on freeze drying isolates are provided. Test solutions consisted of a range of Brain Heart Infusion (BHI) broths supplemented with either sodium chloride, sucrose or glycerol to control water activities in the range 0.99 to 0.81. Incubation of test solutions in a shaking water bath was found to be unsatisfactory because extracellular capsular material formed and interfered with sampling. Test solutions were incubated. at 30°C without shaking in medical flats laid horizontally. Samples were plated on BHI agar using a Spiral Plate Maker (SPM) and were manually enumerated after incubation at 30°C. Statistical analysis of a comparison between the SPM and a traditional surface spread method showed that the two gave significantly different results at the 5% level of probability. However, regression analysis showed a high positive correlation between results by the two methods. Results of growth rates and lag times at various water activities are shown. The same general effect of ay on growth was seen in all three test systems. Plots of growth rates against aw showed negative binomial distributions. Maximum growth rates were at 0.97 to 0.98 aw and the limiting ay's for growth were 0.83 to 0.84 in sodium chloride, 0.81 to 0.82 in sucrose and approximately 0.81 to 0.84 in glycerol test systems. Growth rates were generally highest in sodium chloride, intermediate in sucrose and lowest in glycerol. Plots of lag times against aw showed exponential relationships. Below 0.98 ay, lag times were longest in glycerol, intermediate in sodium chloride and shortest in sucrose. Isolate M10 was characterized as a salt tolerant, glycerol intolerant organism but with unusual tolerance of low aw with sucrose as the aw controlling solute. A discussion on possible mechanisms of tolerance of low aw is presented. Finally, proposals for further work are discussed.