Moisture Loss from Agar Plates During Incubation

Abstract

Composition of media, quantity of medium in petri plates, incubator relative humidity, placement of plates on upper or lower shelf and time in the incubator were important variables in evaporative losses of weight of agar in Petri plates during incubation. However, the most important variable was temperature. Standard Methods for the Examination of Dairy Products (2) states for the Standard Plate Count that "agar in plates should not lose more than 15% of its weight during 48 h of incubation." Presumably, rates of colony development could be slowed if the loss of moisture exceeded this value. Since concentration of solute influences vapor pressure, it would be predicted that agar media containing large amounts of solids would lose moisture to the atmosphere more slowly than media containing lesser amounts. Other variables likely to effect rate of evaporation are (a) surface area per unit volume of medium, (b) temperature, (c) humidity and (d) rate of air movement in the incubator. The first of these is related to uniformity in quantity of medium poured, whereas the remaining three may vary with position within the incubator as well as incubator design and operation. This note presents results of a study of the following variables as they influenced loss of moisture from plates during incubation: temperature and time of incubation, position within the incubator and amount and type of medium in the plates. MATERIALS AND METHODS Plate Count Agar and Staphylococcus 110 Agar were formulated as per the Compendium of Methods for the Microbiological Examination of Foods Q). Each medium was pi petted in volumes of 12 and 17 ml per ;Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 8820. plate into Petri dishes (10 x 1.5-cm) preweighed to the nearest mg. Sufficient plates were poured to provide two each per medium per volume per position (top and bottom shelves) per incubator temperature (10, 21, 32, 37, 45 and 55 C). Plates were weighed after 0, 8, 24, 32, 48, 56 and 72 h of incubation. Amounts of moisture evaporated were calculated as the difference between initial weights and weights after the respective times of incubation. There were two replications. Data were subjected to analysis by the general linear models and analysis of variance procedures ofSAS (I). Incubators used in the study were as follows: 10 C, Lab-Line BOD type with circulating fan and external condensate drain; 21 C, Freas BOD type with circulating fan and internal condensate collection tray; 32 C, Freas BOD type with circulating fan; 37, 45 and 55 C, bench type gravity convection incubators with bottom shelves 15 to 20 em above heating elements and top shelves 25 em below ceilings. Temperatures were controlled within± 0.5 C in the BOD type incubators and ± 1 C in the convection-heated ones. Incubators contained only the plates being tested, and no source of other moisture was available except in the 21 C incubator which contained the internal condensate drip pan. Relative humidity was monitored continuously and simultaneously in each incubator over one 48-h incubation period. Belfort hydrothermo-graphs were each standardized under the same ambient air conditions. Relative humidities were as follow: 10 C, 42o/o, 21 C, 68 ± 3%; 32 C, 34o/o; 37 C, steadily increased from 34 to 40%; 45 C, steadily increased from 18 to 22%; 55 C, steadily increased from 22 to 24%. RESULTS AND DISCUSSION Figure 1 shows that at temperatures from 32 to 55 C the rate of moisture loss varied logarithmically with temperature. The rate of change in moisture loss from plates held at 32 through 55 C averaged 43 mg/day/C for PCA and 38 mg/day/C for SllO agar. Quantities lost from Plate Count Agar (PCA) were significantly higher than from Staphylococcus 110 agar (SllO) at both 45 and 55 C. This difference in rates of evaporation would be predicted because the vapor pressure of SllO agar would be considerably below that of PCA. The former contains 9.2% sugar and salt plus 3.25o/oproteinaceous substances, whereas PCA contains only 0.1 o/o and 3o/o of these components, respectively. Both contain 1.5% agar. In pure water the 7.5o/o NaCI of SllO agar would lower the vapor pressure by 34 mm.