Hiroshi Fujikawa

Fractal Growth of Bacillus subtilis on Agar Plates

Bacteria have been shown to grow with various morphologies under different conditions on agar plates. A Bacillus subtilis strain was inoculated on the plate surface and incubated at 35°C. Colonies grew two-dimensionally with random branches, similar to clusters of the diffusion-limited aggregation (DLA) model. The colony patterns were analyzed and found to be self-similar with a fractal dimension of 1.716±0.008, in excellent agreement with the expected value of the DLA model. Interior branches were observed to stop growing in spite of their open neighborhood during the incubation period, implying the existence of a screening effect. These results clearly suggest that the colony pattern of the organism was formed through the DLA process. Moreover, the colonies were found to grow radially with almost regular branches on agar plates with moist surfaces, reminiscent of “dense radial” morphology.

Bacterial Fractal Growth in the Concentration Field of Nutrient

A Bacillus subtilis strain is found to grow through the diffusion-limited aggregation (DLA) process on agar plates. The organism is spotted on the agar plate containing a low concentration of peptone as a single nutrient and incubated at 35°C. The colony pattern grown on the plate surface is self-similar with the fractal dimension of 1.73±0.02. Bacterial DLA branches are shown to grow in a concentration field of nutrient from the fact that they grow predominantly in the direction of higher nutrient concentration. The colonial morphology varies with the nutrient and agar concentrations of an agar plate, including DLA, a round type, dense branching morphology (DBM), and a spreading without openings. Two neighboring colonies repulse each other in DLA and DBM types only. On an agar plate containing glycerol the colony becomes remarkably round, quite different from the DLA morphology.

Modeling Surface Growth of Escherichia coli on Agar Plates.

ABSTRACT Surface growth of Escherichia coli cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H. Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.

Study of the Biological Activities of Toxic Shock Syndrome Toxin-1

The mitogenic and interleukin 2 (IL 2) production‐inducing effects of toxic shock syndrome toxin‐1 (TSST‐1) on murine lymphocytes were investigated. TSST‐1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome.

Characteristics of a multicomponent first-order model for thermal inactivation of microorganisms and enzymes

There are several mathematical models that express different types of non-linear thermal inactivation of microorganisms and enzymes. Among the models the characteristics of a multicomponent first-order model were studied on a calculative simulation. For the initial period of inactivation the pattern of the model was predominantly affected by the kinetics of the component with the highest initial concentration. For longer periods of inactivation the pattern was predominantly affected by the kinetics of the component whose rate constant was the lowest. It was also found that the observation period affected the apparent pattern of the model. The estimation method of parameter values of the model from inactivation curves was then studied.

Periodic growth of Bacillus subtilis colonies on agar plates

Abstract Bacillus subtilis colonies show periodic growth on agar plates. The organism has been observed to show several colony morphologies including diffusion-limited aggregation (DLA) type, dense branching morphology (DBM), Eden type, and spreading without producing openings. The agar concentration for the periodic growth is higher than that of DBM and lower than that of DLA or Eden type. The nutrient (peptone) concentration for the periodic growth is higher than that of DLA and DBM and lower than that of Eden type. The colony grows towards a place with higher peptone concentration. These findings suggest that the diffusion of nutrient particles, i.e. the concentration gradient of peptone particles at the growing perimeter of a colony, would be essentially involved in the periodic growth. The distance between concentric rings of a colony is constant and intervention between two colonies is not observed, unlike the Liesegang ring.

New Blue Pigment Produced by Pantoea agglomerans and Its Production Characteristics at Various Temperatures

ABSTRACT A bacterium capable of producing a deep blue pigment was isolated from the environment and identified as Pantoea agglomerans. The pigment production characteristics of the bacterium under various conditions were studied. The optimal agar plate ingredients for pigment production by the bacterium were first studied: the optimal ingredients were 5 g/liter glucose, 10 g/liter tryptic soy broth, and 40 g/liter glycerol at pH 6.4. Bacterial cells grew on the agar plate during the incubation, while the pigment spread into the agar plate, meaning that it is water soluble. Pigment production was affected by the initial cell density. Namely, at higher initial cell densities ranging from 106.3 to 108.2 CFU/cm2 on the agar plate, faster pigment production was observed, but no blue pigment was produced at a very high initial density of 109.1 CFU/cm2. Thus, the cell population of 108.2 CFU/cm2 was used for subsequent study. Although the bacterium was capable of growing at temperatures above and below 10°C, it could produce the pigment only at temperatures of ≥10°C. Moreover, the pigment production was faster at higher temperatures in the range of 10 to 20°C. Pigment production at various temperature patterns was well described by a new logistic model. These results suggested that the bacterium could be used in the development of a microbial temperature indicator for the low-temperature-storage management of foods and clinical materials. To our knowledge, there is no other P. agglomerans strain capable of producing a blue pigment and the pigment is a new one of microbial origin.

Effect of Native Microflora on the Growth Kinetics of Salmonella Enteritidis Strain 04-137 in Raw Ground Chicken

Effects of native microflora (NM) on growth kinetics of Salmonella Enteritidis strain 04-137 were studied in raw ground chicken. First, samples of ground chicken with high and low levels of NM (10(7.1) and 10(4.9) CFU/g, respectively) were spiked with Salmonella at doses ranging from 10(1) to 10(4) CFU/g. The growth kinetics, including the rate constant of growth, r, and the lag period, were similar, but the maximum cell level, N(max), was higher at higher initial Salmonella doses for both NM levels. Second, samples of ground chicken with high and low NM levels (10(6.8) and 10(4.7) CFU/g, respectively) were spiked with Salmonella and then stored at various constant temperatures ranging from 8 to 32°C. Both N(max) and r for Salmonella were higher at higher temperatures for both NM levels. Although r for total bacteria, which consisted of NM and Salmonella, was also higher at higher temperatures, N(max) was constant at all temperatures for both NM levels. Further, Salmonella growth was compared among samples of ground chicken with high and low NM levels and samples of sterilized chicken. Salmonella growth, characterized by both N(max) and r, was highest in sterilized chicken, followed by chicken with the low NM level. Our growth model successfully described and analyzed the growth of Salmonella and total bacteria in chicken at constant temperatures; using the data obtained, the model also successfully predicted the growth of Salmonella and total bacteria in chicken stored at dynamic temperatures. Our study clarified the effects that different doses of NM in ground chicken had on the growth kinetics of the Salmonella strain and demonstrated the usability of the growth model for foods with NM.

Comparison of capillary and test tube procedures for analysis of thermal inactivation kinetics of mold spores.

Characteristics of capillary and test tube procedures for thermal inactivation kinetic analysis of microbial cells were studied for mold spores. During heating, capillaries were submerged in a water bath and test tubes were held with their caps positioned above the level of the heating medium. Thermal inactivation curves of Aspergillus niger spores in capillaries at around 60 degrees C consisted of a shoulder and a fast linear decline, whereas curves in test tubes consisted of a shoulder, a fast linear decline, and a horizontal tail. There were no significant differences in values of the rate and the delay of fast declines in curves between the procedures. Some experiments were done to clarify the cause for tailing with test tubes. There were no tails with test tubes whose inner walls were not contaminated by A. niger spores, suggesting that tails arise from A. niger spores contaminating the inner walls of test tubes. Temperature of the inner wall at the level of a heating medium was lower than that of the medium. Further, there were no tails for test tubes submerged in the heating medium. These results showed that the reason for survival of contaminants on the upper wall of test tubes was that cells were not subjected to sufficient inactivation temperature. Finally, thermal inactivation curves of A. niger spores in capillaries at various constant temperatures were studied. Curves consisted of a shoulder and a fast linear decline at 57 degrees C and above, whereas curves at below 57 degrees C consisted of a shoulder, a fast linear decline, and a sloping tail.

Contamination of microbial foreign bodies in bottled mineral water in Tokyo, Japan.

A total of 292 imported and domestic bottled mineral waters (90 brands) obtained from consumers and retailers were examined, by eye, for observable microbial foreign bodies. Fungal and bacterial foreign contaminants were found in 45 samples of water (20 brands) and in 14 samples of water (10 brands), respectively. Of the samples of water found to be contaminated, 41 (22 brands) were imported and 18 (8 brands) were produced domestically. Of 22 brands that were contaminated, 20 (91%) had been sterilized by at least one method. Forty‐eight (98%) of 49 samples confirmed with foreign bodies were less than 1 year old. Among the moulds isolated the most predominant genus was Penicillium, followed by Acremonium and Cladosporium. The samples that contained fungi were less contaminated by bacteria than those that contained observable bacterial foreign bodies.