Bacterial culture and microwave treatment.

E. coli wild-type strain K-12 (3) was obtained from the Korean Collection for Type Cultures, and Bacillus subtilis KM107 (24) was obtained from the stock culture in our laboratory. E. coli was grown in Luria broth (1% Bacto Tryptone, 0.5% yeast extract, 1% NaCl) (33), and B. subtilis was grown in nutrient medium (0.3% Bacto beef extract, 0.5% Bacto Peptone) (35). The bacteria were cultured in 500 ml of the liquid medium at 37°C for 15 h on a rotary shaker (150 rpm). Cells were harvested by centrifugation and washed twice with a sterile 0.9% NaCl solution. The cell pellets were resuspended in a 0.9% NaCl solution at a cell concentration of 10⁹ to 10¹⁰ CFU/ml, which was used for the microwave radiation.

For the microwave heating, a 2,450-MHz microwave oven (MR301M; LG Electronics, Inc., Changwoon, Korea) was used. The cell suspensions were divided into 500-ml plastic beakers and maintained at 20°C. The plastic beakers with the cell suspensions were placed individually in the center of the oven and exposed to microwaves at full power (600 W). The temperature changes in the suspensions were monitored with a fluoroptic thermometer (950 channels; Luxtron Co., Santa Clara, Calif.). After microwave radiation, the suspensions were stored at 4°C for the following experiments. Figure ​Figure11 shows the correlation between the microwave radiation time and the temperature changes in the bacterial cell suspensions. A linear increase in the temperature relative to exposure time was observed, which was consistent when the microwave radiation was repeated.

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FIG. 1

Changes in the temperature of bacterial cell suspensions relative to microwave exposure time. A bacterial cell suspension in 0.9% NaCl was exposed to microwave radiation at 600 W, and its temperature changes were monitored.

Measurements of viable cell counts and nucleic acid and protein amounts.

The microwave-radiated cell suspensions were serially diluted with a sterile 0.9% NaCl solution and spread on Luria-Bertani agar (E. coli) or nutrient agar (B. subtilis) plates. The plates were incubated at 37°C for 24 h, and cells were enumerated. Cell density was measured at 600 nm using a spectrophotometer (CE393; Cecil Instruments, Cambridge, United Kingdom). The amount of protein released from the microwave-treated cells was measured at 595 nm by the method of Bradford (6). Bovine serum albumin was used as the standard protein. The nucleic acid content of the supernatants was directly measured at 260 nm using a UV spectrophotometer (U200; Hitachi Co., Tokyo, Japan). All the experiments were carried out in triplicate.

Electron microscopy.

After the cells were treated by microwave radiation, the shape of the cells was examined by electron microscopy. The cells were fixed at 24°C for 60 min with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany), dehydrated with a serial concentration of ethanol, and then dried on a critical point dryer (HCP-2; Hitachi Co.). The dried cell samples were coated with gold (26), and examined using a scanning electron microscope (S-4100; Hitachi Co.). For transmission electron microscopy, dehydrated cells were embedded in a medium type LR white resin (Sigma Chemical Co., St. Louis, Mo.), which was polymerized at 60°C for 24 h (26). The polymerized samples were sliced with an ultramicrotome and observed using a transmission electron microscope (Hitachi Co.).

Inactivation of bacterial cells by microwave radiation.

The inactivation patterns of the microwave-radiated cells were investigated using cell suspensions (10⁹ to 10¹⁰ CFU/ml) of E. coli, a gram-negative bacterial strain, and B. subtilis, a gram-positive strain (Fig. ​(Fig.2).2). The viable counts in both cell suspensions were found to dramatically diminish relative to an increase in the microwave heating temperatures. Treatment up to 80°C resulted in an approximate 5-log reduction of the viable count in both strains compared to the initial counts. The highest reduction ratio in the viable counts was observed when the temperature was increased from 50 to 60°C, which was a ca. 3-log reduction in E. coli organisms (from 1.1 × 10⁸ to 2.5 × 10⁵ CFU/ml) and a ca. 2-log reduction in B. subtilis organisms (from 3.3 × 10⁶ to 1.6 × 10⁴ CFU/ml). When the microwave heating temperature exceeded 60°C, the amount by which viable counts were reduced dramatically decreased. When the temperature was increased from to 60 to 80°C, the viable counts were reduced only by factors of 10 in the E. coli and 3 in the B. subtilis cell suspensions. Therefore, it is assumed that microwave heating for microbial inactivation is highly efficient up to a temperature of 60°C, yet not as effective at higher temperatures.

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FIG. 2

Changes in viable count and cell density in E. coli (left panel) and B. subtilis (right panel) cell suspensions relative to the temperature produced by microwave radiation. The temperature of 20°C shown on the x axis represents the temperature of cell suspensions before microwave treatment. ●, viable cell count; ■, cell density.

Although E. coli cells were slightly more sensitive to microwave radiation than B. subtilis cells when the temperature was increased from 50 to 60°C, B. subtilis cells were more sensitive than E. coli cells when the temperature was increased from 40 to 50°C. A temperature increase from 40 to 60°C resulted in a ca. 3.23-log reduction in the E. coli and a ca. 3.66-log reduction in the B. subtilis viable cell counts, indicating that B. subtilis cells are more inactivated by microwave heating than E. coli in this temperature shift. Because the cell wall of gram-positive bacteria is generally much thicker and stronger than that of gram-negative bacteria, it was expected that B. subtilis would be more resistant to microwave radiation than E. coli. However, B. subtilis was found to be more sensitive than E. coli when the temperature was increased from 40 to 60°C.

Interestingly, it was observed that cell density in both cell suspensions did not decrease in spite of a significant reduction in the viable counts. This may be due to the fact that the microwave-treated cells were not completely lysed even when they were inactivated by microwave radiation, and thus the cell density did not decrease.

Leakage of cell materials caused by microwave processing.

Another general indication of heat damage to microorganisms is the leakage of nucleic acid and protein from cells. Microwave-injured cells have often been reported to release ninhydrin-positive material, purines, and pyrimidines into a suspension (23). Nucleic acid and its related compounds, such as pyrimidines and purines, are well known to absorb UV light at a wavelength of 260 nm. The presence of these materials in a suspension indicates damage to the cell at the membrane level. Furthermore, similarly injured cells are also known to release intracellular proteins into a suspension.

The amount of nucleic acid released into the cell suspension was analyzed by measuring the absorbance at 260 nm (Fig. ​(Fig.3A).3A). The two bacterial strains showed similar patterns in their release of nucleic acid. The amount of leaked nucleic acid from the cells grew relative to an increase in the microwave-heated temperature of the cell suspension. However, the leakage of nucleic acid from B. subtilis was higher than that from E. coli. This result would seem to imply that B. subtilis suffered greater membrane damage than E. coli. The amount of protein released into the cell suspension was also analyzed in both strains (Fig. ​(Fig.3B).3B). Microwave heating up to 40°C resulted in no significant differences in the amount of protein leaked from the cells. However, when the treatment temperature exceeded 40°C, substantial differences in the amount of leaked protein were observed. These results indicate that most of the microwave-heated cells were ghost cells from which intracellular materials were released into the cell suspension. The protein release pattern of the two bacterial strains was the reverse of the nucleic acid release pattern; the amount of leaked protein in B. subtilis was found to be much lower than that in E. coli. In particular, a low level of protein leakage was observed when B. subtilis cells were heated to 60°C, a temperature observed to be sufficient for a 5-log reduction in the viable count. The reason for this is still unknown.

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FIG. 3

Nucleic acid and protein amounts released into the cell suspension from microwave-radiated bacterial cells relative to the temperature produced by microwave radiation. After the E. coli (●) and B. subtilis (■) cell suspensions were heated to the temperature shown on the x axis, the amounts of nucleic acid and protein in the supernatant of the cell suspension were measured.

Effect of microwave radiation on the surface structure of bacterial cells.

The opposite release patterns for the release of nucleic acid and protein in two bacterial strains prompted us to examine the surface structure of microwave-radiated cells (Fig. ​(Fig.4).4). The untreated cells and cells heated up to 70°C were examined using a scanning electron microscope, and the shapes of their surface structures were compared. It was found that untreated E. coli cells had a smooth surface, while most of the microwave-radiated cells exhibited severe destruction. The surfaces of the microwave-heated cells were damaged and had become rough and swollen. However, all the B. subtilis cells exhibited the same smooth surface. Whether the cells were microwave heated or not, no damage to their surface structures was observed. This result suggests that the microwave-radiated cells remained unlysed in suspension, although they were inactivated by the radiation. Furthermore, the damage to the surface structure of E. coli cells may not, therefore, be the main reason for inactivation by microwave heating.

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FIG. 4

Scanning electron microphotograph of untreated and microwave-radiated (up to 70°C) bacterial cells.

Sensitivity of microwave-heated cells to SDS.

In order to investigate the sensitivity of microwave-injured cells to lysis by sodium dodecyl sulfate (SDS), microwave-heated cells in 0.9% NaCl were incubated at 37°C with shaking (150 rpm) in the presence of 0.1% SDS, and the cell density was monitored at 600 nm (Fig. ​(Fig.5).5). For E. coli, the density of the microwave-heated cell suspension was dramatically reduced within an hour of incubation in the presence of SDS, but it did not decrease significantly in the absence of SDS. In the case of the untreated cell suspension, no significant reduction in the cell density was observed during 4 h of incubation, regardless of the presence of SDS. These results support the conclusion that most of the cells inactivated by microwave radiation remain unlysed in a cell suspension in the absence of SDS. In addition, they are also highly sensitive to lysis by SDS.

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FIG. 5

Sensitivity of untreated and microwave-radiated bacterial cells to SDS. E. coli and B. subtilis cell suspensions in 0.9% NaCl, both untreated and microwave heated up to 70°C, were incubated at 37°C with shaking (150 rpm). Changes in cell densities were then monitored at 600 nm, both with and without the presence of 0.1% SDS. ●, untreated cells in the absence of SDS; ○, untreated cells in the presence of SDS; ■, microwave-radiated cells in the absence of SDS; □, microwave-radiated cells in the presence of SDS.

When the experiment was repeated using microwave-heated B. subtilis cells, different results were obtained. In the absence of SDS, the cell density in both the untreated and microwave-heated cell suspensions slightly decreased in a similar pattern. Unexpectedly, however, the cell density in both cell suspensions slightly increased in the presence of SDS. Why cell density increased is still unknown. Although ambiguous results were obtained for the reaction of B. subtilis cells to SDS, it was obvious that microwave-heated B. subtilis cells were not affected by SDS. It was predicted that microwave-injured E. coli cells would be sensitive to SDS and that untreated cells would be resistant. However, in the case of B. subtilis, both untreated and microwave-heated cells were unexpectedly resistant to SDS. This may be due to the fact that the cells of B. subtilis, a gram-positive bacterium, were not lysed even in the presence of SDS because of their thick and rigid cell wall structure.

Effect of microwave radiation on the intracellular components of cells.

To investigate the effect of microwave heating on intracellular components, cells that were microwave heated up to 70°C were examined using a transmission electron microscope (Fig. ​(Fig.6).6). When microwave heated, both types of bacteria showed several dark spots in their cytoplasm. However, no dark spots were observed in the untreated cells, suggesting that the dark spots were the result of microwave heating. Heat treatment has been known to cause protein denaturation and aggregation in cytoplasm as well as to induce heat shock proteins (39, 40). Therefore, the dark spots are thought to be aggregated proteins caused by microwave heating. The two bacterial strains showed similar results for protein aggregation regardless of their cell wall structure, which suggests that protein aggregation may participate somehow in microbial inactivation caused by microwave heating. Further studies on the induction of heat shock proteins are in progress to elucidate whether microwave heating induces heat shock proteins in bacterial cells.

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FIG. 6

Transmission electron microphotograph of untreated and microwave-radiated (up to 70°C) bacterial cells.

This work was supported by a research grant from the Living System Laboratory, LG Electronics Inc. We are highly grateful for this support.