I. Introduction

Microwave radiation has gained popularity in the food industry for various processes such as thawing, drying, and baking foods [1]. The inactivation of microorganisms [2-7] by microwave radiation has great potential in the pasteurization of foods [8, 9]. The short heating and exposure time of microwaves is less destructive to food than normal conventional heating [10]. Various intrinsic and extrinsic parameters may influence the effect of microwaves on bacterial cultures. Microwaves create pores in the bacterial membrane, resulting in cell leakage [11] and death. The effect of microwaves has been studied on bacteria, such as Streptococcus faecalis [10,12,13], Salmonella [14], Escherichia coli and Listeria spp [15, 16].

Microwaves are electromagnetic waves with frequencies between 300 MHZ to 300 GHZ. A conventional microwave oven passes non-ionizing microwave radiation at a frequency of 2.45 GHZ (wavelength of 122mm) through food that cause “dielectric heating” as water, sugar and food molecules absorb these wsaves [16, 17]. Water molecules are electric dipoles with a positive charge at one end and a negative charge at the other end. When water molecules absorb microwaves, the molecules rotate in order to align their dipoles with the alternating electric field of the microwaves. This molecular movement generates heat, and when one rotating molecule strikes another, the heat is dispersed [17, 18]. This principle of dielectric heating can be applied to eliminate the bacteria from the culture media not only by reducing the time required to sterilize but also by limiting the damage to the media components caused by high temperatures in the autoclave. Thus, microwaves find several applications in biology ranging from staining to sample processing to extraction [19]. The damaging effect of ionizing microwave radiations from mobiles on proteins has been recently studied [20].

In this paper, we have studied the influence of a few parameters such as intensity of microwave radiation, time of exposure of the cells to microwaves, bacterial cell type, sporulation in bacterial cells, age of the bacteria, the amount of culture exposed to microwave radiation, and presence of various media components on the survival of bacterial cells exposed to microwave radiation.

II. Materials and methods

A. Bacterial Strain

E. coli and B. subtilis were selected for the study. These were grown in a 250 ml Erlynmayer flask containing 50ml of sterile nutrient broth. The E. coli and B. subtilis cultures were incubated in a New Brunswick Scientific environmental shaker (150 rpm) at 37°C and 28°C respectively for 24hours.

B. Microwave Oven

The microwave oven used was a Panasonic NNS235WF with a power of 800W. There were 5 modes of operation, Low – 10%, Defrost – 30%, Medium – 55%, High-Med – 75%, High – 100%, each with a different intensity of microwave radiation.

C. Standardization Of Bacterial Cells

E. coli and B. subtilis cells grown in sterile nutrient broth were washed twice in sterile phosphate buffered saline (pH7.4). The cell density for each culture was adjusted to an optical density 0.3 at 680 nm using a Perkin Elmer Lambda 25 spectrophotometer. The cell suspension at this density was used throughout the study.

D. Standardization of Microwave Intensity And Time For Bacterial Sterilization

To optimize the power and time required for the complete knockout of the bacterial cultures, the 24hour old cells of E. coli and B. subtilis were each taken in a sterile petriplate in a volume of 10ml. The cultures were exposed to 5 different intensities for a varying time such as 5, 8, 10, 15, 20, 30, 40 seconds respectively.

The culture suspension after being exposed to the microwave radiations was streaked on sterile nutrient agar. The plates containing E. coli and B. subtilis were incubated at 37°C and 28°C respectively for 24hours.

Results were recorded on the basis of visible growth obtained on nutrient agar plates. The increase in cell growth was expressed as increasing values from 1 to 7. In case of absence of growth, zero value was assigned. The same key has been used throughout the paper.

E. Effect Of The Culture Volume On Bacterial Sterilization

Varying amounts of E. coli cell suspension (10ml, 15ml, 20ml) were exposed to medium intensity microwave radiation for a period of 5, 8, 10, 20 and 30 seconds. The cell survival was studied by streaking the microwave exposed culture on sterile nutrient agar plates and incubating them at 37°C for 24hours.

F. Effect Of Age Of The Culture On Bacterial Sterilization

E. coli cell of varying age (24, 96, 336, 672 hours) was exposed to medium intensity microwave radiation for a period of 5, 8, 10, 20 and 30 seconds. The cell survival was studied by streaking the microwave exposed culture on sterile nutrient agar plates and incubating them at 37°C for 24hours.

G. Effect Of Intrinsic Media Components On Bacterial Sterilization

Sterile solutions of Glucose (1% w/v), Ammonium Chloride (0.2%) and Yeast Extract (3%) were individually added to sterile phosphate buffered saline (pH 7.4). E. coli and B. subtilis were suspended in phosphate buffered saline containing these media components. The cell suspensions of E. coli were exposed to medium intensity microwaves for a time of 5, 8, 10 and 12 seconds and those of B. subtilis were exposed to high intensity microwaves for a time of 15, 20, 25 and 30 seconds, to study their effect on cell survival. The cell survival was studied by streaking the microwave exposed culture on sterile nutrient agar plates and incubating them at 37°C and 28°C for E. coli and B. subtilis respectively for 24hours.

III. Results and discussiont

A. Standardization of Microwave Intensity And Time For Bacterial Sterilization

Standardization was done for E. coli and B. subtilis cells to determine the optimal intensity of microwave radiations and the time of exposure required for complete bacterial sterilization. The results obtained are depicted in Fig.1A and B.

Figure 1.
figure 1figure 1

The effect of microwave intensity and time of exposure on (A) E. coli and (B) B. subtilis cells

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It is clearly observed that the E. coli cells were sensitive towards the microwave radiations and were completely killed with medium intensity of the radiation when exposed for 10 seconds. However, the B. subtilis cells were observed to be more resistant towards the microwave radiation and were completely killed when exposed to high intensity for 40 seconds. This is due to the cell type, where E. coli is a gram negative bacterium, while B. subtilis is a gram positive bacterium. Also B. subtilis is known to form the dormant forms ie spores, which are extremely resistant to the harsh environmental condition. These results signify that B. subtilis is also resistant towards microwave radiations.

An increase in cell growth was observed in the B. subtilis cells at 15 second exposure. This could be due to breaking of the dormancy of the culture, which resulted in the increased cell growth.

B. Effect Of The Culture Volume On Bacterial Sterilization

The Effect of the culture volume on the survival of E. coli cells is seen in Fig. 2. As the volume of the culture increased the resistance towards sterilization increased. 10ml of the culture was completely killed in 8 seconds, but 20ml of the culture resisted being killed even after 10 seconds of exposure. However, it got killed completely at 20 seconds exposure.

Thus, we can concluded that as the volume of the solution being exposed to microwaves increases, the efficiency of microwaves in eliminating micro-organisms decreases and a longer exposure time is required to achieve successful knockout of bacterial cells.

Figure 2.
figure 2

The effect of culture volume on the survival of E. coli cells exposed to microwave radiations

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C. Effect Of Age Of The Culture On Bacterial Sterilization

The effect of the culture age on the survival of E. coli cells is seen in Fig. 3. It is observed that a one day (24hour) old culture was able to survive a 5 second exposure and completely killed after 8 seconds exposure to medium intensity microwaves. However, a 2 week (336 hours) and one month (672 hours) old cultures survived a 20 second microwave exposure. These could be completely killed after a 30 second exposure to medium intensity microwaves. It can hence be inferred that as the age of a bacterial culture increases, the efficiency of microwaves in eliminating these bacteria decreases. Older cultures are more resistance towards the effects of microwave radiations as compared to the young cultures.

Figure 3.
figure 3

The effect of culture age on the survival of E. coli cells exposed to microwave radiations

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D. Effect Of Intrinsic Media Components On Bacterial Sterilization

The effect of different media components was different on the two cultures (Fig. 4 A and B). The E. coli culture when exposed to medium intensity microwaves, exhibited a complete sterilization after 10 seconds exposure. However, in presence of ammonium chloride a shielding effect was seen, as the culture resisted the effects of microwave radiations. However, in the B. subtilis cells, presence of glucose resulted in an early sterilization of the culture. This suggests that the presence of glucose enhances the sterilization effect of microwaves.

Figure 4.
figure 4figure 4

The effect of some media components on the survival of (A) E. coli and (B) B. subtilis cells exposed to medium intensity microwaves over a period of time

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IV. Conclusion

Microwaves are extremely efficient in removing the harmful bacteria from food and other materials. However, the efficiency of these microwaves in completely killing the bacterial cultures depended on several factors like the age of the culture, volume of the culture exposed to the microwaves, the intensity of the radiations and also the media components that shield or enhance the microbial death.

Acknowledgements

The authors thank Prof. R. N. Saha, Director, BITS Pilani, Dubai Campus for the constant encouragement and support rendered throughout the project.