**4. Cell status by flow cytometry**

Flow cytometry (FCM) is a well-established and powerful analytical tool that has led to many revolutionary discoveries in cell biology and cellular-molecular disease diagnosis and, more recently, has been used to analyze physiological responses of bacteria [33, 34]. In FCM, cells are first introduced to a high-speed (up to 5–20 m/s) laminar flow stream, and after being focused into single file, they are subjected to laser-induced fluorescence, and/or forward and sideways scattered light is detected using photodetector arrays with spectral filters. More recently, FCM has been used to characterize distinct physiological conditions in bacteria including their responses to antibiotics and other cytotoxic chemicals [33]. Once the control of bacterial cells or fungal conidia has been applied, an accurate technique is required to measure the effectiveness of the silver nanoparticles. Flow cytometry is one of the most reliable techniques for detecting and counting living cells and to measure their viability.

When studying response to antibacterial agents such as silver nanoparticles, viability can be evaluated as an indicator of antibiotic susceptibility. There are now reagents available that allow assays of membrane permeability and potential by measuring the production of a fluorescent metabolite from a nonfluorescent precursor [33, 34].

Besides monitoring susceptibility to antibacterial activity, information can be obtained using FCM that can establish mechanisms of antibacterial drug

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**Figure 2.**

*Silver Nanoparticles Offer Effective Control of Pathogenic Bacteria in a Wide Range of Food…*

activity [35–40]. Traditional culture-based techniques cannot do this [41]. The use of fluorescent probes to detect specific cell changes provides a unique tool for interrogating bacteria permeability and changes in membrane potential [42] (**Figure 2**). DNA content and metabolic activity [42] are useful indicators of cell viability and

The accuracy of cell counting depends on fluorescent staining. The choice of a fluorescent dye should take into account factors such as membrane permeability, photostability, pH and sensitivity to temperature [43, 44]. The total bacterial count is a key quality criterion for food or beverages [45] and a useful tool for detecting the presence of microbes within matrices. Williams et al. [46] used this technique to detect *E. coli* O157:H7 in raw spinach. The presence of plant pathogens during crop growth has been investigated by several authors. Day et al. [47] used FCM to detect and quantify *Phytophthora infestans* sporangia. A study of colonization of root-associated bacteria in rice was carried out by Valdameri et al. [48]. Otherwise, Golan et al. [49] counted *Pectobacterium carotovorum* subsp. *carotovorum* cells tagged with green fluorescent protein (GFP) in *Ornithogalum dubium* seedlings to detect resistant cultivars. The application of FCM is useful to create the bases for predic-

Cell viability assays can distinguish between live and dead cell populations and so correlate with other cell functions or treatments. Many companies offer a wide range of viability dyes, including fixable and non-fixable types and ones specific to bacterial or yeast viability tests. FCM can be applied to monitor the efficacy of treatments to reduce contamination of water [43] and foods and beverages [45, 50] by determining the viability of residual microorganisms. In agriculture FCM can be used to test the effectiveness of antibiotics and antifungals against plant pathogens. The advantage of live FCM cell counts compared to plate counts is that FCM allows the determination of several different morbidity stages between living and dead cells. Some of these are membrane integrity, esterase activity, membrane potential, electron transport, total cells, GFP expression, active/dead, mitochondrial activity,

tive models of spore germination, infection and disease development.

*Fluorescent probes to detect specific bacterial cell changes as an indicator of cell viability.*

intracellular pH and carotenoid content [51–53].

*DOI: http://dx.doi.org/10.5772/intechopen.89403*

thus of antibiotic susceptibility.

#### *Silver Nanoparticles Offer Effective Control of Pathogenic Bacteria in a Wide Range of Food… DOI: http://dx.doi.org/10.5772/intechopen.89403*

activity [35–40]. Traditional culture-based techniques cannot do this [41]. The use of fluorescent probes to detect specific cell changes provides a unique tool for interrogating bacteria permeability and changes in membrane potential [42] (**Figure 2**). DNA content and metabolic activity [42] are useful indicators of cell viability and thus of antibiotic susceptibility.

The accuracy of cell counting depends on fluorescent staining. The choice of a fluorescent dye should take into account factors such as membrane permeability, photostability, pH and sensitivity to temperature [43, 44]. The total bacterial count is a key quality criterion for food or beverages [45] and a useful tool for detecting the presence of microbes within matrices. Williams et al. [46] used this technique to detect *E. coli* O157:H7 in raw spinach. The presence of plant pathogens during crop growth has been investigated by several authors. Day et al. [47] used FCM to detect and quantify *Phytophthora infestans* sporangia. A study of colonization of root-associated bacteria in rice was carried out by Valdameri et al. [48]. Otherwise, Golan et al. [49] counted *Pectobacterium carotovorum* subsp. *carotovorum* cells tagged with green fluorescent protein (GFP) in *Ornithogalum dubium* seedlings to detect resistant cultivars. The application of FCM is useful to create the bases for predictive models of spore germination, infection and disease development.

Cell viability assays can distinguish between live and dead cell populations and so correlate with other cell functions or treatments. Many companies offer a wide range of viability dyes, including fixable and non-fixable types and ones specific to bacterial or yeast viability tests. FCM can be applied to monitor the efficacy of treatments to reduce contamination of water [43] and foods and beverages [45, 50] by determining the viability of residual microorganisms. In agriculture FCM can be used to test the effectiveness of antibiotics and antifungals against plant pathogens. The advantage of live FCM cell counts compared to plate counts is that FCM allows the determination of several different morbidity stages between living and dead cells. Some of these are membrane integrity, esterase activity, membrane potential, electron transport, total cells, GFP expression, active/dead, mitochondrial activity, intracellular pH and carotenoid content [51–53].

**Figure 2.** *Fluorescent probes to detect specific bacterial cell changes as an indicator of cell viability.*

*Pathogenic Bacteria*

Disruption of biofilms is another effect of AgNPs. The anti-biofilm action of ~8 nm AgNPs on Gram-negative bacteria has been demonstrated [31]. The outer membrane of Gram-negative contains aquaporins (water-filled channels) which are involved in the transport of Ag ions into the cell where they exert their antibacterial

Flow cytometry (FCM) is a well-established and powerful analytical tool that has led to many revolutionary discoveries in cell biology and cellular-molecular disease diagnosis and, more recently, has been used to analyze physiological

responses of bacteria [33, 34]. In FCM, cells are first introduced to a high-speed (up to 5–20 m/s) laminar flow stream, and after being focused into single file, they are subjected to laser-induced fluorescence, and/or forward and sideways scattered light is detected using photodetector arrays with spectral filters. More recently, FCM has been used to characterize distinct physiological conditions in bacteria including their responses to antibiotics and other cytotoxic chemicals [33]. Once the control of bacterial cells or fungal conidia has been applied, an accurate technique is required to measure the effectiveness of the silver nanoparticles. Flow cytometry is one of the most reliable techniques for detecting and counting living cells and to

When studying response to antibacterial agents such as silver nanoparticles, viability can be evaluated as an indicator of antibiotic susceptibility. There are now reagents available that allow assays of membrane permeability and potential by measuring the production of a fluorescent metabolite from a nonfluorescent

Besides monitoring susceptibility to antibacterial activity, information can be obtained using FCM that can establish mechanisms of antibacterial drug

**206**

effects [32].

**Figure 1.**

**4. Cell status by flow cytometry**

*Mode of action of silver nanoparticles in the bacterial cell.*

measure their viability.

precursor [33, 34].
