Plant seedlings of peas, tomatoes, and cucumbers exude compounds that are needed for growth and chemoattraction of Rhizobium leguminosarum bv. viciae 3841 and Azospirillum brasilense Sp7.

This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

Migration Rates on Swim Plates Vary between Escherichia coli Soil Isolates: Differences Are Associated with Variants in Metabolic Genes.

This study investigates migration phenotypes of 265 Escherichia coli soil isolates from the Buffalo River basin in Minnesota, USA. Migration rates on semisolid tryptone swim plates ranged from nonmotile to 190% of the migration rate of a highly motile E. coli K-12 strain. The nonmotile isolate, LGE0550, had mutations in flagellar and chemotaxis genes, including two IS3 elements in the flagellin-encoding gene fliC. A genome-wide association study (GWAS), associating the migration rates with genetic variants in specific genes, yielded two metabolic variants (rygD-serA and metR-metE) with previous implications in chemotaxis. As a novel way of confirming GWAS results, we used minimal medium swim plates to confirm the associations. Other variants in metabolic genes and genes that are associated with biofilm were positively or negatively associated with migration rates. A determination of growth phenotypes on Biolog EcoPlates yielded differential growth for the 10 tested isolates on d-malic acid, putrescine, and d-xylose, all of which are important in the soil environment. IMPORTANCE E. coli is a Gram-negative, facultative anaerobic bacterium whose life cycle includes extra host environments in addition to human, animal, and plant hosts. The bacterium has the genomic capability of being motile. In this context, the significance of this study is severalfold: (i) the great diversity of migration phenotypes that we observed within our isolate collection supports previous (G. NandaKafle, A. A. Christie, S. Vilain, and V. S. Brözel, Front Microbiol 9:762, 2018, https://doi.org/10.3389/fmicb.2018.00762; Y. Somorin, F. Abram, F. Brennan, and C. O'Byrne, Appl Environ Microbiol 82:4628-4640, 2016, https://doi.org/10.1128/AEM.01175-16) ideas of soil promoting phenotypic heterogeneity, (ii) such heterogeneity may facilitate bacterial growth in the many different soil niches, and (iii) such heterogeneity may enable the bacteria to interact with human, animal, and plant hosts.

A Wash of Ethyl Acetoacetate Reduces Externally Added Salmonella enterica on Tomatoes.

The continuously high numbers of food-borne disease outbreaks document that current intervention techniques are not yet satisfactory. This study describes a novel wash for tomatoes that can be used as part of the food processing chain and is designed to prevent contamination with serovars of Salmonella enterica. The wash contains ethyl acetoacetate (EAA) at a concentration of 8% in H2O. This wash reduced live bacterial counts (on Salmonella Shigella agar) of externally added S. Newport MDD14 by 2.3 log, counts of S. Typhimurium ATCC19585 by 1.5 log, and counts of S. Typhimurium FSL R6-0020 by 3.4 log. The naturally occurring background flora of the tomatoes was determined on plate count agar. The log reduction by EAA was 2.1. To mimic organic matter in the wash, we added 1% tomato homogenate to the 8% EAA solution. Prior to using the wash, the tomato homogenate was incubated with the EAA for 2 h. In the presence of the tomato homogenate, the log reductions were 2.4 log for S. Newport MDD14 and 3 log for S. Typhimurium FSL R6-0020. It seems like tomato homogenate did not reduce the efficacy of the EAA wash in the two S. enterica serovars tested. We propose the use of EAA as a wash for tomatoes to reduce bacterial counts of S. enterica well as naturally occurring background flora.

Variants of SARS CoV-2: mutations, transmissibility, virulence, drug resistance, and antibody/vaccine sensitivity.

Severe acute respiratory syndrom coronavirus-2 (SARS CoV-2) is the causative agent of coronavirus disease-19 (Covid-19) which has been designated a worldwide pandemic by the World Health Organization on March 11, 2020. Since that time, the virus has mutated and an assortment of variants have been successful at establishing themselves in the human population. This review article describes the SARS CoV-2 genome, hot spot mutations, variants, and then focuses on the Delta variant, finishing up with an update on the Omicron variant. The genome encompasses 11 open reading frames, one of which encodes the spike or S protein that has been the target for vaccines and some of the drugs because of its role in attachment to the human host cell, as well as antibodies. Mutations in the S protein that are common among several of the variants include D614G that increases transmissibility and viral load and is often associated with P323L on the RNA dependent RNA polymerase. N501Y is a mutation in the receptor binding domain of the S protein that increases binding to the ACE-2 receptor on the human host cells by 10 fold. The discussed variants carry combinations of these and other mutations and are classified by the World Health Organization as variants of concern, variants of interest, and variants under monitoring. All variants are characterized by increased transmissibility (relative to the original SARS CoV-2), which is the reason for their ability to establish themselves. Several but not all variants are more resistant to antiviral drugs and less susceptible to antibodies/vaccines. The Delta variant that dominated the world until November 2021 causes an increased risk for hospitalization and death, but is still very susceptible to the current vaccines. The most recent variant, Omicron, is characterized by increased transmissibility and decreased antibody susceptibility.

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

The Food Anti-Microbials ß-Phenylethylamine (-HCl) and Ethyl Acetoacetate Do Not Change during the Heating Process.

ß-Phenylethylamine hydrochloride (PEA-HCl) and ethyl acetoacetate (EAA) are anti-microbials with applications in food processing. As food anti-microbials, the compounds will have to withstand the cooking process without changing to toxic compounds. With this Communication, we address the question of whether PEA and EAA are altered when heated to 73.9 °C or 93.3 °C. A combination of gas chromatography and mass spectrometry was used to analyze solutions of PEA(-HCl) or EAA in beef broth or water. In addition, the anti-microbial activity of PEA-HCl and EAA was compared between heated and unheated samples at a range of concentrations. The gas chromatograms of PEA(-HCl) and EAA showed one peak at early retention times that did not differ between the heated and unheated samples. The mass spectra for PEA and EAA were near identical to those from a spectral database and did not show any differences between the heated and unheated samples. We conclude that PEA(-HCl) and EAA formed pure solutions and were not altered during the heating process. In addition, the anti-microbial activity of PEA-HCl and EAA did not change after the heating of the compounds. Regardless of temperature, the minimal inhibitory concentrations (MICs) for PEA-HCl were 20.75 mmol mL-1 for Escherichia coli and Salmonella enterica serotype Typhimurium. For EAA, the MICs were 23.4 mmol mL-1 for E. coli and 15.6 mmol mL-1 for S. enterica.

Current State of the First COVID-19 Vaccines.

SARS CoV-2 and its associated disease COVID-19 has devastated the world during 2020. Masks and social distancing could be efficient if done by large proportions of the population, but pandemic fatigue has decreased their efficacy. Economic shut downs come with large price tags and cannot be a long term solution either. The announcements by three vaccine manufacturers in November that their vaccines are 90% or more effective has given hope to at least those in the population who plan to get vaccinated as soon as a scientifically and medically sound vaccine becomes available. This review summarizes the underlying design strategies and current status of development of the nine vaccines that were in phase III trial on 8 November 2020. Contracts between vaccine manufacturing companies and governments aim at distributing the vaccine to a large part of the world population. Questions remain how the temperature sensitive mRNA vaccines will be transported and/or stored and how vaccination will be prioritized within each country. Additionally, current contracts do not cover all countries, with a serious gap in Africa and South America. The second part of this review will detail current distribution plans and remaining challenges with vaccine accessibility and acceptance.

Microbes in Our Food, an Ongoing Problem with New Solutions.

Despite an increasing number of techniques that are designed to mitigate microbial contamination of food and the resulting food borne disease outbreaks, the United States and many other countries across the world continue to experience impressive numbers of such outbreaks. Microbial contamination can occur during activities that take place in the pre-harvest environment or in the processing facility post-harvest. Current treatments of food that are aimed at reducing bacterial numbers may be only partially effective because of the development of bacterial resistance, the formation of bacterial biofilms, and inactivation of the treatment compound by the food products themselves. This Special Issue will include basic research approaches that are aimed at enhancing our understanding of how contamination occurs throughout the food processing chain, as well as more immediate and applied approaches to the development and use of novel anti-microbials to combat microbes in food. Novel techniques that aim to evaluate the efficacy of novel anti-microbials are included. Overall, we present a broad spectrum of novel approaches to reduce microbial contamination on food at all stages of production.

Efficacy of ß-phenylethylamine as a novel anti-microbial and application as a liquid catheter flush.

With this study, we introduce a liquid flush for catheters and other tubing-based applications that consists of a solution of ß-phenylethylamine (PEA) in tryptic soy broth. The initial experiments in multiwell polystyrene plates were conducted with Escherichia coli K-12 to assess the effectiveness of PEA at reducing planktonic growth, as well as the biomass and adenosine triphosphate (ATP) content of biofilm; PEA reduced these growth parameters as a function of increasing concentration. This effect was also seen in mutants of PEA catabolism, which leads us to believe that the PEA effect is due to PEA itself and not one of its degradation products. Since PEA reduced planktonic growth and biofilm when added at the time of inoculation, as well as at later time points, we propose PEA as a novel compound for the prevention and treatment of biofilm. PEA reduced planktonic growth and the ATP content of the biofilm for five bacterial pathogens, including an enterohemorrhagic E. coli, two uropathogenic E. coli, Pseudomonas aeruginosa and Staphylococcus aureus. A major finding of this study is the reduction of the ATP content of biofilm that formed in silicone tubing by periodic flushes of PEA. This experiment was performed to model antibiotic-lock treatment of an intravenous catheter. It was found that 10 mg ml-1 of PEA reduced the ATP content of biofilm of five bacterial strains by 96.3 % or more after 2 weeks of incubation and three treatments with PEA. For P. aeruginosa, the reduction in ATP content was paralleled by an identical percentage reduction in viable cells in the biofilm.

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development.

Two-component signaling is a specialized mechanism that bacteria use to respond to changes in their environment. Nonpathogenic strains of Escherichia coli K-12 harbor 30 histidine kinases and 32 response regulators, which form a network of regulation that integrates many other global regulators that do not follow the two-component signaling mechanism, as well as signals from central metabolism. The output of this network is a multitude of phenotypic changes in response to changes in the environment. Among these phenotypic changes, many two-component systems control motility and/or the formation of biofilm, sessile communities of bacteria that form on surfaces. Motility is the first reversible attachment phase of biofilm development, followed by a so-called swim or stick switch toward surface organelles that aid in the subsequent phases. In the mature biofilm, motility heterogeneity is generated by a combination of evolutionary and gene regulatory events.

Spontaneous mutations in the flhD operon generate motility heterogeneity in Escherichia coli biofilm.

BACKGROUND: Heterogeneity and niche adaptation in bacterial biofilm involve changes to the genetic makeup of the bacteria and gene expression control. We hypothesized that i) spontaneous mutations in the flhD operon can either increase or decrease motility and that ii) the resulting motility heterogeneity in the biofilm might lead to a long-term increase in biofilm biomass. RESULTS: We allowed the highly motile E. coli K-12 strain MC1000 to form seven- and fourteen-day old biofilm, from which we recovered reduced motility isolates at a substantially greater frequency (5.4 %) than from a similar experiment with planktonic bacteria (0.1 %). Biofilms formed exclusively by MC1000 degraded after 2 weeks. In contrast, biofilms initiated with a 1:1 ratio of MC1000 and its isogenic flhD::kn mutant remained intact at 4 weeks and the two strains remained in equilibrium for at least two weeks. These data imply that an 'optimal' biofilm may contain a mixture of motile and non-motile bacteria. Twenty-eight of the non-motile MC1000 isolates contained an IS1 element in proximity to the translational start of FlhD or within the open reading frames for FlhD or FlhC. Two isolates had an IS2 and one isolate had an IS5 in the open reading frame for FlhD. An additional three isolates contained deletions that included the RNA polymerase binding site, five isolates contained point mutations and small deletions in the open reading frame for FlhC. The locations of all these mutations are consistent with the lack of motility and further downstream within the flhD operon than previously published IS elements that increased motility. We believe that the location of the mutation within the flhD operon determines whether the effect on motility is positive or negative. To test the second part of our hypothesis where motility heterogeneity in a biofilm may lead to a long-term increase in biofilm biomass, we quantified biofilm biomass by MC1000, MC1000 flhD::kn, and mixtures of the two strains at ratios of 1:1, 10:1, and 1:10. After 3 weeks, biofilm of the mixed cultures contained up to five times more biomass than biofilm of each of the individual strains. CONCLUSION: Mutations in the flhD operon can exert positive or negative effects on motility, depending on the site of the mutation. We believe that this is a mechanism to generate motility heterogeneity within E. coli biofilm, which may help to maintain biofilm biomass over extended periods of time.

Lessons in Fundamental Mechanisms and Diverse Adaptations from the 2015 Bacterial Locomotion and Signal Transduction Meeting.

In response to rapid changes in their environment, bacteria control a number of processes, including motility, cell division, biofilm formation, and virulence. Research presented in January 2015 at the biennial Bacterial Locomotion and Signal Transduction (BLAST) meeting in Tucson, AZ, illustrates the elegant complexity of the nanoarrays, nanomachines, and networks of interacting proteins that mediate such processes. Studies employing an array of biophysical, genetic, cell biology, and mathematical methods are providing an increasingly detailed understanding of the mechanisms of these systems within well-studied bacteria. Furthermore, comparisons of these processes in diverse bacterial species are providing insight into novel regulatory and functional mechanisms. This review summarizes research presented at the BLAST meeting on these fundamental mechanisms and diverse adaptations, including findings of importance for applications involving bacteria of medical or agricultural relevance.

Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

Screening the mechanical stability of Escherichia coli biofilms through exposure to external, hydrodynamic shear forces.

The desire to attain a deeper understanding of the fundamental aspects governing the mechanical properties of biofilms has become more prominent in recent years. This has largely been due to the realization that these sessile microbial communities often withstand environments where hydrodynamic turbulence and shearing forces are considerable. In the present study, Escherichia coli K-12 was used as a model system to develop a laboratory technique that can be used to quickly screen the mechanical integrity or stability of laboratory cultivated bacterial biofilms when exposed to such external, hydrodynamic shear forces. The screening method utilizes a custom-built, automated water jetting apparatus to generate and precisely apply a pressurized stream of water directly to biofilms cultured in multi-well plates. An optimized set of water jetting parameters was determined to resolve subtle to moderate differences in the mechanical stability of isogenic strains of E. coli K-12 as a function of percent biofilm removal. Mutations in both flagella biosynthesis (fliA) and acetate metabolism (ackA and ackA pta) were shown to impair the mechanical integrity of 24-h biofilms, while a "housekeeping" strain deficient in arginine metabolism (argD) exhibited a mechanical stability profile comparable to the parent strain.

OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli biofilm.

BACKGROUND: Biofilms are communities of bacteria that are characterized by specific phenotypes, including an increased resistance towards anti-microbials and the host immune system. This calls for the development of novel biofilm prevention and treatment options to combat infectious disease. In Escherichia coli, numerous global regulators have been implicated in the control of biofilm associated cell surface organelles. These include the flagellar regulator FlhD/FlhC, the osmoregulator EnvZ/OmpR, and the colanic acid activator RcsCDB. Using flow cell technology and fluorescence microscopy, we determined the temporal expression from flhD::gfp, ompR::gfp, and rcsB::gfp in E. coli biofilm, as well as the impact of the negative regulation of flhD by OmpR and RcsB. Spatial gene expression was investigated from flhD::gfp. RESULTS: The temporal gene expression profile for flhD yielded an early peak at 12 h, a minimum of expression at 35 h, and a second increase in expression towards 51 h of biofilm development. In contrast, the ompR profile showed a peak at 35 h. A mutation in ompR abolished time dependence of flhD expression after the initial growth period of 12 h. Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well. Spatially, expression of flhD was highest in the outermost layer of the biofilm in the parent strain. In ompR and rcsB mutants, flhD was expressed throughout the biofilm. Mutations in both, ompR and rcsB increased flhD expression throughout all temporal and spatial experiments. This increase was paralleled by reductions in biofilm amounts at four tested time points. CONCLUSION: Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.

Acetate metabolism and Escherichia coli biofilm: new approaches to an old problem.

Current antibiotics continue to lose effectiveness for infectious diseases, especially in cases where the bacteria from a biofilm. This review article summarizes control mechanisms for bacterial biofilm, with an emphasis on the modification of signal transduction pathways, such as quorum sensing and two-component signaling, by externally added metabolic intermediates. As a link between central metabolism and signal transduction, we discuss the activation of two-component response regulators by activated acetate intermediates in response to signals from the environment. These signals constitute 'nutrients' for the bacteria in most cases. Depending on the identity of the nutrient, biofilm amounts may be reduced. The nutrient may then be used for the development of both novel prevention and treatment options for biofilm-associated infectious diseases.

ß-phenylethylamine, a small molecule with a large impact.

During a screen of bacterial nutrients as inhibitors of Escherichia coli O157:H7 biofilm, the Prüß research team made an intriguing observation: among 95 carbon and 95 nitrogen sources tested, ß-phenylethylamine (PEA) performed best at reducing bacterial cell counts and biofilm amounts, when supplemented to liquid beef broth medium. This review article summarizes what is known about PEA. After some starting information on the chemistry of the molecule, we focus on PEA as a neurotransmitter and then move on to its role in food processing. PEA is a trace amine whose molecular mechanism of action differs from biogenic amines, such as serotonin or dopamine. Especially low or high concentrations of PEA may be associated with specific psychological disorders. For those disorders that are characterized by low PEA levels (e.g. attention deficit hyperactivity disorder), PEA has been suggested as a 'safe' alternative to drugs, such as amphetamine or methylphenidate, which are accompanied by many undesirable side effects. On the food processing end, PEA can be detected in food either as a result of microbial metabolism or thermal processing. PEA's presence in food can be used as an indicator of bacterial contamination.

The role of activated acetate intermediates in the control of Escherichia coli biofilm amounts.

A previous study postulated that acetate metabolism was a metabolic sensory mechanism that related information about E. coli's environment to the formation of biofilms (Prüß et al., Arch. Microbiol. 2010). Considering that mutants in pta ackA (no acetyl phosphate) and ackA (high acetyl phosphate) exhibited similarly increased biofilm amounts and three dimensional structures, the hypothesis for this study was that acetyl Co-A was a more likely mediator of the acetate effect than acetyl phosphate. The effect of acetate metabolism on biofilm amounts was detailed by using single carbon sources rather than the previously used mixed amino acid medium, as well as mutations in additional genes that contribute to acetate metabolism (ldhA, pflA, pflB). In summary, the mutations in ackA, pta ackA, and ldhA increased biofilm amounts in the presence of maltose, D-trehalose, D-mannose, and L-rhamnose, all of which get converted to acetyl-CoA. The ackA mutant also exhibited increased biofilm amounts in the presence of inosine and thymidine. The mutation in pflA decreased biofilm amounts in the presence of maltotriose, uridine, D-serine, and acetate. Since ackA, pta ackA, and ldhA mutants are expected to exhibit increased intracellular acetyl-CoA levels, and pflA and pflB mutants likely exhibit decreased acetyl-CoA concentrations, we believe that acetyl-CoA is the activated acetate intermediate that controls biofilm amounts.

Regulation of cell division, biofilm formation, and virulence by FlhC in Escherichia coli O157:H7 grown on meat.

To understand the continuous problems that Escherichia coli O157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC of E. coli K-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of an E. coli O157:H7 parent strain to that of its isogenic flhC mutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in the flhC mutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity of E. coli O157:H7 on meat by interfering with the signal transduction pathways.

A new method to determine initial viability of entrapped cells using fluorescent nucleic acid staining.

Entrapped bacterial cells are widely used in several biotechnological applications. Cell entrapment procedures are known to affect the viability of bacterial cells. To determine the effect of entrapment procedures on viability of bacterial cells, dissolution of the entrapment matrices using chelating agents or heat is required immediately after the entrapment is completed. Chelating agents and heat applied in the matrix dissolution reduce cell viability and in turn hinder accurate quantification of viable cells. In this study, a method to determine the effect of entrapment procedure on bacterial cell viability which involves entrapping cells directly onto glass slides was developed. The developed method showed less viability reduction than the methods requiring matrix dissolution. The percentage of live cells in the culture before entrapment ranged from 54% to 74%, while the percent of live cells after entrapment determined by the developed method was 39-62%.