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Vibriosis in shrimp farms poses potential risks to the industry's sustainability and the consumers' health. Little is known about the dynamics and variation of Vibrio spp--population in shrimp production in Sarawak, Malaysia. The apparent prevalence of three Vibrio species, V. parahaemolyticus, V. cholerae, and V. alginolyticus, were investigated in water (n=12), sediment (n=12), shrimp (n=12), and effluent (n=12) samples collected from two ponds in a coastal shrimp farm during one production cycle. Multiplex-PCR using specific primer sets showed the presence of the three Vibrio species. Based on the results, V. parahaemolyticus was the most prevalent in all four samples, with a contamination rate of 97.92% (95% CI: 89. 10 to 99.89%), while V. cholerae and V. alginolyticus had a contamination rate of 47.92% (95% CI: 34.37 to 61.67%) and 25.0% (95% CI: 14.92 to 38.78%), respectively. High Vibrio load in the shrimp farm is due to favourable environmental factors, such as optimal temperatures, salinity, and pH ranges for the growth of these species. The study's findings offer important preliminary insights into the prevalence and distribution of these pathogenic Vibrio spp., within a shrimp farm in Kuching, Sarawak. This study serves as a potential model for monitoring Vibrio spp. prevalence in other shrimp farms across Sarawak, thereby addressing the scarcity of data on prevalence in the region. [ABSTRACT FROM AUTHOR]

Introduction: Water is one of the important natural resources available for mankind for its survival. In rural Karnataka, there are 1,182,990 wells, out of which 131,305 are covered wells, and 1,051,685 are uncovered. This highlights the threat to safe and potable drinking water. Aim: To assess the physical, chemical, and microbiological qualities of well water situated in the field practice area of Mangaluru, Karnataka, India. Materials and Methods: A cross-sectional study was conducted in the year 2019, between July and August, in the rural area of Kinya Panchayat, Ullal Taluk, Dakshina Kannada District. This area serves as the field practice area of Yenepoya Nursing College, Yenepoya deemed to be University. This pilot project was a time-bound study, and all twenty wells available were conveniently included in the study and comprised dug and ring residential and government wells utilised by residents for drinking and domestic purposes. Water collection followed World Health Organisation (WHO) guidelines. Baseline data included the age of the wells, colour, odour, and chlorination of the well water. Chemical parameters such as pH levels and bacteriological assessments to identify the presence of Escherichia coli, H2S, and Most Probable Number (MPN) bacterial counts were conducted using laboratory tests. Results: The colour of all 20 (100%) well water sources met WHO standards. The water was odourless in all 20 (100%) sources. The mean age of the wells was five years. The majority, 17 (85%) of the wells, were never chlorinated. Moreover, 17 (85%) of the well water sources had a pH level of ≤7, while 3 (15%) had a pH level of >7. E. coli bacteria were present in three-fourths of the well water (17 wells, 85%). No other organisms were found during testing. Ten (50%) of the well water sources had a bacterial count of 351-550 MPN {Most Probable Number (MPN) of bacteria} per 100 mL, 7 (35%) had a count of 151-350 MPN per 100 mL, and 3 (15%) had a count of less than or equal to 150 MPN per 100 mL. The results indicate that 17 (85%) of the wells’ water was not satisfactory for potable purposes, while 3 (15%) were deemed satisfactory. Conclusion: Water sources in the studied area were found to be contaminated, with most of the well water sources not meeting satisfactory potable standards. This underscores the importance of raising awareness, implementing routine monitoring, and conducting consistent and periodic examinations of drinking water, as well as disinfection processes, to address this issue.

Introduction: Water is one of the important natural resources available for mankind for its survival. In rural Karnataka, there are 1,182,990 wells, out of which 131,305 are covered wells, and 1,051,685 are uncovered. This highlights the threat to safe and potable drinking water. Aim: To assess the physical, chemical, and microbiological qualities of well water situated in the field practice area of Mangaluru, Karnataka, India. Materials and Methods: A cross-sectional study was conducted in the year 2019, between July and August, in the rural area of Kinya Panchayat, Ullal Taluk, Dakshina Kannada District. This area serves as the field practice area of Yenepoya Nursing College, Yenepoya deemed to be University. This pilot project was a time-bound study, and all twenty wells available were conveniently included in the study and comprised dug and ring residential and government wells utilised by residents for drinking and domestic purposes. Water collection followed World Health Organisation (WHO) guidelines. Baseline data included the age of the wells, colour, odour, and chlorination of the well water. Chemical parameters such as pH levels and bacteriological assessments to identify the presence of Escherichia coli, H2S, and Most Probable Number (MPN) bacterial counts were conducted using laboratory tests. Results: The colour of all 20 (100%) well water sources met WHO standards. The water was odourless in all 20 (100%) sources. The mean age of the wells was five years. The majority, 17 (85%) of the wells, were never chlorinated. Moreover, 17 (85%) of the well water sources had a pH level of ≤7, while 3 (15%) had a pH level of >7. E. coli bacteria were present in three-fourths of the well water (17 wells, 85%). No other organisms were found during testing. Ten (50%) of the well water sources had a bacterial count of 351-550 MPN {Most Probable Number (MPN) of bacteria} per 100 mL, 7 (35%) had a count of 151-350 MPN per 100 mL, and 3 (15%) had a count of less than or equal to 150 MPN per 100 mL. The results indicate that 17 (85%) of the wells’ water was not satisfactory for potable purposes, while 3 (15%) were deemed satisfactory. Conclusion: Water sources in the studied area were found to be contaminated, with most of the well water sources not meeting satisfactory potable standards. This underscores the importance of raising awareness, implementing routine monitoring, and conducting consistent and periodic examinations of drinking water, as well as disinfection processes, to address this issue. [ABSTRACT FROM AUTHOR]

Low level of rifampicin‐resistant E. coli is frequently used in food safety research as a model microorganism and is typically enumerated using traditional Most Probable Number (MPN) method. To simplify the process, we developed and validated a modified MPN method based on lactose peptone broth (LPB) containing rifampicin and bromocresol purple, the broth changes to a yellow color due to pH shifts induced by lactose fermentation from E. coli. E. coli TVS353 were prepared in following suspensions including healthy cells, injured cells, competitive bacteria with or without rifampicin resistance, and unknown natural microflora. E. coli TVS353 was quantified by traditional MPN method and our improved method. At a predicted level of 1 log CFU/mL, there was no significant difference in the MPN index of LPBR compared with TSB and TSBR in these suspensions (p > 0.05). Confirmation results generated from this new color change MPN method were analogous to traditional MPN methods. [ABSTRACT FROM AUTHOR]

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements. [ABSTRACT FROM AUTHOR]

Red guava can be processed into a juice drink, but it does not rule out the possibility that fruit juice contains disease-causing microorganisms. This study aims to determine the number of Escherichia coli and TPC/Total Plate Count bacteria in red guava fruit juice sold in the Keboansikep, Gedangan, Sidoarjo. The method used is descriptive using a survey technique with a cross-sectional approach. Laboratory experiments are carried out, namely analyzed in the microbiology laboratory sampling using a saturated sampling technique. Samples were examined to compare the analysis results obtained compared to INS 3719:2014 concerning the Quality of Fruit Juice Drinks. Samples were examined using Escherichia coli analysis based on INS 01-2332.1-2006 with the Most Probable Number (MPN) method and Total Plate Count/TPC analysis based on INS 01-2332.3-2006 with the pour plate method. The test results showed that the Escherichia coli analysis obtained 100% of the samples showing positive with the lowest MPN index of 9 APM/ml to the highest >1100 APM/ml. The results of the PNA analysis obtained 7% of all samples, indicating that it was safe and met the requirements, namely sample 7A with a TPC value of 4.9 x 103 CFU/ml, while 93% of all samples showed that it was unsafe or did not meet the requirements based on INS 3719: 2014. The sample containing the most microbes was sample 5A with a TPC value of 5,1 x 105 CFU/ml. [ABSTRACT FROM AUTHOR]

ABSTRACT Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects.IMPORTANCEWe created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Water contamination with faecal matter is usually the main cause of microbial waterborne diseases. Such diseases are an alarming situation for small cities in developing countries like India. In this research, to check the microbiological status of drinking water in Solan, Himachal Pradesh (India), water samples were collected from baories/stepwells (n = 14), handpumps (n = 9), and the municipal water distribution system (MWDS) (n = 2) in alternative months of the year (covering three main seasons). In 6 months, 150 samples were collected, and they were all examined for the presence of total coliforms and other bacterial pathogens. The associations between the isolates' ecological and seasonal prevalence were also examined. The coliforms were detected by the Most Probable Number (MPN) method, whose range was noticed from the 2–540/100-ml MPN index. The colony forming unit (CFU) count for different samples at the base log 10 value ranged from 3.03 to 6.19. Different genera isolated and identified were Escherichia coli, Salmonella enteric subsp. enterica, Pseudomonas spp., Klebsiella spp., and Staphylococcus aureus. Overall, 74% of the isolates identified in water samples were from the Enterobacteriaceae family. E. coli was about 42.67% (n = 102), followed by Salmonella enterica subsp. enterica 20.92% (n = 50), Staphylococcus aureus 13.38% (n = 32), Pseudomonas spp. 12.55% (n = 30), and Klebsiella spp. 10.46% (n = 25) amongst the total of 239 isolates. The seasonal impact and the dependency of the occurrence of bacteria on one another were determined to be insignificant in the Spearman correlation test. These results showed that external factors (anthropogenic activities) are mainly responsible for the presence of these bacteria in water resources. The occurrence of bacterial isolates has been noticed in all water samples, irrespective of collecting site or season. [ABSTRACT FROM AUTHOR]

The provision of potable water is a global challenge. Infections caused by drinking contaminated water are a regular occurrence in developing countries. This study was carried out to determine Gram-negative bacterial distribution and antibiotic resistance in potable water from hand-dug wells within Iwo, Nigeria. Thirty hand-dug wells were randomly selected within Iwo for sampling carried out between October and December 2018. Bacteria identification was carried out using standard methods. The most probable number (MPN) and antibiotic resistance profile as well as Multiple Antibiotic Resistance Index (MARI) for these isolates were determined in addition to studying their haemolysis patterns on blood agar. Results showed that all the water samples from these hand-dug wells were highly contaminated. The highest value >1,100+ was recorded for 21 samples. In addition, 11 genera of bacteria were isolated: Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Salmonella, Serratia and Pseudomonas. Antibiotic resistance to cefixime and cefuroxime were 92.6 and 90.9%, respectively. One hundred and sixty-nine (96.6%) isolates had a MARI greater than 0.2 and all showed haemolysis. Ingestion of this contaminated water has major public health implications. Hence, it is advisable that every individual should embark on in-house water treatment to avoid water-borne diseases. HIGHLIGHTS Isolation and identification of Gram-negative bacteria from well water.; All the wells were grossly contaminated with coliform bacteria.; A high percentage of the bacteria were resistant to at least three classes of antibiotics.; Multi-antibiotic resistance index was greater than 0.2 in 96.6% of the isolates.; The isolates harboured other virulence genes in addition to antibiotic resistance.;

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Raw materials associated with foodborne illness outbreaks are rarely available for evaluation. The levels and distribution of Salmonella were determined in naturally contaminated raw cashews linked to a salmonellosis outbreak associated with a fermented cashew cheese analog. Two unopened 22.7-kg boxes from a single lot of cashew kernel pieces were each divided into seven approximately equal units, 14 in total. Three 10-g subsamples per unit (n = 21) were evaluated for aerobic plate count (APC), coliform counts, and Escherichia coli counts, and 10 50-g subsamples per unit (n = 70) were enriched for the presence of Salmonella. Presumptive Salmonella-positive colonies were confirmed using CHROMagar Salmonella and real-time PCR (invA) and then serotyped using antigenic methods and genome sequencing prediction tools. APC and coliform counts ranged from 1.81 to 5.47 (mean 2.44 ± 0.63) log CFU/g and 0.60 to 5.20 (mean 1.74 ± 0.80) log CFU/g, respectively. Salmonella was recovered from four units in Box 1 and all seven units in Box 2. One of the 10 subsamples was positive in all but four of the positive units; one (Box 1) and three (Box 2) units had two positive subsamples. The level of Salmonella in the two boxes combined was 0.0023 most probable number/g (95% confidence interval [0.0014, 0.0038]). Salmonella Urbana was isolated from three of five positive subsamples in Box 1 and eight of 10 positive subsamples in Box 2. Salmonella Fresno and Vinohrady were unique to single subsamples from Box 1, and Salmonella Nima was isolated from two subsamples from Box 2. Of the four serovars recovered, Salmonella Urbana and Salmonella Vinohrady were in common with outbreak-associated clinical or product isolates. Understanding the distribution and concentration of Salmonella in naturally contaminated cashews provides important information for hazard analysis and risk assessments for soaked and fermented cashew products.

Objective: This study is designed to evaluate the microbiological safety concern of drinking water of the 50 schools of Chattogram city, Bangladesh, and to investigate the sanitation and hygiene conditions of the schools, which are directly related to the health of thousands of students. Methods: In this study, pour plate method used for total viable count (TVC), most probable number (MPN) method used for the total coliform count (TCC) and fecal coliform count (FCC), and four selective media used to isolate pathogenic bacteria from drinking water samples, and then identified by using morphological, cultural, and biochemical tests. To investigate the water, sanitation, and hygiene conditions a structured questionnaire was used. Results: A total of 46% samples were found above the acceptable limit (> 500 CFU/ml) for TVC count, 52% samples had coliform, and 28% samples had fecal coliform, which exceeded the WHO guideline value. We categorized 50 studied schools into five groups and found the highest TVC (67%) and TCC (83%) in the water samples of city corporation schools. E. coli, Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, and Yersinia were isolated and identified from drinking water samples. In most schools, drinking water reservoirs were found impure and contaminated by various bacteria. In government primary schools, a toilet is used by 143 students, whereas in private English medium schools, a toilet is used by 30 students and found clean. Hand-washing soap was supplied in only 14% of government primary schools, but 100% in private English medium schools. Conclusion: Our findings suggest that drinking water in most of the schools was found contaminated by pathogens. Poor hygiene, sanitation, and contaminated drinking water seem responsible for different kinds of diseases. Moreover, this study indicates the necessity for raising awareness about drinking water, hygiene, and sanitation facilities of schools, which should monitor at regular intervals.

The problem on food health is the high amount of bacterial contamination of food served by food industries. The quality of beef can be affected by the level of bacterial contamination in meat. Coliform is a group of bacteria used as a sanitary hygiene indicator. The purpose of this study is to measure the value of MPN Coliform in beef at Krian’s abattoir of district Sidoarjo. Thirty samples were taken from the abattoir of Krian, Sidoarjo. Most Probable Number was used to determine the level of Coliform's contaminate beef. The results showed that all beef samples at abattoir were contaminated with Coliform. The average contaminant levels was 706,76 MPN/g. Twenty six samples exceed the maximum limit of Coliform bacteria contamination in accordance with Indonesian National Standard (SNI). The highest Coliform MPN value is ≥ 1600 MPN / g, whereas the lowest MPN Coliform value is 8 MPN/g.

Coliform pollution for the last three decades in major river systems of the world has resulted in far ranging impacts on water quality. In this context, the present study aimed to assess the levels of indicator bacteria like total Coliform (TC), fecal Coliform (FC) and fecal Streptococcus (FS) in major watersheds of Kashmir valley. Sampling was carried out for a period of 2 years (summer 2017 to spring 2019) along several upstream, midstream and downstream reaches of Jhelum River Basin (JRB), while analysis was carried out by multiple tube fermentation technique involving Most Probable Number (MPN). Major highlights of the results revealed high levels of TC, FC and FS among downstream sites with pronounced seasonal variations between summer and winter. TC was highest at all the reaches and during all the seasons followed by FC and FS. Non-metric multidimensional scaling (NMDS) revealed more variation in Coliform count among reaches as compared to seasons. Mantle test revealed that environmental factors like observable environmental pressure (OEP) (r: 0.235, p < 0.0001), DO (r: 0.2815, p < 0.0001) and temperature (r: 0.04419, p = 0.0104) had prominent effect on Coliform distribution as compared to geographical factors. The study thus highlights the prevalence of Coliform bacteria along JRB resulting from fecal sources. Due to growing urbanization and lack of adequate sewage treatment facilities, there is an increase in the levels of Coliform bacteria along downstream reaches especially those residing within lower Jhelum and Dara watershed, which could jeopardize water quality and public health. [ABSTRACT FROM AUTHOR]

To assess the biodegradation potential of polycyclic aromatic hydrocarbons (PAHs) in sediments, sediment microcosms were constructed with sediments collected from six lake zones with different trophic statuses in Taihu Lake. The presence and concentration of PAH-degrading bacteria (PDB) were estimated by the most probable number (MPN) method. After 85 d of aerobic and anaerobic incubation, spiked PAHs (phenanthrene, pyrene, and benzo[a]pyrene) were partially degraded by indigenous sediment microorganisms. Large differences in PAH degradation were observed depending on the molecular size of the PAHs. The PAH removal efficiency in sediments under aerobic conditions was higher than that under anaerobic conditions. MPN analyses showed a higher abundance of degrading microflora in the high PAH-contaminated sites than in the low PAH-contaminated sites. Moreover, the anaerobic PDB populations in the sediments from the six different sites were much higher than those of aerobic PDB. The PAH biodegradation capability in sediments was associated with the geochemical conditions and bacterial populations. PDB showed a broad spatial distribution, thereby implying that they played an important role in the natural attenuation and cycling of PAHs in Taihu Lake. This work indicates that PAHs remain a concern in Taihu Lake sediments and can provide useful information for further bioremediation of PAH-contaminated sediments. [ABSTRACT FROM AUTHOR]

Objective: This study is designed to evaluate the microbiological safety concern of drinking water of the 50 schools of Chattogram city, Bangladesh, and to investigate the sanitation and hygiene conditions of the schools, which are directly related to the health of thousands of students. Methods: In this study, pour plate method used for total viable count (TVC), most probable number (MPN) method used for the total coliform count (TCC) and fecal coliform count (FCC), and four selective media used to isolate pathogenic bacteria from drinking water samples, and then identified by using morphological, cultural, and biochemical tests. To investigate the water, sanitation, and hygiene conditions a structured questionnaire was used. Results: A total of 46% samples were found above the acceptable limit (> 500 CFU/ml) for TVC count, 52% samples had coliform, and 28% samples had fecal coliform, which exceeded the WHO guideline value. We categorized 50 studied schools into five groups and found the highest TVC (67%) and TCC (83%) in the water samples of city corporation schools. E. coli, Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, and Yersinia were isolated and identified from drinking water samples. In most schools, drinking water reservoirs were found impure and contaminated by various bacteria. In government primary schools, a toilet is used by 143 students, whereas in private English medium schools, a toilet is used by 30 students and found clean. Hand-washing soap was supplied in only 14% of government primary schools, but 100% in private English medium schools. Conclusion: Our findings suggest that drinking water in most of the schools was found contaminated by pathogens. Poor hygiene, sanitation, and contaminated drinking water seem responsible for different kinds of diseases. Moreover, this study indicates the necessity for raising awareness about drinking water, hygiene, and sanitation facilities of schools, which should monitor at regular intervals. [ABSTRACT FROM AUTHOR]

Marina and Baruna Beach are widely recognized as popular beach tourism destinations in Semarang. Its proximity to the main beach estuary causes them to significantly impact marine tourism quality. In addition, their exposure to waste may be a potential threat to biological pollution. Coliform and Escherichia coli are used as bioindicators to monitor the quality of marine tourism due to their high correlation to faecal contamination and pathogenic microbes. Samples collected were beach sand and seawater from Marina Beach, Baruna Beach and the estuary of Semarang Banjir Kanal Barat. Data were collected following the presumptive and confirmed test, followed by the conversion to MPN Table. Data obtained is combined with Government Regulations No. 22 of 2021 in Appendix VIII, specifically seawater quality standards for marine tourism. The results of this study reveal high contamination of coliform and E. coli in both beaches. Coliform result in the swimming zone at Marina Beach was 2800 MPN/100ml and at Baruna Beach was 16000 MPN/100ml. Meanwhile, Escherichia coli in the swimming zone area at Marina Beach was 400 MPN/100ml and at Baruna Beach was 1700 MPN/100ml. Overall, these results show that the contamination rate from both swimming zones is higher than the recommended seawater quality standard for marine tourism Wisata Pantai Marina dan Pantai Baruna menjadi kawasan wisata pantai yang populer di Kota Semarang. Lokasinya yang berdekatan dengan muara sungai utama di Kota ini berdampak terhadap kualitas wisata pantai. Paparan limbah berpotensi menyebabkanpencemaran biologis. Monitoring terhadap bakteri coliform dan bakteri Escherichia coli dapat digunakan sebagai bioindikator kualitas wisata pantai, karena keberadaannya berkorelasi dengan cemaran feses dan mikroba patogen. Sampel berupa pasir pantai dan air laut diambil dari lokasi Pantai Marina, Pantai Baruna, dan muara Sungai Banjir Kanal Barat Kota Semarang. Pengujian sampel dilaksanakan dengan presumtif test dan confirmed test, selanjutnya dikonversi dengan Tabel MPN. Data yang didapatkan akan dipadukan dengan PP Nomor 22 Tahun 2021 pada Lampiran VIII, yaitu baku mutu air laut untuk wisata bahari. Hasil penelitian menunjukkan tingginya kontaminasi bakteri coliform dan bakteri Escherichia coli dikedua pantai. Hasil coliform pada kawasan swimming zone pada Pantai Marina 2800 MPN/100ml dan Pantai Baruna 16000 MPN/100ml, serta hasil Escherichia coli pada kawasan swimming zone pada Pantai Marina 400 MPN/100ml dan Pantai Baruna 1700 MPN/100ml. Kawasan swimming zone pada wisata Pantai Marina dan Pantai Baruna mempunyai hasil yang melebihi baku mutu air laut untuk wisata bahari.

Background: The coliform bacteria group is a group of bacteria used as an indicator of food sanitation, one of which is in dairy products. Objective: This study aimed to determine the relationship of milking techniques with the number of Escherichia coli in raw milk compared to the bacterial contamination limit set by the National Standardization Agency. Methods: The design of this study was a descriptive study. Raw milk samples were taken from Lucky Farm and several traditional dairy farms in the KUD Kertajaya area, Kediri Regency, East Java. This study uses the most probable number (MPN) test. Results: The results showed 11 (73.3%) of raw milk samples using conventional milking techniques exceeding the maximum limit of E. coli bacterial contamination determined by the National Standardization Agency (< 3 MPN/mL). However, all 15 samples, or 100% of raw milk samples using machine milking techniques, were negative for E. coli and met the maximum bacterial contamination limit requirements. Conclusion: The milking techniques affect E. coli contamination limits in raw milk. The milk samples taken using machine milking techniques had better results with MPN values ​​< 3 MPN/mL than those taken using conventional milking techniques.

The United States Pharmacopoeia (USP) presents two approaches for showing non-inferiority of an alternate qualitative microbiological method versus a compendial method. One approach compares the positive rates for the alternate and compendial methods at one spike level, while the other one compares multiple most probable number (MPN) estimates from a multi-spike design using a t-test. In this paper, we discuss these approaches under certain assumptions and propose a third approach that can be used for both single and multiple dilutions, which we call the generalized MPN (gMPN) approach. Simulations, using Poisson distributed numbers of microorganisms in test samples, confirm that the USP approach based on rates is not suitable, that the USP approach based on MPNs is appropriate for non-inferiority, but the gMPN approach outperforms the MPN-based approach and is therefore recommended. [ABSTRACT FROM AUTHOR]

Measuring the abundance of microbes in a sample is a common procedure with a long history, but best practices are not well-conserved across microbiological fields. Serial dilution methods are commonly used to dilute bacterial cultures to produce countable numbers of colonies, and from these counts, to infer bacterial concentrations measured in colony-forming units (CFUs). The most common methods to generate data for CFU point estimates involve plating bacteria on (or in) a solid growth medium and counting their resulting colonies or counting the number of tubes at a given dilution that have growth. Traditionally, these types of data have been analyzed separately using different analytic methods. Here, we build a direct correspondence between these approaches, which allows one to extend the use of the most probable number method from the liquid tubes experiments, for which it was developed, to the growth plates by viewing colony-sized patches of a plate as equivalent to individual tubes. We also discuss how to combine measurements taken at different dilutions, and we review several ways of analyzing colony counts, including the Poisson and truncated Poisson methods. We test all point estimate methods computationally using simulated data. For all methods, we discuss their relevant error bounds, assumptions, strengths, and weaknesses. We provide an online calculator for these estimators.Estimation of the number of microbes in a sample is an important problem with a long history. Yet common practices, such as combining results from different measurements, remain sub-optimal. We provide a comparison of methods for estimating abundance of microbes and detail a mapping between different methods, which allows to extend their range of applicability. This mapping enables higher precision estimates of colony-forming units (CFUs) using the same data already collected for traditional CFU estimation methods. Furthermore, we provide recommendations for how to combine measurements of colony counts taken across dilutions, correcting several misconceptions in the literature., Competing Interests: The authors declare no conflict of interest.

The study aimed to identify the total number of E. coli used in fresh cow milk in KPSP Ijen Makmur. The sample was used by as many as 16 samples from a group of cattle farmers. E. coli analyzed using Most Probable Number (MPN), 3 series of tubes. Before continuing the MPN test the milk must be diluted, 25 ml of milk was poured into the dilution of the 225ml peptone water buffered then homogenized for 2 minutes. MPN consist of presumptive coliform test if positive was found of gas and was cloudy, presumptive faecal coliform test positive was found in the gas and was cloudy, confirmed E.coli test if there was a black colony with or metallic green, continued by a biochemical test with red ring-positive Indole, Methyl Red positive the color is red, negative Voges Paskauer if there is no change in color, and negative citrate will turn green. Results showed that from the 16 samples of fresh milk used there were 7 samples of fresh milk that exceeded the contamination limit E. coli or < 3 apm /ml. Conclusion, number of E. coli in fresh milk at KPSP Ijen Makmur 43.75% of the total sample exceeded maximum contamination.

Foodborne salmonellosis is commonly associated with poultry and poultry products, necessitating continued development of pre- and postharvest food safety interventions and risk management strategies. Evaluation of technologies and strategies is limited by availability of cost-effective, rapid laboratory methods. The objective of this study was to evaluate a commercial qualitative PCR assay and its novel quantitative application to detect and enumerate Salmonella in poultry ceca as an analytical matrix. Ceca were collected at harvest, the contents were homogenized, and paired samples were evaluated with buffered peptone water (BPW) and BAX MP+Supplement (MPS) preenrichment broths followed by PCR screening with a BAX System Q7 PCR and by culture isolation. Additional ceca were inoculated with Salmonella to develop a standard curve for the BAX System SalQuant quantitative PCR application (QA), and estimates were obtained by the QA and most-probable-number (MPN) methods. For preenrichment media, PCR outcomes were equivalent to those of culture isolation for detecting Salmonella in ceca with 95.65 and 87.88% sensitivity and 82.00 and 100.00% specificity (P = 0.074) for BPW and MPS, respectively. However, at the sample level, BPW performed significantly worse (47.92%) than did MPS (68.75%) for overall isolation of Salmonella (P < 0.0001). After standard curve development, the mean QA estimates obtained for the inoculated samples were 1.14 (95% confidence interval [CI]: 0.62 to 1.66), 1.79 (1.50 to 2.08), 2.91 (2.65 to 3.17), and 3.76 (3.26 to 4.25) log CFU/mL for each targeted inoculation of 1.0, 2.0, 3.0, and 4.0 log CFU/mL, respectively, and were within or comparable to the 95% CI values of paired MPN estimates. These data support the use of MPS for the detection and isolation of Salmonella enterica from poultry ceca when screening with PCR and indicate that QA may be useful as an alternative tool to estimate Salmonella loads in poultry ceca, which may support preharvest food safety interventions. [ABSTRACT FROM AUTHOR]

Seasonal variation plays an important role in the occurrence of diarrheal diseases and distinct seasonal occurrence of diarrheal diseases, with bacterial diarrhea occurring more during the warm seasons and viral diarrhea occurring during the drier cool months, has been observed due to seasonal variation. Time-series cross-sectional study was conducted from January to December 2018 among 553 under-five children to assess seasonal variation of diarrheal diseases and its association with the drinking water quality in a resettlement colony of Delhi. The prevalence of diarrhea was 40.7% and was highest during the rainy season (67.6%). Majority of the water samples were found to be unsatisfactory for human consumption during the rainy and summer seasons. Significant and moderate relationship was found between the seasonal variation and occurrence of diarrhea (r = 0.728, P < 0.05) and most probable number count (r = 0.50, P < 0.05), respectively. Understanding the environmental factors that influences the occurrence of diarrheal diseases is warranted.

Objective: Drinking water analysis is an important quality monitor and forms an integral part of the infection control policy of every hospital. On an average daily basis, 1750 liters of drinking water are used by patients and hospital staff. Presence of contaminating microorganisms in drinking water should therefore be identified at the earliest and necessary action be taken in order to prevent outbreaks through contaminated water. This study was conducted to compare two methods for drinking water analysis and document benefits of the new method. Methods: This cross sectional study was conducted over a period of two years at a tertiary care hospital in Chennai. A newer rapid method of drinking water analysis was evaluated for two years from July 2017 to June 2019. Parallel testing with conventional multiple tube method was performed. All samples collected from various sources during the study period were subjected to parallel testing by multiple tube method (conventional method for most probable number) and presence absence method (new method). Results: The new commercial kit for drinking water analysis comprehensively detects all water borne bacterial pathogens associated with gastroenteritis within 18 hours. The major advantage is marked reduction in turnaround time by 54 hours. False positive/false negative results were not encountered compared to the standard conventional method. Conclusion: Safer, healthier and high quality of water is ensured for use at an earlier and cost effective as well as environment friendly system. There was marked reduction in turnaround time, cost of testing, manpower usage, reagent wastage, waste generation using the kit based presence absence method. The new commercial kit can effectively replace the conventional method as an effective screening tool for drinking water analysis.

The present study aimed to evaluate the efficacy of a depuration system equipped with UV-irradiation to control Vibrio vulnificus infection such as septicemia (or sepsis) using alive oysters. After 6 h of bioaccumulation of V. vulnificus, Pacific oyster Crassostrea gigas were found to be contaminated by > 8.0 log MPN/g of V. vulnificus cells. After 60 h of depuration, the V. vulnificus cell number significantly decreased to < 4.0 log MPN/g. The present depuration process meets the standard effectiveness in reducing V. vulnificus cells by > 3.52 log and < 30 MPN/g as recommended by the National Shellfish Sanitization Procedure Molluscan Shellfish Control guidelines. Furthermore, no significant changes in pH value and glycogen content indicate that the depuration process did not affect the freshness and quality of the oyster samples. The present study could help control any potential infection associated with the consumption of raw oysters without losing their quality. [ABSTRACT FROM AUTHOR]

• A new calibration-free, non-invasive, and accurate microbial total viable count (TVC) detection method (MPN-TDLAS method) was innovatively proposed. • The MPN-TDLAS method combines the MPN counting method with the TDLAS technique, which not only achieves the advantages of calibration-free and a large measurement range but also overcomes the shortcomings of large subjective errors. • The minimum detection limit of the homemade CO 2 sensor system is analyzed to be 76.4 ppm at a path length of 3.6 cm. • The MPN-TDLAS method showed a good correlation with the standard plate count ISO: 4833: 2013 method for TVC (R2 = 0.99). • The result of the quality control (QC) sample detection was satisfactory (Z-score was less than 2), which demonstrated that the MPN-TDLAS method is well-suited for food microbiology testing applications. Total viable count (TVC), as an important indicator in the microbiological detection. A calibration-free, non-invasive, and accurate TVC detection method was developed using the most probable number (MPN) method and tunable diode laser absorption spectroscopy (TDLAS). In the experiment, a tunable distributed feedback (DFB) laser with a center wavelength of 4989.97 cm−1 was used to measure CO 2 emitted from the microorganisms in a sealed culture bottle. Then, the thresholds of negative and positive culture bottles in the MPN-TDLAS method were established by the features of the Staphylococcus aureus (S. aureus) growth curve. The experimental results revealed that the MPN-TDLAS method has a good correlation (R2 = 0.99) with the TVC of the ISO:4833:2013 standard plate counting method, and the result of the quality control (QC) sample was satisfactory (Z-score was less than 2), which demonstrated that this method is well-suited for food microbiology testing applications. [ABSTRACT FROM AUTHOR]

Water contaminated by human and animal excreta may contain a range of pathogenic microorganisms such as viruses, bacteria and protozoa. During the bathing season, water samples are taken and analyzed for Escherichia coli and intestinal enterococci. The aim of these study is to assess the quality of bathing waters along Black Sea Coast in Varna Region from 2012 to 2016. Materials and Methods: The information about microbiological monitoring of coastal bathing waters in Region Varna is received from Regional Health Inspection – Varna. Results and discussion: From May 2012 to August 2016, 1117 water samples were collected and analyzed in the Laboratory of Sanitary Microbiology of Regional Health Inspection – Varna. In the region of Varna, there is a tendency to increase the number of bathing areas with excellent water quality. In Avren Municipality, bathing water was classified as good and excellent. In Shkorpilovtsi – Tsentralen plazh, the bathing water was excellent quality. In Byala Municipality there was a deterioration in the quality of the seawater. During 2012-2016 two of these 17 sites – Riviera, located in resort Golden Sands and St. Elias located in St. St. Konstantin and Elena had excellent bathing water quality. The water indicators of Ofitserski plazh- Varna and Tsentralen plazh, Zlatni pyasatsi - PSVO were poor, while the indicators of Varna -Yuzhen plazh were satisfying or good. Conclusion: According to the European Environment Agency, the development and improvement of community water supply and collecting system will have a positive effect on the quality of bathing water.

Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects., Importance: We created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil., Competing Interests: The authors declare no conflict of interest.

Food is one of components that can cause illness, so it must be safe. An outbreaks of food poisoning 19% happened in school and 78.57% happened to elementary school children. Hygiene and sanitation are important factors related to food safety. This study was aimed to analyze correlation between behavior of hygiene and sanitation of food vendors with food safety by using most probable number of coliform indicator. This was an observational study used cross sectional design, involving 25 food vendors and 25 food sample which were selected randomly. Result showed that 40% of the snacks were contaminated by Coliform (>10 Juml./g). The highest contamination of Coliform was >1.100 Juml./g and was found on four food snacks sold in the canteen. Categories of hygiene knowledge score of vendors ware moderate (52%). The majority of vendors ware included in the moderate hygiene attitude score category (52%), and most of the vendors ware included in the moderate hygiene practice score category and moderate environment sanitation score category (56%, respectively). Statistical analysis using Spearman’s rho test showed that there ware a significant correlation between knowledge of hygiene, practice of hygiene, and environment sanitation of vendors with food safety using indicator most probable number of coliform (p=0.022, p=0.005, p=0.000, respectively). Whereas no significant correlation found between attitude of hygiene with contamination of Coliform (p=0.088). It was Found as much as 40% of foods are contaminated by coliform and should be awared, observations of food processing needs to be applied to prevent coliform contamination of school children’s snacks.

Pickled ready-to-eat meat (PRTEM) is a meat product that is treated with various seasonings and then cooked. PRTEM is a popular food consumed mostly in China and some Asian countries. Since this food is considered ‘ready to eat’, once it is contaminated by foodborne pathogens such as Salmonella, the prospect for significant morbidity, mortality, and immeasurable economic losses can occur. Here we investigated the prevalence and concentration of Salmonella in 107 PRTEM samples collected from Shaanxi, China during 2015–2016. Furthermore, we analyzed the serotype, antibiotic susceptibility, and presence of antibiotic resistance genes and amino acid mutations in 219 Salmonella isolates, followed by subtyping of 115 representative isolates. The average detection rate of Salmonella-positive PRTEM was 58.9%, and the average most probable number (MPN) of Salmonella in positive samples was 2.27 logMPN per gram of sample (range: 2.10–2.43). Ten serotypes were identified from the 219 Salmonella isolates, with S. Thompson (37.9%) and S. Indiana (20.5%) being predominant. The remaining serotypes were S. Typhi (7.8%), S. Typhimurium (7.3%), S. Mbandaka (6.9%), S. Albany (6.4%), S. Blockley (5.5%), S. Infantis (4.1%), S. Escanaba (3.2%), and S. Dusseldorf (0.5%). All isolates were resistant to ceftiofur (100%), while most of them were resistant to ciprofloxacin (99.1%), amoxicillin-clavulanic acid (97.7%), trimethoprim-sulfamethoxazole (96.4%), ampicillin (92.3%), sulfisoxazole (92.2%), tetracyclines (90.4%), and nalidixic acid (90.4%), respectively. A single mutation of Ser83Phe (27.1%) and double mutations of Ser83Phe-Asp87Gly (25.9%) in GyrA were detected in 85 isolates, whereas mutations of Thr57Ser (63.9%) and Ser80Arg (36.1%) in ParC were detected in 122 isolates. qnrB, oqxAB, aac(6′)-Ib, and qnrA were present in 50 (22.8%), 48 (21.9%), 26 (11.9%), and 1 (0.5%) isolate(s), respectively. Pulse field gel electrophoresis results revealed that those isolates recovered from the same type of PRTEM or the same sampling place shared identical or similar DNA profiles, antibiotic resistance phenotypes, and even plasmid-mediated quinolone resistance encoding genes. The findings indicate that Salmonella is commonly prevalent in PRTEMs at high concentrations in Shaanxi, China. More attention should be paid to the processing and storage of this ready-to-eat food to prevent bacterial contamination and foodborne outbreaks.

The random variable of passenger journeys between any two stopping points on a route (passenger correspondence) represents a discrete variable. For each value of the discrete random variable, its probability is calculated. As a solution to the problem of determining the number of corresponding passengers between two stopping points on a route, we take the most probable value of the random variable. [ABSTRACT FROM AUTHOR]

Tofu is one of the products that are often consumed in the community because the price is cheap and easy to obtain. Tofu has high protein and water content and pH is close neutral. This makes tofu a suitable medium for growing pathogen microbial in food. The presence of pathogen microbial in tofu can cause health problems for those who consume them. Objectives This research was conducted to observe the microbial content in tofu snacks in three Medan city markets. Method This research was conducted by conducting laboratory tests and the results obtained in the form of a description of microbial content in the sample of tofu. The content of coliform microbes in tofu samples was tested using the Most Probable Number (MPN) method and biochemical identification. The tofu sample was selected using total sampling method from three markets in the city of Medan. Results A total of 15 tofu samples from three markets were analyzed in this study. From the MPN test, almost all samples had MPN levels exceeding the limits set in the Regulation of the Badan Pengawas Obat dan Makanan No. 13 of 2019, which is below 3 MPN / g. Five samples of tofu (33.3%) of E.coli bacteria, 9 samples (60%) of Klebsiella sp and 1 sample (6.7%) contained Enterobacter sp. Conclusions Bacteria found in this study were Escherichia coli, Klebsiella sp and Enterobacter sp. The presence of microbial content in the tofu sample that exceeds the threshold must be a concern of various parties in the supervision of snacks being sold. [ABSTRACT FROM AUTHOR]

Objective: Drinking water analysis is an important quality monitor and forms an integral part of the infection control policy of every hospital. On an average daily basis, 1750 liters of drinking water are used by patients and hospital staff. Presence of contaminating microorganisms in drinking water should therefore be identified at the earliest and necessary action be taken in order to prevent outbreaks through contaminated water. This study was conducted to compare two methods for drinking water analysis and document benefits of the new method. Methods: This cross sectional study was conducted over a period of two years at a tertiary care hospital in Chennai. A newer rapid method of drinking water analysis was evaluated for two years from July 2017 to June 2019. Parallel testing with conventional multiple tube method was performed. All samples collected from various sources during the study period were subjected to parallel testing by multiple tube method (conventional method for most probable number) and presence absence method (new method). Results: The new commercial kit for drinking water analysis comprehensively detects all water borne bacterial pathogens associated with gastroenteritis within 18 hours. The major advantage is marked reduction in turnaround time by 54 hours. False positive/false negative results were not encountered compared to the standard conventional method. Conclusion: Safer, healthier and high quality of water is ensured for use at an earlier and cost effective as well as environment friendly system. There was marked reduction in turnaround time, cost of testing, manpower usage, reagent wastage, waste generation using the kit based presence absence method. The new commercial kit can effectively replace the conventional method as an effective screening tool for drinking water analysis. [ABSTRACT FROM AUTHOR]

Pickled ready-to-eat meat (PRTEM) is a meat product that is treated with various seasonings and then cooked. PRTEM is a popular food consumed mostly in China and some Asian countries. Since this food is considered 'ready to eat', once it is contaminated by foodborne pathogens such as Salmonella , the prospect for significant morbidity, mortality, and immeasurable economic losses can occur. Here we investigated the prevalence and concentration of Salmonella in 107 PRTEM samples collected from Shaanxi, China during 2015–2016. Furthermore, we analyzed the serotype, antibiotic susceptibility, and presence of antibiotic resistance genes and amino acid mutations in 219 Salmonella isolates, followed by subtyping of 115 representative isolates. The average detection rate of Salmonella -positive PRTEM was 58.9%, and the average most probable number (MPN) of Salmonella in positive samples was 2.27 logMPN per gram of sample (range: 2.10–2.43). Ten serotypes were identified from the 219 Salmonella isolates, with S. Thompson (37.9%) and S. Indiana (20.5%) being predominant. The remaining serotypes were S. Typhi (7.8%), S. Typhimurium (7.3%), S. Mbandaka (6.9%), S. Albany (6.4%), S. Blockley (5.5%), S. Infantis (4.1%), S. Escanaba (3.2%), and S. Dusseldorf (0.5%). All isolates were resistant to ceftiofur (100%), while most of them were resistant to ciprofloxacin (99.1%), amoxicillin-clavulanic acid (97.7%), trimethoprim-sulfamethoxazole (96.4%), ampicillin (92.3%), sulfisoxazole (92.2%), tetracyclines (90.4%), and nalidixic acid (90.4%), respectively. A single mutation of Ser83Phe (27.1%) and double mutations of Ser83Phe-Asp87Gly (25.9%) in GyrA were detected in 85 isolates, whereas mutations of Thr57Ser (63.9%) and Ser80Arg (36.1%) in ParC were detected in 122 isolates. qnr B, oqx AB, aac(6′)-Ib , and qnr A were present in 50 (22.8%), 48 (21.9%), 26 (11.9%), and 1 (0.5%) isolate(s), respectively. Pulse field gel electrophoresis results revealed that those isolates recovered from the same type of PRTEM or the same sampling place shared identical or similar DNA profiles, antibiotic resistance phenotypes, and even plasmid-mediated quinolone resistance encoding genes. The findings indicate that Salmonella is commonly prevalent in PRTEMs at high concentrations in Shaanxi, China. More attention should be paid to the processing and storage of this ready-to-eat food to prevent bacterial contamination and foodborne outbreaks. [ABSTRACT FROM AUTHOR]

Microbiological and geochemical data are presented to characterize the hydrogeochemistry and to investigate extant microbial life in fracture waters 2.4 km below surface, at the Kidd Creek Observatory in Canada. Previous studies identified the world's oldest groundwaters with mean residence times on the order of millions to billions of years trapped in fractures in Precambrian host rock here. In this study, major ion chemistry, δ18O and δ2H isotopic signatures and dissolved gases in the fracture waters are shown to be distinct from potential contamination end-members, demonstrating the fracture waters are not impacted by waters used in mining operations. A previous work on sulfur isotope signatures suggested a longstanding indigenous population of sulfate-reducing bacteria in these highly reducing fluids and sufficient sulfate to support microbial activity. Here, we report the first evidence for extant visible and cultivable microbial life at this location. Anaerobic metabolisms were investigated using the Most Probable Number (MPN) method. The fracture fluids contained extant cells at low biomass density (∼103–104 cells/mL) and showed a strong response from autotrophic sulfate-reducers and alkane-oxidizing sulfate reducers. These lines of evidence provide the interpretational framework (chemical, hydrogeologic, and microbiologic) essential to the on-going genomic and metagenomic investigations at the Kidd Creek Observatory – the world's most longstanding location for investigation of subsurface fluids and deep life at such profound depth. [ABSTRACT FROM AUTHOR]

To challenge ballast water treatment system (BWTS) efficacy for organisms in the size-class 10–50 μm, intake concentration during tests must reach certain minimum requirements. Often, natural concentrations are too low to meet intake requirements and standard test organisms (STOs) are added. We tested the robustness of Tetraselmis suecica and Odontella sp. to a range of UV-treatments to explore fluences needed to meet the IMO discharge standard (<10 org. ml−1) evaluated using two viability assessment methods. To meet discharge standards, fluences of >1000 mJ cm−2 were required using vital stain whereas 135–500 mJ cm−2 were needed using regrowth assays. Besides, results suggest that T. suecica and Odontella sp. were at least as robust as natural algae towards UV-treatments. We suggest the advantageous use of these species as STOs in test water to support intake water requirements and to obtain more conservative validation of UV-based BWTS to ensure more environmental protective procedures. Display Omitted • The use of standard test organisms for safer validation of UV-based BWTSs • Higher robustness of STOs towards UV treatments compared to natural algae • MPN method more suitable than vital stain method for validation of UV-based BWTS • IMO intake concentration requirement can be fulfilled with standard test organisms. [ABSTRACT FROM AUTHOR]

Ultraviolet radiation (UV) and total residual oxidant (TRO) treatments have been used to control microorganisms in drinking water, and are commonly used in ballast water management systems designed to reduce the transport and delivery of invasive organisms. Because treatments may not result in immediate cell death but may render organisms unable to reproduce, standard approaches to determine if a cell is living or dead (i.e., based on movement or cellular function) may not identify treated organisms. This scenario particularly applies to UV-treated cells that are rendered non-viable but still living. Thus, alternative approaches are needed to determine an organism's viability (ability to reproduce). This study evaluated a cell proliferation assay measuring DNA concentrations in 96-well plates to determine proliferation of UV- and chlorine-treated organisms. Treatments included UV doses of 300 and 800 mWs cm-2 or an applied ClO- concentration of 10 mg L-1. To determine optimal cell proliferation thresholds and detection limits, two phytoplankton species, Tetraselmis marina (UV and ClO-) and Prorocentrum micans (ClO-), were evaluated immediately following treatment and after 1-, 5-, and 14-day incubations in light or dark conditions. T. marina exhibited cell proliferation in all UV treatments, although no cell proliferation was detected for T. marina or P. micans in any ClO- treatment. This type of assay allows for increased sample replication, smaller sample volumes, quicker preparation, and faster measurement than traditional growth assays. In conclusion, DNA staining assays may be useful to detect changes in cell proliferation over time in the context of ballast water testing. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Technological transference from laboratory scale to rural area in a developing country. • Energy available as Biogas to replace traditional fuels in non-interconnected areas. • Use of a low cost technique to estimate quantity and quality of microbial community in inoculum. • Structure, dynamics and balance are indicators of microbial population behavior in inoculum. Abstract The aim of this work was to use an inexpensive technique, such as the most probable number, MPN to quantitatively and qualitatively examine microbial groups in anaerobic digestion, AD of cattle manure, CM. Different diluents and mixing ratios were evaluated in 100 mL reactors using biomethane potential, BMP, as output variable. MPN tests were performed to samples from a 23 L tank and 6.5 m3 low-cost tubular digester, LCTD. To monitor LCTD stability, volatile solids, VS, specific methanogenic activity, SMA, pH, volatile fatty acids, VFA, and chemical oxygen demand, COD were measured. Microbial characterization highlighted the optimal population essential to initiate the anaerobic digestion process. Bacteria domain were predominant over Archaea, with average abundance values of 78% and 22%, respectively. SMA of 0.0486 g COD g−1 SV d−1 was obtained at laboratory-scale and LCTD reached 83% of this value. Conditions selected are cost-effective to establish a biodigester in rural areas. [ABSTRACT FROM AUTHOR]

The most probable number (MPN) assay is used to enumerate viable phytoplankton in the 10-50 μm size range for Type Approval of ballast water management systems. However, its validation has been questioned by the United States Coast Guard, which does not accept it as an alternative to its required method. To address key elements of method validation, three research laboratories used the same experimental protocols to enumerate viable organisms from uniformly viable and heat-killed cultures of three species of phytoplankton. Statistical analysis of the ratio of MPN estimates of viable organisms to measurements from flow cytometry was consistent with predictions based on perfect adherence to the method's assumptions. These include reliable detection of all viable organisms and no false-positive results. The validation confirmed established estimates of the MPN method's precision and demonstrated reproducibility, having been transferred to and implemented by two of the laboratories for the first time. [ABSTRACT FROM AUTHOR]