scholarly journals Colourless agar for enhancing colour contrast between microbial colonies and agar

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Deionized Water ◽  
Good Growth ◽  
Automated Identification ◽  
E Coli ◽  
Colour Contrast ◽  
Varied Background ◽  
Ammonium Compounds ◽  
Standard Techniques

Lack of colour contrast hampers automated identification of colonies on agar, given that many microbial colonies are of the same colour as the background colour of most agar: beige colour. On the other hand, a colourless agar could increase the colour contrast between the agar background and microbial colonies. But, the challenge in preparing a colourless agar comes from the formation of coloured compounds when sugars and ammonium compounds are sterilized together in an autoclave. Hence, by separating glucose and ammonium compounds into different solutions for autoclave sterilization, a method was developed for preparing colourless agar that remained colourless even with 1 g/L of yeast extract supplementation. Specifically, three separate solutions were used in reconstituting the colourless agar at ~48 oC after individual sterilization. Solution A comprised glucose, MgSO4 and agar powder; Solution B contained NH4Cl, K2HPO4, KH2PO4, and NaCl; and Solution C was yeast extract solution for providing vitamins and trace elements needed by microorganisms unable to grow in minimal medium. Reconstituted colourless agar could be poured into agar plates using standard techniques and had a viscosity similar to many commercial agar. Composition of the colourless agar medium was [g/L]: D-Glucose, 2.0; NH4Cl, 0.5; K2HPO4, 0.5; KH2PO4, 0.1; NaCl, 0.5; MgSO4.7H2O, 1.0; Yeast extract, 1.0; Agar, 15.0. On observation against varied background images, the formulated colourless agar lacked the yellow tinge present in LB Lennox agar and had greater optical transparency. Good growth of common bacteria such as Escherichia coli DH5α (ATCC 53868), Bacillus subtilis NRS-762 (ATCC 8473) and Pseudomonas protegens Pf-5 (ATCC BAA-477) was observed in both liquid and solid versions of the formulated colourless agar medium. Specifically, colonies of E. coli DH5α, B. subtilis NRS-762 and P. protegens Pf-5 exhibited similar morphology and characteristics compared to growth on LB Lennox agar. Use of the formulated agar medium in cultivating microbial flora present in deionized water recovered many types of colonies, which suggested that the medium was able to support a variety of microbial species. Collectively, by separately sterilizing sugars and ammonium compounds, a method was developed for preparing colourless agar which helped improve colour contrast for colony identification and counting. The formulated colourless agar could find use in various microbial ecology studies seeking to profile microbes from different environmental niches.

scholarly journals Colourless agar for enhancing colour contrast between microbial colonies and agar

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Deionized Water ◽  
Good Growth ◽  
Automated Identification ◽  
E Coli ◽  
Colour Contrast ◽  
Varied Background ◽  
Ammonium Compounds ◽  
Standard Techniques

Lack of colour contrast hampers automated identification of colonies on agar, given that many microbial colonies are of the same colour as the background colour of most agar: beige colour. On the other hand, a colourless agar could increase the colour contrast between the agar background and microbial colonies. But, the challenge in preparing a colourless agar comes from the formation of coloured compounds when sugars and ammonium compounds are sterilized together in an autoclave. Hence, by separating glucose and ammonium compounds into different solutions for autoclave sterilization, a method was developed for preparing colourless agar that remained colourless even with 1 g/L of yeast extract supplementation. Specifically, three separate solutions were used in reconstituting the colourless agar at ~48 oC after individual sterilization. Solution A comprised glucose, MgSO4 and agar powder; Solution B contained NH4Cl, K2HPO4, KH2PO4, and NaCl; and Solution C was yeast extract solution for providing vitamins and trace elements needed by microorganisms unable to grow in minimal medium. Reconstituted colourless agar could be poured into agar plates using standard techniques and had a viscosity similar to many commercial agar. Composition of the colourless agar medium was [g/L]: D-Glucose, 2.0; NH4Cl, 0.5; K2HPO4, 0.5; KH2PO4, 0.1; NaCl, 0.5; MgSO4.7H2O, 1.0; Yeast extract, 1.0; Agar, 15.0. On observation against varied background images, the formulated colourless agar lacked the yellow tinge present in LB Lennox agar and had greater optical transparency. Good growth of common bacteria such as Escherichia coli DH5α (ATCC 53868), Bacillus subtilis NRS-762 (ATCC 8473) and Pseudomonas protegens Pf-5 (ATCC BAA-477) was observed in both liquid and solid versions of the formulated colourless agar medium. Specifically, colonies of E. coli DH5α, B. subtilis NRS-762 and P. protegens Pf-5 exhibited similar morphology and characteristics compared to growth on LB Lennox agar. Use of the formulated agar medium in cultivating microbial flora present in deionized water recovered many types of colonies, which suggested that the medium was able to support a variety of microbial species. Collectively, by separately sterilizing sugars and ammonium compounds, a method was developed for preparing colourless agar which helped improve colour contrast for colony identification and counting. The formulated colourless agar could find use in various microbial ecology studies seeking to profile microbes from different environmental niches.

scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Agar medium enables spatially-resolved simultaneous cultivation of different microbes in a simple and relatively low-cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. Nevertheless, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilisation, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilisation, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesise them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas fluorescens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilisation; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Solid medium enables spatially-resolved simultaneous cultivation of different microbes in a simple and relatively low-cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. Nevertheless, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilisation, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilisation, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesise them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilisation; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Solid medium enables spatially resolved simultaneous cultivation of different microbes in a simple and relatively low cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. However, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilization, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilization, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesize them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilization; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

334. A simple agar medium for the growth of lactic streptococci: the role of phosphate in the medium

1946 ◽  
Vol 14 (3) ◽  
pp. 283-290 ◽  
Author(s):  
G. J. E. Hunter
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Yeast Extract ◽  
Active Substance ◽  
Agar Medium ◽  
Good Growth ◽  
Paper Pulp ◽  
Buffering Effect ◽  
Growth Promoting

A cheap, easily prepared and relatively uniform agar medium, containing beef extract, lactose, peptone, yeast extract and phosphate, is described. It gives excellent results with lactic streptococci and a good growth with many other organisms.The role of phosphate and agar in the medium and the effect of filtration is discussed. It is suggested that the value of phosphate is due partly to its buffering effect and partly to its combination with some constituent of agar to form a substance which has a growth-promoting action on the lactic acid bacteria. This active substance is largely removed from the medium if filtration through paper pulp is practised.

Microbiological performance and salmonella dynamics in a wastewater depuration pond system of southeastern spain

1995 ◽  
Vol 31 (12) ◽  
pp. 239-248 ◽  
Author(s):  
Ana Emparanza-Knörr ◽  
Francisco Torrella
Keyword(s):  
Fecal Coliform ◽  
No Reduction ◽  
Agar Medium ◽  
Microbiological Quality ◽  
Total Coliform ◽  
Fecal Coliforms ◽  
Selective Media ◽  
E Coli ◽  
The Village ◽  
Final Effluent

The Salmonella presence and the microbiological quality indicators, total and fecal coliforms and coliphages of E. coli C, have been studied in a overloaded wastewater lagoon system treating urban wastewatrers of the village of Guardamar del Segura (Alicante, Spain). Classical microbiological technology to detect salmonellae was used, including pre-enrichment, enrichment, selective media plating and biochemical and serological confirmation. Water was physicochemically characterized according to COD, SS, temperature, pH and dissolved oxygen. The selective migration step through Rappaport-Vassiliadis semisolid agar medium was essential for the consistent detection of Salmonella in the different lagoon effluents. Total and fecal coliform levels of up to 105-106 MPN/100 ml were detected in the final effluent. High coliphage concentrations of 103-104 pfu/ml were also found in the effluent waters. Salmonella was always detected in 100 ml samples and eventually reached an order of value of 103 MPN/100 ml. Total coliform reduction was higher in the anaerobic ponds whereas fecal coliforms were more efficiently eliminated in the facultative (mostly “anoxic”) lagoons. Coliphage reduction was higher in the facultative lagoons when compared to the anaerobic ponds. On many occasions, no reduction or eventual increment of the concentration of salmonellae was detected in the effluents from the anaerobic ponds compared to concentrations of the patohogen in the influent raw wasterwaters. The possibility exists for a capacity of Salmonella to multiply in the anoxic phase of the wastewater treatment, but the presence of microorganisms in raw sewage waters which could maskSalmonella detection with the enrichment methodology employed cannot be ruled out.

scholarly journals Phenotypic and Genotypic Properties of Fluoroquinolone-Resistant, qnr-Carrying Escherichia coli Isolated from the German Food Chain in 2017

2021 ◽  
Vol 9 (6) ◽  
pp. 1308
Author(s):  
Katharina Juraschek ◽  
Carlus Deneke ◽  
Silvia Schmoger ◽  
Mirjam Grobbel ◽  
Burkhard Malorny ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Point Mutations ◽  
Beta Lactamase ◽  
Resistance Development ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
German Food ◽  
Sequence Types ◽  
Ammonium Compounds ◽  
Additional Point

Fluoroquinolones are the highest priority, critically important antimicrobial agents. Resistance development can occur via different mechanisms, with plasmid-mediated quinolone resistance (PMQR) being prevalent in the livestock and food area. Especially, qnr genes, commonly located on mobile genetic elements, are major drivers for the spread of resistance determinants against fluoroquinolones. We investigated the prevalence and characteristics of qnr-positive Escherichia (E.) coli obtained from different monitoring programs in Germany in 2017. Furthermore, we aimed to evaluate commonalities of qnr-carrying plasmids in E. coli. We found qnr to be broadly spread over different livestock and food matrices, and to be present in various sequence types. The qnr-positive isolates were predominantly detected within selectively isolated ESBL (extended spectrum beta-lactamase)-producing E. coli, leading to a frequent association with other resistance genes, especially cephalosporin determinants. Furthermore, we found that qnr correlates with the presence of genes involved in resistance development against quaternary ammonium compounds (qac). The detection of additional point mutations in many isolates within the chromosomal QRDR region led to even higher MIC values against fluoroquinolones for the investigated E. coli. All of these attributes should be carefully taken into account in the risk assessment of qnr-carrying E. coli from livestock and food.

Effects of Water Source, Dilution, Storage, and Bacterial and Fecal Loads on the Efficacy of Electrolyzed Oxidizing Water for the Control of Escherichia coli O157:H7

2004 ◽  
Vol 67 (7) ◽  
pp. 1377-1383 ◽  
Author(s):  
S. M. L. STEVENSON ◽  
S. R. COOK ◽  
S. J. BACH ◽  
T. A. McALLISTER
Keyword(s):  
Escherichia Coli ◽  
Bactericidal Activity ◽  
Storage Temperature ◽  
Uv Light ◽  
Tap Water ◽  
Organic Matter Content ◽  
Water Source ◽  
Deionized Water ◽  
Escherichia Coli O157 ◽  
E Coli

To evaluate the potential of using electrolyzed oxidizing (EO) water for controlling Escherichia coli O157:H7 in water for livestock, the effects of water source, electrolyte concentration, dilution, storage conditions, and bacterial or fecal load on the oxidative reduction potential (ORP) and bactericidal activity of EO water were investigated. Anode and combined (7:3 anode:cathode, vol/vol) EO waters reduced the pH and increased the ORP of deionized water, whereas cathode EO water increased pH and lowered ORP. Minimum concentrations (vol/vol) of anode and combined EO waters required to kill 104 CFU/ml planktonic suspensions of E. coli O157:H7 strain H4420 were 0.5 and 2.0%, respectively. Cathode EO water did not inhibit H4420 at concentrations up to 16% (vol/vol). Higher concentrations of anode or combined EO water were required to elevate the ORP of irrigation or chlorinated tap water compared with that of deionized water. Addition of feces to EO water products (0.5% anode or 2.0% combined, vol/vol) significantly reduced (P < 0.001) their ORP values to <700 mV in all water types. A relationship between ORP and bactericidal activity of EO water was observed. The dilute EO waters retained the capacity to eliminate a 104 CFU/ml inoculation of E. coli O157:H7 H4420 for at least 70 h regardless of exposure to UV light or storage temperature (4 versus 24°C). At 95 h and beyond, UV exposure reduced ORP, significantly more so (P < 0.05) in open than in closed containers. Bactericidal activity of EO products (anode or combined) was lost in samples in which ORP value had fallen to ≤848 mV. When stored in the dark, the diluted EO waters retained an ORP of >848 mV and bactericidal efficacy for at least 125 h; with refrigeration (4°C), these conditions were retained for at least 180 h. Results suggest that EO water may be an effective means by which to control E. coli O157:H7 in livestock water with low organic matter content.

scholarly journals The Survey of Microbial Quality of the Dry Sample, Extract and Brewing of some Medicinal Plants

2014 ◽  
Vol 6 (4) ◽  
pp. 478-482
Author(s):  
Razieh VALIASILL ◽  
Majid AZIZI ◽  
Maasome BAHREINI ◽  
Hossein AROUIE
Keyword(s):  
Medicinal Plants ◽  
Pathogenic Bacteria ◽  
Agar Medium ◽  
Total Count ◽  
Plate Count ◽  
Microbial Quality ◽  
Salmonella Spp ◽  
E Coli ◽  
Wide Range

Medicinal plants may be exposed to a wide range of microbial contamination during pre- and post- harvest stages and they can present high microbial counts. In this study, the microbial quality of 44 samples of dry herbs namely: mint (Menthaspp.), lemon balm (Melissa officinalis), summer savory (Satureja hortensis), zataria (Zataria multiflora), Indian valerian (Valeriana wallichii), their brewing and extracts were analyzed. Total count using plate count agar medium (PCA), coliform count by Violet Red Bile Agar (VRBL), Enterobacteriacea by Violet Red Bile Glucose (VRBG) were evaluated. Medium Baird-Parker agar (BP) medium and Tryptone Bile X-Gluc (TBX) medium were used for the isolation and enumeration of Staphylococcus aurous and E. coli spp. respectively. Furthermore, Xylose Lysine Deoxycholate agar medium (XLD) and Bismuth Sulfite Agar medium(BSA) were used for detection of Salmonella spp. Fungal and mold contamination was assessed using yeast extract glucose chloramphenicol agar. The results showed that the contamination of the samples with total count (100%) and Enterobacteriaceae (85%), total coliform (83%), mold and yeast (98%) and E. coli ssp. (2.27) were detected, including in the study samples the absence of pathogenic bacteria like Staphylococcus aurous, Salmonella spp. Moreover, the extract had a lower microbial load in comparison to dry herb samples. Also, the lowest and the highest of contamination rates were observed for Indian valerian and zataria, respectively. According to the results, there is a need to control the environmental conditions and improve hygiene in the production process; even more, it is recommended to choose a suitable decontamination method for disinfection during packing medicinal plants and during post-packing manipulation and transport.

scholarly journals A Clean-In-Place Type Sanitation Method Validation for a Benchtop Meat Grinder

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
C. E. Rayfield ◽  
R. Jadeja ◽  
S. Billups
Keyword(s):  
Lactic Acid ◽  
Foodborne Pathogens ◽  
Peracetic Acid ◽  
Deionized Water ◽  
Cross Contamination ◽  
Direct Plating ◽  
Water Ice ◽  
E Coli ◽  
Different Types ◽  
Low Levels

ObjectivesThis research is designed to validate a novel clean-in-place type antimicrobial ice-based meat grinder sanitation method.Materials and MethodsFour different types of antimicrobial ice were prepared from peracetic acid (PAA, 350 mg/L) and combination PAA with 2% FreshFX® (PAAF), 2% Paradigm® (PAAP) and 2% lactic acid (PAAL). The grinders were inoculated by processing 400 g beef trim containing 400 μL of E. coli O157:H7 or S. Typhimurium DT 104 suspensions at 8.4 to 8.7 (high inoculation) and 5.3 to 5.5 (low inoculation) log CFU/mL. Each meat grinder was then treated by processing 1000 g of antimicrobial ice and 500 mL of corresponding antimicrobial solution. At the end of each treatment, 400 g un-inoculated beef was processed through the meat grinder, and the resulting ground beef was then analyzed for the presence of target pathogens by direct plating and after enrichment. Efficacies of antimicrobial ice-based treatments were compared with 1000 g deionized water ice + 500 mL deionized water (DI), and no treatment (NT) controls.ResultsAll antimicrobial ice treatments were able to reduce cross-contamination to non-detectable levels from the meat grinders inoculated at the low levels of pathogens, but after enrichment, target pathogens were detected in all the samples. Recoveries from the meat grinder inoculated with high levels of pathogens ranged from 5.95 to 3.50 log CFU/g and 5.86 to 3.46 log CFU/g for E. coli O157:H7 and S. Typhimurium DT 104, respectively. All antimicrobial ice treatments were significantly (p ≤ 0.05) more effective in reducing cross-contamination in comparison of NT and DI controls. The microbial reductions achieved by different antimicrobial ice treatments were not significantly (p ≤ 0.05) different from each other.ConclusionThe antimicrobial ice-based meat grinder sanitation technique could effectively reduce foodborne pathogens from meat grinders without needing meat grinder disassembly.

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