E. coli was identified in accordance with the International Organization for Standardization guidelines (ISO 16649-2, 2001). Twenty-five grams of the samples were emptied in a stomacher bag (Bag Mixer®DOA 20550) and added to 225 ml of peptone buffered water. The stomacher bag with the sample was then placed in a bag mixer machine and mashed for 3 min. Afterward, 0.1 ml of the test sample was transferred into the tubes with the use of a sterile pipette. Then, the mixture was incubated at 37°C for 24 h [13 ]. The identification of E. coli was performed in accordance with the International Organization for Standardization guidelines, with the use of the most probable number technique (ISO 16649-2 2003). The tubes exhibiting gas production were recorded as positive, and a loop-full from each positive gas tube was transferred to a separate tube with MacConkey Broth (Oxoid, UK). E. coli confirmation was achieved by observing the gas production and acidification during growth in MacConkey Broth (Oxoid, UK). The positive results were streaked onto tryptone bile glucuronic agar (TBX agar, Oxoid, UK), and the plates were incubated at 37°C for 24 h. The pink colonies were counted using a colony counter-digital machine (Lasec, South Africa) and further subjected to indole and catalase tests.
Macconkey broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
MacConkey broth is a culture medium used in microbiology for the isolation and differentiation of Gram-negative enteric bacteria, particularly members of the Enterobacteriaceae family. It contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria, while allowing the growth of Gram-negative bacteria.
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Lab products found in correlation
Eosin methylene blue agar, by Thermo Fisher Scientific (3 mentions)
Tryptone soy broth (tsb), by Thermo Fisher Scientific (2 mentions)
Peptone water, by Thermo Fisher Scientific (2 mentions)
Emb agar, by Thermo Fisher Scientific (2 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Tbx agar, by Thermo Fisher Scientific (1 mentions)
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (1 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (1 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
Rappaport vassiliadis broth, by Thermo Fisher Scientific (1 mentions)
Lactophenol cotton blue stain, by Hardy Diagnostics (1 mentions)
Brain heart infusion, by Thermo Fisher Scientific (1 mentions)
Macconkey agar plate, by Thermo Fisher Scientific (1 mentions)
Brilliant green lactose bile broth, by Thermo Fisher Scientific (1 mentions)
Jenway 3540 ph and conductivity meter, by Cole-Parmer (1 mentions)
Tryptone soy agar, by BD (1 mentions)
Microflex lt, by Bruker (1 mentions)
Tsa sb, by BD (1 mentions)
Ceftazidime, by Merck Group (1 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
17 protocols using macconkey broth
1
Isolation and Identification of E. coli
2
Bacterial and Fungal Propagation Protocols
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The bacterial isolates were propagated after Soliman et al. (2021b , 2021c ). Escherichia coli O157: H7 suspension (2.0 × 104 CFU.ml-1) was propagated into Mac-Conkey broth (Oxoid™) at 44°C/24 hours, eosin methylene blue agar (OxoidTM) at 37°C/24 hours, and tryptone soya broth (Oxoid™) providing 1.7 × 1011 CFU. ml-1 suspension. Lyophilized S. Typhimurium (3.4 × 102 CFU) was propagated into Rappaport-Vassiliadis broth (Oxoid™) at 37°C/24 hours, xylose lysine deoxycholate agar (Oxoid™) at 37°C/24 hours, and tryptone soya broth providing 1.5 × 106 CFU.ml-1 suspension.
The fungal isolates were propagated following Soliman et al. (2021b) . Aspergillus niger and C. albicans clinical isolates were propagated into sabouraud dextrose broth (SDB; HIMEDIA®) at 37°C/24–72 hours, sabouraud dextrose agar (SDA; Oxoid™) at 37°C/24 hours, identified with lactophenol cotton blue stain (Hardy Diagnostics®), and resuspended in SDB broth, providing 2.5 × 108 CFU.ml-1 suspensions.
The fungal isolates were propagated following Soliman et al. (2021b) . Aspergillus niger and C. albicans clinical isolates were propagated into sabouraud dextrose broth (SDB; HIMEDIA®) at 37°C/24–72 hours, sabouraud dextrose agar (SDA; Oxoid™) at 37°C/24 hours, identified with lactophenol cotton blue stain (Hardy Diagnostics®), and resuspended in SDB broth, providing 2.5 × 108 CFU.ml-1 suspensions.
3
Isolation and Identification of E. coli
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10 g of lettuce, soil, and manure were weighed into well-labelled sterile zip lock bags and 90 ml of peptone water (Oxoid, UK) was added and pulsified using a pulsifier (PUL 100E; Stuart Scientific Co. Ltd., UK) for 30 s. One millimeter (1 ml) of the resultant stock solution was inoculated in 5 ml of MacConkey broth (Oxoid, UK) and incubated for 24 ± 2 hours at 44°C. 1 ml of the overnight positive culture was transferred into test tubes containing 5 ml of Tryptone Soy Broth (Oxoid, UK) and incubated for 24± 2 hours at 44°C. A few drops of Kovac's Indole Reagent (LOBA CHEMIE PVT. Ltd., India) were added to each positive tube. All tubes that retained the red color ring following simple agitation were selected, and a loop full streaked on Eosin Methylene Blue Agar (EMBA) (Oxoid, UK) and incubated for 24± 2 hours at 37°C. Colonies with a shiny green metallic appearance were confirmed as E. coli and stored in Brain Heart Infusion (Oxoid, UK) with 20% glycerol.
4
Isolation of Cefotaxime-Resistant Bacteria from Poultry Feces
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The samples were collected between September 2015 and February 2016. A total of 240 fecal droppings were collected aseptically from 11 commercial poultry farms from five states in South Western Nigeria i.e., Oyo, Ondo, Ogun, Ekiti and Osun. Immediately after collection, 1g of each fecal sample was homogenized in 9 mL of MacConkey broth (Oxoid, Cheshire, UK) supplemented with cefotaxime (2 µg/mL) and incubated at 37 °C for 24 h. The incubated broth was plated on MacConkey agar plates (Oxoid, Cheshire, UK) supplemented with cefotaxime (2 µg/mL) (Tamang et al., 2013 (link)) at appropriate dilutions and incubated at 37 °C for 24 h. All possible distinct colonies with different morphology that grew on MacConkey plates were sub cultured to get pure cultures which were stored appropriately.
5
Plating Efficiency Assay Protocol
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For plating efficiency tests, strains were grown overnight in LB medium at 37°C, serially diluted 10-fold, and then replica plated on LBA or MacConkey agar (MacConkey broth from Oxoid with 1.5% (w/v) agar). Plates were then incubated overnight at 37°C.
6
Assessing Coliform Contamination in Water
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A modified most probable number (MPN) method was used to assess the microbiological quality of the water, specifically coliform contamination [9 (link),10 ]. Briefly, five ten-fold serial dilutions were made from each water sample by inoculating 1 ml of undiluted water sample into 9 ml of MacConkey broth (Oxoid, UK). This was continued until a dilution of 1 x 10−5. A total of 30 tubes (five tubes for each dilution) were prepared for each sample. The inoculated broths were incubated at 44°C for 48 hours for the culture of thermotolerant coliforms. After incubation, each tube was examined and those that were positive (production of acid and gas) were counted. The number of positive and negative tubes in each of these three sets was noted in order and these data were used to estimate the coliform content using a five-tube MPN table.
7
Coliform Detection in Water Samples
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Presumptive coliform test was performed using MacConkey broth (Oxoid) [25 ]. All the tubes contained Durham tubes before sterilization. The first set of three tubes had 10 ml of sterile double strength broth and the second and the third sets had 10 ml of single strength broth. The three sets of tubes would receive 10 ml, 1 ml and 0.1 ml quantities of water samples using sterile syringes respectively and were incubated for 48 h at 37 °C. Tubes showing gas formation were considered presumptive coliform positive. The most probable number (MPN) was then estimated from the table for three tube test [22 ].
Confirmatory testing was carried out by transferring a loop full of culture from each tube which showed acid and gas in the presumptive test and inoculating it in to Brilliant Green Lactose Bile (BGLB) broth (Oxoid) and Escherichia coli (E. coli) medium. The inoculated tubes were incubated at 37 °C for 48 h for total coliforms and 44.5 °C for 24 h for thermotolerant coliforms in water bath. Then, after 24–48 h of incubation, samples were monitored for gas production and standard biochemical testing was performed to identify water contaminants up to genus level [25 ].
Confirmatory testing was carried out by transferring a loop full of culture from each tube which showed acid and gas in the presumptive test and inoculating it in to Brilliant Green Lactose Bile (BGLB) broth (Oxoid) and Escherichia coli (E. coli) medium. The inoculated tubes were incubated at 37 °C for 48 h for total coliforms and 44.5 °C for 24 h for thermotolerant coliforms in water bath. Then, after 24–48 h of incubation, samples were monitored for gas production and standard biochemical testing was performed to identify water contaminants up to genus level [25 ].
8
Juice pH and Microbial Analysis Protocol
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The pH of juice samples was measured by using Jenway 3540 pH and Conductivity Meter (Bibby Scientific Ltd, Staffordshire, UK) after calibration using standard buffer solutions of pH 7.0 and pH 4.0 [33 ]. Microbiological evaluation was done by establishing TVC and most probable number (MPN). Total viable count for enumeration of microorganism in raw juice at 37 and 45 °C applied a protocol described by Tanzania Bureau of standards and ISO/FDIS 8261 (E) [35 , 36 ] as was described for the raw milk. For the determination of total coliform count (TCC) (coliform and faecal coliform bacteria), MPN, the presumptive test for coliforms was carried out using MacConkey broth (Oxoid Ltd, Basingstoke, UK) in the three test tube method as described by WHO [39 ]. The three tubes with 10 ml of broth were inoculated with 1 ml from 10−1, 10−2, 10−3, 10−4 and 10−5 prepared dilutions and incubated at 37 °C for 24 h for coliforms and at 45 °C for faecal coliforms as described by WHO [39 ]. All tubes showing gas production were observed and recorded. The MPN tables for three tubes dilution were used to report the result of the presumptive MPN of coliform bacteria per ml. E. coli was confirmed in samples as described above for the milk samples.
9
Screening for 3GCs-R Enterobacteriaceae
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The inoculated Tryptone Soy Broth (Oxoid) medium was transferred into 5 mL of MacConkey Broth (Oxoid) containing 8 mg/L of ceftazidime (Sigma-Aldrich, Merck, St. Louis, MO, USA) and incubated at 37 °C for 24 h under agitation. Then, a loopful (10 μL) of the mixture was plated onto MacConkey Agar (BioCen, BioCubaFarma, Bejucal, Cuba) supplemented with 8 mg/L of ceftazidime (Sigma-Aldrich) for the screening of 3GCs-R Enterobacteriaceae and reincubated overnight. Lactose-positive (pink colonies) were selected and streaked three times on selective MacConkey Agar plates to obtain pure culture. Single pink colonies from each selective plate were streaked onto Tryptone Soy Agar plates containing 5% sheep blood (TSA-SB; Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37 °C. The colonies were identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS, Microflex LT; Bruker Daltonics, Billerica, MA, USA) and frozen at −80 °C in glycerol stocks.
10
Quantifying Fecal Contamination in Well Water
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The quality of the samples was determined using the multiple tube fermentation technique as described by Cheesbrough [13 ]. A three-tube most probable number (MPN) method was used to determine faecal contamination of well water using MacConkey broth (Oxoid Ltd., Basingstoke Hampshire, England) as the culture medium. Samples of 50 ml, 10 ml and 1 ml of water were inoculated into corresponding dilution tubes with inverted Durham’s tubes and incubated at 37 °C for 24 h. The tubes were observed for growth and gas production, and the MPN of coliforms in 100 ml of water was determined by referring to McCrady’s table and interpreted as “Excellent”, “Acceptable”, “Unacceptable” and “Grossly polluted”.
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