The bacteriological cultivation for E. coli was performed in accordance with standard procedures [34 ]. Briefly, each rectal swab was directly cultured on sterile MacConkey agar (Mast group Ltd, Merseyside, UK) and incubated at 37 °C for 24 h. Four lactose-fermenting colonies from each sample were separately sub-cultured and biochemically confirmed using tryptophan broth for indole test, methyl red for MVP test and citrate agar for citrate utilization test. The biochemically confirmed E. coli (indole positive, MR positive, VP negative and citrate negative) were stored in brain heart infusion broth (Mast group Ltd, Merseyside, UK) with 20 % glycerol at - 20 °C until needed for DNA extraction.
Macconkey agar
Manufactured by Mast Group
Sourced in United Kingdom
MacConkey agar is a selective and differential culture medium used for the isolation and identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It is designed to inhibit the growth of Gram-positive bacteria while allowing the growth of Gram-negative bacteria.
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4 protocols using macconkey agar
1
Isolation and Identification of E. coli
2
Microbial Analysis of Minced Beef
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Microbial analysis was conducted in the stored minced beef samples [47 ]. The samples (10 g) were aseptically transferred to a stomacher bag containing 90 mL of peptone saline diluent (1.0 g peptone and 8.5 g sodium chloride in 1 L of distilled water) and homogenized for 60 s. A serial 10-fold dilution series was prepared. Different bacterial counts were determined using different specific selective media [48 ,49 (link)]. The total viable count (TVC) was assessed on Plate Count Agar (PCA, Merck, Darmstadt, Germany) at 25 °C after 72 h. Psychrotrophs were assayed on PCA (Merck, Darmstadt, Germany) at 7 °C after 10 days. Coliform bacteria were determined on MacConkey agar (Mast Group, Merseyside, UK) with a double layer of the same medium incubated at 37 °C for 24 h. Microbiological data were expressed as the logarithms of the number of colony forming units (CFU g−1).
3
Bacterial Quality Assessment of Milk
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The bacterial quality assessment of milk was carried by the viable bacterial count method to enumerate colonyforming units per millilitre (cfu/ml) of the milk samples. Briefly, serial dilutions of milk in peptone water from 10 -1 to 10 -5 in duplicates were made and cultured on standard plate count agar at 37 °C for 24-48 h. The number of colonies between 30 and 300 colonies per plate was counted and expressed as cfu/ml (Cowan et al. 1993) .
For identification of bacteria, about 50 μl of the milk sample was inoculated onto nutrient agar (Oxoid, Hampshire, England) and incubated at 37 °C for 18-24 h. Briefly, a discrete single colony was inoculated for each of the three selective media, namely Mac-Conkey agar (Mastgroup, Merseyside, UK), MSA agar (Oxoid, Hampshire, England) and Xylose-lysine-deoxycholate medium (HiMedia, India). The culture plates were incubated at 37 °C for 18-24 h to isolate bacteria. These were then characterized using standard morphological and biochemical tests (Cowan et al. 1993) . Pure cultures stored as glycerol stocks were used for further analysis.
For identification of bacteria, about 50 μl of the milk sample was inoculated onto nutrient agar (Oxoid, Hampshire, England) and incubated at 37 °C for 18-24 h. Briefly, a discrete single colony was inoculated for each of the three selective media, namely Mac-Conkey agar (Mastgroup, Merseyside, UK), MSA agar (Oxoid, Hampshire, England) and Xylose-lysine-deoxycholate medium (HiMedia, India). The culture plates were incubated at 37 °C for 18-24 h to isolate bacteria. These were then characterized using standard morphological and biochemical tests (Cowan et al. 1993) . Pure cultures stored as glycerol stocks were used for further analysis.
4
Environmental Screening for NICU Pathogens
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During the outbreak, the routine fortnightly conducted environmental screening was intensified to weekly from mid-August 2016 until October 2016. Various environmental samples (Supplementary Table S1) were collected from high-touch surfaces of NICU as recommended by the hospital infection prevention and control (IPC) committee. A sterile individually packed cotton swab was taken, immersed in sterile normal saline, and horizontally wiped across a given sampling surface for 10e30 s. The moist swab was returned to the container and transported to the microbiology laboratory at room temperature. The swab was inoculated on to MacConkey agar (Mast Group Ltd, Bootle, UK) and 5% blood agar (Mast Group Ltd), and incubated at 35 AE 2 C for 18e24 h. The bacterial identification was carried out manually using the standard microbiological methods [11] .
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