Keywords

Thermo Scientific (ISO) Chromogenic Coliform Agar, Coliforms, waterproof

Goal

To compare the performance of Thermo Scientific Chromogenic Coliform Agar (ISO) (Thermo Fisher Scientific) and Chromocult Coliform Agar (Merck); two cultures media formulated according to ISO 9308-1: 2014 – Enumeration of  Escherichia coli and coliform bacteria, Part 1: Membrane filtration method for waters with low bacterial background1.

Abstract

Thermo Scientific (ISO) Chromogenic Coliform Agar was evaluated together with Chromocult Coliform Agar to compare the performance of the two media for coliform detection. Coliform organisms were enriched in water samples that were then vacuum filtered in a membrane, placed in each medium and incubated according to ISO 9308-1: 2014. Non-coliform organisms were scratched directly on the two media. scientific thermos Chromogenic Coliform Agar (ISO) proved to be effective isolate coliforms from water samples.

Introduction

Chromogenic Coliform Agar (ISO) is a medium for the detection and enumeration of coliforms in food and water samples; the formulation is described in ISO 9308-1: 2014. The publication of ISO 9308-1: 2014 superseded ISO 9308-1: 2000, which reduced the time to result and included the use of a chromogenic medium. The method is based on membrane filtration, subsequent culture on chromogenic coliform agar mean and calculation of the number of target organisms in the sample.

The method is suitable for waters with a low number of bacteria that will cause less than 100 total colonies on agar medium for chromogenic coliforms. These can be drinking (potable) water, disinfected pool water, or finished drinking water from water treatment plants.

The study

Organism preparation

Thirty-seven coliform organisms (including 5 Citrobacter freundii, 1 Enterobacter aerogenes, 2 Enterobacter cloacae, 21 Escherichia coli, 4 Klebsiella aerogenes and 4 Klebsiella pneumonia) and 17 non-coliform organisms (including 2 Aeromonas hydrophilia, 2 Bacillus spp., 1 Candida albicans, 3 Enterococcus spp., 1 Pseudomonas aeruginosa, 2 Salmonella spp., 4 Staphylococcus spp. Y 2 Streptococcus spp.) Were included in the study.

All organisms were cultured from -80 ° C freezer storage on tryptone soy agar; The cultures were incubated at 36 ± 2 ° C for 18-24 hours. Three to five colonies were selected from each culture plate and inoculated in Nutrient Broth No. 2; The broth was then incubated at 36 ± 2 ° C for 18-24 hours. After incubation, inoculated Nutrient broths were diluted in sterile saline to produce a Standard 0.5 McFarland suspension.

Coliform tests

One hundred ml volumes of saline were inoculated with each coliform organism to achieve an inoculum level of 50-150 CFU in 10 ml according to the following method:

  • Transfer 150 µl of each 0.5 McFarland suspension to the first 100 ml saline bottle (bottle A). Invert bottle A three times.
  • Transfer 1 ml from bottle A to a second 100 ml saline solution (bottle B). Invert bottle B three times.
  • Transfer 1 ml from bottle B to a third 100 ml saline solution (bottle C). Invert bottle C three times.

Ten ml samples were taken from each organism from bottle C. Vacuum filtered using 47 mm diameter 0.45 µm cellulose Nitrate membrane filters according to ISO 9308-1: 2014. The membrane filters were placed in the centre of the surface. Thermo Scientific (ISO) Chromogenic Coliform Agar, Chromocult Coliform Agar plus Tryptone Soy Agar Plates
in duplicate.

Vacuum filtration equipment was cleaned after each 10 ml sample filtration by vacuum filtration 25 ml sterile saline between samples. Thermo Scientific (ISO) Chromogenic Coliform Agar and Chromocult coliform agar plates were incubated at 36 ± 2 ° C for 20-24 hours. After incubation, the colony Counts, colony size, and colony colour were recorded.

Non-coliform tests

One 10 µl loop of each 0.5 McFarland standard suspension inoculated directly into Thermo Scientific Chromogenic Coliform Agar (ISO), Chromocult Coliform Agar plates plus tryptone soy agar. All the media were incubated at 36 ± 2 ° C for 20-24 hours. Following incubation, the amount of growth, colony and colony size colour was recorded.

Results

The recovery percentage of all coliform isolates was calculated using tryptone soy agar as a control plate; Suburb Thermo Scientific Chromogenic Coliform Agar Counts (ISO) and Chromocult Coliform Agar were compared with Colony count of Tryptone Soy Agar. The percentage recovery of all coliforms plus percentage recovery of E. coli was only calculated for both Chromogenic media.

Coliform Colony Size in Thermo Scientific Chromogenic Coliform Agar (ISO) and Chromocult Coliform Agar were observed during the study. The mean size of the colony (in mm) was calculated for all coliforms and only for E. coli;

Coliform Colony Color in Thermo Scientific Chromogenic Coliform Agar (ISO) and Chromocult Coliform Agar was also observed. E. coli is expected to grow dark blue/purple colonies in both Thermo Scientific Chromogenic Coliform Agar (ISO) and Chromocult Coliform Agar. Everything other coliforms are expected to grow from pink to red Thermo Scientific Chromogenic Coliform Agar Colonies (ISO) and as salmon red in Chromocult Coliform Agar.

Discussion

Thermo Scientific (ISO) Chromogenic Coliform Agar showed statistically significantly higher percentage recovery of all coliform isolates (p = 0.03) and E. coli isolated (p = 0.01) from the membrane filtration method than Chromocult Coliform Agar. Cologne colour in Thermo Scientific (ISO) Chromogenic Coliform Agar more accurately met the ISO expected cologne colour 9308-1: 2014 than Chromocult Coliform Agar.

The colony size of all coliforms and E. coli isolates were statistically significantly higher (both p = <0.01) in Thermo Scientific (ISO) Chromogenic Coliform Agar than Chromocult Coliform Agar.

Conclusion

Thermo Scientific (ISO) Chromogenic Coliform Agar Tested to give superior recovery of coliform isolates while providing a more reliable cologne colour compared to Chromocult Coliform agar.