Jul 23, 2025
Screening protocol for colistin resistance using CHROMagar™ COL-APSE medium
- Soe Yu Naing1
- 1Utrecht University
- Soe Yu Naing: Project Manager | PhD Candidate
External link: https://youtu.be/KC1x9u4d02g
Protocol Citation: Soe Yu Naing 2025. Screening protocol for colistin resistance using CHROMagar™ COL-APSE medium. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlky4o1g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 23, 2025
Last Modified: July 23, 2025
Protocol Integer ID: 223073
Keywords: LMIC, Antimicrobial resistance, Colistin, Screening, COINICDE, Microbiology, Affordable, Easy, CHROMagar, CHROMagar™ COL-APSE, protocol for colistin resistance, colistin resistance, apse medium colistin resistance, antimicrobial resistance monitoring, chromagar screening, scalable tool for antimicrobial resistance monitoring, colistin, resistant isolate, differentiation between colistin, resistant gram, screening protocol, selective chromagar col, large numbers of isolate, practical for routine surveillance effort, negative bacteria, routine surveillance effort, isolate
Funders Acknowledgements:
JPIAMR
Grant ID: COINCIDE
ZonMw
Grant ID: COINCIDE
International Development Research Centre (IDRC)
Grant ID: COINCIDE
the Research Foundation – Flanders (FWO)
Grant ID: COINCIDE
Canadian Institutes of Health Research (CIHR)
Grant ID: COINCIDE
Abstract
Colistin resistance presents a major challenge in combating multidrug-resistant Gram-negative bacteria. To address this concern, a screening protocol employing the selective CHROMagar COL-APSE medium has been developed. This medium enables differentiation between colistin-susceptible and colistin-resistant E. coli strains based on growth characteristics and distinct colony coloration. The method is well-suited for screening large numbers of isolates, making it practical for routine surveillance efforts. Its applicability across both low- and middle-income countries (LMICs) and high-income countries (HICs) enhances its global relevance, offering a cost-effective and scalable tool for antimicrobial resistance monitoring. Additionally, a tailored spot-test protocol was developed to complement the CHROMagar screening, providing a rapid and adaptable approach for preliminary identification of resistant isolates.
Materials
• CHROMagar‱ COL-APSE medium plates prepared according to the manufacturer's protocol (Appendix: Manufacture leaflet) Plates can be stored for up to 1 month under refrigeration (2/8 °C) if properly prepared and protected from light and dehydration
• fresh 24-hour cultures of the E.coli isolates grown on blood agar
• MCR-1 control strain for quality control purposes.
• Tubes with sterile saline NaCl (0.85%) to prepare 0.5 McFarland suspension
• Vortex
• Densitometer for measuring bacterial density
• Sterile wooden-cotton swab
Troubleshooting
1.Background
Colistin resistance has become a significant concern in the fight against multidrug-resistant Gram-negative bacteria. To address this issue, a screening protocol utilizing CHROMagar‱ COL-APSE medium has been developed. This selective agar medium allows for the differentiation of colistin-susceptible and colistin-resistant strains based on growth and distinctive colony colors.
2. Aim
To efficiently screen a large batch of E. coli isolates obtained from fecal samples for colistin resistance using CHROMagar‱ COL-APSE medium.
3. Materials needed
• CHROMagar‱ COL-APSE medium plates prepared according to the manufacturer's protocol
Plates can be stored for up to 1 month under refrigeration (2/8 °C) if properly prepared and protected from light and dehydration
• fresh 24-hour cultures of the E.coli isolates grown on blood agar
• MCR-1 control strain for quality control purposes.
• Tubes with sterile saline NaCl (0.85%) to prepare 0.5 McFarland suspension
• Vortex
• Densitometer for measuring bacterial density
• Sterile wooden-cotton swab
4. Procedures
Note: If the agar plate has been refrigerated, allow to warm to room temperature
before inoculation
Work with batches of strains as a total of 8 strains can be tested per plate
Include 1 positive control and 1 negative control strain on a plate on the day of testing regardless of the number of test strains
Make a pie of 8 on CHROMagar plates and label them carefully
Prepare 0.5 McFarland bacterial suspension from fresh 24-hour cultures of the E.coli isolates, grown on blood agar
Use a wood-cotton swab to immerse into a 0.5 McFarland bacterial suspension, and make sure to remove
excess liquid by pressing and turning the swab against the inside of the tube
Use the wood-cotton swab to spot inoculate the bacterial suspension on CHROMagar plates
(8 grid as a pie)
Incubate in aerobic conditions at 35-37 °C for 18-24 hours
On the next day, check the results and take a photograph of the plates
Record positive and negatives strains and include the results of the control strains
If the results of the control strains are not correct, all strains of that day has to be repeated
In case of any doubt or possible contamination, retest the strain(s)
The scheme for colistin resistance screening using selective CHROMagar COL-APSE medium to detect E. coli isolates resistant to colistin
Figure 1. Screening protocol using a selective CHROMagar COL-APSE medium to detect E. coli isolates resistant to colistin.
5. Interpretation of colistin resistance
Growth of E.coli with dark pink to reddish color (as in figure below) is
considered positive. Absence of growth is considered negative.
Example of colony appearance of colistin-resistant E.coli
6. Quality Control
Please perform Quality Control according to the use of the medium and the local QC
regulations and norms. Good preparation of the medium can be tested, isolating
the following ATCC strain: MCR-1 E. coli NCTC 13846 displaying typical colony appearance of dark pink to reddish.
Please perform the calibration of densitometer regularly.
7. Video Protocol
Protocol references
Abdul Momin MHF, Bean DC, Hendriksen RS, Haenni M, Phee LM, Wareham DW. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens. J Med Microbiol. 2017;66(11):1554-1561. doi:10.1099/jmm.0.000602
Acknowledgements
The COINCIDE study is funded by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR)’s 2021 call on “One Health interventions to prevent or reduce the development and transmission of AMR. The participating countries are supported by the following agencies: the International Development Research Centre (IDRC) for Indonesia, the Netherlands Organisation for Health Research and Development (ZonMw) for the Netherlands, the Research Foundation – Flanders (FWO) for Belgium, and the Canadian Institutes of Health Research (CIHR) for Canada.