• Logo
  • SBMUJournals

Comparison of Multiple Tube Fermentation and Polymerase Chain Reaction Methods for the Detection of Coliforms in Freshwater

Ramin Mazaheri Nezhad Fard, Fahimeh-Sadat Sayyed Asgari, Iraj Ashrafi, Sorayya Gharibi, Mohammad Reza Khani




Background and objective: Detection of microbial pathogens in water is one of the major health issues. Escherichia coli species are used as indicators of fecal contamination in water microbial detection. In this study, efficacies of two methods of multiple tube fermentation and polymerase chain reaction have been compared for the detection of coliforms (especially Escherichia coli) in water.

Material and methods: To compare multiple tube fermentation and polymerase chain reaction methods, 15 water samples were collected from five different sources (three gutter, six well, three tap and three bottled mineral water samples). The samples were cultured in lactose broth media to achieve the most probable number of bacteria. Furthermore, acetate cellulose filter method was used for the bacterial DNA extraction to investigate lacZ (indicating the presence of coliforms) and uidA (indicating the presence of Escherichia coli) genes.

Results and conclusion: Based on the results of multiple tube fermentation, eight (53.3%) and six (40%) samples were contaminated with coliforms and Escherichia coli, respectively. Furthermore, polymerase chain reaction results showed that ten (66.7%) and eight (53.3%) samples contained coliforms and Escherichia coli, respectively. Results have suggested that polymerase chain reaction is much faster, more accurate and more sensitive than traditional methods (e.g. multiple tube fermentation) for the detection of coliform contaminated water. Moreover, several types of bacteria can be tracked simultaneously by M-PCR.

Conflict of interest: The authors declare no conflict of interest.


▪ Coliforms, Escherichia coli ▪ Freshwater ▪ Multiple tube fermentation ▪ Polymerase chain reaction


Stevens M, Ashbolt N, CunliffeD. Recommendation to change the use of coliforms as microbial indicators of drinking water quality, Australian Government. National Health and Medical Research Council. 2003.

Rompre A, Servais P, Baudart J, de-Roubin MR, Laurent P. Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Methods 2002; 49: 31–54. DOI: 10.1016/S0167-7012(01)00351-7.

Deshmukh RA, Joshi K, Bhand S, Roy U. Recent developments in detection and enumeration of waterborne bacteria: a retrospective mini review. Microbiology Open 2016; 5: 901–922. DOI: 10.1002/mbo3.383.

Olsen SJ, Miller G, Breuer T, Kennedy M, Higgins C, Walford J. A waterborne outbreak of Escherichia coli O157:H7 infections and hemolytic uremic syndrome: implications for rural water systems. Emerg Infect Dis 2002, 8: 370–375. DOI: 10.3201/eid0804.000218.

Soltan Dallal MM, Sepehri M, Hosseini M, Tabatabaei Bafrouei A, Deilami Khiabani Z. Determination of genotype variation of Escherichia coli in well water of Tehran’s parks by Multiplex PCR. Pejouhandeh 2011; 16: 226–233.

Bowen KK. Detection of fecal contamination using molecular methods. Independent Research Projects 2016; available at http://digitalcommons.augustana.edu/umcindependent/2.

Nasr Isfahani B, Fazeli H, Babaie Z, Poursina F, Moghim S, Rouzbahani M. Evaluation of polymerase chain reaction for detecting coliform bacteria in drinking water sources. Adv Biomed Res 2017; 6: 130. DOI: 10.4103/2277-9175.216783.

Olesen B, Neimann J, Bottiger B, Ethelberg S, Schiellerup P, Jensen C. Etiology of diarrhea in young children in Denmark: a case-control study. J Clin Microbiol 2005; 43: 3636–3641. DOI: 10.1128/JCM.43.8.3636-3641.2005.

Wohlsen T, Bates J, Vesey G, Robinson WA, Katouli M. Evaluation of the methods for enumerating coliform bacteria from water sample using precise reference standards. Lett Appl Microbiol 2006; 42: 350–356. DOI: 10.1111/j.1472-765X.2006.01854.x.

Nurliyana MR, Sahdan MZ, Wibowo KM, Muslihati A, Saim H, Ahmad SA, Sari Y, Mansor Z. The Detection Method of Escherichia coli in Water Resources: A Review. Journal of Physics Series 2018; 995: 012065. DOI: 10.1088/1742-6596/995/1/012065.

Edberg SC, Rice EW, Karlin RJ, Allen MJ. Escherichia coli: the best biological drinking water indicator for public health protection. Symp Ser Soc Appl Microbiol 2000; 29: 106S–116S.

Procop GW. Molecular diagnostics for the detection and characterization of microbial pathogens. Clin Infect Dis 2007; 45: 99–111. DOI: 10.1086/519259.

Horakova K, Mlejnkova H, Mlejnek P. Direct detection of bacterial fecal indicators in water samples using PCR. Water Sci Technol 2006: 54: 135–140.

Fricker EJ, Fricker CR. Application of the polymerase chain reaction to the identification of Escherichia coli and colforms in water. Lett Appl Microbiol 1994; 19: 44–46. DOI: 10.1111/j.1472-765X.1994.tb00900.x.

Bej AK, McCarty SC, Atlas RM. Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction; comparison with defined substrate and plating methods for water quality monitoring. Appl Environ Microbiol 1991; 57: 2429–2432.

Molina F, Lopez-Acedo E, Tabla R, Roa I, Gomez A, Rebollo JE. Improved detection of Escherichia coli and coliform bacteria by multiplex PCR. BMC Biotechnology 2015; 15: 48. DOI: 10.1186/s12896-015-0168-2.

Iqbal S, Robinson J, Deere D, Saunders JR, Edwards C, Porter J. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water. Lett Appl Microbiol 1997; 24: 498–502. Doi: 10.1046/j.1472-765X.1997.00160.x.

Juck D, Ingram J, Prevost M, Coallier J, Greer C. Nested PCR protocol for the rapid detection of Escherichia coli in potable water. Can J Microbiol 1996; 42: 862–866.

Abtahi H, Ghannadzadeh MJ, Salmanian AH, Ghaznavi Rad E, Karimi M, Molaei N. Improvement of PCR in detection of coliform in water pollution. AMUJ 2008; 11: 1–7.

Ghannadzadeh MJ, Abtahi H, Salmanian AH, Ghaznavi Rad E, Karimi M. Effects of micro-filter in detection of coliform in tap water by PCR. JIUMS 2009; 17: 10–15.

Rees G, Pond K, Kay D, Bartram G, Domingo JS editors. Safe management of shellfish and harvest water. Chapter 7: Real-time monitoring technologies for indicator bacteria and pathogens in shellfish and harvesting waters. London: UK, IWA Publishing; 2010 (WHO).

DOI: https://doi.org/10.22037/afb.v6i2.22484


  • There are currently no refbacks.