Coliform Detection

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Background

http://vulgariz.com/wp-content/uploads/2011/05/escherichia-coli-entero-hemorragique.jpg

Coliform bacteria are Gram-negative bacteria, which can ferment lactose.
They are commonly found in nature and in feces of warm-blooded animals.
This class of bacteria is used to determine fecal contamination of water, not because they are dangerous (although some are), but because they indicate the presence of other pathogenic organisms of the same origin (like viruses, protozoa and many multicellular parasites).
They are also easy to culture.


Escherichia Coli (E.Coli) is the most studied member of this familiy and is found in the intestinal tract of many animals including humans. It is mostly harmless, but some strains express virulence factors that are dangerous.

Fig25 1 E.Coli Diarrheal.jpg
Image taken from here.

 

These pathogenic strains are transmitted via oral absorption of fecal-contaminated water for example (they can be found in food too). One of these strains is the Shiga-toxin E.Coli (STEC), which is found in  in ruminant guts and is mainly known in the case of food contamination. This is one example of illness-causing E.Coli strain.

 

Fig25 2 E.Coli Diarrheal.jpg
Image taken from here.

 

Coliform bacteria intoxication often leads to bloody diarrhea and vomiting (gastroenteritis, dysentry), but some pathogenic strains can cause complications such as hemolytic uremic syndrome (HUS). HUS consists of clot-formation thus blocking arteries and destroying red blood cells, leading to reduced blood flow and ischemia (lack of oxygen and glucose due to a reduced blood supply to tissues).

 

Methods

As we just said, identification of coliform bacteria gives indication on fecal contamination of water. For this measure, the approved method is the HPC (heterotrophic plate count), which involves several test conditions depending on what microorganism we want to detect (heterotrophs include bacteria, yeasts and moulds, and are characterized by their need for organic carbon to grow) in addition with other tests. The standard conditions are: temperature (20-40°C), incubation time (few hours to days or even weeks) and nutrient concentration (low/high).

"In water considered safe, this value lies between 0 and 100 cfu/ml" (Egli) Studies found that this method may underestimate the quantity of bacteria, thus it is important to continue ameliorating the procedures, as well as developing new methods for water safety control.

The presence of microorganisms is in general determined by high HPC values, which are found in stagnant regions. “In piped water supplies, for example, the HPC is required to be less than 300 colony-forming units (cfu)/ml. The presence of E. coli or enterococci is regarded as evidence of fecal contamination; accordingly, a 100 ml water sample has to be shown to be completely free of E. coli and enterococci.” (Egli)

To have an overview of what was possible to do, we compiled a table of the latest methods used for E.Coli detection.

Recap Ecoli.png

The criteria we took in consideration were :

  • detection range
  • time scale
  • cost
  • waste

Another important parameter was the additional « hardware » needed to accomplish the different experiments.

Among the methods available, we distinguished different classes :

  • PCR-based methods (time consuming)
  • Colorimetry (lot of chemicals)
  • Bioluminescence
  • Florescence – spectrometry

For the PCR methods, we can see that the limiting aspect is the incubation time (several hours). The Colorimetry method requires a lot of chemical reagent that we would have to process, so this raises concern about the waste handling. have then Bioluminescence and Fluorescence, that both seem to match our criteria. Meaning that they have a quite good detection range and a small time requirement.

The technique that retained our attention is Fluorescence, because it matches our criteria, but also   because the device we would build to detect the fluorescence allows to detect Arsenic eGFP fluorescence as well. For coliform detection, it is based on the intrinsic fluorescence of bacteria that originates from their chemical components. Aromatic amino acids (tryptophan, tyrosine, phenylalanine), nucleic acids and co-enzymes are native fluorophores that excite with UV light.

More specifically, if we excite E.Coli at 280-290nm, they show fluorescence in the 300-400nm range, which corresponds to tryptophan residues. E.Coli use Trp to produce indole, which work as extracellular signal.

 

“New methods for assessing the safety of drinking water, research reports”, T. Egli, Eawag News 65e/December 2008

 

Plating Method


Using Chromogenic Media

Chromogenic media allows differentiation of coliform bacteria based on their metabolic enzymes.
So far, we have used the Endo Agar for the coliform detection assay.

Endo Agar pH7.4 **
g/L
Dipotassium phosphate 3.5
Peptone 10.0
Agar 15.0
Lactose 10.0
Sodium sulfite 2.5
Basic fuchsin* 0.5

The media contains Lactose, which serves as a carbohydrate source. Bacteria that can cleave lactose, such as E. coli, ferment glucose and produce acetaldehyde. The aldehyde reaction with the Sodium Sulfite and Basic Fuchsin regenerates the color of the fuchisin dye that build up in the colony turning the colony red.
Lactose-fermenting bacteria produce pink to red colonies with or without a metallic sheen.
Lactose non-fermenting bacteria form clear, colorless colonies.

Note:
* Production of Basic Fuchsin is known to causes cancer
** Recipe taken from Practical Handbook of Microbiology - book details in reference section
*** Ox bile can be added (or Sodium deoxycholate 0.01%, Sodium lauryl sulfate 0.005%) to inhibit the growth of gram-positive bacteria.

Water Collection Specific to Coliform Detection

  • All samples sent for microbiological analyses must always be collected in sterile containers (by autoclave).
  • Always leave an air space of at least 2.5 cm between the surface of the liquid and the container lid.
  • The necessary aseptic conditions must be respected when a sample is collected (e.g. use a rubber gloves and mask, avoid inserting fingers or other objects in the mouth or on the lid of the container and minimize exposure of the container to air at the time of sampling); This is also intended to avoid E.coli contamination to the sampling personnel.
  • Carefully close all containers tightly after sampling.
  • Make a detailed record of all samples as soon as possible after sampling.

For sample preservation

  • Refrigerate samples 1- 4°C before analysis.

Optional

  • for halogen neutralization - add 0.5 mL of a 10% sodium thiosulfate solution per 1 L of sample.
  • for chelation of trace elements - add 0.3 mL of a 15% EDTA solution per 100 mL of sample.

Suggested sample holding times

  • 6h - surface water and ground water
  • 30h - treated drinking-water sources


BRILLIANCE E. COLI/COLIFORM AGAR from Oxoid Microbiology Products.

Documentation

  • Lifepatch's Quantification of E. coli contamination in water entry on biodesign.cc
  • Lifepatch's DIY automatic colony counter entry on biodesign.cc
  • Lifepatch's swab test with school children entry on biodesign.cc
  • Hammerdirt's extensive documentation on hackuarium wiki


Using PCR


Molecular techniques can offer more rapid analysis as no growth waiting time is required, and as it is an amplification method, can be more sensitive.

PCR Primers

Maheux et al summarizes the criteria for PCR assays

  • rapidity (this also includes sample preparation, PCR, and results analysis)
  • specificity (ability to target only the desired species)
  • ubiquity (ability to detect all strains of the targeted species)
  • analytical detection limit

Specificity for Coliforms:

Simple PCR

Multiplex PCR
To increase the specificity for E. coli, increased target gene numbers can be considered. for example, Horakova et al. used 4 of the biochemical hall mark genes for E. coli to make a multiplexing PCR assay (4-plex) so that in the test strains, only E. coli and not other bacteria would have 4 PCR products (4 bands on the gel):

* uidA (codes for beta-D-glucuronidase, GUS, observed in ~94% of E. coli)
* lacZ (codes for beta-D-galactosidase - beta-gal, which cleaves lactose into glucose and galactose, necessary for lactose fermentation)
* lacY (codes for lactose permease - for lactose transport across cytoplasmic membranes)
* cyd (codes for cytochrome bd complexes, for respiration under low aeration conditions)

Documentation

References, Resources

  • Horakova et al. Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase. Journal of applied microbiology, 105(4), (2008) pp.970–976.
  • Maheux et al. Analytical comparison of nine PCR primer sets designed to detect the presence of E. coli/Shigella in water samples. Water Research 43 (2009) pp. 3019-3028.
  • Rompré et al. Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J. Microbiological Methods 49 (2002) pp. 31-54. pdf
  • Myers, D.N. FECAL INDICATOR BACTERIA 7.1
  • “New methods for assessing the safety of drinking water, research reports”, T. Egli, Eawag News 65e/December 2008