E. coli Identification Process: What Is the Purpose of Elevated Temperature (42–44 °C)?
Escherichia coli (E. coli) is one of the most important indicator organisms used in microbiology to assess water, food, and environmental hygiene quality. The detection and confirmation of E. coli involve specific biochemical and physiological characteristics that distinguish it from other coliform bacteria. One such distinctive feature is its ability to grow and ferment lactose at an elevated temperature of 42–44 °C. But why exactly is this temperature used, and what does it signify? Let’s explore the complete scientific reasoning and process behind this selective identification step.
🔬 Introduction to E. coli
Escherichia coli is a Gram-negative, rod-shaped, facultatively anaerobic bacterium belonging to the Enterobacteriaceae family. It is commonly found in the intestinal tract of warm-blooded animals, including humans. While most strains are harmless, some can cause diseases like diarrhea, urinary tract infections, and foodborne illnesses.
In microbiological testing, E. coli is considered a fecal indicator organism. Its presence in water, food, or pharmaceutical samples indicates potential fecal contamination and possible presence of pathogens.
🧪 E. coli Identification Process Overview
The E. coli identification process typically involves several sequential steps to confirm the organism’s presence. These include presumptive, confirmatory, and completed tests. The main principle is based on the ability of E. coli to ferment lactose with gas production and grow at elevated temperatures.
1. Presumptive Test
Samples are inoculated into a selective medium such as Lauryl Tryptose Broth (LTB) or Lactose Broth and incubated at 35–37 °C for 24–48 hours. Gas formation in the Durham tube indicates the presence of coliforms.
2. Confirmatory Test
From positive presumptive tubes, a loopful of culture is transferred to Brilliant Green Lactose Bile Broth (BGLB) and incubated at 44 ± 0.5 °C for 24–48 hours. Gas formation in this medium confirms the presence of fecal coliforms (thermotolerant coliforms).
3. E. coli Confirmation Test
Further confirmation is performed using media such as EC Broth or EC-MUG Broth (4-methylumbelliferyl-β-D-glucuronide), incubated at 44 ± 0.5 °C. Fluorescence under UV light or indole production in tryptone water confirms E. coli identity.
🌡️ Why Elevated Temperature (42–44 °C)?
The incubation temperature of 42–44 °C plays a crucial role in differentiating thermotolerant E. coli from other coliform bacteria that cannot grow under such conditions.
Here’s the scientific reasoning behind it:
- 1. Selectivity: The elevated temperature acts as a selective condition that inhibits the growth of non-fecal coliforms like Enterobacter, Klebsiella, and Citrobacter species, which are less heat-tolerant.
- 2. Specificity for Fecal Origin: E. coli originating from the intestines of warm-blooded animals can tolerate and grow at 44 °C. Thus, growth at this temperature indicates fecal contamination.
- 3. Confirmation of Thermotolerance: The ability of the organism to ferment lactose and produce gas at 44 °C confirms it as a thermotolerant coliform, primarily E. coli.
- 4. Reduction of False Positives: Incubating at this elevated temperature eliminates other coliforms that might show positive reactions at 35 °C but are not of fecal origin.
🧫 Principle Behind Elevated Temperature Testing
The principle of E. coli detection at 44 °C relies on the organism’s enzymatic adaptability and thermotolerance. The enzyme β-galactosidase enables lactose fermentation even under elevated temperatures. This property helps to confirm E. coli, distinguishing it from other coliform bacteria that lose enzymatic activity above 40 °C.
⚙️ Media Commonly Used in E. coli Identification at 44 °C
| Medium Name | Purpose | Incubation Condition |
|---|---|---|
| EC Broth | Selective medium for detection of fecal coliforms and E. coli | 44 ± 0.5 °C for 24–48 hours |
| EC-MUG Broth | Contains fluorogenic substrate MUG to detect β-glucuronidase activity in E. coli | 44 ± 0.5 °C for 24 hours |
| BGLB Broth | Used for confirmatory coliform test before E. coli confirmation | 44 ± 0.5 °C for 24–48 hours |
| EMB Agar / MacConkey Agar | Used for isolation and colony morphology study (metallic sheen colonies) | 35–37 °C for 24 hours |
🔍 Biochemical Confirmation Tests for E. coli
After incubation at elevated temperature, suspected colonies are subjected to biochemical tests for final identification:
- Indole Test: Positive (red ring after adding Kovac’s reagent)
- Methyl Red Test: Positive (red color after adding methyl red indicator)
- Voges-Proskauer Test: Negative
- Citrate Utilization Test: Negative
- Gas and Acid from Lactose: Positive
This pattern (IMViC: ++--) confirms E. coli.
📘 Scientific Reasoning – Why 44 °C and Not 37 °C?
Most coliforms grow optimally around 35–37 °C, which is the general body temperature of humans. However, E. coli can also grow at slightly higher temperatures up to 44–45 °C due to its thermotolerant enzymes and intestinal adaptation.
This elevated temperature is used to simulate intestinal conditions and selectively isolate fecal E. coli from environmental or water samples where other coliforms may also exist.
💡 Key Points Summary
- E. coli is identified based on its ability to ferment lactose and grow at elevated temperature (42–44 °C).
- 44 °C incubation differentiates fecal coliforms (E. coli) from environmental coliforms.
- Media like EC Broth or EC-MUG Broth are used for confirmation at elevated temperature.
- Biochemical profile (IMViC: ++--) confirms the organism as E. coli.
✅ Conclusion
The use of elevated temperature (42–44 °C) in the E. coli identification process is not arbitrary—it is a scientifically established parameter that ensures accurate differentiation between fecal and non-fecal coliforms. By growing E. coli at this temperature, laboratories can confirm its thermotolerance and fecal origin with high specificity. This method forms a cornerstone of water, food, and pharmaceutical microbiological testing and is recommended by international standards such as USP, IS, ISO, and WHO guidelines.
📚 References
- APHA Standard Methods for the Examination of Water and Wastewater
- USP <61> and <62> – Microbial Examination of Nonsterile Products
- ISO 9308-1:2014 – Water Quality: Detection and Enumeration of E. coli
💬 About the Author
Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.
📧 Contact: siva17092@gmail.com
Mobile: 09505626106
