Detection of Escherichia coli (E. coli)_A Complete Procedure (ISO 9308-1 & ISO 16649)

Summary

This video demonstrates a critical microbiological test for detecting E. coli. The process involves seven key steps, from preparing media and diluents to sample preparation, enrichment, and streaking methods on various agar plates like Chromocult Coliform, EMB, and Sorbitol MacConkey Agar. The video guides viewers through the procedures of incubating the plates, identifying violet, blue, metallic green, and pink colonies, which confirm the presence of E. coli. With detailed instructions and essential lab equipment, the video aims to educate viewers on becoming laboratory experts in E. coli detection.

Takeaways

• 🔬 The video demonstrates an important microbiological test: detection of E. coli.
• 🧪 The test involves seven steps, using various lab equipment and materials like incubators, microwaves, and petri dishes.
• ⚖️ Chromocult coliform agar is prepared by dissolving 2.65g of dehydrated media in 100ml distilled water, boiling it, and cooling it into sterile petri dishes.
• 🌡 EC broth and EMB agar are also prepared with specific measurements of media and autoclaved at 121°C for 15 minutes.
• 🥄 A sample preparation involves mixing 25g of sample with peptone-sodium chloride solution in a sterile stomacher bag, followed by dilution.
• 🦠 E. coli enrichment is done by transferring 1ml of the sample into EC broth tubes, incubating them at 37°C for 24 hours.
• 🔥 After incubation, the enrichment culture is streaked on Chromocult coliform agar plates using an inoculating loop.
• ⏳ The plates are incubated again for 24 hours at 37°C, and violet or blue colonies indicate possible E. coli presence.
• ✅ Confirmation of E. coli is done by streaking the presumptive colonies on EMB and SMAC agar plates, observing metallic green sheen and pink colonies respectively.
• 🔍 The presence of E. coli is confirmed by observing violet colonies on Chromocult agar, metallic green sheen on EMB agar, and pink colonies on SMAC agar.

Q & A

What is the primary purpose of the video?

The video demonstrates the steps for detecting E. coli using microbiological techniques.

How many steps are involved in detecting E. coli in this experiment?

There are seven main steps involved in detecting E. coli in this experiment.

What are the main materials required to conduct the E. coli detection experiment?

The required materials include balance mission, microwave oven, biological safety cabinet, incubator, stomacher mission, stomacher bag, autoclave, micropipette, petri dish, paper tips, spatula, inoculating loop, pepton, sodium chloride, chromocult coliform agar, EC broth, eosin methylene blue (EMB) agar, and sorbitol MacConkey (SMAC) agar.

What is the first step in preparing the media for detecting E. coli?

The first step is to prepare chromocult coliform agar by dissolving 2.65 grams of dehydrated agar media in 100 ml of distilled water, then boiling the media and pouring 15 ml into sterile petri dishes.

What is the purpose of the EC broth in the experiment?

EC broth is used for the selective enrichment of E. coli from the sample to increase the concentration of the target bacteria for further testing.

How is the sample prepared before enrichment?

The sample is prepared by taking 25 grams of it, diluting it with 225 ml of autoclaved and cooled peptone salt solution, then mixing it using a stomacher machine.

What is done after the sample enrichment step?

After enrichment, the culture is inoculated on chromocult coliform agar plates using a sterile inoculating loop. The plates are then incubated at 37°C for 24 hours.

How is the presence of E. coli confirmed after incubation?

The presence of E. coli is confirmed by observing violet and blue colonies on chromocult coliform agar plates. For further confirmation, presumptive E. coli colonies are inoculated on EMB agar and SMAC agar plates and incubated.

What are the characteristic colony appearances of E. coli on different agar plates?

E. coli forms violet and blue colonies on chromocult coliform agar, metallic green sheen on EMB agar, and pink colonies on SMAC agar.

Why is it important to use multiple agar plates (chromocult, EMB, and SMAC) for confirmation?

Using multiple agar plates provides stronger confirmation of E. coli presence, as different agars allow the bacteria to display distinct colony characteristics, making the identification more reliable.