Uji Coliform - MPN Tahap 2 & 3 (Uji Penegas & Uji Kepastian)

Summary

This script outlines a comprehensive procedure for detecting E. coli in water samples using a series of microbiological tests. It details the use of media like Brilliant Green Lactose Broth (BGLB) and Eosin Methylene Blue (EMB) agar for presumptive and confirmatory tests. Positive results are determined by color changes and gas production, with the final confirmation showing green metallic colonies on EMB agar. The process involves multiple dilution steps, incubation, and observation for clear interpretation of results, ultimately leading to the quantification of E. coli in the sample.

Takeaways

• 😀 The test begins with the observation of the sample color in the initial presumptive test. If the sample is negative for E. coli, the color remains red. If positive, the color turns yellow and gas is produced.
• 😀 A positive result in the presumptive test indicates a possible presence of E. coli, prompting a confirmation test to be conducted using Brilliant Green Lactose Broth (BGLB) medium.
• 😀 The confirmation test is performed using a liquid form of BGLB, and samples from positive presumptive test tubes are used for inoculation into this medium.
• 😀 After inoculation, the samples are incubated at 44°C for 24 to 48 hours. If gas bubbles or a color change occurs, the test is positive for E. coli.
• 😀 The BGLB medium turns a darker green color when the test result is positive, indicating a stronger reaction.
• 😀 A sample showing no positive result (no gas bubbles or color change) in the confirmation test is considered negative, and the analysis continues with further confirmation.
• 😀 In the final confirmation step, Eosin Methylene Blue (EMB) agar is used for inoculation and incubation at 30°C for 24 to 48 hours, where the appearance of metallic green colonies confirms the presence of E. coli.
• 😀 EMB agar is critical because it allows for a clear visual indication of E. coli presence, as positive colonies appear as greenish metallic.
• 😀 After the incubation period, the presence of green colonies confirms the presence of E. coli, and the result is considered conclusive.
• 😀 The final step involves calculating the number of E. coli present based on the results from different dilution tubes, which helps estimate the concentration of E. coli in the sample.

Q & A

What is the purpose of the initial test using Brilliant Green Lactose Broth (BGLB)?

The initial test using Brilliant Green Lactose Broth (BGLB) is to determine if the sample contains E. coli. If the sample is positive for E. coli, the media will turn yellow, and gas will be produced, which is observed in a Durham tube.

How is the confirmation of E. coli performed after the initial test?

After the initial test, the sample is further tested using the BGLB medium. If the sample shows a yellow color and gas production in the Durham tube, it is considered positive, and the test moves to the confirmation phase.

What are the conditions for incubation during the testing process?

The samples are incubated at 44°C for 24-48 hours. If no changes occur at 24 hours, the incubation time is extended to 48 hours to observe any color or gas production changes.

What does the appearance of a green metallic sheen on Eosin Methylene Blue (EMB) Agar indicate?

The appearance of a green metallic sheen on EMB Agar indicates a positive result for E. coli, as this color change is caused by the reaction between E. coli and the EMB medium.

Why is EMB agar used in the confirmation test for E. coli?

EMB agar is used in the confirmation test for E. coli because it contains chemicals that interact with E. coli, causing it to produce a distinctive green metallic sheen, which is a clear indicator of E. coli presence.

What is the role of the Durham tube in this experiment?

The Durham tube is used to capture gas produced during the fermentation process in the broth. A positive result is indicated by the presence of gas in the Durham tube and a color change in the media.

How are the samples prepared for the confirmation phase using EMB agar?

For the confirmation phase, positive samples from the previous step are inoculated onto EMB agar plates using the streaking method. The samples are then incubated at 30°C for 24-48 hours to observe the development of colonies.

What does a result of zero positive tubes in a sample dilution indicate?

A result of zero positive tubes in a sample dilution, such as the 10^-3 dilution, indicates that no E. coli is present in the sample at that concentration.

What is the purpose of the MPN calculation in this process?

The MPN (Most Probable Number) calculation is used to estimate the concentration of E. coli in a sample based on the number of positive results observed in the test tubes at different dilutions.

How does the dilution factor affect the number of positive results in this test?

The dilution factor affects the number of positive results because as the sample is diluted more (e.g., 10^-1, 10^-2, 10^-3), fewer positive results are expected. The MPN calculation uses these results to estimate the bacterial concentration.