Lab 6: Microbial Analysis of Water for Fecal Contamination

• Water sample bottle

• Membrane filtration apparatus (funnel, base, clamp)

• Sterile membrane filters (0.45 μm pore size, gridded)

• Sterile forceps

• M-Endo Agar LES medium (prepared)

• Sterile Petri dishes

• Sterile distilled water or phosphate-buffered saline (for rinsing)

• Bunsen burner or alcohol lamp

• Incubator

• Sterile graduated cylinder or pipette (for measuring water sample)

Water quality is critical for public health. Fecal contamination of water sources can introduce various harmful pathogens. Instead of testing for all pathogens, indicator organisms are used. Coliforms are a group of bacteria (Gram-negative, rod-shaped, non-spore-forming) that ferment lactose, found in intestines and the environment. Their presence indicates potential contamination. While some coliforms are environmental, Escherichia coli (E. coli) is specifically a fecal coliform, reliably indicating recent fecal contamination and thus the potential presence of enteric pathogens.

The Membrane Filtration Technique (MFT) is employed to concentrate bacteria from a water sample onto a sterile membrane filter. This filter is then placed on a selective and differential culture medium, such as M-Endo Agar LES. This medium supports the growth of coliforms while inhibiting other bacteria. Coliforms on M-Endo Agar LES ferment lactose, producing acetaldehyde, which reacts with basic fuchsin and sodium sulfite in the medium to form a characteristic metallic sheen (greenish-gold) or red colonies, allowing for their enumeration. Incubation at specific temperatures facilitates bacterial growth into visible colonies, which are then counted to determine the bacterial load in the original water sample.

M-Endo Agar LES – Chemical Composition

1. Sterilized the workspace and lit a Bunsen burner for aseptic conditions.

2. Placed a sterile membrane filter on the filtration unit.

3. Poured 100 mL of water sample into the funnel and applied vacuum filtration.

4. Rinsed the funnel with 20 mL sterile water to ensure complete bacterial transfer.

5. Transferred the filter onto M-Endo Agar LES (avoiding air bubbles).

6. Incubated the plate at 35°C for 24 hours.

Colonies appeared red/pink on M-Endo Agar LES, indicating the presence of total coliforms. A distinct metallic sheen, typical for E. coli, was not observed. The presence of colored colonies signifies lactose fermentation by coliforms.

No numerical counting of colonies was performed in this specific lab session, therefore CFU/100 mL was not calculated. In a typical quantitative analysis using MFT, each distinct colony on the membrane filter would be counted, and the count would be used to calculate the Colony Forming Units per 100 mL of water sample using the formula:

Where:
\( N \) = Number of colonies counted
\( V \) = Volume of sample filtered (mL)

• The red/pink colonies confirm total coliform presence, suggesting possible fecal contamination.

• According to WHO standards, drinking water should have 0 coliforms/100 mL in 95% of samples and ≤10 coliforms/100 mL in any single sample.

We observed multiple red/pink, it implies that coliform organisms were present in the water sample. Without actual enumeration, we cannot state the exact count, but the visual presence of numerous colonies suggests that the water sample likely does not comply with the WHO standards for drinking water, as it appears to contain more than zero, and potentially more than 10 coliform organisms per 100 mL. The presence of any coliforms, particularly if above the acceptable limit, signifies a potential sanitary risk. This poses a health risk, as it may allow entry of harmful, disease-causing microbes, making the water unsafe without further treatment.

The microbial analysis of the water sample using the Membrane Filtration Technique with M-Endo Agar LES revealed the presence of coliform bacteria, as evidenced by the formation of red/pink colonies on the membrane filter. This finding suggests that the water sample is microbiologically contaminated and likely does not meet the drinking water quality standards set by WHO.

1. Source Water Protection: Implement measures to protect raw water sources from both human and animal fecal contamination.

2. Effective Water Treatment: Ensure proper and consistent application of all water treatment stages, especially filtration and disinfection, to remove or inactivate microorganisms.

3. Distribution System Integrity: Regularly inspect and maintain the water distribution network to prevent leaks, cross-connections, and ingress of contaminants.

4. Continuous Monitoring: Establish a robust monitoring program for bacteriological parameters, performing frequent tests to detect contamination promptly.

5. Public Health Interventions: In cases of confirmed contamination, issue public health advisories (e.g., boil water notices) and take immediate corrective actions to restore water safety.

• Maintain strict aseptic techniques at all times to prevent contamination.

• Ensure all glassware and equipment are properly sterilized before use.

• Handle membrane filters with sterile forceps only.

• Accurately measure the water sample volume for filtration.

• Incubate plates at the correct temperature for the specified duration.

• Dispose of all contaminated materials safely and according to laboratory protocols.

• Wear appropriate personal protective equipment (PPE), including lab coats and gloves.

1. What are E. coli? Are they harmful to human beings? Why is their presence tested in the waters to be supplied for domestic consumption?

E. coli: Escherichia coli (E. coli) are Gram-negative, rod-shaped bacteria that are part of the fecal coliform group. They are typically found in the intestines of warm-blooded animals, including humans.

Harmful to Human Beings? Most strains of E. coli are harmless commensals in the human gut. However, some strains are pathogenic and can cause severe illnesses, ranging from mild diarrhea to life-threatening conditions like hemorrhagic colitis, hemolytic uremic syndrome (HUS), and urinary tract infections. Examples of pathogenic strains include Enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), and Enteropathogenic E. coli (EPEC).

Why is their presence tested in the waters to be supplied for domestic consumption? E. coli is considered the most specific indicator of recent fecal contamination in water. Its presence strongly suggests that the water has been contaminated with human or animal feces. Since fecal matter can contain a wide array of disease-causing pathogens (bacteria, viruses, parasites) that cause waterborne diseases (e.g., cholera, typhoid, giardiasis, cryptosporidiosis), testing for E. coli provides a reliable and cost-effective way to assess the potential health risk associated with consuming the water. Its absence indicates that the water is likely free from fecal contamination and therefore safer for consumption.

2. What is coliform index?

The Coliform Index refers to a measure used in water quality assessment to indicate the extent of coliform bacterial contamination. It is essentially a quantitative expression of the number of coliform organisms present in a given volume of water. It is typically expressed as:

• Most Probable Number (MPN) Index: A statistical estimate of the number of coliforms per 100 mL of water, derived from the results of a series of fermentation tubes in the Multiple Tube Fermentation (MTF) technique.

• Colony Forming Units (CFU) per 100 mL: A direct count of viable coliform bacteria that form colonies on a membrane filter, expressed as CFU per 100 mL of water, as determined by the Membrane Filtration Technique.

The coliform index provides a quantitative basis for comparing water quality against regulatory standards and assessing the potential risk of fecal contamination.

3. Define MPN.

MPN (Most Probable Number) is a statistical method used to estimate the concentration of viable microorganisms in a sample, particularly in water and wastewater analysis, when direct plate counting is not feasible or desired (e.g., for very low concentrations or when dealing with complex matrices).

The MPN method involves inoculating multiple sets of tubes with a serially diluted sample. Each tube contains a selective liquid growth medium (e.g., lactose broth for coliforms). After incubation, the number of positive tubes (i.e., tubes showing bacterial growth and gas production, indicating fermentation) at each dilution is recorded. These results are then compared to a statistical table (MPN table) to determine the “most probable number” of microorganisms present in the original sample, usually expressed per 100 mL. It is a statistical estimate rather than an exact count, relying on probability theory.