When examining water samples for bacterial content, direct microscopic observation often yields minimal visible bacteria due to their small size and low concentrations. To overcome this challenge and accurately assess the bacterial population in water, scientists employ the filtration technique. This method is pivotal for both quantifying bacteria per unit volume of water and analyzing their morphological characteristics.

The core of the filtration process involves passing the water sample through membrane filters. These filters are designed with pores small enough to capture bacteria while allowing water to pass through. This concentration of bacteria on the filter surface facilitates a more detailed and comprehensive analysis than direct microscopy alone.

Following filtration, the membrane filter containing the trapped bacteria is transferred to a nutrient-rich agar plate. This step is critical for viable counting, a process where individual bacteria grow into visible colonies. Each colony originates from a single bacterium or a cluster thereof, making it possible to estimate the number of viable bacteria in the original water sample. The growth of these colonies on agar plates not only aids in quantifying the bacterial population but also allows for the observation of various morphological characteristics of the bacteria, such as shape, size, and arrangement.

This filtration and viable counting method is a rapid, efficient, and widely used approach for sampling bacteria from water. It provides essential information for water quality assessments, environmental monitoring, and public health protection, making it a fundamental technique in microbiological water analysis.

Requirement for Sampling of Bacteria From Water

• Water Sample: The starting point of the process involves collecting a representative sample of water from the source being tested. This sample should be collected in a sterile container to prevent contamination and should accurately reflect the water’s typical conditions.
• Membrane Filter Assembly: This apparatus is central to the filtration technique used in bacterial sampling. It consists of a setup that holds the membrane filter in place and allows the water sample to be filtered under controlled conditions.
• Millipore Filter (0.45 µm): The Millipore filter, with a pore size of 0.45 micrometers, is crucial for trapping bacteria while allowing water and smaller particles to pass through. This pore size is optimal for capturing a wide range of bacteria without getting clogged by larger particles.
• Side Arm Flask: This component of the filtration assembly serves as a receiving vessel for the filtrate (the liquid passing through the filter). It is typically connected to the membrane filter assembly and helps maintain a sterile environment for the filtered water.
• Suction Pump: The suction pump creates a vacuum that pulls the water sample through the membrane filter. This mechanism is vital for ensuring efficient filtration and trapping of bacteria on the filter surface.
• Nutrient Agar Plates (pH 6.8): After filtration, the membrane filter with the captured bacteria is placed on nutrient agar plates. The specific pH of 6.8 is conducive to bacterial growth, allowing the development of visible colonies from the trapped bacteria. Each colony arises from a single bacterium or a cluster, facilitating the enumeration and further analysis of the bacterial content in the water sample.

Procedure

1. Setup of Filtration Assembly: Begin by placing a Millipore filter within the membrane filter assembly. This setup should then be securely connected to a side-arm flask. The Millipore filter, typically with a pore size of 0.45 µm, is critical for effectively trapping bacteria while allowing the water to pass through.
2. Connection to Suction Pump: The side-arm flask is then connected to a suction pump. This pump will generate a vacuum necessary for drawing the water sample through the Millipore filter, ensuring efficient filtration and retention of bacterial cells on the filter surface.
3. Filtration of Water Sample: Introduce the water sample into the membrane filter assembly, allowing it to pass through the Millipore filter under the influence of the vacuum created by the suction pump. This step ensures that bacteria present in the water are captured on the filter.
4. Transfer of Filter Paper: Once filtration is complete, carefully remove the Millipore filter paper, now containing the trapped bacteria, from the assembly. This filter is then gently placed onto the surface of a nutrient agar medium within a Petri plate. The nutrient agar provides a conducive environment for bacterial growth, supporting the development of visible colonies from the captured bacteria.
5. Incubation: The Petri plates, now containing the filters with the bacterial samples, are incubated at a controlled temperature of 28°C. This temperature is generally optimal for the growth of a wide range of bacterial species. The plates are left in the incubator for a period of 24 hours to allow sufficient time for bacterial colonies to form.
6. Observation of Results: After the incubation period, the plates are removed from the incubator and examined for bacterial growth. The presence of bacterial colonies on the filter surface indicates the presence of bacteria in the original water sample. These colonies can be counted and analyzed further to determine the types and quantities of bacteria present.

Result

The outcome of the bacterial sampling process from water is quantified by counting the number of bacterial colonies that emerge on the nutrient agar plates after incubation. These colonies are expressed in terms of Colony-Forming Units per milliliter (CFUs/ml) of the original water sample. This measurement provides a tangible indication of the bacterial load within the water.

Each visible colony on the agar plate is presumed to have originated from a single bacterium or a closely associated cluster of bacteria present in the water sample. By counting these colonies, scientists can estimate the number of viable bacterial cells that were in the water sample before filtration.

The CFU/ml calculation is crucial for assessing water quality and safety. It helps in determining whether the water meets health and safety standards for drinking, recreational activities, or other uses. High CFU/ml values may indicate contamination and potential health risks, prompting further investigation or immediate remedial action.