Enumeration of indicator organism s.pptx

Enumeration of indicator organisms from water samples

1. Enumeration of indicator organisms

2. Enumeration methods MPN -most probable number Membrane filteration method

3. Membrane filtration method principle: In this technique sample is passed through the membrane using a filter funnel and vacuum system. Any organisms in the sample are concentrated on the surface of the membrane. Membrane filters have a known uniform porosity of predetermined size (generally 0.45 µm ) .

4. Procedure Collect the sample and make any necessary dilutions. Select the appropriate nutrient or culture medium. Dispense the broth into a sterile Petri dish, evenly saturating the absorbent pad. Flame the forceps, and remove the membrane from the sterile package. Place the membrane filter into the funnel assembly. Flame the pouring lip of the sample container and pour the sample into the funnel. Turn on the vacuum and allow the sample to draw completely through the filter. Rinse funnel with sterile buffered water. Turn on the vacuum and allow the liquid to draw completely through the filter. Flame the forceps and remove the membrane filter from the funnel. Place the membrane filter into the prepared Petri dish. Incubate at the proper temperature and for the appropriate time period. Count and confirm the colonies and report the results.

5. Uses of membrane filer technique: It is used to analyze a series of dilutions of water samples collected upstream and downstream from an outlet of a sewage treatment plant. A total count of coliform bacteria determines the potability of the water sources. Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate solutions, certain antibiotics, and other heat-labile substances) can be sterilized by membrane filtration. The pharmaceutical and cosmetics industries typically focus on monitoring their process water for Pseudomonas species.

6. Advantages of MF method: Results are available in a shorter period of time Larger volumes of sample can be processed Because of the high accuracy of this method, the results are readily reproducible. Allows isolation and enumeration of discrete colonies of bacteria Allows for removal of bacteriostatic or cidal agents that would not be removed in Pour Plate, Spread Plate, or MPN techniques. It involves less preparation than many traditional methods, and is one of a few methods that will allow the isolation and enumeration of microorganisms.

7. Total coliforms and fecal coliforms: They are the members of the family Enterobacteriaceae . Coliforms may include bacteria of the following genera: Escherichia, Enterobacter, Klebsiella , Citrobacter and Serratia . Fecal coliforms are the subset of total coliforms which are found within the digestive tract and shed through feces.  Bacteria belonging to the genera Escherichia and  Enterobacter  are considered as fecal coliforms.

8. Detection of total coliforms and fecal coliforms: Total coliforms and fecal coliforms are detected using selective and differential culture medium. For the detection of total coliforms mEndo agar LES is used and for fecal coliforms mFC agar is used.

9. Detection of total coliforms using mEndo agar LES Membrane filter is incubated at 35°C for 22-24 hours for the detection of total coliforms. Coliforms typically will produce metallic (golden) sheen. Detection of fecal coliforms using mFC agar Membrane filter is incubated at 44.5°C for 22-26 hours in mFC agar for the detection of fecal coliforms. Fecal coliforms form blue colonies in this medium.