INTRODUCTIONMicroorganisms:-Organisms found in surface water and wastewater include bacteria, fungi, algae, protozoa, plants and animals and viruses which can only be observed microscopically.

Microorganisms

Prokaryote (Simplest cell structureSize: 0.2 -3.0µm)

Eukaryote(more complexSize: 2-100 µm)

(e.g. bacteria, blue-green algae and archaea)

(e.g. protozoa, fungi, rotifers, green algae, plants & animals)

DETECTION TECHNIQUES

Why Detect Microorganisms?

• Determine cause of infection

• Appropriate treatment (antibiotics, antivirals, vaccination )

Rapid detection leads to earlier intervention

Exclusion of infectious causes

Three Major Steps Involved

Recovery &

Concentration

Purification &

Separation

Assay &

Characterization

Methods for Identification of bacteria

1. Direct microscopic count2. Pour & spread plate counts3. Membrane filtration (MF)4. Multiple tube fermentation

Direct Microscopic Count

1. Microscopically by using Petroff-Hauser counting chamber• Difficult to differentiate b/w live & dead

cells• Acridine orange (Vital) stain to

differentiate b/w live & dead cells2. Electronic particle counter is an alternative

• Used to culture, identify & enumerate the bacteria

• Sample of wastewater to be tested is diluted • Small amount of dilution is mixed with a warned

liquid (agar) culture, poured into a culture dish, allowed to solidify and incubated under controlled conditions

• Finally separate distinct bacterial colonies are formed on the plates after incubation and the results reported as colony-forming unit per unit volume (cfu/ml)

Pour & Spread Plate Counts

Membrane-Filter (MF) Technique

• Known volume of water is passed through a membrane filter

• Bacteria are retained on filter • Membrane filter is placed in contact with an agar for

growth of specific bacteria• After incubation, colonies are formed and then

counted

• This method is based on the principle of dilution to extinction• Concentration of total coliform bacteria are reported as most

probable number per 100ml (MPN/100ml)

Multiple-Tube Fermentation

Other methods of bacteria identification

1. Presence – absence test2. Heterotrophic Plate Count

Identification of Specific Bacteria & Protozoa

1. Fluorescence method2. Nucleic acid probe method

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