Fish Microbiology and Quality Assurance

Unit 3 - Enumeration of microorganisms in foods

3.2.1 Direct counting methods
Microorganisms present in the food can be counted by observing the food sample directly or retaining the microorganisms on a filter paper by filtering the sample and then observing under microscope.

A. Direct microscopic count (DMC)
DMC involves detecting the presence of microorganisms in food by microscopic observation. It is a simple method and easy to perform.
DMC is performed by making a smear of food specimen/cultures on to microscopic slides, staining with appropriate dye and viewing and counting all cells using microscope under oil immersion objective. This method can be used only when microorganisms are present in large numbers (106/ml). It is commonly used in dairy industry for assessing microbial quality of raw milk and other dairy products.
Advantages

• Rapid and easy enumeration
• Can be employed to any foods (Ex: dried/frozen foods)
• Simple to perform
• Cell morphology can be assessed
• Efficiency can be increased by using florescent probes

Disadvantages

• Requires tiresome counting under microscope causing fatigue to analyst
• Counts both viable and non-viable cells
• Food particles may interfere with counting and mistaken for microorganisms
• Cells may not be distributed uniformly (single cells/ clumps)
• Some cells may not take up stain and missed while counting
• DMC counts are always higher than standard plate count
• Requires dilution of sample

B. Direct counting on membrane filters
Membrane fillers with pore size smaller (0.45 um) than bacteria retain bacteria and the retained bacteria can be counted using microscope.
Procedure involved

• Concentrating/collecting bacteria on polycarbonate filters by filtering known volume of homogenized sample.
• Staining and counting of retained bacteria.
• Placing the membrane on a nutrient agar media or absorbent pad saturated with culture media of choice, and incubating
• Following growth , colonies are counted

Advantages

• Well suited for samples containing low numbers of bacteria
• Facilities concertinaing bacteria by filtering large volume of sample
• Only small volume of food samples can be used for a single membrane
• Efficiency of membrane filter method can be increased by staining with florescent dyes (Ex. acridine orange) and observing under epiflorescence microscope (DEFT: Direct Epiflorescence Filter technique). Viable cells fluoresce green and are counted. Non viable cells appear orange. Acridine orange is an metachromatic fluorochrome which binds to double stranded DNA of viable bacterial cells .
• Can be used to enumerate microorganisms from a variety of foods (fresh fish, meat, fish/ meat products, water samples etc).