Fish Microbiology and Quality Assurance (2+2)

Unit 3 - Enumeration of microorganisms in foods

3.2.2 Culture based methods
Culture methods involve examination of microorganisms in food by encouraging them to multiply in a liquid or solid media. On solid agar media bacteria develop as colonies and counting such viable colonies gives microbial load in foods.
Enumerating microorganisms by culture based methods can be done by using plate count methods or MPN technique.

Culture media
A wide variety of media with varied composition capable of supporting the growth are available for the cultivation of different microorganisms.
The composition of the media varies depending on;

• Group/type of microorganism to be studied
• Overall purpose of the study
• Whether to grow wide range of microorganisms or specific types
• Resusitation of damaged but viable cells
• Type of diagnostic information required

Ex: General purpose media: Plate count agar
Lactose broth: For Escherichia coli
Seective media: Baird parker agarfor Staphylococcus
Bismuth sulphite agar for Salmonella
TCBS for Vibrios


A. Plate count method
This method is variously referred to as total plate count (TPC), standard plate count (SPC) or aerobic plate count (APC). It is most widely used conventional method for determining viable cells or colony forming units (CFU) in foods.
SPC involves blending/ homogenizing the sample, serially diluting in appropriate diluent, plating in or on suitable agar media, incubating at appropriate temperature for a given time, and counting visible colonies as CFU.

Principle involved
SPC is based on the principle that each viable bacterial cells multiples and grows in to a visible colony. Thus, counting number of colonies gives an idea about bacterial cells present in a sample. Counts determined by taking average of replicate plates showing 30-300 colonies.

Factors affecting SPC

• Sampling method employed.
• Distribution of microorganisms in food.
• Nature of food biota
• Nutritional adequacy of plating media
• Incubation temperature and time
• Type of diluent used
• Presence of other competing organisms etc.
• Plating on selective media for specific organisms is limited by degree of inhibition and effectiveness of selective/ differential agents employed.

SPC can be performed by pour plate method or spread plate method (surface plating method)

a). Pour plate method
Appropriate dilution of the sample (1 ml) is mixed with agar medium, allowed to set, incubated at appropriate temperature and colonies developed are counted. Here colonies develop both on surface and subsurface of agar plate.

Proper mixing of sample with agar medium is necessary so as to get isolated colonies which can be done by 2 ways.

• One ml of appropriate sample dilution is added to petri-plate and about 15 ml of agar medium is added and mixed by rotating the plate in clockwise and anticlockwise direction.
• One ml of appropriate sample dilution is added to test tube containing about 15 ml of molten agar medium, mixed by rolling the tube between the palm and poured to petri-plates, allowed to set and incubated.

b). Spread plate method
Diluted sample (0.1 ml) is spread on the surface of pre-poured, hardened agar plates using glass rod, incubated at appropriate temperature and colony developing on surface counted.

Advantages

• Suitable for heat sensitive psychrotrophs in food as they do not come in contact with molten agar.
• Enables providing colony features useful in presumptive identification especially on selective media.
• Favours strict aerobes on surface, but microaerophils grow slowly

Disadvantage

• Problem of spreaders and colony crowding makes the enumeration difficult.

B. Most probable number (MPN) technique
MPN is suited for enumerating the presence of low numbers of microorganisms in foods. This involves inoculating replicate tubes of appropriate liquid media (3 or 5 tube) with three different sample sizes/ dilutions of the material to be studied and incubating at appropriate temperature. Then the absence or presence of growth is observed and MPN table consulted to get probable number of organisms in the sample. MPN numbers are generally higher than SPC.
Advantages

• Relatively a simple method and assay to perform
• Results are comparable from one laboratory to another
• Specific group of organism determined by use of specific media.
• Suitable for detecting organisms present in low numbers
• Method of choice for coliform detection

Disadvantages

• Requires use of large number of glassware and large volume of sample
• Can not observe colony morphology
• Lack of precision