Techniques Used For The Determination of Microorganisms In Food

Techniques used for the determination of microorganisms in food

Various techniques can be used for determination of microorganism in food some of them give total count (viable, non-viable ) , while others give only viable count .

Viable count

Standard plate count(SPC)/ Aerobic plate count

The most common procedure for the enumeration of microorganisms is the viable plate count . In this method , several dilutions of a sample containing viable microorganism are plate into a suitable growth medium . The suspension is either spread onto the surface of an agar plate(spread plate method) or is mixed with molten agar poured into the plate and allow to solidify(pour plate method), the plates are then incubated under conditions that permit microbial develops. Such colonies can be seen without the aid help of microscopes . It is assured that each bacterial colony arises from an individual cell that has under born undergone multiple cell division . Therefore by counting numbers of colonies and accounting the dilution faster,the number of bacteria in the original sample can be determined.

Type of SPC

1. Pure plate techniques :In this method , food is firstly serially diluted in an appropriate diluent . The measured volume of sample from the diluted tube is placed in sterile Petri plate melted agar at 44-45°c is mixed with it . After homogenous mixing of the sample with melted agar ,it is kept for solidification . Then the Petri plate are incubated at the appropriate time and temperatures plate containing colonies between 30-300 are selected and a number of the organism in an original food sample .Since psychrophile can not temperatures of melted agar. This technique is not suitable for them . In this method, both surface and sub-surface colonies are developed both surface colonies are difficult to isolate.
2. Spread plate techniques : In this method , the appropriately diluted sample is placed on the surface of the solid fine agar . Then the drop of sample is spread over agar surface using bent glass rod plate is incubated for sufficient time and temperature , then a number of colonies are counted . Calculation of no.of organism is done similarly as in pour plate technique . This method is also suitable for psychrophiles and in this method, only surface collines are developed.
3. Streaky plate :In this techniques, transfer loop is used to spread the specific volume of specimen over a surface of solidified agar . The transfer is done by calibrated loop and differential media can be used to select growth specific organism .

Advantages of SPC

• It gives viable count .
• It is extremely sensitive i.e extremely low and high .
• The microbial population can be counted.

Disadvantage of SPC

• If the suspension is not a homogeneous and contains an aggregate of the cell , the colony count will be lower than the actual number of microorganism.
• If the suspension contains different type of microorganism cell of them can not grow in the same medium and under the same condition

Membrane filter

This technique is particularly important to analyze microorganism in liquid food. In which microbial content is too low. In this method measured the volume of liquid is filtered through a membrane filter of specific porosity 0.4μm then filter pad is removed and placed on the surface of the agar plate and the incubated . Microorganisms grow on the surface of membrane filter to from the colony . Then total no.of organism in an original sample is calculated nutrient or selective agar media can be used for microbial growth .

Most probable number(MPN) multiple tube test

It is statistical techniques to determine no.of organism in the sample . It gives a most probable number but not the actual number of the organism . Turbidity ,gas production and acid production are observed to determine microorganisms. This method is based on three steps.

• Presumptive step
• Conform or confirmatory step
• Completed test

Total count ( viable /non-viable)

In the method, these are number different between dead and viable cells . Total cells are counted in this method.

Direct microscopic count (DMC)

In this method , the result is obtained faster than most another method because the incubation period is not required .

Measured volume of free sample

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⇓Spread no clean grease free slide over specific area

Stained with appropriate dye

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Observed under microscope and number of organism counted.

Figer: procedure of DMC

The procedure of DMC is given in above figure . In the case of liquid , the direct smear is made . For solid food, t must be first diluted . Fatty food must be defatted (removing of fat ) in xylene or acetone before preparation of smear . The xylene /acetone is then removed by dipping it in ethanol . In this method number or microorganisms in microscopic field ae counted directly . This technique is widely used to assess the quality of raw milk and other dairy product.

Breed count method

It is on the example of DMC . This method was initially developed by R.S breed . In this method ,0.001 ml of sample is spread over the 1cm² area on solid . If the sample is fatty it should be defeated with xylene or acetone . Excess xylene or acetone is then removed by dipping it in ethane . The slide is dried fixed and stained with appropriate dye observed under a microscope . The average no.of microorganisms per microscopic field are calculated . Then the area of one microscopic field is determined from which no.of microorganisms in the original sample is calculated. It is not practical to count entire field . So only a few microscopic field are counted to determine average no.of organism in the sample .

Wilson′s chart

Advantage of DCM

• It is simple and rapid techniques
• Morphology as well as gram reaction of microorganisms spare production etc and be stored .
• The prepared slides can be stored and maintained as the permanent record .
• Very small amount of sample is needed .

Disadvantage of DCM

• DCM can not distinguish viable and nonviable stable from the microorganism .
• Food practical is not always distinguishable from a microorganism .
• Some microorganisms don't take stain and may not be counted.
• Staining procedure it so;f can wash the same microorganism from slides.
• It is very difficult to count microorganism when the initial load is very high.

Electronic counter

In this method, the standard volume of a suitable dilution of suspension is placed in electronic counter . The mechanic has the small aperture through which microorganism can pass . The passage of microorganisms through aperture causes. Alternating in electric resistance across it which is recorded as an implueses . This impulses is counter by suitable circuit in counted no.of impulses from fixed volume of sample is used to calculated no.of organism in an original food sample .

References

Frazier, WC and Westhoff DC. Food Microbiology. 3rd. New Delhi: Tata McGraw-Hill Publishing Company Limited, 1986.

Jay, MJ. Moderen Food Microbiology. Ed. India. 3rd. Delhi: CBS Publications and Distributions, 1987.