• Various techniques can be used for determination of microorganisms in food.
• Some of them give total count (viable+ non-viable) while others give only viable count.

1. Standard plate count (SPC):

• Standard plate count gives viable count of organism present in food.
• Procedure of performing standard plate count is given above in figure.
• The number of organisms in original food is determined by counting the colony on agar plate.
• Two major assumptions of SPC are:
  • Microorganisms in suspension are separated as single cell so that each colony is developed from single cell.
  • All viable cells placed on medium will multiply and produce a colony
• Incubation time and temperature for different microorganisms:
  • Psychrophilesà 7^oC, 7 days
  • Mesophilesà 35^oC, 24-48hrs
  • Thermophilesà 55^oC, 48hrs
• Advantages:
  • It gives viable count.
  • It is extremely sensitive i.e. extremely low and high microbial population can be counted.
• Disadvantages:
  • If the suspension is not homogenous and contain aggregate of cells, the colony count will be lower than the actual number of microorganisms.
  • If the suspension contains different types of microorganisms, all of them cannot grow in the same medium and under the same condition.

Types of Standard plate count (SPC) method:

1. Pour plate technique:
   • In this method, food is firstly serially diluted in appropriate diluent.
   • Then, measured volume of sample from diluted tube is placed in petriplate.
   • Melted agar at 44-45^oC is mixed with it.
   • After homogenous mixing of sample with melted agar, it is kept for solidification.
   • Then the petri plates are incubated at appropriate time and temperature.
   • Plate containing colonies between 30-300 is selected and number of colonies are counted.
   • Now, number of organisms in original food sample is calculated by the following formula:
     • Colony forming units (CFU/ml) = (Number of colonies/volume of sample ) x dilution factor
   • Since psychrophiles cannot survive temperature of melted agar, this technique is not suitable for them.
   • In this method, both surface and subsurface colonies are developed.
   • Subsurface colonies are difficult to be isolated.
   • Spread plate technique:
   • In this method, appropriately diluted sample is placed on the surface of solidified agar.
   • Then the drop of sample is spread over agar surface using bent glass rod.
   • Plate is incubated for sufficient time and temperature, then number of colonies are counted.
   • Calculation of number or organisms is done similarly as in pour plate technique.
   • This method is suitable for psychrophile also and only surface colonies are developed.
2. Streak plate technique:
   1. In this technique, a transfer loop is used to spread the specific volume of specimen over a surface of solidified agar.
   2. The transfer is done by calibrated loop of specific volume.
   3. Sometimes, selective and differential media can be used to select growth of specific organism.

2. Membrane filter technique:

• This technique is particularly important to analyze microorganisms in liquid food in which microbial content is too low.
• In this methods, major measured volume of liquid filtered through membrane filter of specific porosity.
• Then filter pad is removed and placed on the surface of agar plate and then incubated.
• Microorganisms grow on surface of membrane filter to form colony.
• Then total number of organisms in original sample is calculated.
• Nutrient or selective agar media can be used for microbial growth.

3. Most probable number (MPB) method:

• It is statistical technique to determine number of organisms in sample.
• It gives most probable number but not the actual number.
• Turbidity, gas production and acid production are observed to determine microorganisms.
• This method is based on 3 steps:
  • Presumptive test
  • Confirmed or confirmatory test
  • Completed test

4. Direct microscopic count (DMC):

• In this method, there is no difference between dead and viable cells.
• Total cells are counted.
• In this method, the result is obtained faster than most other methods because incubation period is not required.
• Procedure of Direct Microscopic Count is given in above chart.
• In case of liquid food, direct smear is made.
• For solid food, it must be first divided up to 10^-1.
• Fatty foods must be defatted in xylene or acetone for preparation of smear.
• The xylene/acetone is then removed by dipping it in ethanol.
• In this method, number of microorganisms in microscopic field are counted directly.
• This technique is widely used to assess the quality of raw milk and other dairy product.
• Breed count method:
  • It is an example of Direct Microscopic Count.
  • This method was initially developed by R.S Breed.
  • In this method, 0.01ml sample is spread over 1cm square area on slide.
  • If sample is fatty, it should be defatted with xylene or acetone.
  • Excess xylene or acetone is then removed by dipping it into ethanol.
  • Then slide is dried, fixed and stained with appropriate dye and observed under microscope.
  • Average number of microorganisms per field is counted.
  • Then area of microscopic field is determined from which number of microorganisms in original sample is calculated.
  • It is not practical to count entire field.
  • So, only few microscopic fields are counted to determine average number of organisms in sample.

• Advantages of DMC:
  • It is simple and rapid technique.
  • Morphology as well as gram reaction of microorganism spore production etc. can be observed in microscope.
  • Very small amount of sample is needed.
  • The prepared slides can be stored and maintained as permanent record.
• Disadvantages of DMC:
  • DMC cannot distinguish viable and non-viable cells.
  • Food particles are not always distinguishable from microorganism’s cell.
  • Some microorganisms do not take stain and may not be counted.
  • It is very difficult to count microorganisms when the initial load is very high.

5. Electronic counter:

• In this method, standard volume of suitable dilution of suspension is placed in electronic counter.
• The machine has small aperture through which microorganisms can pass.
• The passage of microorganisms through aperture causes alteration in electric resistance across it which is recorded as on impulses.
• These impulses are counted by suitable circuit in the counter.
• Number of impulses from fixed volume of sample is used to calculate number of organisms in original food sample.

6. Dye reduction test:

• Two dyes are commonly employed in dye reduction test to estimate viable number of organisms.
• Methylene blue reduction test:
  • Methylene blue reduction test is commonly used to determine number of viable organisms in raw milk.
  • In this method, methylene blue is mixed with raw milk or incubated.
  • Microorganisms present in milk reduce methylene blue to form leucomethylene blue so that milk becomes blue to colorless.
  • The time of decolorization of milk is indicative of number of viable organisms.
  • If number of organisms are higher it is decolorized in shorter time and vice-versa.
  • In this method microbial quality of milk assessed by reduction time.
• Resazurin reduction test (rapid test):
  • It is an example of rapid dye reduction test use to determine number of viable organism in food such as raw milk.
  • In this test, resazurin dye is mixed with raw milk. Microorganism present in milk reduce resazurin such that its color changed from stale blue to pink or colorless.
  • If the number of microorganism is higher, dye is reduced in shorter time and vieversa
  • Therefore, microbial load of milk can be predicted by reduction time of resazurin. In this method, result is obtained within 10 mins.
• Advantages of dye reduction test:
  • It is simple, easier, and inexpensive test.
  • Only viable cells actively reduce the dye. So, that number of viable organism can be predicted.
• Disadvantages of dye reduction test:
  • Not all microorganisms reduce the dye equally.
  • They are not applicable for food that contain reducing substances such as reducing enzymes unless special steps are employed.

Detection of Microorganisms in foods: methods and techniques

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