How to detect microorganisms in food ?
- 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 7oC, 7 days
- Mesophilesà 35oC, 24-48hrs
- Thermophilesà 55oC, 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:
- 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 44-45oC 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
- Colony forming units (CFU/ml) = (Number of colonies/
- 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.
- Streak plate technique:
- In this technique, a transfer loop is used to spread the specific volume of specimen over a surface of solidified agar.
- The transfer is done by calibrated loop of specific volume.
- 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.
| Average number of microorganisms | Number of fields to be counted |
| 0-3 | 64 |
| 4-6 | 32 |
| 7-12 | 16 |
| 13-25 | 8 |
| 26-50 | 4 |
| 51-100 | 2 |
| >100 | 1 |
- 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.