FM Assignment

FACULTY OF ENGINEERING SCIENCES AND TECHNOLOGY
Hamdard Institute of Engineering & Technology

Hamdard University
Hamdard University
Hamdard University
Hamdard University
Hamdard University
METHODS AND TECHNIQUES FOR THE

DETERMINATION OF MICROORGANISMS IN FOOD:
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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.
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 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:
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 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:
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 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:

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 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.
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 Plate containing colonies between 30-300 is selected and numbers 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
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 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.
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 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.
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 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 filters technique:

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 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.
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 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.
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 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.

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 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.
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 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.
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 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.
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 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
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7-12 16
13-25 8
26-50 4
51-100 2
>100 1
Wilson’s Chat
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 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.
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 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.
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5. 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.
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 In this method, methylene blue is mixed with raw milk or incubated.

 Microorganisms present in milk reduce methylene blue to form leuco
methylene 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 is higher it is decolorized in shorter time and vice-
versa.
 In this method microbial quality of milk assessed by reduction time.
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 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. Microorganisms present in
milk reduce resazurin such that its color changed from stale blue to pink or
colorless.
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 If the number of microorganism is higher, dye is reduced in shorter time and

vice versa
 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.
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 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 contains reducing substances such
as reducing enzymes unless special steps are employed.
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Hamdard University

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FACULTY OF ENGINEERING SCIENCES AND TECHNOLOGY

Hamdard Institute of Engineering & Technology

METHODS AND TECHNIQUES FOR THE

Various techniques can be used for determination of microorganisms in food.

1. Standard plate count (SPC):

 The number of organisms in original food is determined by counting the

 Psychrophilesà 7oC, 7 days

 If the suspension is not homogenous and contain aggregate of cells,

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.

 Plate containing colonies between 30-300 is selected and numbers of

volume of sample ) x dilution

 Since psychrophiles cannot survive temperature of melted agar, this

 Spread plate technique:

 Streak plate technique:

2. Membrane filters technique:

 This technique is particularly important to analyze microorganisms in liquid

 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.

 This method is based on 3 steps:

4. Direct microscopic count (DMC):

 In this method, there is no difference between dead and viable cells.

 Total cells are counted.

 In this method, number of microorganisms in microscopic field are counted

 Breed count method:

 It is not practical to count entire field.

5. Dye reduction test:

 Methylene blue reduction test:

 In this method, methylene blue is mixed with raw milk or incubated.

 Resazurin reduction test (rapid test):

 If the number of microorganism is higher, dye is reduced in shorter time and

 Advantages of dye reduction test:

 Disadvantages of dye reduction test:

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