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774 vues5 pagesPour Plate Method
Pour plate method to enumerate microbe in a sample
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0% ont trouvé ce document utile (0 vote)
774 vues5 pagesPour Plate Method
Pour plate method to enumerate microbe in a sample
Transféré par
Noviana HussenCopyright
© © All Rights Reserved
Nous prenons très au sérieux les droits relatifs au contenu. Si vous pensez qu’il s’agit de votre contenu, signalez une atteinte au droit d’auteur ici.
Formats disponibles
Téléchargez aux formats PDF ou lisez en ligne sur Scribd
23/2020 Pour plate Method: Principle, Procedure, Uses, and (Dis) Advantages - Learn Microbiology Online
Pour plate Method: Principle, Procedure,
Uses, and (Dis) Advantages
Calculation: Number of cotonies on plate x reciprocal of dilution of sample = number of bacteria/m
(For example, if 32 colonies are on a plate of "0000 dilution, then the count is $2 x 10,000 = 320,000 bacteria/m! in sample.)
Pour plate method is usually the method of choice for counting the number of colony-forming bacteria
present in a liquid specimen. In this method, fixed amount of inoculum (generally 1 mi) from a broth/sample
is placed in the center of sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15ml) is then
poured into the Petri dish containing the inoculum and mixed well. After the solidification of the agar, the
plate is inverted and incubated at 37°C for 24-48 hours.
Microorganisms will grow both on the surface and within the medium. Colonies that grow within the medium
CFU/mL= CFU * dilution factor * 1/aliquot
For accurate counts, the optimum count should be within the range of 30-300 colonies/plate. To insure a
countable plate a series of dilutions should be plated.
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Calculation: Number of colonies on plat «reciprocal of ition of sample = numberof bacteria
(For example. if 32 colonies are ona pate of asm dition, then the count ts 32 10,000 = 320,000 bactria/m! in sample.)
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The pour plate method of counting bacteria is more precise than the streak plate method, but, on the
average, it will give a lower count as heat sensitive microorganisms may die when they come contact with hot,
molten agar medium.
Uses:
The pour plate technique can be used to determine the number of microbes/ml in a specimen, It has the
advantage of not requiring previously prepared plates, and is often used to assay bacterial contamination of
food stuffs.
4, Sterile Petri dishes
5. Flame
6. Colony counter with magnifying glass
7. Sterile capped 16150 mm test tubes
8, Pipettes of various sizes (e.g. 01, 1.0 and 2.0 ml)
Procedure of Pour plate technique
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1, Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL. (Follow serial (x)
dilution technique) ~
2. Inoculate labeled empty petri dish with specified mL. (0.1 or 1.0 mL) of diluted specimen
Note: for the detail description regarding use of pipette, inoculation of sample, dilution technique etc follow the
reference 1.
Pouring the molten agar and incubat
1. Collect one bottle of sterile molten agar (containing 15 mL of melted Plate Count Agar or any other
standard culture media) from the water bath (45°C).
2. Hold the bottle in the right hand; remove
the cap with the little finger of the left
hand.
3. Flame the neck of the bottle,
4. Liftthe lid of the Petri dish slightly
with the left hand and pour the
sterile molten agar into the Petri dish
and replace the the lid,
5, Flame the neck of the bottle and replace
the cap. Pouring the molten agar medium
6. Gently rotate the dish to mix the culture
and the medium thoroughly and to ensure that the medium covers the plate evenly. Do not slip the
‘agar over the edge of the petri dish,
7. Allow the agar to completely gel without disturbing i, it will take approximately 10 minutes.
8, Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.
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Pour plat Method: Principle, Procedure, Uses, and (Dis) Advantages - Learn Microbiology Online
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‘Convriaht © 2007 Pearson Education, inc. publishing as Benjamin Cumminas.
‘Overview of Pour plate method and spread plate method
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Calculate CFU/mL using the formula: CFU/mL= CFU * dilution factor * 1/aliquot
(the volume of diluted specimen (aliquot) is either 0.1 or 1.0 mL)
Disadvantages of Pour plate method
1. Preparation for pour plate method is time consuming compared with streak plate/and or spread plate
technique.
2. Loss of viability of heat-sensitive organisms coming into contact with hot agar.
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