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The plate count method relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown.
In the Plate Count Method, the sample of drug product to be tested and Soybean-Casein Digest Broth is poured into a Petri dish. [4] The Petri dish is then incubated. The most probable number method (MPN) can also be performed for products considered to have a low bioburden [ clarification needed ] .
The pour plate technique is the typical technique used to prepare plate count agars. Here, the inoculum is added to the molten agar before pouring the plate. The molten agar is cooled to about 45 degrees Celsius and is poured using a sterile method into a petri dish containing a specific diluted sample.
When the quantity of bacteria is unknown, dilutions should be made to at least 10 −8. Three plates are needed for each dilution series, for statistical reasons an average of at least 3 counts are needed. The surface of the plates need to be sufficiently dry to allow a 20μl drop to be absorbed in 15–20 minutes.
Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius, or the human body temperature, for cultures from humans or animals, or lower for environmental cultures). After the desired ...
The spread plate method wherein the sample (in a small volume) is spread across the surface of a nutrient agar plate and allowed to dry before incubation for counting. [11] The membrane filter method wherein the sample is filtered through a membrane filter, then the filter placed on the surface of a nutrient agar plate.
The 96-well plate was then incubated two hours in the plate reader, set to shake and take readings every five minutes. During this incubation time, the seed culture was kept on ice. For a calibration curve, 1 mL of seed culture was added to 1.5 mL of PMH after the two hour incubation to generate a suspension of 10 8 CFUv/mL.
To enumerate the growth, bacteria can be suspended in molten agar before it becomes solid, and then poured into petri dishes, the so-called 'pour plate method' which is used in environmental microbiology and food microbiology (e.g. dairy testing) to establish the so-called 'aerobic plate count'.