Pour Plate Method – and remelting Agar

The pour plate method is often used to count the number of organisms present in a liquid sample, generally Colony Forming Units / ml (CFU/ml). 1 ml of sample (neat or diluted) is pipetted into the middle of a sterile petri dish, and cooled, still molten agar is poured into the dish and mixed well. The media / sample is allowed to gel, and then inverted and incubated as required to allow organism growth, in and on the surface of the media, and a final organism count is obtained.

When a dilution series is tested, the final count is calculated after considering the sample dilution and sample volume added to the plate showing between 30 – 300 colonies following incubation. The pour plate method is considered more precise than the streak plate method. Colonies need to be counted carefully, as surface colonies will appear as on a streak plate, while those in the media are much smaller in size. An illuminated colony counter, or magnifying glass may be required. Heat sensitive organisms may be killed when the molten media is added, especially if the media is added at a high temperature.

Media can be made ready for use and held at < 50 degrees celsius (preferably 44 degrees celsius – 46 degrees celsius) in a water bath for no more than 3 – 4 hours. Prolonged time in a molten state can affect agar performance in different ways, such as reduce selectivity, reduce gel strength, degrade nutrients.

Media can also be made in advance and allowed to gel in bottles which can then be remelted before use. The agar should not be melted more than one time, using one of the following methods:

Autoclave or Water Bath
Steaming at around 100 oC. Loosen the cap on the agar bottle and place it into a water bath. Water temperature should remain at around 100°C. Leave it in the water bath until the agar is completely melted. While wearing heat-protective gloves, carefully remove the hot bottle and let it cool to < 50°C and hold for use as above before pouring.

Loosen the cap on the agar bottle before microwaving. Heat in one minute intervals on low power until all of the agar is melted. Between intervals, gently swirl the bottle to make sure the agar is melting evenly. While wearing heat-protective gloves, carefully remove the hot bottle and let it cool to <50°C and hold for use as above before pouring.

Caution - Microwave power output can vary significantly by model. If using the microwave, it is very important you use a low setting only, heat for 1 minute only and then gently swirl and repeat until all agar is melted. You need to avoid hot spots that microwaves can produce, as this will damage the media.

Through Microbiologics Inc, we can supply a range of Qualitative and Quantitative Control organisms to use when monitoring the quality and performance of your pour plate testing. See attached for the product range and alignment with ISO 11133.

This information is provided as a guide, and relevant standards and manufacturer’s instructions should be followed.

Microbiologics Food Saftey Organisms - July 2017
Pour Plate Method - Best Practices