SOP: Inoculation of Bacteria by the Pour Plate Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

In the pour plate method the sample is diluted in a series of steps, a measured volume of each dilution is mixed into molten agar, and the mixture is poured into a Petri dish. On setting, the organisms are distributed through and on the agar, growing into colonies that can be counted or examined. Replicate plates of each dilution are prepared so the result does not rest on a single plate.

Apparatus required

• Water bath (45–50°C)
• Sterile pipettes
• Petri dishes
• Test tubes containing 15 ml of suitable agar medium
• Glass marking pencil

Reagents

a. Quarter-strength Ringer solution — the diluent in which the material is taken up and serially diluted. Flag: the book gives the preparation of this solution in its “Saline and Ringer Solution” section; that preparation is not given in this chapter.

b. Suitable agar medium, tubed in 15-ml portions and melted.

Procedure — Pour plate

Melt several tubes containing 15 ml of medium and hold them in a 45–50°C water bath to cool

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Take the material to be examined in quarter-strength Ringer solution and prepare 1/10, 1/100 and 1/1,000 dilutions in the same solution

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To 15 ml of medium at 45°C add 1 ml of a dilution and mix by rotating the tube between the palms of the hands

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Pour the mixture into a Petri dish and make replicate pour plates of each dilution for incubation at suitable temperatures

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Allow the medium to set, invert the plate, and incubate

Procedure — Inoculating a liquid medium from a solid culture

Pick the colony from the solid culture with a long straight wire (mounted on a holder) without a loop

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Hold the tube to be inoculated inclined almost to the horizontal position and deposit the inoculum on the wall of the tube just above the surface of the liquid at the lower end of the tube

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Return the tube to the vertical position — done properly, the inoculum will now be below the surface of the broth

Note: for adding large amounts of liquid inoculum, use a sterile pipette. Mark every tube and plate with the nature of the inoculated material and the date, using a glass marking pencil or a pasted label.

Precautions

• Keep the molten medium at 45°C when adding the inoculum, as the book specifies.
• The pour plate cannot be used for counting purposes: a little of the diluted inoculum can stay clinging to the test tube wall.
• Always prepare replicate pour plates of each dilution.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.