SOP: Preparation of Streak Plates for Bacterial Cultures
GIFSQ — Global Institute for Food Safety & Quality
Principle
A mixed culture cannot be studied directly: the organisms must be separated into discrete colonies, each grown from one cell or clump. Streaking spreads a small inoculum across a solid medium so thinly that individual cells are deposited apart; each pass across a new area thins the inoculum further, leaving isolated colonies that can be picked for pure cultures.
Apparatus required
- Platinum loop — No. 23 S.W.G. platinum wire, 2.5 in. long, one end fused into a glass rod or fixed to a suitable holder, the free end bent into a closed loop; the loop is bent so the wire makes an angle of 60° with the perpendicular (nichrome wire may be used instead)
- Bent glass rod, 3 mm in diameter, bent at right angles (for the rotating plate method)
- Bunsen burner or spirit lamp (for sterilizing the loop)
- Ethyl alcohol (for sterilizing the bent glass rod)
- Petri dishes containing solidified sterile agar medium (see the companion SOP on preparation of bacteriological media)
- Glass marking pencil or labels
Procedure — Simple streak
Sterilize the loop in a flame until the whole length is red hot
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Dip the loop into the broth culture and take up a small quantity of culture (the inoculum)
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Touch the surface of the plate lightly with the loop, holding the wire so the whole loop lies in contact with the medium, and streak the inoculum across the plate
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Label the plate (nature of the material, date) and incubate
Procedure — Cross streak
With a previously sterilized bent wire loop, transfer one loopful of the inoculum near the rim of the plate and make parallel streaks over a segment (area A)
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Flame the loop, let it cool, then make parallel streaks from area A over area B, touching the path made by the loop in area A; take care that the streaks do not overlap
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Flame the loop again, let it cool, and streak through area C in the same way, and so on — each pass across a new area thins the inoculum further
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Label and incubate
Note: two loops may be used instead of one — while one is cooling, the other is in use.
Procedure — Rotating plate
Sterilize the bent glass rod by dipping it in alcohol and flaming it; repeat twice
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Transfer a loopful of the inoculum onto the surface of the plate
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Sterilize the rod again, let it cool, and remove the cover of the dish
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Rotating the dish with one hand, move the bent short limb of the rod over the surface of the medium in a to-and-fro motion with the other hand
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Replace the cover; without flaming the rod, streak a second plate in the same manner
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Label and incubate
Note: with any of the above methods, the resulting culture should show a number of discrete colonies.
Procedure — Incubation
Incubate the inoculated cultures, tubes and plates at the specified temperature (25°, 37°, 40° or 55°C) for the specified length of time
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Incubate agar plates in the inverted position — with the lid of the plate underneath; the inverted plates may be set on a clean sheet of blotting paper in the incubator
Precautions
- Flame the loop until red hot and let it cool before touching the culture; a hot loop kills the inoculum.
- In the cross streak, do not let the streaks overlap — overlap defeats the dilution.
- Mark every tube and plate with the nature of the inoculated material and the date, using a glass marking pencil or a pasted label.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.