Spreading Technique for Plating Bacteria

Spreading Technique for Plating Bacteria

See also: Sterile Technique: Delivery of Liquids by Pipet, and Use of Displacement Pipets

70% EtOH in squirt bottle, wipettes
Wax pencil
Labeled samples in test tube rack
agar plates labeled on bottoms
displacement pipets and sterile tips
(or sterile 0.1, 0.2 or 1.0 mL pipets)
Turntable 95% EtOH in 250 mL beaker (1/2 full)
Spreader, made from bent glass rod
Flame (Bunsen burner or alcohol lamp)
Plastic bucket for used pipets
Incubator, set for correct temperature

1. Construct a table in your notebook with the following information for each plate: plate type, sample source, dilution (if appropriate) volume plated, and a column to enter colony counts. Label plate bottoms at edge in small print with wax pencil: date, initials, sample source, dilution factor (if any), volume plated.(IF you are using glass plates, clean thebottoms with 95% EtOH if necessary.)

2. Flame mouth of culture tube (hold lid in crook of little finger of pipetting hand. Do not place lid on desk.) If using glass pipets, pass through flame briefly.


3. With tip of sterile pipet just below the surface of the liquid, withdraw the desired volume of sample.

4. Flame lip of culture tube, replace cap, set in rack. Keep pipet horizontal so that fluid does not dribble out.

5. Deliver desired aliquot of sample to the surface of the plate(s) without gouging plate. Blow out to ensure total transfer to the plate.

6. Place contaminated tip into used pipet receptacle containing dilute Lysol or other antiseptic.

7. Shake off excess EtOH from spreader, pass quickly through flame to burn off the rest.


8. After flaming has stopped, touch spreader to inside of the top of petri dish to cool (if necessary), then, turning the turntable slowly with one hand, hold spreader like a rake in the other hand and rotate spreader on surface of agar in the opposite direction. Do not press hard enough to damage agar surface. Continue this action until the fluid has been absorbed. You can feel the spreader drag slightly, and reflected light will show the liquid to be absorbed.


9. Replace spreader into 95% EtOH, invert plates, place in an incubator set at the desired temperature for one to two days.


10. Count the colonies on the plates, record the numbers in the table in your notebook (set up in step #1.).

11. Calculate the number of colony forming units/mL (C.F.U./mL)or /100 mL in the original sample, enter in notebook. The calculation is performed thus:
C.F.U/mL original sample = C.F.U./plate x (1/mL aliquot plated) x dilution factor

For example
89 colonies on plate x 1/0.2 mL (aliquot) x 106 (dilution factor) = 4.45  x 108 CFU/mL

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