SOP: Sterilization of Glassware for Bacteriological Examination

GIFSQ — Global Institute for Food Safety & Quality

Principle

Glassware must be completely sterile before it touches a culture; any surviving organism would contaminate the work. Heat destroys the organisms: dry heat in a hot air oven for dry-wrapped items, steam under pressure in an autoclave for bottles, and direct flaming for inoculating wires, whose small size makes them sterilizable in the flame in seconds.

Apparatus required

• Hot air oven
• Autoclave
• Copper or stainless steel cylinders with lids (for pipettes)
• Cotton pads (for lining the pipette cylinder and its cover)
• Kraft paper (for wrapping pipettes and Petri dishes)
• Cylindrical tinned copper boxes (alternative container for Petri dishes)
• Paper strips, 3 × 0.5 in. (for stoppered bottles)
• Absorbent cotton wool (for plugging tubes)
• Bunsen burner or spirit lamp (for flaming needles and loops)

Procedure — General rules for the hot air oven

Place only dry articles in the oven, and start with the oven cold

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Raise the temperature steadily to the required level and hold it for the full sterilization time

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Cool the oven before taking the articles out

Procedure — Pipettes

Thrust a cotton plug into the mouth of each pipette before sterilization

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Place the pipettes in copper or stainless steel cylinders with a lid, with a pad of cotton at the bottom of the container and on the inside of the cover to protect the tips from damage

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Sterilize in the hot air oven at 160°C for 1 hr, or at 140°C for 2 to 3 hr

Note: if suitable pipette containers are not available, wrap each pipette in kraft paper in a spiral manner and sterilize in the hot air oven; such wrapped pipettes stay sterile for a long time.

Procedure — Petri dishes

Wrap the dishes in kraft paper in sets of 3 to 5

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Sterilize in the hot air oven at 160°C for 1 hr

Note: the dishes may also be sterilized without any wrapping in cylindrical tinned copper boxes.

Procedure — Sampling and dilution bottles

For bottles with stoppers, insert a strip of paper 3 × 0.5 in. between the stopper and the neck of the bottle, so the stopper cannot jam and the glass cannot crack on cooling

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Keep at least half the strip projecting outside the bottle, so the paper can be pulled out without touching the rim when the stopper is removed

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Sterilize in the hot air oven or in the autoclave; in the autoclave, sterilize at 15 psig for 20 min

Procedure — Tubes

Plug the tubes with absorbent cotton wool, inserting the plug to a depth of 1.25–1.5 in. into the tubes

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Leave enough of the plug projecting out of the mouth to protect the rim from dust

Note: the plug is right if the tube can hang from the cotton tuft and support its own weight.

Procedure — Inoculating needles and loops

Pass the article through a Bunsen flame or the flame of a spirit lamp until sterilized

Precautions

• Never put wet articles into a hot air oven, and never remove articles from a hot oven — always cool first.
• When using the hot air oven, follow the time-temperature pairs exactly: 160°C for 1 hr or 140°C for 2 to 3 hr for pipettes, 160°C for 1 hr for Petri dishes.
• Keep wrapped pipettes and dishes wrapped until the moment of use so they stay sterile.

References

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