Sterile graduated pipettes

Sterile graduated pipettes of 10, 5, 2 and 1 ml, sterile capped 7.5 x 1.3 cm tubes/ small screw-capped bottles, Pasteur pipettes, an overnight broth culture of the test and control organisms (same as for disc diffusion tests), the required antibiotic in powder form, the required solvent for the antibiotic, sterile distilled water - 500 ml and suitable nutrient broth medium. A suitable rack to hold 22 tubes in two rows, i.e., 11 tubes in each row. 

Prepare flow hood (see Sterile Technique above) and supply one 15 ml conical vial (labeled CHO cells), approximately five 10 ml graduated pipettes, 1000 and 200 pi pipetmen and a waste beaker. 

This type of transfer is slightly more complicated than the transfer of a colony from solid to liquid media hut is as important as these techniques are used to enumerate microorganisms. To perform these procedures, graduated pipettes are used to transfer specihc volumes to dilution blanks or to pour/spread plates (Section 14.3). Where reusable glass pipettes are used, these should be washed and placed into a metal canister for autoclaving and subsequent drying in an oven prior to use. Alternatively, sterilized disposable pipettes can be used for transfers (Section 18.7). 

Pipettes and Petri Dishes. It is preferable to use presterilized disposable pipettes and petri dishes. They are not only more economical to use, but the method of sterilizing and sealing is generally better than that accomplished in most laboratories. Pipettes should be 1 ml serological type graduated in 0.1 ml. If concentrates are to be sampled, the large bore pipette for viscous liquids should be specified. Petri dishes should be standard 100 x 15 mm. 

When preparing a dilution series, it is important to maintain aseptic conditions (Section 13.8). For example, dilution blanks must be flamed when opened to transfer sample (Fig. 13.2). Sterile 1.0 mL pipettes (0.1 mL graduations) or variable volume pipettors equipped with sterile plastic tips must be used to aseptically transfer liquid between dilution blanks. Sterile pipettes or pipette tips should be used only once, and then a new pipette or tip is used. Pipettors can be sterilized by wiping down their surfaces with 70% v/v ethanol and should be calibrated yearly. 

---