Tips for Proper Pipette handling and Maintenance. D. Muruganand, Ph.D ILQA - Bangalore, Feb 2014

Transcription

1 Tips for Proper Pipette handling and Maintenance D. Muruganand, Ph.D ILQA - Bangalore, Feb 2014

2 Liquid handling tools Manual pipettes Electronic pipette and dispenser Automated systems

3 Principles of Pipetting Air-cushion Positive displacement

4 Air-cushion pipette Piston Air cushion Liquid No contact between piston and sample

5 Air-cushion pipetting principle Plunger pressed Plunger Released Piston moves down Piston moves up Air cushion Partial vacuum

6 Manual air-cushion pipettes Research Research plus Reference Reference 2

7 Electronic air-cushion pipettes Research pro Xplorer

8 Manual versus electronic pipette Imprecision Manual Electronic Constant speed (electronic pipette) Spring loaded tip cone High reproducibility

9 Air-cushion pipetting techniques Forward pipetting Reverse pipetting

10 Forward pipetting Applications Recommended for standard aqueous solutions: > Water > Buffer > Diluted saline solutions > Diluted acids and alkalis

11 Forward pipetting technique 1 st stop 2 nd stop Press to 1 st stop. Immerse tip a few mm into liquid. Release button slowly. Tip fills up Press to 2 nd stop To dispense

12 Reverse pipetting Applications Recommended for: > High vapor pressure liquids > Viscous liquids

13 Reverse pipetting technique 2 nd stop 1 st stop Press to 2 nd stop. Immerse tip a few mm into liquid. Release button slowly. Tip fills up. Press to 1 st stop to dispense. Some liquid will remain in the tip.

14 Pipetting high vapor pressure liquids High vapor pressure liquid Liquid evaporates Pressure builds up Liquid drips For even higher accuracy and precision, use positive displacement system. Pre-wet tip Reverse pipetting

15 Type of pipettes Air-cushion Positive displacement

16 Positive displacement principle Absence of air-cushion Higher accuracy and precision Tip with integrated piston Air cushion Liquid

17 Pipetting high vapor pressure liquids Air-cushion Positive displacement Liquid evaporates pressure builds up Absence of air-cushion Liquid drips No liquid dripping

18 Pipetting viscous liquids Piston wipes down along the cylinder wall during dispensing minimal residual liquid in the tip

19 Pipetting viscous liquids Effect of viscous liquid on pipetting accuracy and precision Volume (µl) 52 Pipetting 50 µl of 85% glycerol Pipettieren von 87 % Glycerol Volumen [µl] Vorwärts Forward pipetting Pipettieren Reverse Pipettieren pipetting Multipette plus plus Anzahl der Pipetting Pipettierungen (No.) [Nr.]

20 Multiple Dispensing Advantage > Highly reproducible > Larger volume range > Easy to handle

21 Factors affecting pipetting performance Pipette Pipette tip Liquid Accuracy Cleaning and maintenance System (air-cushion vs positive displacement, manual vs electronic) Quality (material properties, molding) Correct fit (intact sealing) Size (air-cushion volume) Aqueous Viscosity Vapor pressure Density

22 Factors affecting pipetting performance Environment Temperature Humidity Air pressure Operator Experience, skill Pipetting techniques Strain (RSI)

23 Effect of hand temperature Holding the pipette continually; warming effect Influence of hand temperature on pipetted volume with a 100 µl pipette 1) Gloves were worn. 2) Pipette was taken out of hand during weighing.

24 Effect of liquid temperature Inaccuracy at different liquid temperatures; calibration at 22 C Inaccuracy (%) Liquid temperature ( C)

25 Effect of holding angle & immersion depth Holding angle Immersion depth 1 cm 3 cm 3-4 cm Inaccuracy % % 1-1.2%

26 Optimum immersion depth Optimum immersion depth for different pipetting volumes Volume (µl) Optimum immersion depth (mm)

27 Contamination scenarios 1. Aqueous solutions, buffers, acids and alkalis 2. Organic solvents 3. Potentially infectious liquids and cell cultures 4. Nucleic acids 5. Proteins 6. Radioactive substances 27

28 1. Aqueous solutions, buffers, acids and alkalis Disassemble pipette lower parts Rinse contaminated lower parts with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston (if necessary) Assemble pipette 28

29 2. Organic solvents Disassemble lower parts Allow liquid to evaporate Alternatively: Rinse contaminated lower parts with mild detergent Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston Assemble pipette Do not use concentrated detergent! Do not spray detergent into the pipette nose cone! 29

30 3. Potentially infectious liquids and cell cultures > Alcoholic disinfectants > Ultra-violet (UV) irradiation > Autoclaving 30

31 Alcoholic disinfectants Dampen clean cloth / lint-free tissue / soft sponge with 70% ethanol / 60% isopropanol Wipe contaminated parts Allow to dry (max. 60 C or air dry) Lubricate piston (if necessary) Assemble pipette Do not spray alcohol into the pipette nose cone! 31

32 Ultra-violet (UV) irradiation > Suitable as a general surface microbial decontamination. > Eppendorf pipettes are stable against UV light. > Accuracy and precision are NOT affected. > Research and Reference slight change in the color of the pipette plastic surface. 32

33 Autoclaving Allow to dry & cool to room temperature before use If necessary, disassemble lower parts and remove inactivated contaminant using a normal laboratory disinfectant solution Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston (if necessary) Assemble pipette Autoclaving condition: C, 1 bar, 20 min 33

34 4. Nucleic acids > Sodium hypochlorite (5-10%) > Glycine/HCl buffer (ph 2.0) Autoclaving does not remove traces of nucleic acids! 34

35 Sodium hypochlorite 5-10% Use 5-10% fresh solution Soak contaminated lower parts for min Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston and Assemble pipette 35

36 1x Glycine/HCl buffer (ph 2) Boil contaminated lower parts for min Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston Assemble pipette 36

37 5. Proteins Rinse contaminated lower parts with mild lab detergent Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston Assemble pipette Do not use alcohol Precipitates proteins! 37

38 6. Radioactive substances Rinse contaminated lower parts with radioactive cleaning solutions Rinse well with dh 2 O Allow to dry (max. 60 C or air dry) Lubricate piston Assemble pipette 38

39 Leak test Set pipette to the nominal volume Attach an original Eppendorf tip and pre-wet the tip Aspirate distilled water using forward pipetting Hold the pipette vertically for approx. 30 sec Watch for any droplet formation at the end of the tip

40 Inaccuracy and imprecision Accurate Imprecise Inaccurate Precise Accurate Precise

41 Inaccuracy and imprecision Inaccurate and imprecise Desired volume = 0.5 ml Inaccurate but precise Accurate and precise

42 Frequency of pipette calibration ISO : the pipettes shall be tested at regular intervals, for example, every 3 months but at least once a year. Factors determining the frequency of pipette calibration: 1. Frequency of pipette usage 2. Number of users 3. Aggressiveness of liquids dispensed 4. The acceptable error limits

43 Thank you for your attention