Operation, Cleaning & Calibration of Micropipette 2023

Operation, Cleaning & Calibration of Micropipette 2023:-

A pipette (also spelt pipet) is a laboratory tool used to transport a measured volume of liquid, typically as a media dispenser, in chemistry, biology, and medicine. Pipettes are available in a variety of designs for a variety of applications, with varying degrees of accuracy and precision, ranging from simple single-piece glass pipettes to more complex adjustable or electronic pipettes. Many pipettes work by drawing up and dispensing liquid by creating a partial vacuum above the liquid-holding chamber and selectively releasing this vacuum. The precision of measurements varies greatly depending on the instrument.

OBJECTIVE

• • To lay down the procedure for operation of micropipette.

SCOPE

• • This procedure shall be applicable to micropipette used at microbiology Department.

RESPONSIBILITY

• • Microbiology Personnel shall be responsible for the operation and cleaning of micropipette as per established procedure, maintaining its documents, recording observations and reporting the results.
  • Officer Microbiology or above who is trained or well versed with the procedure shall be responsible for ensuring that the same is carried out correctly and checking of documents for completeness of content.
  • Head – Microbiology/ Designee shall be responsible for the compliance of the SOP.

 ACCOUNTABILITY

• • Head – Microbiology/ Designee shall be accountable for the SOP.

PROCEDURE

Operation:

• • • Set the required volume in volume indicator with the help of thumb wheel or push button.
    • While setting the volume, slowly reach the required setting; make sure not to overshoot the mark.
    • Attach tip to tip holder taking care to avoid contamination. Hold pipette comfortably in one hand with thumb resting on plunger (figure A).
    • Press the plunger down smoothly to the first stop position. At this stage the pipette tip must not be immersed in the liquid (this corresponds to the set volume of liquid) (figure B).
    • Wait hold the pipette vertically and immerse the tip in liquid (2-3 mm).
    • Slowly & smoothly release the push button (to top position) to aspirate the set volume of liquid (figure C).

• • • Ensure that the plunger extends fully to its upper stop position and wait one or two seconds before withdrawing the tip of the pipette from the liquid (figure D).
    • Touch the pipette against the wall of receiving vessel at an angle of about 30° to 45°. Be careful to not immerse the tip in any liquid already in the receiving vessel (figure E).
    • Press the push button slowly and smoothly to the ‘first stop’ (figure F).
    • Wait for at least a second; then press the push button to the ‘second stop’ to expel any residual liquid from the tip (figure G).
    • Keep the push button pressed fully down and (while removing the pipette) draw the tip along the inside surface of the vessel.
    • Release the push button, smoothly (figure H).

Flow Diagram :How to use 

• Reverse pipetting:

  • • A variation of the conventional method, termed reverse pipetting, is often useful when dealing with liquids that have a high vapor pressure or those, which are very viscous.
    • Press the plunger down fully; past the first stop position to the second stop position. At this stage the pipette tip must not be immersed in the liquid (figure I).
    • Immerse the tip of the pipette in the liquid to the correct depth. Keep the pipette in a vertical position and release the plunger in a smooth and uniform manner to draw up the liquid (figure J).

• • • Ensure that the plunger extends fully to its upper stop position. Check the tip to make sure no drops are forming at the orifice (figure K).
    • Touch tip against wall of receiving vessel and expel the contents of the pipette by pressing the plunger down steadily and evenly, but only to the first stop position (figure L).
    • Touch pipette against wall of delivery vessel at correct angle. Be careful to not immerse tip in any liquid already in receiving vessel (figure M).
    • Note that some liquid will be left in the tip after dispensing. This should be returned to the original vessel or discarded as per the requirement (figure N).

• Precautions:

  • • • Clean the pipette carefully.
      • Do not autoclave the pipette.
      • Do not use hard brush for cleaning.
      • Do not reuse the tip.
      • Before use, always check that pipette is clean and calibrated.
      • Press the push button slowly and smoothly to the first stop only, while aspirating the solution.
      • To avoid the parallax errors make sure that the volume indicator & volume marking is in your direct line of vision.
      • Liquid should not touch the upper part of the pipette.
      • Never lay the pipette down when there is liquid in the tip.
      • Never turn the pipette upside down when there is liquid in the tip.
      • Do not pipette liquid having temperature above 70 ºC or below 4 ºC.
      • Always press & release the push button slowly & smoothly.
      • Each new tip should be pre rinsed with the liquid to be pipetted.

• Calibration:

  • Calibration is done by gravimetric testing. Weight the amount of pure water delivered in a single operation of the pipette, and divides the mass obtained by the density of the water thus giving its volume.
  • Variable volume pipettes should be tested at three or more points over their designated range; usually at the maximum volume, at 50 % of the maximum volume and at the lower limit of their range.
  • The room temperature should be stable to within ± 0.5 ºC.
  • Check the temperature of water with calibrated thermometer for the calculation of accurate density.
  • Calibrated balance should be used for weighing appropriate for the pipette volume.
  • Keep a dry, small glass container on the balance & tare its weight.
  • Take the pipette to be calibrated, set the required volume taking all the precautions.
  • Fit the tip in the pipette. Rinse the tip at least 4-5 times with the purified water.
  • Every time discard the water used for rinsing the tip.
  • Again aspirate the set volume & dispense in the glass container kept on the balance.
  • Note down the weight. Again tare the balance to “zero” & repeat above steps again.
  • For each set volume repeat the procedure 10 times.
  • Calculate the mean value of the ten reading for the each selected volume using following formula.
  • Mean value = ∑XN
  • ∑ X = X₁ + X₂…………..X₁₀Xi = result of the weighings.
  • N = number of weighings
  • • Calculate the mean volume, accuracy and coefficient of variation using the following formula.
• Mean volume = Mean value/ density of water
  • Accuracy (A%) = Mean volume – V0 X 100                                                   V₀                                                       
• V₀   = nominal volume

• Acceptance Criteria = Accuracy value must be ±1%.

• Acceptance Criteria:

  Coefficient of variation (CV%) value must be less than 1%.

• • • Record the results in the Annexure-1 “Report for Micropipette calibration”.
    • Set the pipette on the other volume for calibration as per the Table-I & repeat the procedure.
    • If any value is out of limit then recheck all the parameters & recalibrate the pipette as mentioned above.
    •  Calibration Frequency: Quaterly

• • • To use the table below, run down the left column for whole degrees then move across for tenths of a degree.
    • Density of Water (g/cm³) at Temperatures from 0 °C (liquid state) to 30.9 °C by 0.1 °C increase.

 

°C	0.0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9
0	0.999841	0.999847	0.999854	0.999860	0.999866	0.999872	0.999878	0.999884	0.999889	0.999895
1	0.999900	0.999905	0.999909	0.999914	0.999918	0.999923	0.999927	0.999930	0.999934	0.999938
2	0.999941	0.999944	0.999947	0.999950	0.999953	0.999955	0.999958	0.999960	0.999962	0.999964
3	0.999965	0.999967	0.999968	0.999969	0.999970	0.999971	0.999972	0.999972	0.999973	0.999973
4	0.999973	0.999973	0.999973	0.999972	0.999972	0.999972	0.999970	0.999969	0.999968	0.999966
5	0.999965	0.999963	0.999961	0.999959	0.999957	0.999955	0.999952	0.999950	0.999947	0.999944
6	0.999941	0.999938	0.999935	0.999931	0.999927	0.999924	0.999920	0.999916	0.999911	0.999907
7	0.999902	0.999898	0.999893	0.999888	0.999883	0.999877	0.999872	0.999866	0.999861	0.999855
8	0.999849	0.999843	0.999837	0.999830	0.999824	0.999817	0.999810	0.999803	0.999796	0.999789
9	0.999781	0.999774	0.999766	0.999758	0.999751	0.999742	0.999734	0.999726	0.999717	0.999709
10	0.999700	0.999691	0.999682	0.999673	0.999664	0.999654	0.999645	0.999635	0.999625	0.999615
11	0.999605	0.999595	0.999585	0.999574	0.999564	0.999553	0.999542	0.999531	0.999520	0.999509
12	0.999498	0.999486	0.999475	0.999463	0.999451	0.999439	0.999427	0.999415	0.999402	0.999390
13	0.999377	0.999364	0.999352	0.999339	0.999326	0.999312	0.999299	0.999285	0.999272	0.999258
14	0.999244	0.999230	0.999216	0.999202	0.999188	0.999173	0.999159	0.999144	0.999129	0.999114
15	0.999099	0.999084	0.999069	0.999054	0.999061	0.999023	0.999007	0.998991	0.998975	0.998959
16	0.998943	0.998926	0.998910	0.998893	0.998877	0.998860	0.998843	0.998826	0.998809	0.998792
17	0.998774	0.998757	0.998739	0.998722	0.998704	0.998686	0.998668	0.998650	0.998632	0.998613
18	0.998595	0.998576	0.998558	0.998539	0.998520	0.998501	0.998482	0.998463	0.998444	0.998424
19	0.998405	0.998385	0.998365	0.998345	0.998325	0.998305	0.998285	0.998265	0.998244	0.998224
20	0.998203	0.998183	0.998162	0.998141	0.998120	0.998099	0.998078	0.998056	0.998035	0.998013
21	0.997992	0.997970	0.997948	0.997926	0.997904	0.997882	0.997860	0.997837	0.997815	0.997792
22	0.997770	0.997747	0.997724	0.997701	0.997678	0.997655	0.997632	0.997608	0.997585	0.997561
23	0.997538	0.997514	0.997490	0.997466	0.997442	0.997418	0.997394	0.997369	0.997345	0.997320
24	0.997296	0.997271	0.997246	0.997221	0.997196	0.997171	0.997146	0.997120	0.997095	0.997069
25	0.997044	0.997018	0.996992	0.996967	0.996941	0.996914	0.996888	0.996862	0.996836	0.996809
26	0.996783	0.996756	0.996729	0.996703	0.996676	0.996649	0.996621	0.996594	0.996567	0.996540
27	0.996512	0.996485	0.996457	0.996429	0.996401	0.996373	0.996345	0.996317	0.996289	0.996261
28	0.996232	0.996204	0.996175	0.996147	0.996118	0.996089	0.996060	0.996031	0.996002	0.995973
29	0.995944	0.995914	0.995885	0.995855	0.995826	0.995796	0.995766	0.995736	0.995706	0.995676
30	0.995646	0.995616	0.995586	0.995555	0.995525	0.995494	0.995464	0.995433	0.995402	0.995371

 

• • Cleaning:

    • Clean the external surface of the pipette with soft cloth or lint free tissue impregnated with 70% IPA.
    • Wipe the pipette with clean dry cloth.

  • Frequency:

    As & when required.

  • Maintenance:

    • If pipette fails in calibration, discontinue its use & call the service engineer.
    • Any sign of leakage from the tip fitted & the pipette filled with liquid indicates leaking seal, O-rings, or an ill-fitting or inappropriate tip, discontinue its use & call the service manager.

6. ABBREVIATIONS

• • • MIP :           Micropipette
    • MB :           Microbiology
    • µL :           Microliter
    • mL :           Milliliter
    • mm :           Millimeter
    • IPA :           Iso Propyl Alcohol

7. ANNEXURES:

Nil

8. DISTRIBUTION:

 MB

9. REFERENCES:

NA

10.0 REVISION HISTORY:

New

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