Written/Revised By: Bette Ford and Alberto Gregorio/ Revised by: Jennifer Taylor

Johns Hopkins Medical Institutions Flow Volume II

Department of Pathology Effective Date 6/1/2001
Core/Specialty Laboratories
Supersedes 4/10/2009
Digitally signed by MBOROWIT

MBOROWIT DN: cn=MBOROWIT

Date: 2010.12.22 18:31:09
-05'00'

Initial Review (Division Director): Date: (Laboratory Director, if applicable) Date

Annual Review (Director or designee): Date:

This document has been electronically signed Page 1 of 7

Pipettes & Pipette Calibration Check

(Non-Eppendorf Pipettes)
I. Purpose:
The routine operation, calibration and maintenance of pipettes are an integral part
of good total quality control in the Laboratory. Adjustable pipettes are checked
for calibration annually at a minimum. Each pipette is checked at different
designated volumes which bracket the normal operating volumes for that
particular pipette. The delivery volume must be within acceptable tolerance limits
as established by the lab.
Pipettes which are out of calibration must be adjusted and calibration re-
established at each of the designated volumes. Gravimetric method is a reliable
and easy method for testing pipette performance.

II. Cleaning and Calibration Guidelines for Automatic Pipettes (Rainin, Gilson,
MLA and Pipetman).
New pipets or pipets which have just been returned from renovation at the factory
do not need lubricated but must be calibrated prior to being used. All
maintenance and calibrations must be recorded. Each pipet has a serial no. etched
on the barrel of the pipet to be used for identification. If a pipet is cleaned, its
calibration must be checked before it is placed in use.

III. Frequency:
A. Before newly purchased pipettes are placed into service
B. Every six months at a minimum.
C. Anytime a pipette becomes dismantled and repaired
D. As a means of troubleshooting assay problems

IV. Equipment
1. Pipette
2. Pipette QC log sheet
3. Pipette tips for appropriate volumes and pipettes
4. Weigh boats
5. Water, reagent grade, room temperature
6. Centrigrade thermometer
7. A temperature vs. density diagram
8. Mettler-Toledo Analytical Balance
9. Calculator
10. Kim Wipes
11. Silicone Grease
Pipette Calibration Check________________________________________________2

12. Applicator stick and cotton

V. Procedure for Cleaning and Lubricating MLA Pipets:

1. Using diagram and instructions in the manufacturer’s literature
disassemble pipet. This can best be accomplished by placing towel
on work area, disassembling pipet and lining pieces in order they
are removed.

2. Clean outer pipette body and nose piece with an alcohol wipe and
kimwipe. Use a small piece of cotton on applicator stick to clean
out barrel. Be very careful not to lose the cotton plug.

3. Apply a very thin coat of silicone grease with your finger.

4. Reassemble and make sure it delivers properly.

5. Consult Lead Technologist if you encounter a problem.

3. Record the pipette serial number on the pipette QC log sheet for
the pipet that was cleaned.

Note: The manufacturer does not permit the Pipetman Pipet to be taken
apart and cleaned. We may only wipe it down with alcohol or send
it back to the Rainin Company for repairs.

VI. Procedure for Pipet Calibration (weighing pipet volumes)

1. Using the analytical balance and a suitable container, such as a

polystyrene micro weigh boat, tare the weigh boat and pipette tip.
Place the tip on the pipette. Avoid handling tips with your fingers.
Use Kimwipes or tweezers when possible.

2. Pour some room temperature reagent grade deionized water into a

container. Measure the temperature of the reagent grade deionized
water to the nearest hundredth of a degree Celsius. Record the
temperature on the pipette QC log sheet.

3. Determine the density of the deionized water based on the

temperature just measured using the temperature vs. density
diagram. See attached Table (Density of Water). Record the
density in the appropriate space on the pipette QC log sheet.

4. Pipette an aliquot of deionized water for the volume being tested.

Turn the pipette horizontally and remove the tip, being careful not
to lose any water. Weigh the tip and the water and record the
weight on the Pipette Calibration Log Sheet.

5. Perform all measurements in duplicate.

Pipette Calibration Check________________________________________________3

6. Calculate the average volume of delivery by dividing the average

weight of the deionized water (gm) delivered by the density of the
water at room temperature. Record the volume of delivery.

Weight of Water (gm)

Density of water at room temp.

7. Compute the percent tolerance for the designated volumes by

dividing the volume of delivery by the nominal (expected) volume,
Record this number.

Tolerance is calculated as:

Volume Delivered (ml) X 100
Nominal Pipet Volume (ml)

a. If the % tolerance for each of the three designated volumes

Is between 94%-106%, the pipette is considered to be in
calibration and a sticker showing the calibration date
should be affixed to the pipette.
Note: The Becton Dickinson Reversible Pipette should be
between 98-102% according to the manufacturer.

b. If the % tolerance is outside the range for any of the three

measurements, adjustments should be made to the pipette
and a new set of weighings should be made. Repeat steps 1-9.

c. If the above steps fail, take the pipette out of service until it
can be repaired and rechecked & may need returned to the
company for repairs

Plastic Disposable S/P Pipettes:

For Plastic Disposable S/P Pipettes used to prepare reagents or solutions where the
amount added needs to be precise and accurate, pipettes are checked semi-annually to
ensure they deliver +/- 3% of the stated volume.

Follow above steps 2 – 10. If the percent tolerance for each of the three designated
volumes is +/-3%, the disposable pipette is considered to be within acceptable calibration.
If the % tolerance is outside the range for any of the three measurements, a new set of
weighings should be made and repeat steps 2-9 above.
If the above steps fail, please contact a Lead or Supervisor for assistance to determine
appropriate corrective action.

VII. Operation of Automated Pipets (Pipetman and MLA)

A. Delivery Techniques
Pipette Calibration Check________________________________________________4

There are different techniques for filling and discharging automatic pipets
(i.e. MLA).

1. Pick up the exact amount then dispense entirely.

2. You may or may not rinse the tip with the diluent. It is important
that each person maintains a consistent technique throughout a
procedure.

Note: If the plunger on an automatic pipet does not move smoothly, the accuracy
and precision may suffer. Bring this to the attention of the Lead
Technologist. The pipet should be cleaned, lubricated and recalibrated. If
the pipet is not repairable, send for service.

B. Operation of Pipetman Adjustable Pipetor

1. Operating Procedure
a. Set the desired volume by holding the PIPETMAN body in
one hand and turning the volume adjustment knob until the
correct volume shows on the digital indicator. The friction
o-ring of the volume adjustment knob “cold flows” in order
to lock the volume securely. For best volume setting
precision, always approach the desired volume by dialing
downward (at least one third revolution) from a larger
setting volume.

b. Attach a new disposable tip to the shaft of the pipet. Press

on firmly with a slight twisting motion to insure a positive,
airtight seal.

c. Depress the plunger to the FIRST POSITIVE STOP. This

part of the stroke is the calibrated volume displayed on the
digital micrometer.

d. Holding the PIPETMAN vertically, immerse the disposable

tip into the sample liquid to a depth of:
2-4 mm RC-200 (P-1000)
3-6 mm C-5000 (P-5000)

e. Allow the pushbutton to return SLOWLY to the Up

position. Never permit it to snap up.

f. Wait 1 to 2 seconds to insure that the full volume of sample

is drawn into the tip.

g. Withdraw tip from sample liquid. Should any liquid

remain on outside of tip, wipe carefully with a lint-free
cloth taking care not to touch the tip opening.
Pipette Calibration Check________________________________________________5

h. To DISPENSE SAMPLE, place tip end against side wall of

the receiving vessel and depress plunger SLOWLY to the
FIRST STOP. Then depress plunger to SECOND STOP
(bottom of stroke), expelling any residual liquid in tip.

i. With plunger fully depressed, withdraw PIPETMAN from

vessel carefully with tip sliding along wall of vessel.

j. Allow plunger to return to TOP POSITION.

k. DISCARD TIP by depressing tip ejector button smartly. A

fresh tip should be fitted for each sample to prevent
carryover between samples, unless specified otherwise in
the specific procedure.

C. Pipetting Guidelines and Precautions for Pipetman and MLA Pipets

Consistency in all aspects of pipetting procedure will contribute
significantly to optimum reproducibility. Therefore, attention should be
given to the following:

1. SPEED and SMOOTHNESS during depression and release of

control push button.
2. PRESSURE on the control push button at first stop.
3. IMMERSION DEPTH
4. MINIMAL ANGLE from vertical axis

If an air bubble is noted within the tip during intake, dispense the sample
to original vessel, check tip immersion depth, and pipet more slowly. If an
air bubble appears a second time, discard the tip and use a new one to
repeat.

Prevent liquids from being drawn into the PIPETMAN shaft to prevent
possible resultant damage by observing the following precautions:
a. Pipet very slowly, holding the PIPETMAN not greater than
20o from the vertical axis.
b. Never invert or lay the PIPETMAN on its side with liquid
in the tip.
c. Use the special Safety Filters supplied with the P5000 &
P1000 instruments and C-5000 & C-1000 tips.

D. Service Information

1. Pipetman Pipet

Never grease any components of the PIPETMAN.

Service includes repair, cleaning and recalibration. Calibration is certified
Pipette Calibration Check________________________________________________6

to original specifications. Instruments will perform as well as an equivalent new

instrument unless otherwise noted. Determination of warranty status rests solely
with Rainin’s Liquid Handling Service Department. A quote will be provided
prior to repair to obtain a service purchase order number (PO.).

Please follow these steps to send a pipet for repair:

a. Decontaminate pipet completely before shipping.
b. Provide billing & shipping address to the company along with name &
phone number of principle operator.
c. Indicate problems or service required
d. Complete and send an Express Repair Form along with a note stating that
a PO no. will be sent upon receipt of a repair quote.
e. Package securely.
f. Ship FedEx to the following addresses:

RAININ OR GILSON PIPET REPAIR:

Rainin Instrument LLC

Pipette Service
Rainin Road
Woburn, MA. 01801

For further information, direct inquiries to 1-800-662-7027

8:30 am to 5:00 pm EST (M-F).

MLA PIPET REPAIR:

MLA Pipette Repair

270 Marble Avenue
Pleasantville, NY 10570-2982

For further information, direct inquiries to 1-800-431-1720

NOTE: All MLA Pipettes have a lifetime guarantee.

References:
1. Eppendorf Pipette Instruction Manual
2. FACService Tech Notes from Becton Dickinson
3. Instructions for Pipetman Continuously Adjustable Digital Microliter Pipets, Rainin
Instruments Company, Inc., 1982.
4. Instructions for MLA pipets; MLA Pipettes, 270 Marble Ave. Pleasantville, NY. 10570
Pipette Calibration Check________________________________________________7.

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