# Presentation: 021236

## Use and Maintenance of Micro-pipettes

## Introduction

- Automatic pipettes are used to accurately transfer small liquid volumes
- Glass pipettes are not highly accurate for volumes less than 1 milliliter (1 ml), but the automatic pipettes are both accurate and precise
- These are continuously adjustable digital pipettes
- Each pipette can be set to transfer any volume within its own volume range

## The Automatic Pipette

- Select the proper automatic pipette to transfer a specified volume of sample
- Set a specified volume on the pipette volume indicator using the volume adjustment knob
- Read a digital volume setting in both micro liter (μl) and milliliter (ml) units
- Demonstrate the correct technique to accurately transfer a sample of a stock solution to another vessel
- Correctly answer questions based on the material for the Automatic Pipette

**After successful completion of this module the participant will be able to perform following:**

## Parts of the Pipette

**Parts of the Pipette**

**Pipette tips**

## Operating the Micropipette

**Volume Adjustment Knob:**

**Digital Volume Indicator:**

**Pipettors – 3 Volumes:**

**Step 1:  Set the Volume**

**Operating the Micropipette**

## Operating the Micropipette

**Step 1:  (Continued) Read the Volume**

**How to Read the Volume Indicator:**

| 
                           |                            |                           |
| --- | --- | --- |
| (a): P-20 Model
 6.86  l = 0.00686 
or 6.86 x 10-3 ml   | (b): P-200 Model 
132.4  l = 0.1324 
or 1.324 x 10-1 ml | (c): P-1000 Model 
262 l= 0.262 
or 2.62 x 10-1 ml |

## Operating the Micropipette

**Example of tip sizes:**

**Attaching the disposable tip**

**Operating the Micropipette**

**Step 2: Attach the Disposable Tip**

## Operating the Micropipette

**Step 3: Depress the Plunger to the First Stop**

**Step 5: Draw up the sample**

**Step 6:  Pause**

**To aspirate the sample into the tip, allow the pushbutton to return slowly and smoothly to the fully extended UP POSITION. NEVER LET THE PLUNGER SNAP UP! This draws the exact calibrated volume into the tip if the tip remains below the liquid surface during withdrawal. **

**Wait a few seconds to ensure that the full volume of sample is drawn into the plastic tip. WAIT LONGER FOR LARGER VOLUMES. WAIT LONGER FOR MORE VISCOUS ("SYRUP-LIKE") SUBSTANCES. **

**Step 4:  Immerse Tip in Sample**

## Operating the Micropipette

**Step 7:  Withdraw the Tip**

**Remove the tip from the sample liquid. No liquid should remain on the OUTSIDE of the tip. Wipe away any droplets on the outside of the tip with a lint-free tissue, such as KIMWIPES, but only wipe droplets from the side of the tip. NEVER TOUCH THE TIP OPENING or you may absorb part of your sample. **

**Proper Droplet Removal**

**WRONG Droplet Removal**

## Operating the Micropipette

**Step 8:  Dispense the Sample**

**To dispense the sample from the pipette: ****a) Touch the tip end to the side wall of the receiving vessel and ****b) Depress the plunger to the FIRST STOP. ****c) Pause for at least one second-- 1-2 seconds for P-1000, 2-3 seconds for P-5000,**
**    ****or longer for viscous liquids. ****d) Press the plunger to the SECOND STOP (the second point, of greater resistance,**
**    ****at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing**
**    ****out" a glass pipette).**

| 
                                                                   |  |  |
| --- | --- | --- |
| (a) Start Dispensing  | (b) 1st Stop = Dispense | (c) 2nd Stop = Expel |

## Operating the Micropipette

**Step 9:  Withdraw the Pipette**

**With the plunger fully depressed, withdraw the pipet from the receiving vessel carefully, sliding the tip along the wall of the vessel. Holding the tip against the side of vessel is **_**especially important when transferring small volumes of liquid**_**. **

**Step 10:  Release the Plunger**

**Gently allow the plunger to return to the UP position. DO NOT allow it to SPRING BACK! **

**Step 11:  Discard the Tip**

**Discard the tip by **_**depressing the**_** tip ejector button, as shown below. A fresh tip should be used for each sample to prevent sample carryover. **

**Press ejector button to discard tip.**

## Practice with Pipette

- Review material and practice using a pipette
- Use proper pipetting techniques

## Equipment and Supplies

- 3 automatic pipettes
- Several capped sample vials
- Green sample solution to practice volume transfer

##  Step-wise Operation of the Automatic Pipette

**  ****(1) Set the volume**
**  ****(2) Attach disposable tip**
**  ****(3) Depress the plunger to the first stop**
**  ****(4) Immerse tip in sample**
**  ****(5) Draw up the sample**
**  ****(6) Pause**
**  ****(7) Withdraw the tip**
**  ****(8) Dispense the sample**
**  ****(9) Withdraw the pipette**
**(10) Release plunger**
**(11) Discard the tip**

**Notes:**
As you read the following steps, you should manipulate the automatic pipette, 
disposable tips, and the vials of colored solution TO LEARN HOW TO PROPERLY 
HANDLE THE PIPETS.
STEP-WISE OPERATION OF THE AUTOMATIC PIPET
As you read the following steps, you should manipulate the automatic pipette, 
disposable tips, and the vials of colored solution TO LEARN HOW TO PROPERLY 
HANDLE THE PIPETS.
1) SET THE VOLUME: Hold the pipet in one hand while turning the volume 
adjustment knob until the correct volume shows on the digital indicator. 
Proper Grasp to Set Volume
Turn 1/3 revolution above the desired setting and then slowly down to the 
desired setting for optimal accuracy.
2) ATTACH DISPOSABLE TIP: Press disposable tip of PROPER SIZE firmly to the 
shaft of the pipet to ensure a positive airtight seal.

Tip Sizes Attaching Tip
3) DEPRESS THE PLUNGER TO THE FIRST STOP: Using your thumb, depress the plunger 
gently and smoothly until resistance is felt. The point of resistance is the 
first stop. It is that part of the stroke that extends the piston to the 
calibrated volume which is displayed on the digital indicator. 
 Depress Plunger 1st Stop
4) IMMERSION INTO THE SAMPLE: With the pipet in a vertical position, immerse the 
disposable tip into the sample liquid to a depth of at least 3-4 mm 
(millimeters) below the liquid surface for the common pipets, 
Immerse Tip in Sample
5) DRAW UP THE SAMPLE: To aspirate the sample into the tip, allow the pushbutton 
to return slowly and smoothly to the fully extended UP POSITION. NEVER LET THE 
PLUNGER SNAP UP! This draws the exact calibrated volume into the tip if the tip 
remains below the liquid surface during withdrawal.
6) PAUSE: Wait a few seconds to ensure that the full volume of sample is drawn 
into the plastic tip. WAIT LONGER FOR LARGER VOLUMES. WAIT LONGER FOR MORE 
VISCOUS ("SYRUP-LIKE") SUBSTANCES. 

7) WITHDRAW THE TIP: Remove the tip from the sample liquid. No liquid should 
remain on the OUTSIDE of the tip. Wipe away any droplets on the outside of the 
tip with a lint-free tissue, such as KIMWIPES, but only wipe droplets from the 
side of the tip. NEVER TOUCH THE TIP ORIFICE (opening) or you may absorb part of 
your sample. 

8) DISPENSE THE SAMPLE: To dispense the sample from the pipet,

a) Touch the tip end to the side wall of the receiving vessel and 
b) Depress the plunger to the FIRST STOP. 
c) Pause for at least one second-- or longer for viscous liquids. 
d) Press the plunger to the SECOND STOP (the second point, of greater 
resistance, at the bottom of the stroke) to expel any residual liquid in the tip 
(like "blowing out" a glass pipet). 
9) WITHDRAW THE PIPET: With the plunger fully depressed, withdraw the pipet from 
the receiving vessel carefully, sliding the tip along the wall of the vessel. 
Holding the tip against the side of vessel is especially important when 
transferring small volumes of liquid.  

10) RELEASE PLUNGER: Gently allow the plunger to return to the UP position. DO 
NOT allow it to SPRING BACK!
11) DISCARD THE TIP: Discard the tip by depressing the tip ejector button, 
A fresh tip should be used for each sample to prevent sample carryover.
n handling the automatic pipettors. Also, be certain that you can 
BOTH READ AND SET THE PROPER VOLUMES. 

PIPETTING CONSIDERATIONS
Accuracy and Precision: Accuracy means the closeness with which the dispensed 
volume approximates the volume set on the pipette. It is specified as mean 
error, the average deviation of replicate measurements from the expected set 
volume. Precision is the "scatter" or reproducibility of individual measurements 
of the same volume. It can also be expressed as standard deviation. 
Specifications are generally tighter for precision than for accuracy; and, in 
controlled experiments, the smaller precision specification is a good estimate 
of pipetting accuracy. The smaller the mean error and/or standard deviation of a 
measurement, the more accurate and/or precise it is.  

TO SUMMARIZE, relative accuracies are generally about 1% or less and precision 
is less than 0.5 % except when transferring the smallest recommended volume for 
a given pipet model. Us 

## Accuracy and Precision

- Accuracy means the closeness with which the dispensed volume approximates the volume set on the pipette
- Accuracy is specified as mean error, the average deviation of replicate measurements from the expected set volume
- Precision is the "scatter" or reproducibility of individual measurements of the same volume
- Precision can also be expressed as standard deviation

## Accuracy and Precision (Continued)

- Relative accuracies are generally about 1% or less
- Precision is less than 0.5 % except when transferring the smallest recommended volume for a given pipette model
- Using the pipettes to transfer volumes which are below the recommended range will introduce larger errors

## Pippetting Guidelines and Precautions

**For optimal reproducibility, **use the following pipetting procedures:
- (1) Consistent SPEED and SMOOTHNESS when you
-       press and release the PLUNGER
- (2) Consistent pressure on the PLUNGER at the FIRST
-      STOP
- (3) Consistent and sufficient IMMERSION DEPTH
- (4) Nearly VERTICAL POSITIONING of pipette
- (5) AVOID ALL AIR BUBBLES: Since the plastic pipette
-      shaft can be damaged if liquids are drawn beyond the
-      tip into the shaft
- (6) NEVER lay the pipette on its SIDE nor INVERT the   pipette if liquid is in the tip

## Practice with Pipettes

- Practice using the pipette
- Practice setting a few volumes
- Practice reading the digits of set volumes
- Practice drawing up and dispensing samples
- Get the "feel" of the 1st and 2nd stops
- Practice "blowing out" the pipette