Aims : 1.To investigate the skills of manipulating small volumes

2. To investigate the skills of preparing and manipulating laboratory buffers.

Introduction: Small volumes are manipulated in laboratories using the most essential laboratory instrument known as the micropipette. The micropipette measures volumes of liquids in microliter (µL).

The buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. To determine the final pH of the solution a pH meter was used

Materials: NaCl, KCl, Na2HPO4, KH2PO4, pH meter, Magnetic stirrer, Micropipette

Method (A):

i. How to collect sample with a micropipette

1. The cap or the lid of the tube from which the liquid is taken is opened, before picking up the micropipette.

2. The micropipette is held in one hand, almost vertical and the tube is held in the other hand. Both are placed at almost eye level.

3. The plunger of the micropipette is depressed to the first stop and held in that position.4. The tip is dipped into the solution to be pipetted.5. The fluid is drawn into the tip slowly by releasing the plunger.

ii. How to expel a sample from the micropipette

1. The cap or lid of the tube into which the fluid is ejected is opened.2. The micropipette is held in one hand, almost vertical and the tube is held the other hand.

Both are kept at about eye level.3. The micropipette tip is touched to the inside wall of the reaction tube into which the

sample is expelled. A tiny surface of tension is created and the fluid is coaxed out of the tip.

4. The plunger is depressed slowly to the second stop to expel the last bit of the fluid. The plunger is held down in this position.

5. The pipette is slowly removed out of the tube, keeping the plunger depressed to avoid sucking any liquid back into the tip. The thumb is released, when the tipis free of the tube.

6. The tips are always changed for each new reagent that is needed to be pipetted or if any other liquid is touched in the reaction tube. The large gray button on the top of the micropipette is depressed to eject a tip.

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Method (B):

1. All the reagents are dissolved in 800ml distilled H2O.2. The pH is adjusted to 7.4 with HCl.3. The volume is adjusted to 1L with additional distilled H2O and a Phosphate Buffered

Saline (PBS) is prepared.

Results:

1. Preparing 3M of KCl in 100ml.

n= MV1000

=(3 ) (100 )

1000=0.3mol

mass=mol×MW

¿0.3mol×74.56 gmol

¿22.368 g

2. Calculating how to prepare 100 ml 2X solution of PBS from 1L 10X solution of PBS.

m1 v1=m2 v2

(2)(100)=(10)×(v2)

v2=20ml

3. Calculating how to prepare 100 ml 1X solution of PBS from a stock of 500ml 10X solution of PBS.

m1 v1=m2 v2

(1)(100)=(10)×(v2)

v2=10ml

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Discussion (A):

A micropipette is a precise pipette than can have a disposable tip. The tips fitted in are tips that are specifically allocated for the scales of the micropipette. The micropipette works in such way that the volume of the air space in the barrel is adjusted by adjusting the knob volume adjustment knob. Turning the knob left and right adjusts the volume of the air space.

To draw up the specific amount of volume of scale chosen, the plunger is depressed all the way down and the tip is placed in the container of liquid. Releasing the plunger creates vacuum, which in turn forces the liquid with volume of the selected scale to enter via the tip. The withdrawn fluid is expelled by depressing the plunger again. In other words, the volume of air space adjusted is the volume that is taken up by the liquid when the plunger is depressed than released.

The ranges of the micropipette are:

Colour of micropipette Pipetting range (µL)White 0.5 – 10Yellow 10 – 100, 20 – 200Blue 100 – 1000

Ways to read the scales:

The scales are divided into three boxes which displays a specific number.

The above digital readout indicates a reading of 20 µL on a 2-20 µL volume micropipette. The first box represents the number of tens and the second box represents the numbers of ones while the last box displays the number after the decimal point.

 1 2 5

4

Plunger button

Placement of disposable tip Tip ejector button

Digital volume indicator Volume adjustment knob

200

Example: The above digital readout indicates a reading of 12.5 l on a 2-20 l volume micropipette

 2 0 0

The above digital readout indicates a reading of 200 l on a 20-200 volume micropipette. Where the first box represents the number of hundreds, the second box the numbers of tens and the last box is the numbers of ones.

 0 2 3

Example: The above digital readout indicates a reading of 23 l on a 20-200 volume micropipette

 1 0 0

The above digital readout indicates a reading of 1000 l or 1 mL on a 100-1000 l micropipette. Whereby the first box represents the numbers is thousands, the second is the numbers in hundreds and the last box represents the numbers in tens.

 0 5 2

Example: The above digital readout indicates a reading of 520 l on a 100-1000 l micropipette.

Discussion (B):

A buffer is solution of mixture between a weak acid and its conjugate base or a weak base and its conjugate acid. (NaCl – Na2HPO4 / KCl – KH2PO4).

A buffer solution can resists the change in pH when a small amount of acid of base is added to the solution.

The Phosphate Buffered Saline (PBS) is a buffer solution which contains sodium chloride, sodium phosphate, potassium chloride, and potassium phosphate. The PBS is prepared in such way that the osmolarity and the ion concentration in it resembles of those in the human system. That is the PBS can be said to be isotonic to the human cell fluids.

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The PBS that is prepared is manipulated to pH 7.4 that is the pH of the fluids in our cells. In order to get the specific pH, the HCl and NaOH is added. The HCl is added if the pH of the solution is more than 7.4 (basic) while the NaOH is added if the solution is less than 7.4 (acidic).

PBS is useful in the field of cell biology that is to maintain the osmolarity of the cell by having the same ion concentration and pH. PBS is also commonly used as buffer solution in biochemistry experiments to maintain the pH of the proteins. As the increase or decrease in pH can result in the denaturing of the protein.

Conclusion:

1. The manipulation of small volumes was done using micropipette.2. The phosphate buffered saline (PBS), a laboratory buffer was prepared and manipulated.

References:

1. http://www.uri.edu/smile/documents/6-micropipettinglabinstructions

2. http://www.di.uq.edu.au/sparqmicropipette

3. http://en.wikipedia.org/wiki/Buffer_solution

4. http://chemwiki.ucdavis.edu/Physical_Chemistry/Acids_and_Bases/Buffers/

Preparing_Buffer_Solutions

5. http://en.wikipedia.org/wiki/Phosphate_buffered_saline

Prepared by:

(Nagaswitra d/o Manukaran)

DVM 1

178824

Date of submission:

26/09/2014

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