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  2 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, Na 2 HPO 4 , KH 2 PO 4 , 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.  3 Method (B): 1.   All the reagents are dissolved in 800ml distilled H 2 O. 2.   The pH is adjusted to 7.4 with HCl. 3.   The volume is adjusted to 1L with additional distilled H 2 O and a Phosphate Buffered Saline (PBS) is prepared. Results: 1.   Preparing 3M of KCl in 100ml.  ()()          ⁄      2.   Calculating how to prepare 100 ml 2X solution of PBS from 1L 10X solution of PBS.            ()()() (  )      3.   Calculating how to prepare 100 ml 1X solution of PBS from a stock of 500ml 10X solution of PBS.            ()()() (  )       4 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  –   10 Yellow 10  –   100, 20  –   200 Blue 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 2 0 0 Plunger button Placement of disposable tip Tip ejector button Digital volume indicator Volume adjustment knob  5 Example: The above digital readout indicates a reading of 12.5 -20 l volume micropipette 2 0 0 The above digital readout indicates a reading of 200 n 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 n a 20-200 volume micropipette 1 0 0 The above digital readout indicates a reading of 1000 -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 -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  –   Na 2 HPO 4 / KCl  –   KH 2 PO 4 ). 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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