Introductory Experiments On Common Laboratory Instruments Biology Essay

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A biotechnology scientist must perform his/her Laboratory techniques successfully. The success of the experiment depends only on the accuracy in performing the experiment. And Accuracy can be achieved by the experience and also depends on knowing the proper knowledge about the instrument usage and observing the proper techniques involved in the execution of the experiment and the instruments. And some of the common laboratory techniques are discussed below, in the techniques we must observe the use of analytical balance, the usage of the pH meter and knowing how to calibrate, microcentrifuge working, micropipette usage and very important labelling the reagents properly.

PREPARATION OF A BUFFER REAGENT

In this experiment with the help of the analytical balance, micropipette and pH meter buffer reagent must be accurately prepared. The buffer reagent consists of 0.1M sodium chloride and 0.025M citric acid with pH of the buffer solution is maintained at 6.0. The weights of the chemicals must be weighed by using the analytical balance carefully as the instrument is very sensitive. And pH of the solution is maintained by using the pH meter by immersing the pH meter electrode into the solution and noting down the reading displayed by the pH meter regularly. The objectives of the experiment is to prepare 100ml of stock buffer solution in a container which consists of 0.1M of sodium chloride and 0.25M of citric acid with the pH-6 and to know the proper usage of the analytical balance and calibration of the solution pH accurately by using pH meter.

MATERIALS

100ml beaker is needed for mixing the chemicals; disposable pipettes are used for controlling pH putting drop by drop of 3M NaOH for levelling the pH. pH meter with pH electrode is used for measuring the pH by placing it in buffer reagent prepared by using 0.1M Sodium Chloride, 0.25M citric acid monohydrate which are measured using Analytical balance. Additional containers required are volumetric flask 100ml and 100ml screwcap bottles they must be washed thoroughly by using deionised water present in the wash bottle. Standard buffer, Spatulas and gloves are also required.

METHOD

The amounts of chemicals are required for the preparation of 100ml solution can be found out by using molarity equation and known as 0.025M citric acid and 0.1M NaCl.

_Mass of solute (gram)/FWsolute_

M = Volume of solution (liter)

The chemicals are weighed as calculated and transferred to the 100ml beaker and they are dissolved by using 90ml of the distilled water. pH meter is calibrated by using standard buffer solutions on is of pH 4.0 and the other is pH 10.0 at the room temperature.

The solution in the 100ml beaker (NaCl + Citric acid) is mixed well and the calibrated pH meter is placed inside the solution and the mixture pH is slowly and carefully adjusted to 6.0 by adding 3M NaOH drop by drop with the help of the pipette and continuously stirring the solution.

After the solution has reached to the required pH that is 6.0 the solution is transferred to the volumetric flask of 100ml the volume is measured exactly by seeing the measurement marks provided on the surface of the flask.

And the solution is transferred to the screwcapped nalgene bottle to store the solution for the future use and bottle with the solution is properly labelled for clear identification and indication.

RESULT

_Mass of solute (gram)/FWsolute_

M = Volume of solution (liter)

.

Gram = M - FW - volume of solution

Amount of NaCl= NaCl (FW- 58.44)

Gram = 0.1-58.44-0.1

= 0.5844

Amount of Citric Acid = citric acid (FW- 210.14)

Gram = 0.025 - 210.14 - 0.1

= 0.5253

Carefully 3M NaOH is added drop by drop at constant stirring to adjust the pH of the solution to 6.0.in the 100ml beaker.

Firstly before calculation the units must be converted 1mM = 0.001M and 1ml = 0.001litre

Gram = M - FW - volume of solution

NaCl = 58.44 - 0.25 - 0.14

=2.0454

KCl = 74.55 - 0.25 - 0.004

= 0.07455

NaH2PO4.H2O = 137.99 - 0.001 - 0.25

=0.03449

MgSO4 = 120.37 - 0.0009 - 0.25

=0.0270

HEPES = 283.31 - 0.01 - 0.25

= 0.7082

Glucose = 180.2 - 0.025 - 0.25

= 1.1262

All the reagents are taken in to the 250ml of flask and dissolve using distilled water, now set the pH to 7.4 using 2M NaOH in the flask using dropper or pipette.

DISCUSSION

Accurate mixture of the chemical compositions will give the accurate results so we must be careful while weighing the chemicals and mixing them n the proper proportions. One must take care of while he/her dealing with the NaOH as it is very corrosive we must follow some of the health and safety precautions like wearing gloves and goggles when they are handling the chemical.

The pH meter consist of a pH sensitive bulb which is made up of a very thin glass and it is very fragile one must be very careful while handling the equipment. The glass bulb may crack even if it touches the bottom surface of the beaker while testing the pH of the solution. Before using the pH meter it must be calibrated, and we must never allow the pH electrode to get dried up. It must be stored by immersing it in storage solution like 4M KCl solution with pH 4 or 7 buffer solution. While we about to use it, it must be taken out of the storage solution wipe it and calibrate it with the standard solution, wipe the bulb again and immerse it in to the test solution check the reading and again store it in the storage solution.

Analytical balance is a very sensitive device and it is digital it has a capability of weighing the substance up to 0.0001 or even 0.00001 grams and it is very accurate. It must be placed in the dry and the clean area and the area must by level. Over loading the balance more than its capacity may spoil the device. The extra weight of a plate or a paper placed on the balance can be tared. And the precaution must be taken that the air also must not enter in the device while measuring, doors of the device must be closed to gain accuracy.

Labelling is very important on the storage bottle as it tell the whole information about the solution that contains in the bottle the label must have the information about the chemical composition, pH of the solution, when it is made and the batch number if it is made by a group. So that it will be easy for the identification for the future us of the solution for any other experiment.

REFERENCES

Barry Haggett, 2010. Introductory experiment on common laboratory instruments. Learning resources, Analytical and Diagnostic Technologies. [Online]. Available at: http://breo.beds.ac.uk (Accessed: 26 May 2010).

CETRIFUGATION OF SUSPENSIONS

Centrifugation is most widely used technique in different field of research like Biochemistry, Cell and Molecular biology and in the medicine. The technique is used to isolate the cell organelles inside the cell. Centrifugation process is nothing but spinning the samples at very high speed, which result in the separation of the particles based on their density due to the gravitational force and the precipitation will be formed in the bottom of the tube which is also called as pellet. And the remaining solution on the top of the pellet is called as the supernatant. After separation they must be immediately separated by using the micropipette without disturbing the pellet. The centrifuges are two types among which are used in the laboratory purpose. One is the simple centrifuge which is regular size containing regular size centrifuge tubes or centrifuge tips and the other one is micro centrifuge which consists of micro centrifuge tips. They both different in their speed and capacity, the rate of the centrifugation depend on the acceleration that is applied to the sample.

The rotor consists of a covering lid which is interlocked to initiate the spinning of the rotor and acts as a protector from any touch injures during the process. And also from the accidental catastrophically rotor fails. The rotor must be always balanced, samples inside the rotor are placed such a way that they lie opposite to each other with equal masses. If the empty space is available on the opposite of any sample it must be filled with water sample of same mass to equalise the balance of the rotor.

The acceleration rate of the centrifuge is measured in terms of revolutions per minute (RPM) or RCF (relative centrifugal force) and unit of gravity as (g). Some procedures of separation particles and liquid are based on the centrifugal condition which is specified in terms of RCF or unit of gravity but most of the micro centrifuges consist of setting for only RPM. So the relationship between RPM and RCF is

g = (1.118 x 10⁻⁵) RS²

Where g = RCF, R is the radius of the rotor in cm and S is the speed i.e. RPM.

The objectives of the experiment are to know the use of the micropipette to measure the volume and decanting liquid, usage of Micro centrifuge and converting RPM to RCF.

MATERIALS

1000µl and 200µl micropipettes are needed with at least 60 micropipette tips for measurement, 1.5 ml micro centrifuge tube for placing the samples in it to run micro centrifuge 70 in number at least. 2 micro centrifuges for the process, sample A and B containing black and green coloured mixtures respectively and a ruler to measure the radius of the rotor.

METHOD

By using 1000µl micropipette take 1000µl of the will mixed sample for sample A and transfer into the 3 separate 1.5ml micro centrifuge tubes and place them in the rotor in the balanced condition.

Measure the radius of the rotor with the help of the ruler placing at the center of the rotor to the edge of the rotor and note it down the reading and cover the lid of the centrifuge. Set the centrifuge machine for 1 min at the speed of 5000RPM and press the start button. After the centrifuge machine stop after 1min remove and discard the supernatant liquid carefully without disturbing the pellet with the help of the 200µl micropipette.

Now transfer 1500µl of sample from sample B into 2 separate micro centrifuge tube in the same way as explained previously and place them inside the rotor in the balanced condition like opposite to each other. And set the centrifuge machine for 1 min and at the speed of 5000 RPM. And check if there is any separation or not, if the separation is not seen then increase the speed to 10000RPM and remove and discard the supernatant liquid carefully.

Calculate the RCF in terms of g from rotation speeds of the both samples.

RESULTS

For the suspension A (Blue colour)

g = (1.118 - 10-5)RS2

= 1.118 - 5 - 5000 - 5000 / 10000

= 1397.5

For the suspension B (off Green colour)

g = (1.118 - 10-5)RS2

= 1.118 - 5 -10000 - 10000 /100000

= 5590

DISCUSSION

Micro centrifuge is most widely used in the laboratories for the isolation of the cell organelles and macro molecules from the cells it is very simple process.

Care should be taken when placing the micro centrifuge tubes in the rotor they must be arranged in the balanced conditions otherwise the machine will make wobbling sound and there is a chance of burning inside the machine and it may get spoiled. If u heard wobbling sound form the machine immediately push the stop button and place the tubes inside balanced and turn it on again.

If there is no precipitation formed inside the tube change the speed of the rotation RPM (increase the RPM). This can be the reason for the incomplete separation of the precipitation.

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