2.

2 Instrumentation

The instruments in the laboratory included:

 Spectrophotometer

 Hybridization chamber

 Refrigerators

 Oven

 Centrifuges

 Thermocycler

 Weighing balance

 Incubator

 Scintillation counter

 Fluorescent microscope

 Autoclave

 Biosafety cabinet

 Gel drier

2.3.1 Sonicator

This machine was used in the extraction of proteins and it’s prime role was to release cell

contents by shearing the cell membranes.

2.3.1.1 Principle of operation of a sonicator

A sonicator has a probe (a rod) which is connected to a motor that runs on electrical energy to

cause vibration of the probe at very high frequencies, these vibrations cause mechanical shearing

of the cell membranes there by releasing cell contents. The cells to be disrupted are put in a
plastic tube together with a lysis buffer which enhances the disruption. It is into this tube that the

probe is placed in order to shear the cell membranes.

2.3.1.2 Care and maintenance of a sonicator

The machine had to be placed on a firm bench in order to prevent it from falling down and hence

avoiding damage and financial losses due to its break down. A user’s manual provided by the

manufacturer was always adhered to in case of any operations to be done using the machine.

This was of a very great importance especially to new users of the machine to avoid spoiling it

due to less knowledge about its use.

It was ensured that any technical fault on the machine was handled only by technical persons

who had skills in machine repair and maintenance. Any spillage was immediately cleaned up to

prevent contamination of the machine which could lead to rusting of the metallic parts of the

machine.

2.31.3 Safety when using a sonicator

As a matter of electrical safety, the sonicator was always switched off every after use to prevent

it from un necessary exposures to electricity which could damage it due to short circuits

2.3.1.4 Quality assurance and quality control during use of the sonicator.

These were technical measures taken to ensure that pure proteins were obtained from the use of

the machine. These included proper cleaning of the machine after use before the next use to

prevent cross-contamination of the samples.

2.3.2Top pan balance:

This is an instrument used to measure weights of powdered medium prior to preparation as well

as powdered gels.
2.3.2.1 Principle of operation of a top pan balance.

The principle of operation is that this balance has a pan on top which is connected to a scale

which can read up to a specified weight so when the weight to be measured is put on the pan it

will press the pan which is connected to a scale so the indicator of the scale will move according

to the weight to be measured.

2.3.2.2 Adjustments of the top pan balance before use:

Before using the balance, the air bubble had to be centered well in the ring.

The weight of the container to house the sample was determined in order to tare the balance to

zero. The weight of the container had not to exceed 50g

2.3.2.3 Care and maintenance of the weighing balance (top pan balance)

The weighing balance is very, sensitive to vibrations, so it had to be put on a firm flat form or

bench top free from vibrations in order to get accurate results.

After use the pan had to be cleared off any spillage of either media or chemicals which when left

could corrode the pan.

The mains and the machine had to be switched off when ever not in use, and the machine

covered well to avoid accumulation of dust which could interfere with the operation of the

machine resulting into wrong results being obtained.

2.3.2.4 Safety when using a top pan balance

It was always ensured that the machine was placed on strong support to avoid accidents that

could lead to injuries to both the workers and the machine it self.

As a matter of electrical safety, the balance was always switched off every after use to prevent it

from un necessary exposures to electricity which could damage it due to short circuits
2.3.3 Spectrophotometer

A spectrophotometer was used to quantify nucleic acids such as DNA, but can also be used to

quantify proteins.

2.3.3.1Principle of operation of spectrophotometer

Quantification using this method is based on the optical density of the solution containing the

DNA to be quantified. When a beam of light is passed though the solution, some light is

absorbed and the other transmitted, the amount of light that is absorbed is proportional to the

concentration of the absorbing substance in the solution. The machine will then give the results

in terms of optical density which is dependent on the DNA concentration.

2.3.3.2 Care and maintenance of a spectrophotometer

For safety reasons the spectrophotometer was always placed on a firm bench this was to ensure

that it’s not damaged due accidents like falling off the table. Electrical safety was ensured by

making sure the machine was plugged into a power stabilizer and switching it off when ever not

in use. Accumulation of dust on the machine could lead to reduction in the working efficiency of

the machine. This problem was handled by covering the machine when ever not in use with

covers specially made for the purpose.

2.3.3.3 Quality assurance and quality control when using the spectrophotometer

To ensure that accurate values for the quantities of nucleic acids were obtained, blanking was

always done in order to calibrate the machine before carrying out the quantification process. This

was done by running a blank sample that contained no nucleic acids to see whether the results

obtained correlate to the theoretically known idea about the sample

2.3.4 Centrifuge

The micro- haematocrit centrifuge was used in the measurement of packed cell volume. It helps

to increase on the rate of sedimentation of blood.

2.3.4.1 Principle of operation of the Centrifuge.

When force that is greater than gravity is applied to a liquid in a tube placed in a centrifuge, an

outward force pushes the particles in that solution to the extreme of the test tube causing

separation. The heavier particles receive greater force and hence are pushed furthest away from

the center. This force is called centrifugal force and it depends on the speed of rotation of the

rotor of the centrifuge.

2.3.4.1 Adjustment of the Centrifuge for use

The speed and time of the centrifuge were always adjusted according to what was to be

centrifuged.

2.3.4.2 Care and maintenance of the Centrifuge

The centrifuge was always placed on a firm bench free from vibrations to avoid breakage of

tubes during its operation. We also avoided loading the centrifuge with sample to avoid

breakages. Before use, the buckets were checked for presence of broken particles to avoid excess

weight as well as to prevent contamination of samples to be centrifuged. The centrifuge was not

opened before rotation had stopped to prevent breakages and generation of aerosols which could

contaminate the laboratory and the environment. The centrifuge was kept clean by disinfecting it

with 70% alcohol. It was ensured that the centrifuge was tightly closed before it was started, that

was important in avoiding spillages that could easily affect the health of laboratory workers and

clients and physical accidents to the personnel

2.3.5 Scintillation counter

This was a machine used to detect and count radioactive decay or molecules.

2.3.5.1 Principle of operation of a scintillation counter

The sample which is suspected to be radioactive is combined with a liquid scintillation cocktail

or solid scintillator. Decay of a radionuclide produces an ionizing particle. Part of the kinetic

energy of the particle is transferred to the scintillator which converts the energy of the particle

emitted during the radioactive decay process into light which is detected by the liquid scintillator

system. The number of protons produced from one ionizing particle is proportional to its kinetic

energy. The collecting optics of the liquid scintillator system directs the protons emitted to either

of the two photomultiplier tubes. If both photomultiplier tubes are activated by one photon burst,

then one nuclear decay event is registered.

2.3.5.2 Care and maintenance of a scintillation counter

For proper functioning of the machine, it was placed on a firm bench. This was of importance for

safety of the machine in order to prevent it from damages due accidents like falling off the

bench. Overloading of the sample was always discouraged to avoid interference with workings

of the machine. Any radioactive contamination of the machine say due to spillage was

immediately removed by appropriate measures. The users’ manual provided by the manufacture

was always adhered to when ever using the machine. This was of safety importance in order to

prevent the spoilage of the machine due to lack of knowledge. Technical faults on the machine

were always handled by technical persons skilled in machine repair.