Pharos university Faculty of Allied Medical SCIENCE

Clinical Laboratory Instrumentation

(MELI-201)

Dr. Tarek El Sewedy

Lecture 3

Incubators , Microscopes and

Thermo cyclers

Intended Learning Outcomes

By the end of this lecture the student should

learn the basics of the following:

1. Cell Incubators used in Clinical Labs

2. Microscopes

3. Thermo cyclers.

Lecture content

Laboratory Incubators

1. Microscopes.

2. Thermo cyclers

Cell IncubatorsA cell incubator is an apparatus used to grow and maintain

mammalian cell cultures, microbes,or plant cells

The concept of incubation refers to the maintenance of

controlled environmental conditions needed to sustain the

development or growth of cells, eggs, tissues, or whole

organisms.

 The incubator maintains optimal temperature, humidity and

 carbon dioxide (CO2) levels .

Incubator Alarm System

A controlled atmosphere is achieved by using a CO2-monitoring

device, which draws air from the incubator into a sample

chamber, determines the concentration of CO2, and injects pure

CO2 into the incubator to make up any deficiency.

Air is circulated around the incubator by using a fan to keep

both the CO2 level and the temperature uniform.

Mammalian Cell incubator

Bacteriological Incubator

Cell incubators According to design

Cell incubators usually come in one of three types

of designs:

1. Air draft.

2. Dry wall.

3. Water jacket.

1. Air draft incubators circulate air throughout the interior of the incubator to maintain

constant temperatures.

This style of incubator is used for growing large amounts of cells in a

big growth chamber.

These incubators are designed to respond quickly to environmental

needs of the cells.

However, these incubators lose the temperature very quickly and

must consistently adjust the air to control the temperature.

These are the most commonly used type of incubators

2. Dry wall incubatorsPass air within the walls of the incubator.

The walls then radiate the temperature to the growing

chamber. For this reason these incubators are sometimes

called radiant incubators.

They are better at maintaining a constant temperature

because the jacket does not let heat escape through the

walls of the incubator.

These incubators respond slowly to temperature

adjustments compared to Air draft incubators

Water jacketed incubatorsWater-jacket incubators are surrounded by water within the

walls making up the three sides, the top, and the bottom.

Advantages include stable temperature control and

increased security in the event of power failure (due to

water’s natural insulation abilities).

These are usually smaller incubators and work by the

same principles as dry wall incubators.

These incubators also respond more slowly to temperature

changes.

Use of copper in incubators Copper fights contamination: Some CO2 incubators incorporate copper in

their cabinet design to resist contamination. As the copper breaks down, it

releases copper oxide, which destroys microbes present in the chamber.

 Copper is also used inside the water jacket to eliminate contamination

growth over time inside the jacket.

No chemicals are recommended or needed in the water jacket.

Copper is also used around the sample port and the gas injection tube.

Some incubators have a decontamination cycle incorporated where the

temperature can be increased up to 200C

Cleaning of incubators

1. Remove the humidity pan weekly and autoclave

2.  Remove all shelves. Autoclave or wash and disinfect as described.

3. Clean and disinfect all access ports, air bleeds, shaft holes, electrical

pass-through. and any other passages into the incubator.

4. Remove the door gasket and gasket guards. Clean and disinfect.

5. Replace all air and CO2 filters as needed, approximately every six

months or when noticeably dirty.

Microscopes A microscope is an instrument used to see objects that are too small for

the naked eye. The science of investigating small objects using such an

instrument is called microscopy.

In Clinical labs the microscope is an essential instrument for the diagnosis

of disease.

There are many types of microscopes, the most commonly used is the

optical microscope which uses light to image the sample.

Other major types of microscopes are the electron microscope (both the

transmission electron microscope and the scanning electron microscope)

Components of a microscope

The various components of the microscope can be classified into four

systems:

Support system

Magnification system

Illumination system

Adjustment system.

Magnification System• Consists of a system of lenses.

The lenses of the microscope are

mounted in two groups:

• The first group of lenses is at the

bottom of the tube, just above the

preparation under examination (the

object), and is called the objective.

• The second group of lenses is at the

top of the tube and is called the

eyepiece.

Objectives of the microscopes The magnifying power of each objective is shown by a

figure engraved on the sleeve of the lens:

— the x10 objective magnifies 10 times;

— the x40 objective magnifies 40 times;

— the x100 objective magnifies 100 times.

(The X100 objective is usually marked with a red ring to show that it must be used with immersion

oil.)

Immersion oil increases the resolving power by focusing light rays on the sample

Eyepiece Magnification

a x5 eyepiece magnifies the image produced by the objective five times;

a x10 eyepiece magnifies the image 10 times.

If the object is magnified 40 times by the x40 objective, then by five times

by the x5 eyepiece, the total magnification is: 5 x 40 = 200 times

Illumination system Light source It is provided by a lamp built into the microscope beneath the stage, MirrorThe mirror reflects rays from the light source onto the object CondenserThe condenser brings the rays of light to a common focus on the object DiaphragmIs used to reduce or increase the amount of light that passes into the condenser.

Resolving power of a microscope

The resolving power of an objective is its ability to reveal closely adjacent

details as separate and distinct.

The greater the resolving power of the objective, the clearer the image.

A good resolving power of a good medical laboratory microscope is

about 0.25 micrometer (the resolving power of the normal human eye is

about 0.25mm).

Care and maintenance Heavy contamination can be removed with mild soapy solutions.

Grease and oil can be removed with the special cleaning solution with a

50 : 50 mixture of distilled water and 95% ethanol.

The mechanical parts should be periodically cleaned and lubricated with

machine oil to make them run freely.

In hot, humid climates fungi may grow on the microscope, particularly on

the surface of the lenses. This can be prevented by keeping the

microscope under an airtight plastic cover when not in use, together with

a dish filled with blue silica.

Electron microscopes

Is a type of microscope that uses a beam of electrons to illuminate the

specimen and produce a magnified image.

Electron microscopes have a greater resolving power than a light-powered

optical microscope, because electrons have wavelengths about 100,000

times shorter than visible light, and can achieve better than 50 pm

resolution and magnifications of up to about 10,000,000x

The electron microscope uses electrostatic and electromagnetic "lenses" to

control the electron beam and focus it to form an image. These lenses are

analogous to, but different from the glass lenses of an optical microscope that

form a magnified image by focusing light on or through the specimen.

Electron Microscopes

Electron Microscopes

Scanning Electron Microscopes

Thermocyclers A laboratory instrument that repeatedly cycles through a series of

temperature changes required for chemical reactions such as the

polymerase chain reaction or PCR.

PCR is used to make multiple copies of DNA. This process is called

amplification because it can generate million copies of DNA.

PCR is used to produce ample of quantities of DNA when only a small

amount is available. It is a valuable tool for DNA analysis, disease diagnosis,

and genetic engineering.

The technology is regularly used in crime scene analysis to collect DNA from

traces of blood, hair, saliva, or skin.

Thermo cyclers

PCR

Stage one is a hot stage called

denaturation At 90◦C temperature

opens up the DNA for copying.

Stage two is a cooling or annealing that

permits the DNA to attach to chemicals

needed to copy it.

Stage three is a warm temperature

cycle called the extension. It

encourages the growth of the DNA

strand.

The effectiveness of a thermo cycler is its ability to

change temperature rapidly with precision.

Part of the heating and cooling efficiency is due to the

small thin reaction tubes used in the thermo cycler.

Samples of DNA to be copied are placed in minuscule

containers called micro tubes.

The thermo cycler is set to run a particular number of

these cycles depending on the amount of DNA a person

wants to collect.

Applications of PCR

Medicine: Detection of mutations in genes causing tumor.

Infectious disease: early diagnosis of viruses such as AIDS.

Forensic: Human DNA fingerprinting.

Research: isolation of certain DNA or RNA regions.

Thermo cycler Specification

Sample block should have 96x 0.2 ml and have option of dual block of 2 X

48 X .2 ml wells in addition to the traditional micro tubes.

Independent control for all blocks.

High ramp rate of 3-5 °C/ sec.

Temperature range should be 0-100°C with high accuracy.

High Temperature uniformity.

Programmable (up to 1000 programs)

Security features should be password protected.

Option of using the instrument through a PC

USB peripheral compatibility

Assignment

Goseph Adel is selected to make the assignment Different applications of

PCR

The Assignment should be delivered before next lecture

Study questions

Mentions 3 different applications of PCR

Mention the main difference between different types of incubators

Suggesting reading

Encyclopedia of Medical Devices and Instrumentation, 2nd ed. New York: Wiley, 2006