Pharos university Faculty of Allied Medical SCIENCE Clinical Laboratory Instrumentation (MELI-201)

1. Pharos universityFaculty of Allied Medical SCIENCEClinical Laboratory Instrumentation(MELI-201) Dr. Tarek El Sewedy

2. Lecture 3 Incubators , Microscopes and Thermo cyclers

3. Intended Learning Outcomes • By the end of this lecture the student should learn the basics of the following: • Cell Incubators used in Clinical Labs • Microscopes • Thermo cyclers.

4. Lecture content • Laboratory Incubators • Microscopes. • Thermo cyclers

5. Cell Incubators • A 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 .

9. Incubator Alarm System

11. 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 fanto keep both the CO2 level and the temperature uniform.

12. Mammalian Cell incubator

13. Bacteriological Incubator

14. Cell incubators According to design • Cell incubators usually come in one of three types of designs: • Air draft. • Dry wall. • Water jacket.

15. 1. Air draft incubators • circulate air throughout the interior of the incubator to maintain constant temperatures. • Thisstyle 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 quicklyand must consistently adjust the air to control the temperature. • These are the most commonly used type of incubators

17. 2. Dry wall incubators • Pass 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

19. Water jacketed incubators • Water-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.

21. 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. • Copperis 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

22. Cleaning of incubators • Remove the humidity pan weekly and autoclave •  Remove all shelves. Autoclave or wash and disinfect as described. • Clean and disinfect all access ports, air bleeds, shaft holes, electrical pass-through. and any other passages into the incubator. • Remove the door gasket and gasket guards. Clean and disinfect. • Replace all air and CO2 filters as needed, approximately every six months or when noticeably dirty.

23. Microscopes • A microscopeis 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 lightto image the sample. • Other major types of microscopes are the electronmicroscope (both the transmission electron microscopeand the scanning electron microscope)

24. Components of a microscope • The various components of the microscope can be classified into four systems: • Supportsystem • Magnificationsystem • Illuminationsystem • Adjustmentsystem.

25. 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. Magnification System

26. 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 focusinglight rays on the sample

27. EyepieceMagnification • 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

28. Illumination system • Light source It is provided by a lamp built into the microscope beneath the stage, • Mirror The mirror reflects rays from the light source onto the object • Condenser The condenser brings the rays of light to a common focus on the object • Diaphragm Is used to reduce or increase the amount of light that passes into the condenser.

29. 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 clearerthe 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).

30. 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.

31. Electron microscopes • Is a type of microscope that uses a beam of electronsto 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 50pmresolution 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.

32. Electron Microscopes

33. Electron Microscopes

34. Scanning Electron Microscopes

35. 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.

36. Thermo cyclers

37. PCR • Stage one is a hot stage called denaturationAt 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.

38. 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.

39. 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. Applications of PCR

40. 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 be0-100°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

41. Assignment • Goseph Adel is selected to make the assignment Different applications of PCR • The Assignment should be delivered before next lecture

42. Study questions • Mentions 3 different applications of PCR • Mention the main difference between different types of incubators

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