Lab # 1; Basic Microscopy and lab safety

1. Lab # 1; Basic Microscopy and lab safety Medgar Evers College, CUNY Prof. Santos Spring 2014

2. Containment • This term is used to describe safe methods and procedures used for managing infectious materials in the lab where they are being handled or maintained. • 2 types; primary and secondary

3. 3 elements of containment 1- lab practice and technique 2- safety equipment 3- facility design

4. The purpose of containment To reduce or eliminate exposure of lab workers, other people, and the environment to potentially hazardous materials.

5. primary • The protection of personnel and the immediate laboratory environment from exposure to infectious agents. • This is provided by good microbiological techniques and the use of appropriate safety equipment. • The use of vaccines may increase protection.

6. secondary • The protection of the environment external to the lab from the exposure to infectious materials. • This is provided by a combination of facility design and operational practices. • Can you name any facility designs involved here?

7. Biosafety level (BSL) • BSL-1, not likely to pose a disease risk to healthy adults. Example; Bacillus cereus • BSL -2, poses a moderate risk to healthy adults, unlikely to spread throughout the community, and effective treatment is readily available. Example: E.coli

8. BSL-3, Can cause disease in healthy adults, may spread to the community, and effective treatment is readily available. Example: Yersinia pestis • BSL-4, Can cause disease in healthy adults, poses a lethal risk and does not respond to vaccine or anti microbial therapy easily. Example: Lassa virus

9. We will only work with BSL levels 1 and 2. • Please familiarize yourself with these on page 7. The past 2 departmental midterms have included a question about this!

10. AIM • Exercises 1, 2, and 3.

11. Compound light microscope

13. Brightfield microscopes • A brightfield microscope allows light to pass directly to the eye without being deflected by an intervening opaque plate in the condenser.

14. Our microscopes are binocular! The inter-ocular distance is changed by simply pulling apart or pushing together the oculars! • To make diopter adjustments, focus first with the right eye, and then without touching the focusing knobs, turn the diopter adjustment ring on the left ocular until a sharp image is seen.

15. When we first use a microscope, start with the low power dry objective, focus with the coarse adjustment and center your specimen.

16. 3 lens system, eyepiece, objectives and the condenser! • Know the parts of the microscope!

17. A- Body tube- connects the eyepiece with the rotating nosepiece. B-eye piece- the lens the observer looks through. C-rotating nosepiece- holds the objectives and allows you to switch from one to the other. D-objective lens- the lens found on the nosepiece that magnifies the image. Most microscopes contain three; the low power, high power, and the oil immersion lens.

18. E-coarse adjustment knob-allows you to move the stage closer to the objective lens and focus the specimen. When you first place a slide in the microscope, you should always start with low power and focus with the coarse adjustment. F-fine adjustment knob-allows you to move the stage very slowly and finely focus the specimen under high power. Keep in mind that when you move to high power; the amount of light passing through will diminish. You might need to adjust the diaphragm.

19. G-stage- broad flat platform where you place the slide. H-Mechanical stage- a clamping device used to hold and move slide around on the stage. I-diaphragm- a circular flat wheel underneath the stage that allows you to control the amount of light passing through. J-arm- used to carry the microscope and for support. K-light source- either a mirror or a light bulb.

20. Magnification • *To determine the total magnification power, multiply the power of the eyepiece with the power of the objective lens. For example; if the eyepiece is 10x and you are using the high power, which is 40x, then the total magnifying power is 400x. So, it means you are looking at the object 400x closer.

21. Resolution • Resolution- the ability of a microscope to separate and show two points that are very close together. A good microscope has high resolution and magnification. If you buy one of those cheap toy microscopes, it might allow you to see an object 400x closer but it has weak resolution. You will see everything blurry.

22. What is the limit in most compound light microscopes? • The limit of most light microscope is 1000x. • The resolving power is the ability to completely separate 2 points that are close together in a microscope field.

23. Resolving power Resolving power is • d = .5 (wavelength) / NA • d= distance between the 2 points • Wavelength is the wavelength of light used to observe specimen • NA = numerical aperture • NA describes how the condenser concentrates and focuses the light rays from the light source.

24. The greater the NA, the better the resolution, and the smaller the distance between the objects! • The refraction of light decreases the NA, due to less light rays passing through the specimen. The result is that the resolving power is diminished!

26. The use of immersion oil • Prevents the diffraction of light due to the fact that oil has similar refractive index as glass. • Refractive index is the speed at which light travels through a medium. • You should know Air= 1.000293 Water = 1.333 Glass = 1.51714 Oil = 1.52

27. limit of resolution in most light microscope (.2um). This means 2 objects closer than this will appear as one!

28. To maximize resolving power Know steps taken to maximize resolving power such as 1-use a blue filter 2-keep condenser at highest level 3-the diaphragm should not be stopped down too much 4-use oil immersion oil when using the 100x lens to minimize the bending of light rays since oil has the same refractive index as glass.

29. Relationship between working distance and magnification • As the magnification increases, the working distance decrease. • Working distance is the distance between the objective and the specimen.

30. Depth of field is the range of depth that a specimen is in acceptable focus. Look at a cross thread slide and determine which thread is on top! The first thread to come into focus when you raise the stage is the one on top!

31. Letter “e” slide When you look under the microscope, the image appears upside down/ reverse. A series of mirrors and lens is responsible for this.

32. Phase contrast microscope Is the best microscope for observing living unstained cells. It is able to enhance the contrast between a cell and its environment allowing you to see the specimen without staining. Staining kills the cells and sometimes we want to see living cells.