microscopy

Microscopy

Dr. Ashish Jawarkar M.D.

Consultant Pathologist

Parul Sevashram Hospital

Dr. Ashish V. Jawarkar

Types of Microscopes:

1. Compound Light Microscope (what we use most often)

2. Stereoscopes – also known as dissecting scopes

3. Electron Microscopes


Parts of the Microscope

Arm



Light Source

Diaphragm



Stage

Stage Clips



Revolving Nosepiece

Objective Lenses


Parts of the MicroscopeOcular Lens



Coarse adjustment knob

Used only when low power objective is used!!



Fine adjustment knob


Important Vocabulary :

magnification \mag-ne-fe-'ka-shen\ n 1. apparent enlargement of an object 2. the ratio of image size to actual size A magnification of "100x" means that the image is 100 times bigger than the actual object.

resolution \rez-e-loo-shen\ n 1. clarity, sharpness 2. the ability of a microscope to show two very close points separately


Carrying a Microscope



Arm


Steps to Use:

1. Rotate the low power objective into place and make sure the stage is all the way down.

2. Place slide on stage making sure object to be viewed is centered over the hole in the stage. Use the stage clips to hold the slide in place.

3. Turn light on.

4. Focus first with the coarse adjustment knob. Once in focus on low power, turn the nosepiece until the next higher lens is in place.

5. Use FINE adjustment knob ONLY and focus the object.


Techniques of Light Microscopy

• Preparation of Specimens for the Light Microscope:

• 1) Wet Mounts- drop of medium with microbes is spread on a slide

• 2) Smears- microbes from a loopful of medium are spread on a slide, then heat fixed to kill microbes

- heat fixation-


Making a wet mount:


Wet Mounts:

Poorly Done:

Nicely Done:


Principles of Staining

• Stain- dye that binds to a cellular structure and gives it color

• + charge-basic= methylene blue, crystal violet, safranin and malachite green

• - charge-acidic= eosin and picric acid• Simple stain- single dye and reveals basic cell

shapes and structures• Differential stain- 2 or more dyes: Gram stain,

Ziehl-Neelsen acid fast and spore


Gram Stain

• Gram Stain- 1884 crystal violet (+) and iodine and ethanol decolorizer, and counterstained with safranin (-)

• Gram +=purple

• Gram - = red

• Gram non reactive= no stain

• Gram Variable= stain unevenly


Special Staining Procedures

• Ziehl-Neelsen Acid-Fast Stain- 1882 modification of Ehrlich staining method- Acid fast retain red color in cell walls

• Negative staining-capsule is present and won’t take up stain

• Flagellar staining- coats flagella so they can be seen

• Endospore staining- Schaeffer-Fulton stain


Recording what you see:

Include:

1. Figure #: and Title

2. Labeled drawing of the field of view. Label on the right using straight lines which should never cross.

3. Common and scientific name of organism.

4. Magnification you were viewing when you drew the organism: ocular X objective


Remember:

1. If you are seeing perfectly round, clear circles then you just may be looking at air bubbles. Check your slide and try again.

2. Microscopes must always be properly put away.

3. Slides and cover-slips should be washed, dried, and returned to their proper place.

Health & Medicine

microscopy