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

Dr. Ashish V. Jawarkar

Parts of the Microscope

Arm

Dr. Ashish V. Jawarkar

Parts of the Microscope

Light Source

Diaphragm

Dr. Ashish V. Jawarkar

Parts of the Microscope

Stage

Stage Clips

Dr. Ashish V. Jawarkar

Parts of the Microscope

Revolving Nosepiece

Objective Lenses

Dr. Ashish V. Jawarkar

Parts of the MicroscopeOcular Lens

Dr. Ashish V. Jawarkar

Parts of the Microscope

Coarse adjustment knob

Used only when low power objective is used!!

Dr. Ashish V. Jawarkar

Parts of the Microscope

Fine adjustment knob

Dr. Ashish V. Jawarkar

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

Dr. Ashish V. Jawarkar

Carrying a Microscope

Dr. Ashish V. Jawarkar

Parts of the Microscope

Arm

Dr. Ashish V. Jawarkar

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.

Dr. Ashish V. Jawarkar

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-

Dr. Ashish V. Jawarkar

Making a wet mount:

Dr. Ashish V. Jawarkar

Wet Mounts:

Poorly Done:

Nicely Done:

Dr. Ashish V. Jawarkar

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

Dr. Ashish V. Jawarkar

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

Dr. Ashish V. Jawarkar

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

Dr. Ashish V. Jawarkar

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

Dr. Ashish V. Jawarkar

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.