Basics of staining

7/27/2019 Basics of staining

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Microscopy

Dr. Ashish Jawarkar M.D.

Consultant Pathologist

Parul Sevashram Hospital


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Dr. Ashish V. Jawarkar

Types of Microscopes:

1. Compound Light Microscope (what we

use most often)

2. Stereoscopesalso known as dissecting

scopes

3. Electron Microscopes


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Parts of the Microscope

Arm


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Light Source

Diaphragm


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Stage

Stage Clips


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RevolvingNosepiece

Objective

Lenses


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Parts of the MicroscopeOcular Lens


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Coarse adjustment knob

Used only when low power objective is used!!


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Fine adjustment knob


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Important Vocabulary :

magnification \mag-ne-fe-'ka-shen\ n1.

apparent enlargement of an object 2.the ratio of image size to actual sizeA magnification of "100x" meansthat the image is 100 timesbigger

than the actual object.

resolution \rez-e-loo-shen\ n1. clarity,sharpness 2. the ability of a

microscope to show two very closepoints separately


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Carrying a Microscope


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Arm


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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 onlow power, turn the nosepiece until the next higher lens is in

place.

5. Use FINE adjustment knob ONLYand focus the object.


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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, thenheat fixed to kill microbes

- heat fixation-


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Making a wet

mount:


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Wet Mounts:

Poorly

Done:

NicelyDone:


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Principles of Staining

Stain- dye that binds to a cellular structureand gives it color

+ charge-basic= methylene blue, crystalviolet, safranin and malachite green

- charge-acidic= eosin and picric acid

Simple stain- single dye and reveals basiccell shapes and structures

Differential stain- 2 or more dyes: Gramstain, Ziehl-Neelsen acid fast and spore


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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


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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

wont take up stain Flagellar staining- coats flagella so they

can be seen

Endospore staining- Schaeffer-Fulton stain


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Recording what you see:

Include:

1. Figure #: and Title

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

3. Common and scientific name of organism.

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


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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.

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Basics of staining