staining techniques and dark field microscopy

8/3/2019 Staining Techniques and Dark Field Microscopy

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At the end of this presentation..........

Student should know the of

purpose,importace and their uses of Gramand ZN staining.

Also can differentiate between them.

He/she should know Darkfield microscopy

,importances and their advantages &Disadvantages.


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Purpose

To view cellular morphology

What type of cell wall a bacterium has

Gram Positive Thicker peptidoglycan layer and no outer

membrane

Gram Negative

Thinner Peptidoglycan and an out membrane


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Crystal Violet Primary stain; positive stain

water-soluble dye

Stains cell wall purple

For one minute

Iodine Mordant

For three minutes

Ethanol

Decolorizer For 30 seconds

Safranin Counterstain

Gives Pink color

For 1 to 2 minutes


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Gram + Coccus and

Human cells

Gram Coccobacilli and

Human cells


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Purpose To view cellular morphology

Diagnostic purposes

Mycobacterium Mycolic acids: Wax like lipid (hydrophobic)

Requires special stain

Mycobacterium tuberculosis

Mycobacterium leprae


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Carbolfuchsin Primary stain For 8 minutes Red Lipid soluble; phenol

Acid alcohol Decolorizer For about 15 to 20 seconds Removes carbol fuchsin that has not bound to a mycolic

acid. Methylene blue

Counterstain For 30 seconds Cannot penetrate mycolic acid; provides contrast to

non acid fast cells.


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Describes microscopy methods, in both

light and electron microscopy, whichexclude the unscattered beam from theimage. As a result, the field around thespecimen. Dark field microscopes are

used in a number of different ways toview a variety of specimens that arehard to see in a light field unit.


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1. The steps are illustrated in the figure where an upright

microscope is used.2. Light enters the microscope for illumination of the sample.

3. A specially sized disc, the patch stop blocks some light fromthe light source, leaving an outer ring of illumination.

4. The condenser lens focuses the light towards the sample.5. The light enters the sample. Most is directly transmitted,

while some is scattered from the sample.

6. The scattered light enters the objective lens, while thedirectly transmitted light simply misses the lens and is not

collected due to a direct illumination block7. Only the scattered light goes on to produce the image,

while the directly transmitted light is omitted.


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There are multitudes of other ways to use darkfield illumination, often when the specimen isclear or translucent:

1. Living or lightly stained transparent specimens

2. Single-celled organisms3. Live blood samples

4. Aquatic environment samples (from seawaterto pond water)

5. Living bacteria6. Hay or soil samples

7. Pollen samples


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No one system is perfect, and dark fieldmicroscopy may or may not appeal to youdepending on your needs:

Advantages of using a dark fieldmicroscope are:

I. Extremely simple to use

II. Inexpensive to set upIII. Very effective in showing the details of

live and unstained samples


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Disadvantages are:

Limited colors (certain colors will appear, butthey're less accurate and most images will be

just black and white). Images can be difficult to interpret to those

unfamiliar with dark field microscopy.

Although surface details can be very

apparent, the internal details of a specimenoften don't stand out as much with a darkfield setup.


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Thank you for your time

Any Questions

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