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Cell Cio Labpractical 2

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19 views4 pages

Cell Cio Labpractical 2

Uploaded by

ayeshapinky06
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PRACTICAL NO 2

STUDY OF STAINING TECHNIQUES (GRAM STAINING)


AIM

Step by step study procedure of gram staining.

Material required

1. Clean Glass slides 2. Inoculating loop


2. Bunsen burner 4. Bibulous paper

5. Microscope 6. Immersion oil

7. Lens paper and lens cleaner 8. Distilled water

9. 18 to 20 hour cultures of organisms

Reagents

 Primary stain-crystal violet


 Mordant-grams iodine
 Decolourizer-ethyl alcohol
 Secondary stain- safranin

Procedure

Part 1; preparation of the glass microscopic slide

Grease or oil free slide are essential for the preparation of microbial smears. grease or oil from
the fingers on the slide is removed by washing the slides with soap and water. Wipe the slides
with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.

Part 2; Labeling the slides

Drawing a circle on the underside of the slide using a glass ware –marking pen mmay be helpful
to clearly designate the area in which you will prepare the smear .you may also label the slide
with the initial of the name of organism on the edge of the slide care should be taken that the
label should not be in contact with the staining reagents.

Part 3: Preparation of the smear

 Bacterial suspensions in broth: With a sterile cooled loop, place a loop full of the broth
culture on the slide. Spread by means of circular motion of the inoculating loop to about
one centimeter in diameter. Excessive spreading may result in disruption of cellular
arrangement. A satisfactory smear will allow examination of the typical cellular
arrangement and isolated cells.
 Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or
saline solution on the slide. Sterilize and cool the loop again and pick up a very small
sample of a bacterial colony and gently stir into the drop of water/saline on the slide to
create an emulsion.
 Samples Swab: Roll the swab over the cleaned surface of a glass slide.

Please note: It is very important to prevent preparing thick, dense smears which contain an
excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass
through, thus making it difficult to visualize the morphology of single cells. Smears typically
require only a small amount of bacterial culture. An effective smear appears as a thin whitish
layer or film after heat-fixing

Part 4: Heat Fixing

Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the
sample to more readily take up stains.

 Allow the smear to air dry.


 After the smear has air-dried, hold the slide at one end and pass the entire slide through
the flame of a Bunsen burner two to three times with the smear-side up.

Now the smear is ready to be stained.

Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.
Part 5: Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with crystal violet and let stand for 1 minute.

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

4. Gently flood the smear with Gram's iodine and let stand for 1 minute.

5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The
smear will appear as a purple circle on the slide.

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol
drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-
decolorize.

7. Immediately rinse with water.

8. Gently flood with safranin to counter-stain and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

10. Blot dry the slide with bibulous paper.

11. View the smear using a light-microscope under oil-immersion.


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