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PROTOCOL FOR PRACTICALS

PROTOCOL FOR PRACTICALS

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junaidfurhan86
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7 views5 pages

PROTOCOL FOR PRACTICALS

PROTOCOL FOR PRACTICALS

Uploaded by

junaidfurhan86
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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1.

To visualize cells under a light microscope, follow this detailed protocol:

Materials Needed:

 Light microscope
 Glass slides and cover slips
 Staining reagents (e.g., methylene blue, iodine, or crystal violet)
 Pipette or dropper To visualize cells under a light microscope, follow this detailed
protocol:
 Distilled water
 Tissue or blotting paper
 Specimen (e.g., onion peel, cheek cells, or cultured cells)

Procedure:

1. Prepare the Slide:


o Place a small drop of distilled water on the center of a clean glass slide.

Add the specimen (e.g., a thin slice of tissue or a drop of cell suspension) to the water drop
Stain the Specimen (Optional):

 Add a drop of staining reagent to the specimen to enhance contrast and visualize specific
structures.
 Allow the stain to sit for 1-2 minutes, then gently rinse with distilled water to remove
excess stain.

 Cover the Specimen:

 Carefully place a cover slip over the specimen at an angle to avoid air bubbles.

 Set Up the Microscope:

 Turn on the light source and adjust the diaphragm to control the light intensity.
 Place the slide on the stage and secure it with stage clips.
 Focus the Microscope:

 Start with the lowest magnification objective lens.


 Use the coarse adjustment knob to bring the specimen into focus, then fine-tune with the
fine adjustment knob.
 Gradually switch to higher magnification lenses for detailed observation.

 Observe and Record:

 Examine the specimen and note the structures visible under different magnifications.
 Take photographs or draw diagrams for documentation.

2.To prepare a slide for light microscopy, follow this detailed protocol:

Materials Needed:

 Glass slides and cover slips


 Specimen (e.g., tissue, cells, or microorganisms)
 Staining reagents (optional)
 Distilled water
 Pipette or dropper
 Tweezers
 Tissue or blotting paper

Procedure:

1. Clean the Slide:


o Ensure the glass slide and cover slips are clean and free of dust or smudges.
2. Prepare the Specimen:
o For solid specimens (e.g., tissue), slice them thinly to allow light to pass through.
o For liquid specimens (e.g., cell suspensions), use a pipette to place a drop on the
slide.
3. Add Stain (Optional):
o If staining is required, add a drop of the staining reagent to the specimen.
o Allow the stain to sit for 1-2 minutes, then rinse gently with distilled water to
remove excess stain.
4. Mount the Specimen:
o Place the specimen in the center of the slide.
o For liquid specimens, add a drop of distilled water to prevent drying.
5. Apply the Cover Slip:
o Hold the cover slip at an angle and gently lower it onto the specimen to avoid air
bubbles.
6. Remove Excess Liquid:
o Use tissue or blotting paper to absorb any excess liquid around the edges of the
cover slip.
7. Examine Under the Microscope:
o Place the prepared slide on the microscope stage and secure it with stage clips.
o Adjust the focus and light settings to observe the specimen.

3. Detailed protocol for preparing temporary slides to study mitosis from both onion root
tips and buccal mucosa:

1. Onion Root Tip:

Materials Required:

 Onion bulbs
 Glass slides and cover slips
 Aceto-carmine stain (or acetoorcein stain)
 N/10 HCl
 Ethanol (70%)
 Distilled water
 Forceps, blade, and dropper
 Spirit lamp or hot plate
 Watch glass
 Compound microscope
 Blotting paper

Procedure:

1. Growing Root Tips:


o Place an onion bulb in a container with water, ensuring the basal part touches the
water.
o Allow roots to grow for 3–6 days.
2. Fixation:
o Cut 2–3 cm of freshly grown root tips and transfer them to a fixative solution (1
part glacial acetic acid: 3 parts ethanol) for 24 hours.
o Preserve the fixed root tips in 70% ethanol for future use.
3. Hydrolysis:
o Place the root tips in a watch glass and add N/10 HCl.
o Warm gently on a spirit lamp for 3–5 minutes to soften the tissue.
4. Staining:
o Transfer the root tips to a slide and add 2–3 drops of aceto-carmine stain.
o Warm gently to enhance staining and allow it to sit for 10 minutes.
5. Slide Preparation:
o Cut the stained root tip (2–3 mm) and place it on a clean slide.
o Add a drop of water and cover with a cover slip.
o Gently squash the root tip using the blunt end of a pencil to spread the cells.
6. Observation:
o Seal the edges of the cover slip with nail polish or wax.
o Examine under the microscope to observe various stages of mitosis.

2. Buccal Mucosa:
Materials Required:

 Sterile cotton swabs


 Glass slides and cover slips
 Methylene blue stain
 Distilled water
 Dropper
 Blotting paper
 Compound microscope

Procedure:

1. Sample Collection:
o Gently scrape the inner cheek using a sterile cotton swab.
2. Smear Preparation:
o Transfer the collected cells onto the center of a clean glass slide by rolling the
swab.
3. Fixation:
o Allow the smear to air-dry for a few minutes.
o Fix the cells by passing the slide briefly over a flame.
4. Staining:
o Add a drop of methylene blue stain to the smear.
o Let it sit for 1–2 minutes, then rinse gently with distilled water to remove excess
stain.
5. Slide Preparation:
o Place a cover slip over the stained smear, avoiding air bubbles.
6. Observation:
o Examine the slide under the microscope to observe the cells and their nuclei.

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