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RP 02 - Calculating Mitotic Index Using Plant Cells

This document provides instructions for a required practical to prepare stained squashes of plant root tip cells and calculate the mitotic index by identifying stages of mitosis under a microscope. The procedure involves cutting root tip samples, staining them with toluidine blue O, viewing under a microscope at low magnification, and counting the number of cells undergoing mitosis to calculate the ratio of mitotic to total cells. Safety precautions are outlined for using hydrochloric acid, toluidine blue stain, scalpels, and broken glass.

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Hailey Wong
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76 views

RP 02 - Calculating Mitotic Index Using Plant Cells

This document provides instructions for a required practical to prepare stained squashes of plant root tip cells and calculate the mitotic index by identifying stages of mitosis under a microscope. The procedure involves cutting root tip samples, staining them with toluidine blue O, viewing under a microscope at low magnification, and counting the number of cells undergoing mitosis to calculate the ratio of mitotic to total cells. Safety precautions are outlined for using hydrochloric acid, toluidine blue stain, scalpels, and broken glass.

Uploaded by

Hailey Wong
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 PDF, TXT or read online on Scribd
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AQA Biology A-Level

Required Practical 2
Preparation of stained squashes of cells from plant root tips;
set-up and use of an optical microscope to identify the
stages of mitosis in these stained squashes and calculation
of a mitotic index.

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Plant cells undergo mitosis at shoot and root tips in areas called ​meristems. ​Cells in the
meristems are ​totipotent​​ and retain the ability to ​differentiate​​.
The ​mitotic index​​ of a sample is the ​ratio​​ of cells ​undergoing mitosis​​ to the total number of
cells in a ​sample.​​ To find the ​mitotic index​​, cells from the meristem must be viewed under an
optical microscope​​.

Equipment list
● Optical microscope
● Microscope slides and cover slips
● Water bath
● Hydrochloric acid
● Toluidine blue O stain
● Distilled water
● Scalpel
● Forceps
● 100 ml beaker
● Root tip

Method
1. Heat 1 mol dm​-3 HCl
​
at ​60​​°C​​ in a water bath.
2. C​ut a small sample of the ​root tip​​ using a ​scalpel​​.
3. Transfer root tip to ​HCl​​ and ​incubate​​ for 5 minutes.
4. Remove from HCl and wash sample in cold distilled water and ​remove the very
tip ​using a scalpel.
5. Place tip on a microscope slide and add a few drops of ​stain​​ (e.g. ​toluidine blue
O​​). This makes the ​chromosomes visible​​ and will therefore show which cells
are undergoing mitosis.
6. Lower the cover slip down carefully onto the slide. Make sure there are ​no air
bubbles ​in the slide which may distort the image, and that the​ coverslip doesn’t
slide sideways which could damage the chromosomes.
7. Place under a microscope and set the ​objective lens​​ ​on the lowest
magnification.
8. Use the ​coarse adjustment knob​​ ​to move the lens down to just above the slide.

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9. Use the ​fine adjustment knob​​ ​to carefully re-adjust the focus until the image is
clear​​ (you can use a higher magnification if needed).
10. To calculate ​mitotic index​​, ​cells ​undergoing mitosis​​ must be counted (cells
with ​chromosomes visible​​), as well as the​ total number of cells​​.

Risk Assessment
Hazard Risk Safety Precaution In emergency Risk
Level

Hydrochloric May cause Wear eye Wash off skin Low


acid harm/irritation to protection; avoid immediately;
eyes or in cuts contact with skin, tie flood eye/cuts
up long hair with cold water

Toluidine blue May cause Wear eye Wash off skin Low
O stain harm/irritation to protection; avoid immediately;
eyes or in cuts contact with skin flood eye/cuts
with cold water

Scalpel Cuts from sharp Cut away from Elevate cuts; Low
object fingers;use forceps apply pressure;
to hold sample seek medical
whilst cutting, keep assistance
away from the edge
of the desk

Broken glass Cuts from sharp Take care when Elevate cuts; Low
object handling slides and apply pressure;
coverslips; keep do not remove
glassware away glass from
from edge of desk wound; seek
medical
assistance

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