Lab Report
GEN201
Mitosis in Allium cepa root tips
Name: David Beckham
Submitted to TA: zidane
Introduction:
DNA Extraction is the most important mechanism of genetic material isolation across
diverse biological research domains. It is most imperative particularly in molecular
biology investigations or forensic analysis processes conducted in scientific
laboratories. Moreover, DNA extraction is a continuously explored field by the genetic
and biotechnological research communities, with critically sensitive subjects such as
the implementation of genomic studies for personalized medicine, raising
methodological concerns among scientific fraternity. None the lesser, boiling
extraction techniques contributed in a wide range of subjects, including microbial
identification, archaeological genetic research, and forensic diagnostics made
possible through thermal-based molecular investigation techniques.
Aim:
Observe mitosis phases under microscope.
Material:
Biological:
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Allium cepa roots
Chemical:
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Conc HCl
Acetocarmine stain
Fulgen
Ethanol
Tools:
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Light microscope with 10x and 40x magnification
Water bath
Eppendorf
Slides
coverslips
Paper filters
Methodology:
Protocol (1)
Allium cepa root tip preparation
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Allium cepa roots were stored in 70% ethanol in the refrigerator.
Roots were rinsed in an Eppendorf with distilled water one time and carefully
Shaken.
1ml HCl was added into Eppendorf and placed the Eppendorf in a 60°C water
bath for 5 minutes.
HCl was poured off, and the hydrolysed roots were rinsed with distilled water
two times.
(1)
1. Allium cepa root tip was prepared protocol (1)
2. Tissue was transferred to a glass slide using forceps.
3. The tip of the meristematic tissue was cut with a coverslip.
4. Two drops of acetocarmine stain were added on top of the roots, and left for 5
minutes.
5. A coverslip was placed over the tissue on the glass slide.
6. The coverslip was gently pressed with a the bottom of a dropper to spread the
cells into a thin layer.
7. Excess dye was removed with bits of a paper filter.
8. The slide was examined under a 10x and 40x lens.
(2)
1. Allium cepa root tip was prepared protocol (1)
2. The tissue is carefully transferred to a clean Eppendorf filled with 1ml Feulgen
stain using forceps and incubated for 10 minutes.
3. The root tip is removed from the stain and transferred to a clean slide.
4. The dark purple color of the meristematic region is carefully cut using the
coverslip.
5. A coverslip is placed over the tissue and was gently pressed with a the bottom
of a dropper to spread the cells into a thin layer.
6. The slide was examined under a 10x and 40x lens.
Results:
(1)
A blurry sequence of cells was observed (troubleshoot) which was caused by
excessive pressing on the tissue.
Figure 1
Acetocarmine stain
(2)
No results (troubleshoot).
*The tip was unavailable.
Discussion:
In this lab report, we observed the stages of mitosis occurring in the root tips of
Allium cepa. Our main objective was to stain and view several phases of mitosis
such as prophase, metaphase, anaphase, and telophase by using acetocarmine
stain and 8pressing the root tip tissue under a coverslip to spread the cells into a
single layer. However, due to the mistakes realized during slide preparation, the
result was negative because the cells were not clear since they burst, and this did
not permit a good observation of the phases of mitosis. Moreover, the first two steps
of the experiment consisted of inducing root growth of the onion and the preparation
of a fixative solution composed of acetic acid and ethanol. This fixation was
important in maintaining the integrity of cellular structure since any unprepared tissue
in solution would present distorted cellular features. These were then hydrolyzed in 1
ml of HCl in a 60°C water bath for 5 minutes to soften the cell walls. This was to
ensure that individual cells were well separated and could be appropriately
squashed. Tips were stained with the acetocarmine stain, to which chromosomes
have a preference for binding, hence making them visible under the microscope.
Over-staining takes away the excess stain through background coloration that hides
the chromosomes, while under-concentration makes the cell structures too pale or
blurred. According to the protocol, the key steps involved the spreading of the soft
cells of the root tip tissue by pressing with a coverslip. In this technique, controlled
pressure is necessary in order to spread the cells with even boundaries, not hard
enough to rupture the cells. If excessive pressure is applied, the cells rupture into
indistinct cell boundaries. Another good method is to take the eraser end of a pencil
or some other soft tool with a flat surface, and by light, progressive tapping, check
under the microscope whether the cells have spread enough.
Finally, the slide was observed under the microscope if the best results were
accomplished already allowing observation of the different phases of mitosis.
References:
Lab presentation