# Application of the _Aliium_ Test in Toxicity Studies of Lead and Copper: A Cytological Perspective
## 2 Materials and Methods
### Cytological Assessment  
For cytological analyses, the mitotic rate for each root was calculated after examining ten microscopic fields. The cytological analyses involved evaluating the mitotic index of the meristematic tissues at the root tip of each experimental sample. The mitotic index (MI) was expressed as the ratio of dividing cells to the total number of cells.  
\[\text{Mitotic index (MI)}=\frac{Number\;of\;cells\;in\;division}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{1}\]  
During our study, the number of cells in interphase (Figure 1A) was also monitored. Additional analyses involved monitoring the percentage of cells in probleme (Figure 1B), metaphase (Figure 1C), anaphase (Figure 1D), and telophase (Figure 1E).  
For calculating the interphase index (Ii), probleme index (Ip), metaphase index (Im), anaphase index (Ia), and telophase index (It), the following calculation formulas were used:  
\[\text{Interphase index (Ii)}=\frac{Number\;of\;interphase\;cells}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{2}\]  
\[\text{Propphase index (Ip)}=\frac{Number\;of\;probase\;cells}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{3}\]  
\[\text{Metaphase index (Im)}=\frac{Number\;of\;metaphase\;cells}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{4}\]  
\[\text{Anapphase index (Ia)}=\frac{Number\;of\;anapphase\;cells}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{5}\]  
\[\text{Telophase index (It)}=\frac{Number\;of\;telephase\;cells}{Total\;number\;of\; cells\;un\;ysed}\times 100 \tag{6}\]  
Cytological evaluation of the heavy metals was also conducted, during which cell abnormalities and cells deviating from normal characteristics were monitored (Figure 2).  
In the study, cellular abnormalities were observed at all stages of cell division, including aberrations such as chromosomal bridges (Figure 2a), unevenly distributed metaphases (Figure 2b), isolated chromosomes in anaphase (Figure 2c), isolated chromosomes in metaphase (Figure 2d), sticky chromosomes (Figure 2e), lagging chromosomes (Figure 2f,g), and additional instances of lagging chromosomes (Figure 2k) [57].  
Furthermore, aside from the anomalies during the mitotic division process, cellular abnormalities were also observed during the interphase, when the cell was not undergoing mitotic division, such as binucleated cells (Figure 2i) and giant cells (Figure 2j).  
Figure 1: _Allium sativum_ cells at different stages: interphase (**A**), probleme (**B**), metaphase (**C**), anaphase (**D**), and telophase (**E**) (Optika microscope, magnification: 400\(\times\), original images from the Genetics Department).  
Figure 2: _Allium sativum_ cells that exhibit abnormalities and deviate from normal characteristics: (**a**) chromosomal bridges, (**b**) unevenly distributed metaphases, (**c**) isolated chromosomes in anaphase, (**d**) isolated chromosomes in metaphase, (**e**) sticky chromosomes, (**f**,**g**) lagging chromosomes, (**h**) sticky metaphase, (**i**) binucleated cells, (**j**) giant cells, and (**k**) lagging chromosomes (Optika microscope, magnification: 400\(\times\), original images from the Genetics Department).  
To calculate the abnormalities index (%), the number of abnormal cells was quantified relative to the total number of cells evaluated in the microscopic field (1).  
\[\text{Abnormalities index (AI)}=\frac{Number\ of\ abnormal\ cells}{Total\ number\ of\ cells\ analyzed}\times 100 \tag{7}\]  
After determining the mitotic indices and the abnormalities index for all experimental variants, the data were consolidated based on dose and time to evaluate how these indices were influenced. The data were recorded and subjected to statistical analysis to determine the differences in genotoxicity between \(Cu^{+2}\) and \(Pb^{+2}\).