小鼠睾丸生殖细胞标本制作及减数分裂期染色体观察

Isolation and staining of mouse meiotic metaphase chromosomes 小鼠睾丸生殖细胞标本制作及减数分裂期染色体观察xx 2011级生科四班201100140120 同组者：xx【实验目的】1、learn to isolate mouse testes cells and stain with Giemsa.学会分离小鼠睾丸细胞并用Giemsa染液染色2、Observe the number and morphology of mouse chromosomes观察小鼠染色体的数目及形态特征【实验材料】【Reagents】秋水仙素colchicine solution吉姆萨染液Giemsa stains solution生理盐水physiological saline氯化钾溶液KCl solution固定液fixation solution【Tools and equipments】Dissecting tray，beaker，centrifuge tube，microscope，glass slide，pipette，scissors，forceps，copper mesh.解剖盘，烧杯，离心管，显微镜，载玻片，吸管，剪刀，镊子，铜网【实验原理】meiosis is a process of reductional division in which the number of chromosomes per cell is cut in half. In animals, meiosis always results in theformation of gametes, while in other organisms it can give rise to spores. 减数分裂是细胞分裂染色体数减半的过程，在动物中,减数分裂的结果总是在形成配子,而在其他生物可以产生孢子。

Testosterone is the place where the male germ cell develop and mature,it is the organ that produce sperm. After sexual maturation, sex cells of mammals in the testis is always mature partially. Therefore,have an appropriate treatment to testis enables us to get a variety of chromosomes of different process.睾丸是雄性动物生殖细胞发育和成熟的部位，是产生精子的器官。

哺乳动物在性成熟以后，精巢内的性细胞总是在分批分期不断的成熟，因此，对哺乳动物的精巢进行一定的技术处理，随时都可以获得减数分裂过程中各个时期染色体的标本。

Inject an amount of colchicine solution into the abdomen cavity can prevent the formation of the spindle fiber, so that a large amount of cells in the process of meiotic metaphase can be accumulated. By the normal way of making specimen,we can observe the chromosomes of mouse testosterone用适量的秋水仙素溶液注入动物腹腔内，可以阻止分裂细胞纺锤丝的形成，从而积累大量处于分裂中期的细胞。

利用上述原理，通过常规的制片方法，观察小鼠睾丸细胞的染色体。

Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing. However, the DNA that makes up chromosomes becomes more tightly packed during cell division and is then visible under a microscope. Most of what researchers know about chromosomes was learned by observing chromosomes during cell division.一般情况下，细胞核中的染色体是看不见的，然而，在细胞分裂时，DNA使得染色体变粗变短，在显微镜下就可以观察到。

对于染色体的认知大部分都是通过观察分裂时期的染色体而获得的。

Each chromosome has a constriction point called the centromere, which divides the chromosome into two sections, or “arms.” The short arm of the chromosome is labeled the “p arm.” The long arm of the chromosome is labeled the “q arm.” The location of the centromere on each chromosome gives the chromosome its characteristic shape, and can be used to help describe the location of specific genes.每条染色体都有一个溢痕点称作着丝点，着丝点将染色体分为两部分，短臂称作p臂，长臂称作q臂。

着丝点在染色体上的位置使得染色体呈现各异的形态，可以用于描述特定基因的位置。

【实验方法】1、inject colchicine solution 14~16 hours before experiment.实验前14~16小时注射秋水仙素2、Sacrifice a mouse by cervical dislocation method脱臼法牺牲小鼠3、Open the lower abdomen cavity，find its two testes situated in its scrotum. The testes appear as two round，pink balls lying lowest in the body cavity，close to the edge of each side，as depicted in the red circles.打开腹腔下部，找到两个位于阴囊的睾丸。

睾丸是粉红色的小肉球，在腹腔最下方。

4、Put the testes into a small beaker containing 1ml 0.3% KCl solution. Cut up completely with scissors until solution turn into turbid ivory white.将睾丸放入一个盛有1ml 0.3% KCl溶液的小烧杯中，用剪刀将其完全剪碎直至成为象牙白色的悬液。

5、Filter through copper mesh. Transfer the filtrate into a centrifuge tube. Add KCl solution to a total volume of 4 ml.用铜网过滤。

将滤液转移到离心管中，加入KCl 溶液直到总体积为4ml。

6、Incubate at room temperature for 30min to perform hypotonic treatment.在室温下放置30min进行低渗处理。

7、Spin down at 1000r/min for 8 min，remove the supernatant. Resuspend cell pellet with 1 ml fixation solution.1000r/min 离心8min，弃上清，用1ml 固定液重新悬浮。

8、Incubate at room temperature for 5 min.室温下静置5min。

9、Drop the cell suspension onto a clean precooled slide from the height of10 to 15 cm，tap and blow the suspension to spread cells.从10~15cm的空中将细胞悬液滴到一块干净的冷的载玻片上，轻轻敲打、轻吹使细胞分开。

10、Build two slide supports on the glass plate and place the sample slides on top of them，with the sample side down.将两排载玻片排在玻璃板上，将标本面朝下架在上面。

11、Pipette the Giemsa buffer to the space between slides and the glass plate. The liquid should be spread down to the bottom due to capillary absorption.吸取Giemsa 染液加在玻璃板和载玻片之间，由于毛细作用，液体会扩散到玻片底部。

12、When the space is full of staining solution without bubbles,stain for 30 min.当空间中充满了染色液并没有气泡时，染色30min。

13、Get the sample slide off the bridge and erect them to drain the buffer. Wash the back of slide with slow running water. Dry them in the air and store in the container.将样品从桥上取下，直立以使染液流下。

用细小的水流冲洗载玻片的背面。

自然风干，在容器中保存。

14、Observe under the microscope.镜检观察。