Section D Live Dye Partial Organells and Observe Them with Microscope April,2013 Department of Cell Biology School of Basic Medical Sciences Xinjiang Medical University Nafeisha Kadeer

Purposes: Master the method to live dye partial organells--mitochondria and vacuole system. Master the basic structure and distribution of mitochondria, vacuole system in cell.

Contents: 1.Live dye the mitochondria in the rabbit liver cell. 2.Live dye the vacuole system in the swordlike cartilage cell of toad. 3.Observe the Golgi Apparatus.

Materials 1. microscope,slide,coverslip,pipette, tweezers,absorption tissue,dissection plate,dissection needle,dissection scissors,eye scissors,culture vessel,nails,thick plastic gloves; 2. a rabbit,a toad; 3. 1/300Jaun ’ s Green 1/3000Neutral red 0.85% Ringer solution 0.65% Ringer solution

1 、 The method to live dye the mitochondria in the rabbit liver cells Principle Jaun’s Green is a special live dye for mt. The cytochrome oxidase in the mt can oxidize this dye into blue particles, so we can examine the existence of the mt by these blue particles.

Inject air into rabbit’s ear vein vein artery

Method Kill a rabbit (inject air into it’s ear vein), put it on the dissection plate on back, then open it’s abdominal cavity. Cut down a small piece of liver tissue from thinnest rim, put it into cultured dish, with Ringer’s solution wash off the blood. Transfer liver tissue on slide, make it vertial. From it’s bottom add one drop of Jaun’s Green to half-cover it, stained for about 20 minutes. Dispart tissue using tweezer, remove the large blocks tissue. Finally some single cells are left on slide. Add a drop of Ringer’s solution, then cover it with coverslip, absorb excess solution. Observe your sample with microscope.

Observe the slide in the microscope. Using the objective of 10x magnification, then the objective of 40x magnification. We can see there are many blue particles in the cytoplasm of the liver cells, which are arranged tightly. These blue particles show location of mt. Result:

Mitochondria in liver cell nucleus cell membrane mitochondria (high power objective)

2 、 How to live dye the vacuole system in the swordlike cartilage cells of toad Principle In cytoplasm of animal cell, the vesicles enclosed by the membrane all belong to vacuole system (except mt), including Golgi Apparatus 、 Lysosome 、 Endoplasmic Reticulum ， etc. The cartilage cells contain abundant vacuole system. However, these vacuoles are too small to be seen if they are not stained with dye. Neutral red can specially stain the vacuole system dark red and stain cytoplasm and nucleus light red. So we can observe location of vacuole system.

Method Kill a toad, open it’s abdominal cavity, then expose the swordlike cartilage of breastbone. Cut down a small piece of cartilage tissue from its thinnest rim, put it on slide. Add a drop of Neutral red to dye for 15 minutes (not more than 15 min). Remove the dye using the absorbent tissue. Add a drop of Ringer’s solution, add coverslip.Absorb excess solution. Observe your sample with microscope.

Swordlike cartilage of toad swordlike cartilage

Under microscope, we see cartilage cell is oval. There are many rosy vesicles.These rosy vesicles are the vacuole system. Result:

3 、 Observe Golgi Apparatus There are prepared slide of rabbit nerve knot. First observe it using low power. We can see many oval neurons. It is oval and stained yellow. Then observe the neurons using high power. We can see the round and transparent central parts are nucleus of neuron. In cytoplasm there are many dotlike 、 sticklike and threadlike structures scattered and stained dark tan. These dark tan structures are Golgi Apparatus.

(high power objective) nucleus nucleolus Golgi Apparatus