1. Gives a suitable title for Figures 1 and 2.
a. Figure 1: Anatomy of Embryonated Egg
b. Figure 2: Different Site of Inoculation for Different Types of Viruses 2. Explain how to prepare sucrose gradient for purification of viruses. * Sucrose gradients (density gradients) can be done by placing layer after layer of sucrose (gradient media) in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. * The sample is placed on top of the layer and centrifuged. * A discontinuous sucrose density gradient is prepared by layering successive decreasing sucrose densities solutions upon one another. * Example of preparation and centrifugation of a discontinuous gradient containing sucrose solutions from 15-35 %. Figure 1. Setup for the sucrose gradient preparation.
Example of 15-35 % sucrose density gradient preparation
1. The preparation of sucrose solutions as below.
Preparation of sucrose solutions
1. Add 1.5g, 2g, 2.5g, 2.75g, 3g and 3.5 g sucrose to six 15 ml tubes polypropylene centrifuge tubes (Corning) 2. Fill each tube to 10 ml with
a. 50 mM Tris. HCl pH 7.5,
b. 1 mM EDTA,
c. 0.05% lauryl maltoside.
3. Turn the tubes on a rotator for approximately 20 minutes until all the sucrose has dissolved.
2. First, a Beckman polyallomer tube is held upright in a tube stand. 3. Next, a yellow (200 ml) pipettor tip is placed at the end of a blue (1000 ml) pipettor tip. 4. Both are fitting closely and held steady by a clamp stand and the end of the yellow tip is allowed to make contact with the inside wall of the tube. 5. Then, sucrose solutions can be placed inside the blue tip (the solution will enter the tube slowly and steadily), starting with the 35% solution (volumes of solutions are shown below). (Note: if the solutions fail to flow through the tips and into the tube, gently tapping on the wide end of the blue tip).
Volumes of solutions used
| Solution| Volume|
1(top)| Sample (virus)| 0.5 ml|
2| 15 % sucrose | 1 ml|
3| 20 % sucrose | 1 ml|
4| 25 % sucrose | 1 ml|
5| 27. 5 % sucrose | 0.75 ml|
6| 30 % sucrose | 0.5 ml|
7 (bottom)| 35 % sucrose | 0.25 ml|
| Total| 5 ml |
6. Once the 35 % solution has drained into the tube, the 30 % solution can be loaded into the blue tip which will then flow down the inside of the tube and layer on top of the 35 % solution. 7. This procedure is continued with the 27.5%, 25%, 20% and 15% respectively. 8. At the top of the tube, placed the 0.5 ml sample.
Anatomy of the embryonated egg
* It is important to know the anatomy of the embyonated egg for inoculation of various types of viruses. * It also important to know the develoment progress of the embryo in order to observed the physical changes of the embryo done by the virus and the time when the virus infects and kills the embryo. * To examine the structure of embryonated eggs at various stages of development, the entire contents of the egg need to be remove into a Petri dish.
Shell and shell membrane| * The membrane is closely attached to the shell. * Function: act as an exchange system and gaseous and liquid molecules pass in both directions. * This is the reason eggs must be incubated in humid conditions. * Eggs incubated in low humidity will lose moisture and eventually the embryo will die.| Air sac| * Eggs have a rounded and a pointed end. * The air sac is the space at the rounded end and has a function in respiration and pressure adjustments.| Chorioallantoic membrane and allantoic cavity| * The membrane attached to embryo and functions to remove soluble, insoluble and gaseous waste products. * As the embryo develops, the sac increases in size. * The sac contains allantoic fluid which virus is shed after inoculation of the...