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The Anatomy of an Embryonated Egg and Inoculation of Virus

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Question: 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.

Discussion:

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.

| Function | 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 allantoic cavity. * Some viruses are spread by inoculation of the chorioallantoic membrane. This involves placing inoculum on the membrane. | Yolk sac | * This is also attached to the embryo and contains the nutrient-rich yolk. * As the embryo develops, the yolk sac decreases in size to approximately 1 cm diameter 3 days before the embryo hatches. | Amniotic Sac | * This sac surrounds the embryo. * It is filled with liquid and serves to protect the embryo against physical damage as well as functioning as an area of exchange of molecules. * As the embryo develops, the membrane stretches and the amniotic sac is barely visible in the fully developed embryo. | Albumin | * The egg white consists mainly of protein. |

Determining the viability of the embryo

* Under the candling lamp, the embryo appears as a dark shadow with the head as a dark spot. * Healthy embryos will respond to the light by moving. * The blood vessels are well defined in a healthy embryo. * After an embryo has died, the blood vessels start to break down then appear as streaks under the shell when viewed under the candling lamp

* Infertile eggs: * Easy to detect as the egg is clear. * Discard * Early deaths: * The embryo has developed for several days and then died. * Candling reveal a small dark area and disrupted blood vessels. * Often deteriorating blood vessels will appear as a dark ring around the egg. * Discard * Late Deaths: * Often difficult to observed from a viable embryo at the same stage of development. * Absence of movement and breakdown of the blood vessels. * Discard * Viable Embryos: * These move in response to the light and have well defined blood vessels. * The eggs return to the incubator.

Procedure on virus culture

1. The eggs undergo candling to mark the air sac and the point to make a tiny hole. 2. A tiny hole is made in order to let the syringe penetrate the egg shell. This step has to be done very carefully without breaking the inner membrane of the eggs. 3. Then, the eggs will undergo inoculation process. 4. 0.1ml of virus is injected in each egg. 5. The syringe was thrust down vertically through the hole on the shell at 45° and the virus is discharged into the allantoic cavity. 6. The hole is immediately sealed with melted paraffin wax to prevent contamination. 7. Then, the eggs are incubated for a few days depending on the virus used.

Harvesting

1. The harvesting step is to collect the allantoic fluid of the eggs. 2. After the incubation, the eggs have to be chilled in fridge at 4°C for at least 2 hour in order to to kill the embryo and to reduce the contamination of the allantoic fluid with blood during harvesting by harden the blood vessel. This step must be done because it can make the process harvesting easier. 3. The eggs have to be swabbed with 70% alcohol around the sealed tiny hole in order to further sterilize the outer surface of the eggs. 4. Then, the egg’s shell is broken with forceps according to the air sack marking. 5. The embryos that are visibly contaminated are discarded. It can be detected by observe the turbidity of allantoid fluid (Normal allantoid fluid should be clear). 6. Sample of allantoic fluid is taken from each egg using a micropipette with sterile tip or sterile glass Pasteur pipette. 7. The allantoic will be harvested into the sterilized bottle (bijou bottles). 8. Few amount of allantoic fluid (e.g. 3ml) from each bottle will be taken out for sterility test. 9. All sample need to be store in refrigerator until results of sterility test is available. 10. 2 tests need to be done to the sample:

a. Test for bacterial contamination i. Inoculate sample on blood agar or adding 1 drop of sample into a tube of thioglycollate medium and incubate it for 2 days in 37⁰C ii. Observing samples of allantoic fluid by centrifuged or stand overnight at 4°C to allow particles including red blood cells to settle.
(The allantoic fluid should appear clear after centrifugation or standing overnight.)

b. Haemagglutination (HA) test (detect presence of virus). i. Detect by adding a drop of 1% chicken RBC and mix with a drop of allantoid fluid. ii. Positive result (clumping occur) shows that the virus present in sample iii. Negative result (no clumping occur) shows that the virus absent in sample

11. The embryos that either contaminated with bacteria or show negative result in HA test should be discard. 12. Aseptic technique must be use to transfer the clear allantoic fluid supernatant from containers that showed no bacterial growth into a sterile container for storage 13. All the bottles will then store at 4⁰C for further use.
Haemagglutination (HA) test

* Haemagglutination test is done to detect the presence of viral particles. * Viruses will agglutinate chicken red blood cells, forming clumping (haemagglutination visible macroscopically). * This is due to haemagglutinin part of the haemagglutinin/ neuraminidase viral protein bind to receptors on the membrane of red blood cells. * This test does not differentiate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells. * It is necessary to use a specific virus antiserum for the specific types of viruses to inhibit the haemagglutinating activity cause by some other viruses and some bacteria. * Substances that agglutinate red blood cells are referred to as haemagglutinins.

Principle:

1. Negative control well (no haemagglutinin) 2. Positive control well (contains haemagglutinin)

Materials:

* Clean glass microscope slide or a clean white ceramic tile. * 10% suspension of washed chicken red blood cells. * Micropipette and tips, glass Pasteur pipette or a wire loop. * Phosphate buffered saline (PBS). * Negative and positive control allantoic fluid samples. * Sample to be tested for the presence of virus (e.g. allantoic fluid)

Method:

1. 4 separate drops of 10% chicken RBCs are placed onto a glass slide or a white tile. 2. For each drop of blood, add one drop of the control and test samples as follows. 3. Use separate tips, pipettes or a flamed loop to dispense each sample.

Drop | | 1 | Phosphate buffered saline (PBS) solution | 2 | DropNegative control allantoic fluid (no haemagglutinin) | 3 | Positive control allantoic fluid (contains haemagglutinin) | 4 | Unknown sample to be tested |

4. Mix by rotating the slide or tile for 1 minute. 5. Observe and record results. Compare results of the test samples with the control samples.
(Note: If testing many samples at the same time, it is necessary to test the negative and positive control samples only once).

Results:

* The RBCs mixed with the positive control allantoic fluid will clump within 1 minute. * The RBCs mixed with the PBS and negative control allantoic fluid do not clump. * The PBS and negative allantoic fluid controls are used to detect clumping of the red blood cells in the absence of virus. (This is unlikely to occur, but if it does occur, the test is invalid).

Result | Interpretation | Positive | Presence of viral particles that may or may not be infectious. | Negative | Absence of viral particles or presence of viral particles in levels too low to detect |

Negative hemagglutination test
Positive hemagglutination test

2
1
STEP 3: mix by gently rotating the slide for 1 minute and observed the result
Add 1 drop of test sample
Add 1 drop of positive control
Add 1 drop of negative control
Add 1 drop of PBS
STEP 2:
4
3
2
1
4
3
2
1
4
3
3
2
1
4

STEP 1: place 4 drops of 10% RBCs on a slide
STEP 1: place 4 drops of 10% RBCs on a slide

2
1
4
3
STEP 2:
Add 1 drop of test sample
Add 1 drop of positive control
Add 1 drop of negative control
Add 1 drop of PBS

3
2
1
4
No agglutination
Agglutination
STEP 3: mix by gently rotating the slide for 1 minute and observed the result

Agglutination
No agglutination
No agglutination

Sucrose-Gradient Technique

* A sucrose density gradient is created in a centrifuge tube by layering solutions of differing densities. * The sample to be tested is placed on top of the gradient. * Centrifugation causes the various components (fractions) of the sample to sediment differentially. * When the solution is centrifuged, the sedimenting material travels through the gradient at different rates that are related to the sizes and shapes of the molecules. * Large molecules migrate farther in a given period of time than smaller molecules do. * The different fractions appear as bands in the centrifuged gradient. * The different bands can be collected separately by collecting samples from the bottom of the tube at fixed time intervals.

Note: * Difference between a sucrose gradient and the CsCl gradient. * CsCl gradient * The sample being studied have a density somewhere in between the lowest and highest concentrations of CsCl generated in the gradient. * Therefore, at equilibrium they will band at a specific point on the gradient. * Sucrose gradient * The sample being studied are denser than any of the sucrose concentrations used, and at equilibrium they would form a pellet (small button) at the bottom of the tube. * However, they migrate toward the bottom at varying speeds, depending on their size and shape. * By comparing the different positions of each molecule in the gradient at a particular time, the relative sizes of the molecules can be determined.

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