Egg inoculation
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1. There are various routes for inoculating eggs with viruses, including the allantoic, chorio-allantoic, yolk sac, and amniotic routes. The allantoic route through the air sac is most common. 2. For the chorio-allantoic route, two small holes are made - one in the air sac and another in the bloodless chorio-allantoic membrane. Air is passed through the first hole to create an artificial air sac space, and then the vaccine is injected. 3. For the yolk sac route, a longer needle is used to inject the virus seed through the air sac and into the deeper yolk sac area.
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The document provides guidelines for virus isolation techniques using embryonated eggs, outlining various inoculation routes including allantoic, amniotic, chorioallantoic membrane and discussing advantages of different routes for isolating specific viruses. It describes the process of candling eggs to check embryo viability and mark inoculation sites prior to introducing viral samples via needle injection into selected areas. Proper egg storage, cleaning, and incubator maintenance are also covered.
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Viruses are obligate intracellular parasites. They depend totally on their host cells for their existence. Their total host dependence makes it, extremely difficult to get good insight of them natural conditions, because the internal characteristics of the host cells are likely to interfere with the observations. Due to these reasons, it has been found desirable that viruses are cultivated or grown in the laboratory itself.
Laboratory animals
Fertilized Hen’s Egg
Chorioallantoic membrane
Allantoic cavity
Amniotic cavity
Yolk sac
Organ/Tissue/Cell Culture
Growth identified by serological method like neutralization.
Embryonated Egg Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/ Tissue cultures Primary
Diploid/ Secondary
Continuous
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Embryonated Hen’s Egg
Cultivation of Viruses and Bacteria
Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
Amniotic cavity – primary isolation of Influenza virus
Yolk sac – Chlamydia, Rickettsia & some viruses
Embryonated eggs:
The Embryonated hen’s egg was first used for cultivation of viruses by Good Pasteur and Burnet (1931). Cultivation of viruses in organized tissues like chick embryo necessitates a different type of approach.. For all practical purposes they all themselves behave as tissue cultures. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. The egg used for cultivation must be sterile and the shell should be intact and healthy.
F.M. Burnet in the laboratory
in the early 1950's,
was experimenting
on influenza virus genetics,
using the developing hen's egg
Inoculated eggs are candled
daily to see the chicken
embryos inside.
Animals and chick embryo
were the first method that was used
to cultivate virus. This method is rarely
used as it is not convenient.
However, when preparing for bulk virus,
(e.g. antigen or vaccine production)
the usage of chick embryo is useful.
Fertile chicken eggs provide
a convenient, space-saving incubator
for many kinds of animal viruses.
Different viruses can be injected
into an egg at different sites and
the egg can be easily observed
for viral replication throughout
the development of the chicken embryo.
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SUCTIONING OROPHARYNGEAL AND NASOPHARYNGEAL CAVITIES
Nasopharyngeal suctioning is used to remove secretions from the nose and nasopharynx in patients who are unable to effectively clear their upper airway. Bronchiolitis is a common condition for which nasopharangeal suctioning is required.
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Viruses are obligate intracellular parasites. They depend totally on their host cells for their existence. Their total host dependence makes it, extremely difficult to get good insight of them natural conditions, because the internal characteristics of the host cells are likely to interfere with the observations. Due to these reasons, it has been found desirable that viruses are cultivated or grown in the laboratory itself.
Laboratory animals
Fertilized Hen’s Egg
Chorioallantoic membrane
Allantoic cavity
Amniotic cavity
Yolk sac
Organ/Tissue/Cell Culture
Growth identified by serological method like neutralization.
Embryonated Egg Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/ Tissue cultures Primary
Diploid/ Secondary
Continuous
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Embryonated Hen’s Egg
Cultivation of Viruses and Bacteria
Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
Amniotic cavity – primary isolation of Influenza virus
Yolk sac – Chlamydia, Rickettsia & some viruses
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F.M. Burnet in the laboratory
in the early 1950's,
was experimenting
on influenza virus genetics,
using the developing hen's egg
Inoculated eggs are candled
daily to see the chicken
embryos inside.
Animals and chick embryo
were the first method that was used
to cultivate virus. This method is rarely
used as it is not convenient.
However, when preparing for bulk virus,
(e.g. antigen or vaccine production)
the usage of chick embryo is useful.
Fertile chicken eggs provide
a convenient, space-saving incubator
for many kinds of animal viruses.
Different viruses can be injected
into an egg at different sites and
the egg can be easily observed
for viral replication throughout
the development of the chicken embryo.
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Title Media culture Objectives 1 To apply aseptic techniq.pdf
Title:
Media culture
Objectives:
1. To apply aseptic technique.
2. To observe the appearance of different bacteria in different media agar.
3. To explain on the physiology of microbes.
Material/apparatus:
Gloves, Bunsen burner, inoculation loop, incubator, MSA agar, EMB agar, MacConkey agar, blood
agar,
E. coli, E. aerogenes, S. epidermidis, and S. aureus.
Procedure:
1. Label the bottom of petri dish with your name, date and type of media culture.
2. Divide each petri dish into 4 quadrants, by marking the bottom of the dish. Label each
section with the name of bacteria to be inoculated.
3. Using aseptic technique, inoculate all plates except blood agar, with the designated bacteria
by making a single line of inoculation of each bacterium in its appropriate section. Ensure
that the petri dishes are close and the inoculation loop flamed between the inoculations of
different bacteria.
4. Using aseptic technique, inoculate blood agar, with the designated bacteria by making a
single line of inoculation of each bacterium in its appropriate section like in step 3. Upon
completion of each single line of inoculation, use the inoculation loop and make three of four
stabs at a 45 angle across the streak.
5. Incubate the petri dishes in inverted position for 24 to 48 hours at 37C.
6. Observe and record your observation
Results:
Questions:
1. Discuss the appearance of media; with the appearance of bacteria grow on the media.
2. Indicate the specific selective and/or differential purpose of MSA agar, EMB agar, and
MacConkey agar.
3. Explain the purpose of blood in blood agar..
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The document discusses the cultivation of viruses using embryonated eggs. It notes that embryonated eggs are a simpler technique than animal inoculation for growing viruses, as eggs do not have an immune response and contain suitable cells for virus growth. Viruses can be inoculated into different areas of embryonated eggs, including the chorioallantoic membrane, allantoic cavity, amniotic sac, and yolk sac. The allantoic cavity is most commonly used due to its simplicity and ability to produce large quantities of viruses like influenza. Embryonated eggs remain an important method for growing stocks of viruses for research and vaccine production.
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Title Media culture Objectives 1 To apply aseptic techniq.pdf
Title:
Media culture
Objectives:
1. To apply aseptic technique.
2. To observe the appearance of different bacteria in different media agar.
3. To explain on the physiology of microbes.
Material/apparatus:
Gloves, Bunsen burner, inoculation loop, incubator, MSA agar, EMB agar, MacConkey agar, blood
agar,
E. coli, E. aerogenes, S. epidermidis, and S. aureus.
Procedure:
1. Label the bottom of petri dish with your name, date and type of media culture.
2. Divide each petri dish into 4 quadrants, by marking the bottom of the dish. Label each
section with the name of bacteria to be inoculated.
3. Using aseptic technique, inoculate all plates except blood agar, with the designated bacteria
by making a single line of inoculation of each bacterium in its appropriate section. Ensure
that the petri dishes are close and the inoculation loop flamed between the inoculations of
different bacteria.
4. Using aseptic technique, inoculate blood agar, with the designated bacteria by making a
single line of inoculation of each bacterium in its appropriate section like in step 3. Upon
completion of each single line of inoculation, use the inoculation loop and make three of four
stabs at a 45 angle across the streak.
5. Incubate the petri dishes in inverted position for 24 to 48 hours at 37C.
6. Observe and record your observation
Results:
Questions:
1. Discuss the appearance of media; with the appearance of bacteria grow on the media.
2. Indicate the specific selective and/or differential purpose of MSA agar, EMB agar, and
MacConkey agar.
3. Explain the purpose of blood in blood agar..
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The document discusses the cultivation of viruses using embryonated eggs. It notes that embryonated eggs are a simpler technique than animal inoculation for growing viruses, as eggs do not have an immune response and contain suitable cells for virus growth. Viruses can be inoculated into different areas of embryonated eggs, including the chorioallantoic membrane, allantoic cavity, amniotic sac, and yolk sac. The allantoic cavity is most commonly used due to its simplicity and ability to produce large quantities of viruses like influenza. Embryonated eggs remain an important method for growing stocks of viruses for research and vaccine production.
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Meiosis occurs in the megaspore mother cell and microspore mother cell to generate megaspores and microspores respectively. The microspores then undergo mitosis and develop protective outer layers, forming pollen grains. These grains contain sperm cells that can fertilize the female gametophyte within the pistil. The pollen tube grows through the pistil tissues guided by chemicals from the synergid cells within the embryo sac, and releases its contents including sperm for double fertilization of the egg and central cells.
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- Careful planning is required to determine the optimal insertion sites for ventricular and distal catheters based on patient history and anatomy. Meticulous surgical technique and attention to details can reduce complications like infection and blockage.
- The goals of shunt surgery are to achieve normal intracranial pressure and neurological function while
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Egg inoculation
- 1. Various routes of egg inoculation are as follows: 1) Allantoic route A/C- Mostcommon route. Keeping the straight with broad top end and pointed bottom, hole is drilled through air sac by egg driller, passing the tubercular syringethrough air sac and .1ml inoculated in allantoic cavity. Later the hole is sealed with WAX. (Another site of hole can be lateral end of air sac) 2) Chorio- allantoic inoculation: DROP CAM: 2 holes are made , one hole at air sac and other point is determined at the time of candling – by slightly tilting the egg find the site of membrane whereno blood vesselis presentand mark the point.by keep the egg in same tilted position with a rubber bulb pass the air in firsthole created at the air sac space, this will help to create a ARTIFICAL AIRSACSPACEatthe second hole area in the blood less field of chorio-allantoic membrane, seen as flow of somefluid fromthe second hole. Maintain egg in same position pass the tubercular needle to inoculate the .1ml of vaccine seed in the artificial air sac created. Seal the holes with wax and keep the egg in horizontalposition so as to prevent the leak of fluid fromthe egg. 3) Yolk sac inoculation: Keep the egg straight, mark the point on air sac spaceand drill hole, through the air sac pass the 21-22 gauzeneedle and .25mlof virus seed can be inoculated. Long needle is used as yolk sac is deep. Seal the hole with wax. 4) Amniotic route: notroutinely performed, it’s guided by Candler. 5) Intra-venous route: For ARTHROPOD BORNEVIRUS VACCINE. Here the egg shell is skillfully cracked at the site various thevessels streaks are seen in the candling. Slowly remove the shell, this make the vesselvisible, apply the paraffin jelly at the site to make the filed more transparentand clear: for better visualization of vessels. Once vesselfound the are inoculated.
- 2. After inoculating the egg, kept in the egg incubator at the specified temperature and humidity. 24 hrs later the egg is examined by candling for the meticulous inoculation of egg-checking the egg viability. Every day seen for the changes which are proceeding. On 4-5 th day eggs are seen for the dead embryo depicting the virus multiplication