TCID50 Practical Exercise - Science Assignment Help

Assignment Task

Introduction
Virus quantification is an important step at various stages in commercial laboratories producing viral vaccines, or viral antigens, or recombinant proteins using viral vectors, etc. Here, it will be used to determine the presence and quantify the amount of baculovirus in samples from the accident site at the Biotech Company “X”. Plaque assays are the traditional method of quantifying virus. A technically simpler alternative, the 50% Tissue Culture Infective Dose (TCID50) is a simple endpoint dilution assay based upon limiting dilutions. TCID50, a measure of infectious virus titer, quantifies the amount of virus required to infect 50% of host cells i.e. produce cytopathic effects (CPE) or other visible effects in 50% of inoculated cells.
In this experiment, a cell line (Sf21) from Spodoptera frugiperda, an insect in the moth family, will be infected with Baculovirus in samples from around the site of the accident. Baculovirusus (Autographica californica Nucleopolyhedrovirus or AcNPV) infects these cells and other insect cells, but not human cells so is considered safe to handle. The viruses used by the Biotech Company “X” are recombinant viruses engineered to express the green fluorescent protein (GFP) together with other target proteins, investigated in the Western Prac. It is, therefore, a GMO (genetically modified organism) and must not be released to the environment i.e. it must not be taken out of the lab. The GFP is used as a marker to confirm host cell infection instead of cytopathic effects (or CPE) in the TCID50 assay. Thus infection will be determined via visualization of GFP derived from any GFP-expressing AcNPV present.
 

Materials
• A flask of insect cells grown for several days at 27ºC
• Fresh SF900 II medium
• Sample containing recombinant Autographica californica Nucleopolyhedrovirus (or AcNPV) virus
• Serological pipettes (1mL and 10 mL)
• Haemocytometer
• Cover slips
• Inverted microscope
• 96-well plates
• Microfuge and falcon tubes
• Stepper pipettes and sterile tips

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