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BIO60007 Biotechnology

Question:

Haemocytometer Image

Image is for area similar to that shown by green box at left. Count cells in four squares, then multiply by 4 to get cells in large square for calculating cell count.  Note, as we are not in the lab, use just one count, not average of two counts as stated in the manual.

TCID50 Practical Exercise Quantification and size estimation of virus infecting an insect cell culture using TCID50 assay

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.

Week 1: Quantification, size estimation and infection of insect cells

Part A: Sample preparation for cell counting

1. Your demonstrator will have a flask of Sf21 insect cells. They need to be swirled gently to ensure cells are evenly suspended in the cell culture medium.

2. Your demonstrator will use a sterile 1mL serological pipette to sample a small volume of the culture to a tube.

3. Obtain a tube of insect cells from your demonstrator – Handle with care and work in a Class II Biosafety cabinet.

4. Place 10uL of culture against the interface between a haemocytometer and its cover slip so that the suspension runs under the cover slip and just fills the volume under one side of the haemocytometer. 

Week 2: Quantification of infected cells by estimation of TCID50

Part A: Image capture

1. Obtain your plate from your demonstrator and inspect the cells in rows A to H.

2. Obtain at least one image from each row (remember all wells in a row are expected to have similar cells).

3. Include these images in your report.

4. Estimate the size of cells from each row using the software attached to the microscope.

Part B: Observation of GFP activity

1. Turn on the fluorescent light source (please see your demonstrator) and adjust the microscope to allow UV light (visible as blue light) through to the plate – Please be careful with the UV light, as any contact with the UV light may damage your skin and your eyes.

2. Turn down the visible light source (dial on front of microscope).

3. Inspect all the wells of the first row on medium power (10X objective) for fluorescent cells – you may need to turn off the lights or work in dark to reduce ambient light.

4. Once you know what you are looking for (fluorescent cells in the wells), you should use the plate holder and the movement controllers to allow you to “sweep” the wells in each row for fluorescent cells, moving gradually down the plate. You may find it useful to switch between 10 and 20X objective lens to confirm fluorescent cells.

5. Record + for presence and – for absence of any fluorescent cells in each well. When you have a row of wells with no fluorescent cells you should scan the next row only to confirm i.e. it may not be necessary to scan right to the bottom of the plate. See below for calculation of TCID50 from your +/- records.

BIO60007 Biotechnology

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