During the past 16 weeks, we have done a series of experiments involving microbiology. The following is to help you remember the experiments which we’ve done in microbiology. (As such, the procedures are all in active voice)
Cell culture
Learning how to subculture a continuous cell line is integral in any field of microbiology. This experiment basically teaches us how to subculture a continuous cell line, what the functions of solutions such as trypsin, Phosphate buffered saline, and growth mediums are etc. Also, we will see what a confluent monolayer should look like.
Functions of solutions
Phosphate buffered saline (PBS)-> Due to the fact that PBS is isotonic and non-toxic to cells, it is usually used to rinse containers containing cells, dilute substances etc.
Trypsin-> Used to re-suspend cells which have adhered to the cell culture’s dish wall by lysing the bonds between the proteins and the cell culture’s flask wall
Growth medium-> Contains peptide hormones or hormone-like growth factors which promote healthy cell growth; also neutralizes proteases such as trypsin
Summary of procedures
1. Decant medium from cell flask
2. Wash with PBS
3. Add trypsin
4. Incubate the cells in the flask till cells detach
5. Aspirate cell suspension to disperse cell aggregates
6. Transfer some of the suspension into a new cell flask
7. Incubate the flask with cells
Top: A confluent monolayer
Http://www.devicelink.com/mddi/archive/98/04/013.html
Cell counting
Cell counting is also an invaluable tool in microbiology. It is in cell counting that we derive the concentration of cells, in cells/ml from. Using the formula,
Cells/ml = Average cell count per square x dilution factor x 104
Summary of procedures
1. For every 5ml of cells, add 45ml of trypan blue. Mix them and leave them to stand
2. Transfer a small amount of solution onto the haemocytometer
Basically, just count all the cells in the big square in the middle, along with the 4 big corner squares and average them to get your “average cell count per square” value.
http://en.wikipedia.org/wiki/File:Haemocytometer_Grid.png
Top: A haemocytometer chamber.
Red square = 1.0000 mm2
Green square = 0.0625 mm2
Yellow square = 0.040 mm2
Blue square = 0.0025 mm2
All squares have a depth of 0.1 mm if a cover slip is in place
Each big (red) square has a volume of 0.1mm3, or 0.0001cm3. As we want the concentration to be in cm3 (ml), we multiply our value by 10,000 as 0.0001 x 10,000 = 1
Viral infection and amplification
We can culture viruses by getting them to infect host cells. By infecting the host cells with virus, we would be able to observe what the viruses do to the infected hosts. These changes are called cytopathic effects (CPE). As CPE varies from host to host, and from virus to virus, we can use that to determine the species of the virus.
After we get a culture of virus, we should then amplify our virus pool so that we can conduct further research.
Summary of procedures
Viral infection
1. Decant medium from flask and wash monolayer with virus diluent
2. Decant the virus diluent
3. Clean and disinfect the surface of the cryovial with 70% alcohol
4. Transfer the virus from the cryovial to the virus suspension
5. Incubate the virus suspension
6. After 1 hour, remove excess inoculum and rinse monolayer with virus diluent
7. Add maintenance medium
8. Incubate
Viral amplification
9. Decant medium from big flask, then wash with virus diluent
10. Transfer the medium from the original infected flask to the new, big flask
11. After 1 hour, remove the excess inoculum and rinse with virus diluent
12. Add maintenance medium
http://library.thinkquest.org/10607/media/cell-virus.gif
Top: Viral infection
Plaque assay
Since viruses are too numerous to be quantified by direct counting, scientists have come up with plaque assay, which is an indirect method to count viruses. The formula for virus concentration, in plaque forming units (pfu)/ml, is given as,
Virus concentration = (number of plaques) x (dilution factor(s)) pfu/ml
But before we can do the plaque assay, we must first make serial dilutions of a given virus sample.
Also, there is a new solution:
Carboxymethyl cellulose(CMC) solution-> Used to increase the viscosity of the solution
Summary of procedures
Serial dilution
1. Take 10 tubes and label them from 1 to 10. Then add virus diluents into each tube.
2. Transfer x ml of the virus suspension into the tube labeled 1.
3. Transfer the same amount of virus suspension in tube 1 into 2. Repeat till tube 10
Plaque assay
1. Label 2 wells 6, 2 wells 7 and so on until 10. Label the last 2 as controls
2. Decant the medium from the trays of cells and wash them with virus diluent
3. Decant the virus diluent
4. To the well labeled 6, transfer x ml of virus suspension from tube 6. Add the same amount for the other wells. Ex: If you add 0.5ml of virus suspension from tube 6 to well 6, add 0.5ml of virus suspension from tube 7 to well 7
5. Incubate the wells
6. After 1 hour, remove the excess inoculums and rinse the monolayer with virus diluent
7. The maintenance medium and CMC solutions together. Add the same amount of the mixture to each well.
http://upload.wikimedia.org/wikipedia/commons/f/fb/Plaque_assay_dilution_series.jpg
Top: 4-fold dilution series from top-left to bottom-right, stained with crystal violet. Living cells are not stained whereas dead cells are.
ELISA
An ELISA is used to detect the presence of antibodies or antigens in a sample. In our experiment, we used ELISA to test for the presence of a specific antigen. To test for a presence of a specific antigen, we attach its complementary antibody onto the walls of the wells. (This means that we attach an antibody which will “absorb” the antigen onto the walls of the wells)
Please click here to learn about the basics of ELISA.
Summary of procedures
Antigen dilution
1. Add the sample diluted to their correct wells
2. Incubate
Wash procedure
3. After 30 minutes, aspirate the contents of the wells
4. Wash with buffer, let it stand for 1 minute, and decant. Repeat thrice
Add secondary antibody solution
5. Add secondary antibody
6. Incubate for 30 minutes
React substrate
7. Add substrate into well
8. Incubate in dark place for 10 minutes
9. Add appropriate stop solution and shake well
10. Read plate with plate reader
Cell culture
Learning how to subculture a continuous cell line is integral in any field of microbiology. This experiment basically teaches us how to subculture a continuous cell line, what the functions of solutions such as trypsin, Phosphate buffered saline, and growth mediums are etc. Also, we will see what a confluent monolayer should look like.
Functions of solutions
Phosphate buffered saline (PBS)-> Due to the fact that PBS is isotonic and non-toxic to cells, it is usually used to rinse containers containing cells, dilute substances etc.
Trypsin-> Used to re-suspend cells which have adhered to the cell culture’s dish wall by lysing the bonds between the proteins and the cell culture’s flask wall
Growth medium-> Contains peptide hormones or hormone-like growth factors which promote healthy cell growth; also neutralizes proteases such as trypsin
Summary of procedures
1. Decant medium from cell flask
2. Wash with PBS
3. Add trypsin
4. Incubate the cells in the flask till cells detach
5. Aspirate cell suspension to disperse cell aggregates
6. Transfer some of the suspension into a new cell flask
7. Incubate the flask with cells
Top: A confluent monolayer
Http://www.devicelink.com/mddi/archive/98/04/013.html
Cell counting
Cell counting is also an invaluable tool in microbiology. It is in cell counting that we derive the concentration of cells, in cells/ml from. Using the formula,
Cells/ml = Average cell count per square x dilution factor x 104
Summary of procedures
1. For every 5ml of cells, add 45ml of trypan blue. Mix them and leave them to stand
2. Transfer a small amount of solution onto the haemocytometer
Basically, just count all the cells in the big square in the middle, along with the 4 big corner squares and average them to get your “average cell count per square” value.
http://en.wikipedia.org/wiki/File:Haemocytometer_Grid.png
Top: A haemocytometer chamber.
Red square = 1.0000 mm2
Green square = 0.0625 mm2
Yellow square = 0.040 mm2
Blue square = 0.0025 mm2
All squares have a depth of 0.1 mm if a cover slip is in place
Each big (red) square has a volume of 0.1mm3, or 0.0001cm3. As we want the concentration to be in cm3 (ml), we multiply our value by 10,000 as 0.0001 x 10,000 = 1
Viral infection and amplification
We can culture viruses by getting them to infect host cells. By infecting the host cells with virus, we would be able to observe what the viruses do to the infected hosts. These changes are called cytopathic effects (CPE). As CPE varies from host to host, and from virus to virus, we can use that to determine the species of the virus.
After we get a culture of virus, we should then amplify our virus pool so that we can conduct further research.
Summary of procedures
Viral infection
1. Decant medium from flask and wash monolayer with virus diluent
2. Decant the virus diluent
3. Clean and disinfect the surface of the cryovial with 70% alcohol
4. Transfer the virus from the cryovial to the virus suspension
5. Incubate the virus suspension
6. After 1 hour, remove excess inoculum and rinse monolayer with virus diluent
7. Add maintenance medium
8. Incubate
Viral amplification
9. Decant medium from big flask, then wash with virus diluent
10. Transfer the medium from the original infected flask to the new, big flask
11. After 1 hour, remove the excess inoculum and rinse with virus diluent
12. Add maintenance medium
http://library.thinkquest.org/10607/media/cell-virus.gif
Top: Viral infection
Plaque assay
Since viruses are too numerous to be quantified by direct counting, scientists have come up with plaque assay, which is an indirect method to count viruses. The formula for virus concentration, in plaque forming units (pfu)/ml, is given as,
Virus concentration = (number of plaques) x (dilution factor(s)) pfu/ml
But before we can do the plaque assay, we must first make serial dilutions of a given virus sample.
Also, there is a new solution:
Carboxymethyl cellulose(CMC) solution-> Used to increase the viscosity of the solution
Summary of procedures
Serial dilution
1. Take 10 tubes and label them from 1 to 10. Then add virus diluents into each tube.
2. Transfer x ml of the virus suspension into the tube labeled 1.
3. Transfer the same amount of virus suspension in tube 1 into 2. Repeat till tube 10
Plaque assay
1. Label 2 wells 6, 2 wells 7 and so on until 10. Label the last 2 as controls
2. Decant the medium from the trays of cells and wash them with virus diluent
3. Decant the virus diluent
4. To the well labeled 6, transfer x ml of virus suspension from tube 6. Add the same amount for the other wells. Ex: If you add 0.5ml of virus suspension from tube 6 to well 6, add 0.5ml of virus suspension from tube 7 to well 7
5. Incubate the wells
6. After 1 hour, remove the excess inoculums and rinse the monolayer with virus diluent
7. The maintenance medium and CMC solutions together. Add the same amount of the mixture to each well.
http://upload.wikimedia.org/wikipedia/commons/f/fb/Plaque_assay_dilution_series.jpg
Top: 4-fold dilution series from top-left to bottom-right, stained with crystal violet. Living cells are not stained whereas dead cells are.
ELISA
An ELISA is used to detect the presence of antibodies or antigens in a sample. In our experiment, we used ELISA to test for the presence of a specific antigen. To test for a presence of a specific antigen, we attach its complementary antibody onto the walls of the wells. (This means that we attach an antibody which will “absorb” the antigen onto the walls of the wells)
Please click here to learn about the basics of ELISA.
Summary of procedures
Antigen dilution
1. Add the sample diluted to their correct wells
2. Incubate
Wash procedure
3. After 30 minutes, aspirate the contents of the wells
4. Wash with buffer, let it stand for 1 minute, and decant. Repeat thrice
Add secondary antibody solution
5. Add secondary antibody
6. Incubate for 30 minutes
React substrate
7. Add substrate into well
8. Incubate in dark place for 10 minutes
9. Add appropriate stop solution and shake well
10. Read plate with plate reader
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