Aim: To construct a growth curve using aspergillus niger.
Requirements: Master culture of Aspergillus niger
Prepared Potato dextrose agar(PDA) slants
Flasks
Sterile water
Medium composition:
Sucrose 3gm
Sodium nitrate 0.2gm
Potassium dihydrogen phosphate 0.1gm
Magnesium sulphate 0.05gm
Potassium chloride 0.05gm
Ferrous sulphate 0.0001gm
Distilled water 1000ml
pH 5.6
Procedure:
Form master culture of Aspergillus niger, potato dextrose agar slants were incubated at room temp. for 4 days. The medium containing. Above composition was prepared and sterilized. The spores were suspended in sterile water and inoculated in to above medium.this kept on rotary shaker. After two days 10 ml of this broth was used as inoculum for another flask containing 100ml of medium and placed on rotary shaker.
Samples of 10ml were withdrawn using sterile pipette at 24hr interval and transplanted in to clean test-tubes. Samples were collected at 24,48,72,96hr. cell mass of each sample was determined by weighing the cell sediment. After centrifugation and decanting the supernant and weighing the empty tubes later. The differences in the two weight give the cell mass.
Prior to weighing the cell sediment, cells were dried by vacuum drier at 550c overnight. Dry weight of mycelium was found per each sample and noted against sampling hour. A graph plotted taking time on X-axis and weight of dried mycilium on Y-axis. A culture was drawn smoothly through the points, which is the fungal curve.
Observation:
Fungal growth curve:
Result:
As shown in curve we get the growth in the fungus till 72 hrs. Then after there was a decline in the growth of fungus.
Conclusion:
As shown in the graph we can see the four phases in the growth of the fungus.
Lag phase: Here the lag phase is for 24 hrs, which can be observed from the graph. There is a lag in the growth during these hours in fungus.
Log phase: Here the log phase is for 48 hrs in which the growth of fungus slowly increases by utilizing the nutrient from the media.
Stationary phase: After this there was a segment growth of fungus. This is due to lack of sufficient nutrient. Here the stationary phase last for small period of time.
Decline phase: After the stationary phase there is a decline in fungus growth due to total lack of nutrient or formation of some toxic material.
Showing posts with label growth curve. Show all posts
Showing posts with label growth curve. Show all posts
Thursday, April 19, 2007
Growth curve with a monolayer culture in multiwell plates
Outline :
Set up a series of multiwell plates with cultures at three different cell conc and count cells in one plate at daily intervals until the culture reaches plateau phase.
Materials :
monolayer cell culture : like A54 9, Vero , Hela-S3 , 75 cm2 with late log phase
Trypsin 0.25 % crude wit h 10mM EDTA 5ml
Growth medium with 4mM NaHCO3
D-PBSA
Flask 25 cm2
Protocol:
Trypsinize cells as for regular subculture
Dilute cell suspension to 1X105 cells/ml, 3X104 cells/ml, and 1X 104 cells/ml in 25 ml of medium for each conc.
Seed three 12-well plates with 2 ml of 1 X 104 cells/ml suspension to each well of top 4 wells, 2 ml of 3X104 cells/ml to each well of the second row and 2 ml of 1X105 cells/ml to each well of third row. Add cell suspension slowly from center of well so that it does not swirl around well. Similarly do not shake plate to mix the cells, as circular movement of medium will concentrate cells in the middle of well.
Place plates in a humid CO2 incubator or sealed box gassed with 5% CO2.
After 24hr remove first plate from incubator and count cells in three wells at each concentration,
Remove medium completely from three wells containing cells to be counted
Add 0.5ml of trypsin /EDTA to each of three wells
Incubate plate for 15min
Add 0.5ml medium with serum, disperse cells in tryspin /EDTA/medium and transfer 0.4ml of suspension to 19.6 ml of D-PBSA.
Count cells on electronic cell counter
Stain cells in remaining wells at each cell density
Repeat sampling at 48 and 72 hr as in step 5 and 6
Change medium at 72 hr or sooner if indicated by a drop in pH.
Continue sampling daily for rapidly growing cells (i.e.e cells with PDT of 12-24 hr) but reduce frequency of sampling to every 2 days for slowly growing cells (i.e. cells with PDT > 24 hrs) until plateau phase reached.
Keep changing medium every 1,2 or 3 days, as indicated by fall in pH.
Analysis of monolayer growth curves:
Primary count: cells/ml
Cell/flask OR well
Cell concentration: cells/ml of culture medium
Cell density: cells/cm2 of growth surface
Plot cell density (cell/cm2) and cell conc (cells/ml) both on log scale against time on a linear scale.
Determine lag time, PDT and plateau density
Establish appropriate-starting density for routine passage. Repeat growth curve at diff cell conc if necessary
Complete growth curves under different conditions and try to interpret data
Examine stained cells at each density to
1. Determine whether the distribution of cells in flasks/wells is uniform and whether cells are growing up the sides of wells
2. Observe differences in cell morphology as density increases
3. Compare cell-cell interaction in normal and transformed cells
Set up a series of multiwell plates with cultures at three different cell conc and count cells in one plate at daily intervals until the culture reaches plateau phase.
Materials :
monolayer cell culture : like A54 9, Vero , Hela-S3 , 75 cm2 with late log phase
Trypsin 0.25 % crude wit h 10mM EDTA 5ml
Growth medium with 4mM NaHCO3
D-PBSA
Flask 25 cm2
Protocol:
Trypsinize cells as for regular subculture
Dilute cell suspension to 1X105 cells/ml, 3X104 cells/ml, and 1X 104 cells/ml in 25 ml of medium for each conc.
Seed three 12-well plates with 2 ml of 1 X 104 cells/ml suspension to each well of top 4 wells, 2 ml of 3X104 cells/ml to each well of the second row and 2 ml of 1X105 cells/ml to each well of third row. Add cell suspension slowly from center of well so that it does not swirl around well. Similarly do not shake plate to mix the cells, as circular movement of medium will concentrate cells in the middle of well.
Place plates in a humid CO2 incubator or sealed box gassed with 5% CO2.
After 24hr remove first plate from incubator and count cells in three wells at each concentration,
Remove medium completely from three wells containing cells to be counted
Add 0.5ml of trypsin /EDTA to each of three wells
Incubate plate for 15min
Add 0.5ml medium with serum, disperse cells in tryspin /EDTA/medium and transfer 0.4ml of suspension to 19.6 ml of D-PBSA.
Count cells on electronic cell counter
Stain cells in remaining wells at each cell density
Repeat sampling at 48 and 72 hr as in step 5 and 6
Change medium at 72 hr or sooner if indicated by a drop in pH.
Continue sampling daily for rapidly growing cells (i.e.e cells with PDT of 12-24 hr) but reduce frequency of sampling to every 2 days for slowly growing cells (i.e. cells with PDT > 24 hrs) until plateau phase reached.
Keep changing medium every 1,2 or 3 days, as indicated by fall in pH.
Analysis of monolayer growth curves:
Primary count: cells/ml
Cell/flask OR well
Cell concentration: cells/ml of culture medium
Cell density: cells/cm2 of growth surface
Plot cell density (cell/cm2) and cell conc (cells/ml) both on log scale against time on a linear scale.
Determine lag time, PDT and plateau density
Establish appropriate-starting density for routine passage. Repeat growth curve at diff cell conc if necessary
Complete growth curves under different conditions and try to interpret data
Examine stained cells at each density to
1. Determine whether the distribution of cells in flasks/wells is uniform and whether cells are growing up the sides of wells
2. Observe differences in cell morphology as density increases
3. Compare cell-cell interaction in normal and transformed cells
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