Cell Biology
Day 4- pre lab: preparation for protocol
Investigating: Neurospora Crassa Hyphal Tip Growth.
Hypothesis:
Null Hypothesis | Another Hypothesis | 400 nm of Nacodazole will have no effect on Neurospora Crassa Hyphal Tip Growth. | 400 nm of Nacodazole will have an effect on Neurospora Crassa Hyphal Tip Growth. | 400 nm of Nacodazole will have no effect on Neurospora Crassa Hyphal Nuclear position. | 400 nm of Nacodazole will have an effect on Neurospora Crassa Hyphal Nuclear position. | Variables: Fungal growth Tip | Nuclear positioning | Independent on time | Independent on time | Dependent on the distance migrated | Dependent on the distance migrated |
Controls: * Observing the normal movement of Neurospora Crassa Hyphal Tip Growth without 400nm of Nacodazole. * Observing the normal Nuclear position of Neurospora Crassa Hyphal Tip Growth without 400nm of Nacodazole.
Protocol : Investigation of Neurospora Crassa fungal tip growth and nuclear position. 1) From day 3, Neurospora Crassa cushions slides are required 2) Kohler Illumination set up under bright field microscopy. 3) Pick up two agar cushions without bubbles and label them as control 1 and control 2 4) Add 50 ul of growing medium to each control and place a covering slips on the. 5) For 5 minutes, put the slides inside the incubator allowing the medium to transfer into the cell 6) After, place the one of the control under kohler Illumination microscope and start observing. 7) Measure the growth distance of fungal tip growth and record it in table 1. 8) Timer was measured by minutes and microscope based on ocular units. 9) Repeat for control 2
Table 1: Measuring Neurospora Crassa fungal tip growth travel distance for starting at 0 minutes to 10 minutes as 50 ul growth medium was used. Time (min) | Growth distance (o.u) | | | Control 1 | Control 2 | 0 | | | 1 | | | 2 | | | 3 | | | 4 | | | 5 | | | 6 | | | 7 | | | 8 | | | 9 | | | 10 | | |
10) For 5 minutes, put the controls inside the incubator to allow the growth medium to attach to the cell. 11) Using one of the controls that obtained from the incubator. Place it on the microscope stage ( be aware to use low light) then change it to fluorescence ( Fs 14 filter black) and turn up the light intensity to maximum. 12) Observe the Nuclear positioning tip and measure the position from the tip and record the measurement in table 2. 13) Timer was measured by minutes and microscope based on ocular units. 14) Repeat 11- 13 for the other control.
Table 2: Measuring Neurospora Crassa nuclear positioning fungal tip growth travel distance for starting at 0 minutes to 10 minutes as 50 ul growth medium was used.
Investigate the addition of 400 nM of Nacodazole drug to the 50 growth medium for both the hyphal tip growth and the nucleus positioning of Neurospora Crassa.
15) With 15 ul growth medium, add 400 nm of Nacodazole to the agar cushions that contain Neurospora Crassa and place a cover slip on the slide. 16) To get 4 experimental replicates use 4 agar cushions. 17) Put the Nacodazole slide inside the incubator. 18) Focus the specimen using the low light within microscope. 19) Repeat steps 6-8 to get a total of 4 trails.
Table 3: Measuring Neurospora Crassa fungal tip growth travel distance for starting at 0 minutes to 10 minutes as 50 ul growth medium was used with 400 nm of Nacodazole for 4 trails.
20) Repeat steps 15-17 21) Using the specimen obtained from the incubator. Place it on the microscope stage ( be aware to use low light) then change it to fluorescence ( Fs 14 filter black) and turn up the light intensity to maximum. 22) Repeat steps 11-13, for a total of 4 trails and 10 minutes.
Table 4: Measuring Neurospora Crassa n positioning fungal tip growth travel distance for starting at 0 minutes to 10 minutes as 50 ul growth medium was used and 400 nm of Nacodazole for 4 trails.
23) At the end, collect the data and perform statistical analysis to find the accuracy and validity 24) Draw a graph for the tables that include the mean, ±SEM and the t-test values. 25) Use the result to compare with the hypothesis and see whether it agrees with the null hypothesis or with the other hypothesis. 26) To conclude see whether the hypothesis is rejected or accepted.
