Freezing Experiments
Julia Perry
Birky Lab
A Comparison of the Survival Rates of Six Different Genera of Bdelloid Rotifers when Frozen at -80 ° C
Performed 10/10/03-10/13/03
Method
Freezing:
Approximately 50 individuals were placed in 1.5 mL tube from each culture. They were all well fed (had been fed within the last week). Each tube was also filled about 2/3 full of water and food/ debris from the dish that the animals were living in. They were then placed in a freezer at -80 degrees Celsius at 4:30 pm on October 10th.
Defrosting:
The tubes were removed from the freezer at 2:30 pm on October 13th. They had been frozen for 70 hours. The unopened tubes were placed in a beaker of 37° C water for 2.5 hours. They were then moved into a small culture dish and counted for survival rate.
Results:
Table 1: Survival rate of six different genera of bdelloid rotifers when frozen at 80° C
Clade / Label / Count Live / Count Dead / Proportion / Survival PercentagePha- ? / Angl 2/1 / 37 / 12 / 37 /49 / 75.5%
Hab- ? / Rob 2/10d / 10 / 33 / 10/43 / 23.4%
Rot 2 / Ari 1/2 / 35 / 33 / 35/68 / 51.4%
Adi-? / RamO 1/7 / 47 / 3 / 47/50 / 94%
Mac singlet / Rat 2/4 / 33 / 7 / 33/40 / 82.5%
Abr 1 / Angl 2/7 / 39 / 10 / 39/49 / 79.6%
C Squared is equal to 69.9. This is significant at all levels. Based on the data there is a correlation between genus and survival rate.
A Comparison of the Survival Rates of Five Cultures of Philodina when Frozen at -80 ° C for 1 hour, 1 week, 2 weeks and 4 weeks.
Performed 10/22/03-11/25/03
Method:
Freezing:
Approximately 100 individuals were placed in each of 4 1.5 mL tubes from each culture. They were marked as 1 hr, 1 week, 2 weeks and 4 weeks. They were all well fed (had been fed within the last week). Each tube was also filled about 2/3 full of water and food/ debris from the dish that the animals were living in. They were then placed in a freezer at -80 degrees Celsius.
Defrosting:
The tubes were removed from the freezer according to the time they were labeled as. They were then placed in a beaker of 37° C water for 2.5 hours. They were then moved into a small culture dish and counted for survival rate. The tube was rinsed several times with carbonated water to help detach rotifers that may have attached to the side of the tube.
Table 2: Survival rates in proportions of live animals over total animals of Five Cultures of Philodina when frozen at -80° C for 1 hour, 1 week, 2 weeks and 4 weeks.
Label / Clade / 1 hour / 8 days / 16 days / 33 daysAngl 1/2b / Pha ? / 67 / 79 / 96 / 110 / 91 / 97 / 92 / 101
Bel ¼ / Pha 11 / 77 / 100 / 85 / 95 / 91 / 103 / 87 / 93
Bel 1/10 / Pha 11 / 79 / 104 / 94 / 105 / 92 / 100 / 96 / 104
Bel 1/11 / Pha ? / 83 / 111 / 98 / 105 / 92 / 99 / 91 / 102
BirdE 1/1 / Pha 7 / 93 / 114 / 96/ 105 / 87 / 100 / 94 / 104
Table 3: Survival rates in percentages of live animals over total animals of Five Cultures of Philodina when frozen at -80° C for 1 hour, 1 week, 2 weeks and 4 weeks.
Label / Clade / 1 hour / 8 days / 16 days / 33 daysAngl 1/2b / Pha ? / 84.8% / 87.3% / 93.8% / 91.1%
Bel ¼ / Pha 11 / 77.0 % / 89.5% / 88.3% / 93.5%
Bel 1/10 / Pha 11 / 76.0% / 89.5% / 92.0% / 92.3%
Bel 1/11 / Pha ? / 74.8% / 93.3% / 92.9% / 89.2%
BirdE 1/1 / Pha 7 / 81.6% / 91.4% / 87 .0% / 90.4%
See Figure 1
Chi squared analysis test confirms that these are independent results taken from similar environments. The cultures can be lumped together. The Chi Squared value was found to be 1.45. With twelve degrees of freedom it is not significant at any level.
Table 4: Survival Rates In Proportion and Percent vs. Time of all 5 Pha Cultures
1 hour / 8 days / 16 days / 33 daysProportion / 399/508 / 469/520 / 453/499 / 460/504
Percent / 78.5% / 90.2% / 90.8% / 91.3%
See Figure 2
The freezing tolerance of Bel 1/4 in presence and absence or organic material when well fed and when starved
10/30-11/4
“starved” rotifers were washed and placed in a different container with no food for 4 days. Approximately 100 of the animals were placed in each of two 1.5 mL tubes. The tubes were centrifuged and excess water was removed. One of the tubes had water but no food added to it; the other one had chlammy added to the water. From the original, “fed” container, two tubes of 100 rotifers were made. Both were centrifuged and excess water was removed. One of the tubes had the original food from the culture inside of it. The other had been washed and ddH2O was added to the tube.
Table 5: Survival Rate of Well Fed and Starved Bel 1/4 (Pha 11) Rotifers in the Presence and Absence of Food When Frozen for One Day at -80° C
Count Live / Count Dead / Proportion / PercentageFed w/ Food / 87 / 12 / 87 / 99 / 87.9%
Fed no food / 94 / 14 / 94 / 108 / 87.0%
Starved w/ food* / 9 / 3 / 9 / 12 / 75 %
Starved no food* / 15 / 2 / 15 / 17 / 88.2%
* Although 100 individuals were counted when placed into the tube for freezing, only 12 or 17 animals were counted total. This may be because of a loss of animals while centrifuging or pipetting.
A Chi Square test was performed on the two fed and two starved results independently. The null hypothesis was rejected at the 10% significance level. The survival rate is not independent of whether or not food was placed in the tube. Therefore we cannot pool the data.
Survival Rates of Angl 1/2b (no cox 1) frozen at -80° C for one day when the level of water is varied in the tube
Performed 11/6/03-11/7/03
All three tubes contained approximately 100 individuals from the Angl1/2b culture.
Method (for each tube):
No Water: 100 individuals were collected from the culture dish with a micropipette, then centrifuged to collect animals at the bottom. Water was then sucked from the top with the pipet and discarded.
ddH2O: Same method as above except sterile ddH2O was placed in the tube.
Original H20: Animals were left in their original culture water. The tube was filled 2/3 full.
Table 6: Survival rate of rotifers when water content is varied
Live Count / Dead Count / Proportion / PercentageNo Water first trial / 38 / 9 / 38 / 47 / 80.8%
No water second trial* / 90 / 15 / 90 / 105 / 85.7%
ddH2O / 99 / 9 / 99 / 108 / 91.7%
Original Water / 91 / 11 / 91 / 102 / 89.2%
*This was repeated on 11/13 because of the loss of animals (from 100 to 47) in the first trial
A chi square test was performed on the No Water first trial versus the second trial. The result was significant at the 50% level. We cannot pool the first and second trials.
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