Team:Sao Carlos-Brazil/Measurement

Measurement

In contrast to what many teams do, we did not use any fluorescence or absorbance to measure our results. It would be too difficult and inefficient to quantify survival rates using this approach. Therefore, we opted for a measurement technique that we inherited from our partner astrobiologists from USP’s Astrolab based on CFU counting.

The measurement technique we used was applied both to our high altitude survival experiment and our UVC irradiation assays.

Triplicates of desiccated flight samples were resuspended and drop plated (three times for every replicate) on YM solid medium in a 10-fold dilution. This gives a triplicate’s triplicate for CFU counting, maximizing the statistical significance. Sample survival was asserted by counting the colony-forming units of every group. For the flight, different yeast cell dilutions were desiccated with and without melanin. There is no need to count CFUs for all dilutions in a specific group, only the one that is the most adequate to be quantified (one that has a fair number of individual colonies). A lab magnifier should be used to best visualize the CFUs.

For our ultraviolet C irradiation assays using E. coli, samples were constantly agitated while irradiated in saline solution and aliquots were sampled in triplicates for every desired dose. The tested dosed were 0, 50, 100, 200, 400 and 800 joules per square centimeter (J/cm2). For each replicate, three drops were plated on LB medium.

The simple average of CFU counting for all replicates in a group was calculated. The average was then multiplied by the dilution (which gives N) and this number was then divided by N0 (the control average multiplied by the respective dilution). After this treatment, survival rates of all groups are obtained and a bar graph was plotted using these values. This measurement indicates the relative survival rate of the group compared to the control (which will always have a survival rate of 1). Error bars indicate the standard deviation of the average of N/N0 for triplicates of the respective group (all groups have an overall of nine CFU counted drops). Finally, in order to ascertain whether the acquired values are statistically significant, a Two-Way ANOVA test was carried out.

The image above is an example of drop plated desiccated samples after the high altitude flight. CFUs are counted for each drop in one dilution for a specific group. M (no dilution), -1 (10-fold dilution), -2 (100-fold dilution) and -3 (1000-fold dilution) stand for dilutions used for each desiccated spot.

CFU Counting (S288C)
Replicate	Control		Desiccated		Non-Exposed		Exposed
Treatment	No Treatment	Melanin	No Treatment	Melanin	No Treatment	Melanin	No Treatment	Melanin
I	15	N/A	167	189	31	38	0	65
II	19	N/A	177	158	32	30	1	70
III	26	N/A	164	166	33	22	0	63
I	19	N/A	181	172	30	77	0	58
II	18	N/A	138	139	33	65	2	69
III	15	N/A	119	130	26	62	4	57
I	N/A	N/A	119	101	25	76	3	55
II	N/A	N/A	102	120	29	65	2	57
III	N/A	N/A	119	102	35	66	0	54
Average	1,8667E+01	N/A	1,4289E+02	1,4189E+02	3,0444E+01	5,5667E+01	1,3333E+00	6,0889E+01
Dilution Adj	1,8667E+06	N/A	1,4289E+04	1,4189E+04	3,0444E+03	5,5667E+03	1,3333E+01	6,0889E+02
N/N0	1,0000E+00	N/A	7,6548E-03	7,6012E-03	1,6310E-03	2,9821E-03	7,1429E-06	3,2619E-04

CFU counting of S. cerevisiae strain S288C after the high altitude flight.

Bar graph with survival rates of strain S288C. Statistically significant data are displayed as equal letters.

Bar graph with survival rates of E. coli BL21 with part BBa_K3273018 in different doses of ultraviolet C irradiation. Statistically significant data are displayed as equal letters.

References

PULSCHEN, André Arashiro et al. Survival of extremophilic yeasts in the stratospheric environment during balloon flights and in laboratory simulations. Appl. Environ. Microbiol., v. 84, n. 23, p. e01942-18, 2018.