On Monday Dr. Rainey was out of town and his PostDoc Manish ran the lab. He gave a lecture on radiation resistant bacteria and the link between radiation resistance and desiccation tolerance. He had problems with the computer and the projection but Dr. Rainey has now posted this excellent powerpoint presentation from Manish on the BIOL 4126 AirSet page. The information in this powerpoint like all the information in the blog and the AirSet will be useful for the midterm exam.
So, this week we did set up experiments to look at the link if any between gamma radiation resistance and desiccation tolerance.
The lab work on Monday involved plating out aliquots of our strains (which had been suspended in liquid media) after they had been irradiated using a gamma source (cobalt60). The strains had been exposed to doses of 0, 3, 6 and 16 kGy of gamma radiation. On Monday we plated the samples exposed to 0, 3 and 6 kGy and on Wednesday we plated the samples exposed to 16 kGy. The plating involved spotting 10ul of the irradiated sample on the agar surface of the media that the particular strain grows best on. The spots were allowed to air dry in the clean bench before incubation at 25C. We will examine these plates for growth after 20 days incubation.
In order to determine the desiccation tolerance of the strains we took a 10ul aliquot of the control suspension (un-irradiated) and placed it in a 24 well microtitre plate. After the air drying we placed the plate in a desiccation chamber in which the relative humidity is controlled to less than 5%. After up to 6 weeks in the desiccation chamber we will rehydrate the samples and plate them on agar plates to determine tolerance to extended periods of low relative humidity of our test strains.
Picture of the desiccation chamber which we used with the microtiter plate in the back right hand corner, the blue material in the tray in the bottom is the desiccant (silica gel) :
In addition to spotting the samples that had been exposed to a gamma dose of 16 kGy we set up an experiment to test the ability of our strains to produce amylase that will degrade starch. Dr. Rainey provided us with agar plates of the media on which our strains normally grow but to which 10g/l of starch had been added. We did a single streak for a number of our strains based on the number of starch plates available. We incubated the plates at 25C. After 20 days incubation we will flood the plates with iodine solution to detect starch hydrolysis and thus amylase production.
The last tasks of the day involved the dilution series plates of the 3 desert un-irradiated soil samples that we plated 3 weeks ago. We counted all of the colonies on our plates (MA, PCA, 1/10 strength PCA and 1/100 strength PCA) and as a class (with Dr. Rainey’s help) we calculated the Colony Forming Units per gram (cfu/g) of soil. We then compared the results between the samples and Jekeitha put together a very nice excel spread sheet of the results (posted on AirSet in a folder labeled Soil Counts). Dr. Rainey discussed the results with us and explained why there might be different numbers on different media and how this might relate to the ecology of the environments from which the soils originated. We took photographs of selected plates that we will use in our blog this week. Dr. Rainey asked us to include a discussion of the cfu/g results (from the excel sheet in AirSet) in our blog along with what we did in lab.
Dr. Rainey also made it very clear to us that we neede to participate in the blog more and should be posting comments on other students blogs or updates on our own. He said there are technical errors in some of our blogs and we should point these out. I guess we should remember that this blog counts for 15% of our overall grade in this class.
From the cfu/g plates each group selected 10 colonies based on their characteristics that we thought might be representatives of the genera Geodermatophilus, Blastococcus or Modestobacter. We transferred these colonies to new plates of the same media they were isolated on and incubated then at 25C. After they grow and we check their purity we will use 16S rRNA gene sequencing to determine their identity.
So, this week we did set up experiments to look at the link if any between gamma radiation resistance and desiccation tolerance.
The lab work on Monday involved plating out aliquots of our strains (which had been suspended in liquid media) after they had been irradiated using a gamma source (cobalt60). The strains had been exposed to doses of 0, 3, 6 and 16 kGy of gamma radiation. On Monday we plated the samples exposed to 0, 3 and 6 kGy and on Wednesday we plated the samples exposed to 16 kGy. The plating involved spotting 10ul of the irradiated sample on the agar surface of the media that the particular strain grows best on. The spots were allowed to air dry in the clean bench before incubation at 25C. We will examine these plates for growth after 20 days incubation.
In order to determine the desiccation tolerance of the strains we took a 10ul aliquot of the control suspension (un-irradiated) and placed it in a 24 well microtitre plate. After the air drying we placed the plate in a desiccation chamber in which the relative humidity is controlled to less than 5%. After up to 6 weeks in the desiccation chamber we will rehydrate the samples and plate them on agar plates to determine tolerance to extended periods of low relative humidity of our test strains.
Picture of the desiccation chamber which we used with the microtiter plate in the back right hand corner, the blue material in the tray in the bottom is the desiccant (silica gel) :
In addition to spotting the samples that had been exposed to a gamma dose of 16 kGy we set up an experiment to test the ability of our strains to produce amylase that will degrade starch. Dr. Rainey provided us with agar plates of the media on which our strains normally grow but to which 10g/l of starch had been added. We did a single streak for a number of our strains based on the number of starch plates available. We incubated the plates at 25C. After 20 days incubation we will flood the plates with iodine solution to detect starch hydrolysis and thus amylase production.The last tasks of the day involved the dilution series plates of the 3 desert un-irradiated soil samples that we plated 3 weeks ago. We counted all of the colonies on our plates (MA, PCA, 1/10 strength PCA and 1/100 strength PCA) and as a class (with Dr. Rainey’s help) we calculated the Colony Forming Units per gram (cfu/g) of soil. We then compared the results between the samples and Jekeitha put together a very nice excel spread sheet of the results (posted on AirSet in a folder labeled Soil Counts). Dr. Rainey discussed the results with us and explained why there might be different numbers on different media and how this might relate to the ecology of the environments from which the soils originated. We took photographs of selected plates that we will use in our blog this week. Dr. Rainey asked us to include a discussion of the cfu/g results (from the excel sheet in AirSet) in our blog along with what we did in lab.
Dr. Rainey also made it very clear to us that we neede to participate in the blog more and should be posting comments on other students blogs or updates on our own. He said there are technical errors in some of our blogs and we should point these out. I guess we should remember that this blog counts for 15% of our overall grade in this class.
From the cfu/g plates each group selected 10 colonies based on their characteristics that we thought might be representatives of the genera Geodermatophilus, Blastococcus or Modestobacter. We transferred these colonies to new plates of the same media they were isolated on and incubated then at 25C. After they grow and we check their purity we will use 16S rRNA gene sequencing to determine their identity.
does amylase production have anything to do with being radiation resistance/desiccation resistant or is it just a separate part of the experiment?
ReplyDeletei'm sure i have it written down in my lab notebook, but i dont have it with me.
ProbyOne was up early today!
ReplyDeleteNot to do with radiation?desiccation. It is just one of the phenotypic tests we are doing like the temperature of growth, NaCl tolerance etc. This week Eugene prepared media containing stratch for class and so that is why we set it up on Wed. Next Monday we will set up the remaining stratch plates as we did not have enough plates for all 78 strains last day. In some of the IJSEM papers (on AirSet)describing new species of our genera you will see amylase activity or strarch hydrolysis/utilization in the tables comparing the characteristics of the strains and species.
Why do we have to rehydrate the cells that were dessicated? Do these cells go into a dormant state when they are dessicated or are they going about their normal cell business?
ReplyDeleteAlso, my boyfriend asked me a question last night that I thought I should know the answer too. How long is the typical lifespan of a bacterial cell? This is important to know seeing how our soil sample, Golbi-1, is almost 15 years old.
ReplyDeleteWell - who is going to answer Chrti's question?
ReplyDeleteI beleive you rehydrate the cells to re-suspend the bacteria in the well to put in on media. Then you later check it for growth to see if it survived and can come out of its dormant state after the 42 days of dessication.
ReplyDeleteThe lifespan depends on the type of bacteria and the environment. Spore forming bacteria can live for millions of years,and bacteria that are dessication resistant will have longer lifespans than ones that aren't in an arid environment.
The picture of the desiccation chamber looks strangely familiar...
ReplyDeleteAs a former member of the Rainey Lab, it's really great to read all of the experiments this class is doing, as they are all very near (and dear) to my heart. Best of luck in future experiments!
[And rehydrating cells works to both pull them out of the media and to get them started on the road to desiccation recovery...remember that they've had no water for 42 days, so this will be a happy addition to the surviving cells]