Week 4 - Xieyue Xiao

This week was a marathon. I have been preparing the libraries for 16S sequencing of all 267 stool samples from neutropenic patients with leukemia. The microbiology core center in Belfer Research building will help us extract fecal DNAs from our samples and perform the 16S sequencing later on. They will also store the fecal DNA so when we are going to do genomic sequencing on these samples we can make the order directly and use the fecal DNAs we already have. We order the DNA extraction tubes last week, the job of this week for me is just to aliquot ~75mg stool samples into each tube. It also sounded very easy for me at the beginning, but when it times 267 it becomes the workload for a whole week. To process the stool samples, I need to work in the biosafety cabinet which we share with Dr. Walsh's lab. This means I can only use the hood before noon, some days before 11am. So I came very early every day to start the experiment and try to finish preparing all the supplies for the next day's experiment before I leave in the afternoon. To be honest, this work is repetitive and not that interesting but I am really excited about the 16S sequences. We are trying to figure out the colonization of Pseudomonas aeruginosa and also isolate strains from the stool samples using the traditional culturing methods. Even though we have only tried 1/9 of the samples and only one of them has been shown to have P. aeruginosa at the time of sample collection, it is still a little upset to have plated hundreds of different plates and got all negatives. The 16S sequencing will shoot it in another way.  After we get the results back, we will be able to know exactly which patients had P. aeruginosa colonized in their gut and what is the relative abundance. This information will guide our culturing experiments and also give us the possibility of investigating more interesting problems.
As for the culturing method, previously, we plated the samples onto/into four different selective media. Each colony grew on these plates are considered to have resistance to different components and thus help us narrow down. All different kinds of colonies will be subcultured onto blood agar and McConkey agar, we will select the colonies base on their lactose fermentation capability and colony morphology, and winners will be sent to MALDI to finally determine the species. But this pipeline is time-consuming. And considering the chance of P. aeruginosa colonization, we are using too much time and reagents. So this week we introduced a much easier and faster method to further narrow down our range, this is the Oxidase test. Bacteria can be divided base on their capability of oxidase production, and P. aeruginosa is always an oxidase producer. In this experiment, we will put one drop of the reagent on a piece of filter paper, smear a colony on the spot. If the colony is an oxidase producer, it will turn spark blue in less than 20 seconds, otherwise, it will stay the same. We tested all the colonies we picked up previously and there is only one turned blue. But sadly, that colony is from a stool that has been shown to have Pseudomonas fluorescens, also an oxidase producer. But this still proved that we are able to recover things from these stool samples, which is a good sign.
For every sample from each patient at each time points, there are 4 tubes of them, 3 are just stool samples in empty tubes and 1 is stools samples in glycerol solution which is used to protect the bacteria from freezing effects. This means there could be more alive cells in the glycerol tubes. So, for next week's schedule, we will start changing to the glycerol tubes for culturing method and hopefully, we could get the bugs we want!