The Lab Work Begins!

So it has been a pretty intensive 6 weeks of lab work. Ideally I would have liked to keep this blog updated as I was doing the work, however job commitments meant that much of my little spare time has been spent recovering from 18 hour work days. As I am working through my results and writing up my final report I will keep this blog updated regularly with smaller posts relating to what happened chronologically. There are some simple things that could and probably should have been done differently, but this was the first time I have ever done lab work and if others can benefit from my mistakes then I am happy. That being said, there is still plenty to talk about!

As a quick refresher, Chorlton Brewing Company (Manchester) brewed a turbid wort which was then cast to two separate coolship vessels for overnight spontaneous inoculation. The aim of this project was to isolate and identify the yeast and bacteria involved in the fermentation of these beers and assess the viability of the air around a railway arch brewery in Manchester for successful production of lambic-style beer. Sample were taken periodically over three months and stored at 4C until they were sent to me for analysis in May.

The first task was to have a look at the beer under the microscope. It was clearly evident that there were fermentative yeasts and acid-producing bacteria in the beer due to the fact both beers had krausened within two weeks, and that the pH had shown a drop below that of a clean fermentation.

The first stage in identifying the bacteria was using the Gram staining method. This is done by heat fixing a beer sample to a glass slide and staining with crystal violet and counterstaining with safranin-O. Gram positive bacteria (Lactobacillus/Pediococcus) appear as violet under the microscope from being able to retain the original crystal violet stain whereas Gram negative bacteria (Acetobacter/Pectinatus) appear a pink colour due to the safranin-O counterstain and the thin layer of peptidoglycan in their cell wall which prevents retention of the original crystal violet stain. I have some images from a brightfield microscope coming however the (heavily edited) photo above from my phone shows some Gram-positive cocci and maybe the beginning of a chain. I will update with better images when I get them.

 

I examined the beer samples using a haemocytometer to determine estimated cell counts throughout the fermentation. The aim was to use these cell counts to calculate serial dilutions to obtain 30-300 cells which would then be spread on WLN spiked with chloramphenicol (bacteria inhibitor) to give the viable yeasts cells based on the number of colonies that grew on each plate. Bacterial cell counts were assumed to be 10x that of yeast and so diluted by another factor of 10 and were spread on to WLD (WLN spiked with the antibiotic cycloheximide) to give the total viable bacteria cells.

I’ll have a post in another couple of days with some more specific numbers for the serial dilutions and viable cell counts.