Now I have finished analysing the samples within the bacterial collection, I am excited to move onto running a water sample through my planned sample processing procedure and spiral plater plating. Over the next two weeks I hope to really get to grips with the whole procedure from start to finish, work through any issues and refine the process so that when I am working with freshly collected samples I can work quickly and efficiently.
Monday 2nd December
I started the week by preparing membrane lauryl sulfate agar (MLSA) for growth of a test sample with the spiral plater. MLSA is a selective medium used in the detection of suspected coliform organisms within water samples, therefore it is very suitable for the next stage of my research. MLSA is prepared in a similar way to MacConkey agar, however as I only had MLS broth powder I added Technical agar No. 3 in order to make agar plates.
How to make membrane lauryl sulfate agar (MLSA)
- To begin with, weigh out an appropriate amount of MLS broth powder into an appropriate sized conical flask (You can calculate this based on the production information on the side of the container, and on how many agar plates you require).
- As previously mentioned, then add the equivalent amount of technical agar no.3 (15g per 1L, so as I was making 400ml I used 6g).
- Dissolve the powder gradually in distilled water.
- Using cotton wool, plug the top of the flask, cover with grease-proof paper and seal this with autoclave tape. Make sure to label with your initials and the content of the flask, in this case MLSA.
- Autoclave the agar for 45 minutes at 121ºC.
- When it is finished, remove the flask carefully and allow it to cool until comfortable to the touch.
I then poured the plates aseptically as before. Once set I inverted and stacked them all ready to inoculate.
I then decided to have a look over the water filtration system we have in the microbiology and molecular biology teaching lab. I am interested in possibly using this system to filter my water samples prior to growth and colony analysis. I have used this equipment once before as part of my undergraduate degree, however I have never really had chance to play around with it by myself. Using the technical manual I set up all the components of the system and re introduced myself to the filtration process.
Tuesday 3rd December
I began my day by autoclaving a 400ml glass Duran flask, in order to steralise it for sample collection. Once finished and cooled, I headed down to the Brayford Pool to collect a test water sample to work with. Using an extendable collection rod, I attached the Duran flask to the end, removed the cap and collected a water sample from behind the Marina building. When filled I retracted the rod, replaced the lid and headed back to the lab. I decanted the large sample into a few sterile 50ml Falcon tubes to make it easier to work with.
I decided to test the water sample in two ways: Using the spiral plater and using water filtration system.
Spiral plater inoculation
I started by filling the rinsing bath with ethanol and distilled water, before running it through 3 cleaning procedures. I then labelled 5 plates with my initials, B1 (Brayford 1 sample), spiral and the date. Finally, I inoculated the 5 MLSA plates using the exponential setting (To see a video of this procedure click here). I incubated these plates overnight at 30ºC.
Water filtration system membrane filter inoculation
- Begin by labeling plates appropriately (In my case: initials, B1 (Brayford 1 sample), filter and the date).
- Clean the lab bench with ethanol and set up the system within easy reach of a bunsen burner for sterilisation.
- Using a blue bunsen flame sterlise each piece of the system beginning with the membrane filter portion, funnels and then lids.
- Lift the funnel with attached lid up gently and place sterlise membrane filter using flat forceps onto the membrane filter portion.
- Replace the funnel and lid, locking the base into place.
- Lifting each lid, fill the funnels with the water sample to be filtered before replacing the lid (The volume will depend on the sample you are using and organisms you wish to investigate).
- Ensuring the vacuum pump is switched on and is turned up, open the taps under the membrane filter portions.
- Once all the water sample has passed through the filter membrane switch off the vacuum pump.
- Lifting the funnel and lid, use flat forceps to remove the filter membrane and place onto your chosen agar plate (Filter membrane inoculation side up).
- Incubate appropriately (This will depend on your sample/plate).
I incubated both sets of places overnight at 30ºC.
Wednesday 4th December
I started the day by observing both sets of MLSA plates. Unfortunately none of my plates had any growth, so I had a quick brainstorm of reasons why this might have occurred.
Reasons for lack of growth:
- Too little sample and therefore not enough organism – should I use more sample or try growing up sample first to increase organisms present?
- Is MLSA too selective for this initial sample, and the organisms present – should I try using a less selective medium (Nutrient or MacConkey)?
- Could the sample not contain any organisms – due to rapid change in temperature from Brayford to lab?
- Not enough sample used during filtration and therefore not enough organism in filter membrane – should I use more sample or grow up first?
- Membrane filter pores too large (0.45 micron) and therefore letting organisms pass through – should I try using smaller filter pores?
I decided that the most important thing to rule out was that the sample I had collected didn’t contain any organisms. Whether that was due to possible ‘shock’ of organisms when they were brought from the cold water of the Brayford to the warmer lab, or for some other reason. I suspected that the most likely reasons for lack of growth were not enough sample (and therefore organisms) and my medium being too selective.
For these reasons I decided to spread plate 50μl of the original Brayford Pool water sample onto three agar plates: Another MLSA, MacConkey and Nutrient. If any organisms were present I expected to see growth on the nutrient agar plate, due to it’s non selective nature. I included the MacConkey and MLSA out of interest, just to see if the spiral plating or filtration processes prevented growth. After inoculation I incubated the 3 plates overnight at 30ºC.
Thursday 5th December
I began the day by removing my spread plated samples from the incubator and observing them. Unlike before, my MLSA plate had 3 small, round translucent/white colonies with yellow colouring surrounding each.
The MacConkey plate had several different colonies present, including many I have seen before during identifying the bacterial collection (Escherichia coli, Citrobacter, Proteus, Shigella/etc).
As expected the nutrient plate also had several different colonies present, mostly large/small round but also 1 unusual shaped organism.
This confirmed that the sample did have organisms present, and therefore the problem was most likely with the amount of sample, agar and/or procedure. I decided to re try inoculating an agar plate using the spiral plater, this time using a nutrient plate instead. I also decided to inoculate a few glass universals containing nutrient broth with sample.
Inoculating nutrient broth
I inoculated 1 universal with 50μl of the original sample and another with 1000μl, in order to test whether growing up the original sample may be worth the time. I also decided to inoculate a few universals with some of the colonies that grew on the MLSA, MacConkey and nutrient agar plates – just out of interest and to begin practising creating broth samples of single organisms.
- Begin by setting up an aseptic work space (I used the lab cabinet).
- Ensure you have a wire loop (Or disposable plastic loops), plates and labeled glass universals containing nutrient broth within reach.
- If using a wire loop, flame until red hot and allow to cool.
- Remove the lid of your chosen agar plate, carefully take a single colony onto the loop and replace the lid.
- Open the glass universal and swirl the loop into the broth.
- Remove the loop and flame again or dispose in waste then replace the universal lid.
- Incubate appropriately (This will depend on your sample/broth).
I incubated my inoculated broths and agar plate overnight at 30ºC.
Friday 6th December
I removed my plate and broths from the incubator and observed. The spiral plated nutrient agar plate had 1 colony present, which suggested that I need to increase the amount of sample pipetted onto the plate or grow up the sample before inoculating.
Increasing the amount the spiral plater pipettes involves re configuring the machine through a specific and fairly detailed procedure, which I only want to conduct if really necessary. The 50μl broth appeared fairly cloudy but not as much as the 1000μl broth. I also observed the broths inoculated with individual colonies, however due to timing I couldn’t analyse these further.
I feel like I have made a good start with sample processing and using the spiral plater this week. Next week I think it may be worth inoculating broths with a range of sample amounts and then growing these on agar plates to see which is the most appropriate for spiral plating. Unfortunately, as it was the end of the week I couldn’t conduct any identification on the colonies I grew overnight. However I think this will definitely be something I will look into next week, especially in preparation for PCR and gel electrophoresis. I may also need to complete the re configuring procedure for the spiral plater, if pipetting amount is still an issue.
A Year in the Life…