This will be my last week in the lab before the Christmas holidays so I hope to finish off and tie up the first part of the project by Friday. I am starting by carrying on working with the Spiral Plater, perfecting a method and sample preparation. Once I can establish good and consistent growth on NA, I plan to move onto using MAC and/or MLSA. Hopefully finishing the week with some work on the Plate Reader, counting colonies and CFU.
Monday 9th December
I started the week by preparing nutrient agar and broth, in order to inoculate my Brayford water sample. I decided to change the agar I was using from selective (MLSA and MAC) due to the lack of growth I was obtaining. By using nutrient agar I am reducing the selective nature of the agar and therefore should achieve better growth. I prepared the nutrient agar in a similar way to MAC and MLSA.
How to prepare nutrient agar
- To begin with, weigh out an appropriate amount of nutrient agar 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).
- 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 NA.
- 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 poured the plates aseptically, this time using the cabinet in the lab. Once set I inverted and stacked them ready to inoculate.
I then moved onto preparing the nutrient broth, which is made in a similar way to tryptone broth.
How to prepare nutrient broth
- Carefully weigh out your required amount of nutrient broth into a flask (This will depend on how much nutrient broth you need).
- Combine this with your required amount of distilled water and mix until homogeneous.
- Pipette 4ml of the broth into universal glass jars (Again, the amount will be based on how much broth you choose to make).
- Autoclave the broth at 121ºC for 15 minutes.
- Store the universals at 4ºC-10ºC .
Last week I established that my water sample did contain organisms, however the concentration present was not significant enough to obtain growth straight away. Based on the initial samples I ran last week I decided to produce a range of inoculated broths using my collected water sample. I decided to try 10μl, 50μl, 100μl, 500μl and 1000μl. I aseptically inoculated 4 nutrient broth universals with the appropriate amount of Brayford water sample and incubated them overnight at 30°C.
I also decided to inoculate MAC and MLSA plates with 30μl and 50μl of Brayford water sample in order to begin producing a method for organism isolation. I spread plated 2 plates (MAC and MLSA) with 30μl of sample and 2 plates (MAC and MLSA) with 50μl before incubating them overnight at 30ºC.
Tuesday 10th December
I began my day by removing and observing the nutrient broths and MAC/MLSA plates from the incubator.
MAC and MLSA plates
Similar to last week I observed no growth on either MLSA plate (30μl or 50μl), again most likely due to the agar being too selective. Therefore I decided to stop using MLSA for now, until I can achieve good consistent growth on NA and/or MAC agar. Both MAC plates showed some growth, the 30μl plate had 2 small, round pink colonies, with some yellow colour change (Lactose negative). While the 50μl plate had several small, round pink colonies with no colour change (Neither lactose positive or negative).
As I expected the 10μl and 50μl broth samples were clear, while 100μl, 500μl and 1000μl samples were progressively cloudier. The 500μl broth also appeared to contain visible white colonies.
I therefore decided to spiral plate all incubated broths onto NA to see which was the most appropriate in terms of organism concentration. I pipetted 1000μl of each sample into individual eppendorfs to make it easier to the Spiral Plater to intake the sample. I used the same Spiral Plating procedure as before to inoculate 5 plates with each broth sample – 10μl, 50μl, 100μl, 500μl, 1000μl (For a video of the process click here). Before incubating them overnight at 30°C.
I also decided to inoculate MLSA and MAC agar plates with samples, in order to begin organism isolation. While I spread plated MAC with all samples I decided to only inoculate MLSA plates with the more concentrated samples – 100μl, 500μl and 1000μl. Once finished I decided to inoculate nutrient broths with the colonies grown on both MAC agar plates. I inoculated these aseptically then incubated them overnight at 30°C.
Wednesday 11th December
I started my day by observing the NA plates that I spiral plated and incubated overnight. As I expected the 10μl sample showed no growth, while the 50μl, 100μl, 500μl and 1000μl showed considerable growth.
Based on these results I decided to spiral plate broths: 50μl, 100μl and 1000μl – using the same procedure as before. I decided not to use 500μl as it produced growth that was unreadable, most likely due to the visible white growth in the broth.
Instead I decided to repeat the initial nutrient broth dilutions, removing 10μl due to the lack of growth from the sample – 50μl, 100μl, 500μl, 1000μl. Incubating them overnight at 30°C.
I then observed the MLSA plates I inoculated making notes of the colonies present. All plates showed growth: 100μl – some single brown/pink colonies with yellow clearing. 500μl – small brown colonies spread across the whole plate with yellow clearing. 1000μl – orange/brown mucoid colonies across whole plate.
I also observed the MAC plates and made notes of the colonies present. All plates showed growth: 10μl – few small round pink colonies with red agar. 50μl – many small round colonies with yellow agar. 100μl – large pink and some small pink colonies with pink agar. 500μl – large and small pink colonies with pink agar. 1000μl – large orange/brown mucoid and some small colonies.
I then decided to conduct some identification testing on these colonies, including: Indole, Oxidase and Gram. Partly out of interest and partly to continue practicing identification of unknown water collected samples.
I heat fixed slides for each colony of interest then Gram stained them as before. All colonies I processed were Gram negative, rod/bacilli.
I oxidase tested each colony of interest as before. All colonies I processed were oxidase negative.
I inoculated tryptone broth with each colony of interest as before. I then incubated the broths overnight at 30°C.
Thursday 12th December
I began my day by removing and observing NA spiral plates, and making notes of their appearance. All plates had growth similar to previous plates.
The broths I reproduced and re incubated turned out as expected – 50μl fairly clear, 100μl cloudier, 500μl cloudier however 1000μl appeared very cloudy with visible colony growth (Similar to how 500μl appeared before). As I had now managed to produce good consistent growth on NA I decided to use these broths to inoculate MAC and MLSA. Again, I conducted this in the same way as before – using the Spiral Plater.
Finally I completed indole testing on the samples by adding 5 drops of Kovac’s reagent and observing any colour change if present. I ended up with a mixed amount of both positive and negative results.
Friday 13th December
I started the day by observing my MLSA and MAC plates, making notes on their appearance.
50μl – showed some colony clumping in the centre and towards the outside on the right but with single colonies towards the outside.
100μl – some colony clumping in the centre but with single colonies towards the outside.
500μl – good growth with many single colonies.
1000μl – good growth with many single colonies.
50μl – no growth
100μl – several pink colonies in outer spiral.
While I was observing these plates my supervisor brought in the Plate Reader which works alongside the Spiral Plater, so as soon as I had finished I began working through the manual that came with it. Although I haven’t used the plate reader before I found it fairly simple to work and managed to get good readings and CFU results for all plates from Wednesday and Thursday.
I started by practicing the process using the test plate supplied with the Plate Reader, which is a mock agar plate with several black ‘colonies’ on it. You can then adjust the lighting (Above and below) and the settings available on the system – plating, medium, display, lighting and colour. You then select test to see what colonies have been selected. At this point you can adjust settings again, remove or add colonies to the count. You can then select count to save the results in the down section. I then moved onto my own agar plates, placing a NA plate onto the stage before following the steps above to achieve a colony count and CFU.
I carried on using these steps to look at all my spiral plates with growth, continuing to perfect my method and learning new things as I worked through them all. One thing I picked up on was the selecting of light and dark colonies detection, and how this affected what was identified on the agar plate. This also suggested that it may be a good idea to change writing on the bottom of the plates to the rim instead.
I also spent some time discussing and being shown how to use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF), by a member of staff who I worked with on my UROS project between 2nd and 3rd year. Both are techniques used to investigate the proteins present within samples. While SDS-PAGE is mostly concerned with separating proteins by individual size, IEF is more concerned with separating by individual isoelectric point (PI). This equipment is not usually set up in the lab, but it was for a undergraduate practical so I made use of the time as my supervisor suggested I may need to use it in the future. I found both techniques really interesting and challenging, however I look forward to hopefully using them in the new year.
Next year I hope to continue work with the Spiral Plater, Plate Reader and re start work with PFGE in particular with Escherichia coli. Then begin collecting and processing samples from UK cities. Over the Christmas holidays I intend to continue work on my thesis and research methods, as well as planning work for January.
A Year in the Life…