This week I am moving into the final stages of identifying the unknown samples within the bacterial collection. Throughout the week I streak plating out samples in batches that I suspected were the same species of organism, in order to gain single colonies. I then conducted biochemical tests on these single colonies to ensure they had the correct characteristics to be the organism I suspected they were.
Monday 4th November
I began the week by making up more MacConkey agar, and getting it on to autoclave. I hadn’t quite finished spread plating the whole bacterial collection, so I got the final few samples out of the freezer, warmed to room temperature, vortexed and spread plated them. By this point my agar had finished autoclaving and was cool enough to pour. So I poured it aseptically, leaving the plates to set before inverting them.
After this, I reviewed the list I made throughout last week, which consists of all the samples I have spread plated in the collection. From the colonies I observed on the spread plates last week, I placed all the samples into groups based on what organism I suspected they were. This left me with 3 main categories, which I used to group the samples into batches.
I decided to start by streak plating the first group/batch: Lactose positive organisms (Most likely Escherichia coli, Klebsiella, Citrobacter/Enterobacter). However, as it was Monday I only had a small number of MacConkey plates, so I was limited in the amount of samples I could streak plate. I selected as many samples as I could process from the freezer, warmed to room temperature, vortexed and then streak plated them.
How to streak plate
- Begin by setting up an aseptic work space.
- Ensure you have a wire loop, samples and labelled plates within reach.
- Sterlise the wire loop by placing it in the hottest part of the flame, until as much of the wire section as possible turns red hot (You should hold the wire loop almost vertically, but not so much that you burn your fingers).
- Select the sample you wish to streak plate, and carefully open the sample (Don’t put the lid on the bench, and work as quickly as possible), twist the wire loop into the sample a few times.
- Replace the lid of the sample, making sure not to let the wire loop touch anything and that it stays in your sterlised area.
- Lift the lid of the labelled agar plate (Again making sure to not put the lid on the bench, or keep it off for too long), and smear the sample onto the plate as shown below in position 1.
- Flame the wire loop again, allow it to cool slightly then streak the bacteria present in position 1 out onto the plate by passing the loop 5 times through the initial inoculum – creating position 2.
- Continue this process, flaming the wire loop, allowing it to cool briefly before streaking the bacteria further into position 3.
- Repeat the process to create position 4 and then 5.
- Steralise the loop again before putting it back down on the bench.
- Invert the plate and incubate appropriately (This will depend on your sample/plate).
It is very important to flame the wire loop between streaking positions as this reduces the bacterial load, so that by streak 5 single colonies should be visible (This will depend on the concentration of bacteria in your sample). For the same reason it is also important to make sure your final 5 lines don’t touch your original smear in position 1. I have streak plated before, during my degree and also during a recent undergraduate practical I demonstrated for, so this wasn’t too difficult for me but it was good to get to re practise and perfect the technique. The plates were then incubated overnight at 37ºC
Tuesday 5th November
As usual I began my day by making up more MacConkey agar and getting it on to autoclave. I then collected both my spread and streak plates from the incubator. Using the colony counter from before, I observed my spread plates first, making detailed notes and taking photos of both the whole plate and individual colonies. After this I selected plates with single colonies and heat fixed them onto slides before Gram staining.
Once I had finished this the MacConkey agar was ready to pour, so I poured the plates aseptically and left them to set. I then began to observe my first batch of streak plates, again making detailed notes and taking photos of both the whole plate and individual colonies. When I had finished my observations I decided to streak plate the second group/batch: Lactose positive organisms (Most likely Escherichia coli, Klebsiell, Citrobacter/Enterobacter). As I had more agar plates prepared I was able to finish up the rest of the first batch from Monday. I selected these samples from the freezer, warmed to room temperature, vortexed and then streak plated them onto MacConkey plates. Again, I incubated them overnight at 37ºC.
During the end of last week I made detailed lists of all the characteristics of Enterobacteriaceae species that I was likely to have within the collection. This allowed me to decided which biochemical tests I wanted to use to assess the samples I had spread plated. I decided to conduct three biochemical tests that I thought would be most relevant and useful for differentiation: Motility, Oxidase and Indole. At this point I begin setting up two of the three biochemical tests that I wanted to conduct on my samples.
Biochemical Testing: Motility
The API tubes that I am using contain M medium which is used to test the motility of the bacteria inoculated in it. This test is very useful in identification as it allows me to determine whether the organism is motile (Salmonella, Proteus/Escherichia coli) or non motile (Klebsiella/Shigella). I started by preparing my API motility tubes, as they needed to be boiled for 10 minutes before use. I was going to use a water bath, however the small water bath in the lab had a maximum temperature of 60ºC. Therefore I decided to use a tripod, bunsen burner and glass beaker filled with water and manually maintain the temperature instead. While this was heating up to temperature, I followed the instructions supplied with the API test tubes.
How to use API motility test tubes
- Using the white cap, carefully snap off the glass tube protector (It should remain in the lid, but if not dispose of it carefully).
- Boil the tubes for 5-10 minutes.
- Allow the tubes to cool to room temperature.
- Set up an aseptic work space.
- Ensure you have a wire loop, agar plates containing single colonies and room temperature labelled API motility tubes within reach.
- Using a sterlised and cooled wire loop take a single colony from your agar plate, remove the white cap and inoculate the motility tube, stabbing the medium to a depth of around 2/3 of the height of the tube.
- Incubate the tubes for 24-48 hours at 36º+/- 2ºC
- After incubation: A positive result or motile organism is indicated by diffusion from the line of inoculation and turbid medium. A negative result or non motile organism is indicated by growth confirmed to the line of inoculation and transparent medium.
I have never used API test tubes before and I did come across a few problems. The first time I attempted to remove the glass protectors off the tubes I found it quite difficult as the white cap had to be in the right position to snap the glass protector off. Occasionally the glass didn’t remain inside the cap and I had to dispose of it by hand. However after a few goes I got the hang of it and it was fairly easy.
Biochemical Testing: Oxidase
Oxidase testing is used to determine whether bacteria produce certain cytochrome C oxidases. This test is a useful confirmatory test for my identification as most organisms I am likely to be working with are oxidase negative (Salmonella, Klebsiella, Proteus/Escherichia coli), while Pseudomonas is oxidase positive. There are several ways to conduct oxidase testing, however I decided to use a glass slide so that I could remove a colony from the streak plate and the plate could be used again for other tests.
How to conduct an oxidase test
- Begin by preparing the oxidase reagent. Combine designated amount of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD) with distilled water until homogeneous (This will depend on how much reagent you require).
- Set up an aseptic work space.
- Ensure you have a wire loop, glass slides, agar plates containing single colonies and oxidase reagent within reach.
- Sterlise the wire loop, allow to cool briefly and then place 2-3 drops of the oxidase reagent onto the glass slide.
- Sterlise the wire loop again and then pick up a single colony for your chosen agar plate.
- Gently mix the colony with the oxidase reagent until combined on the glass slide.
- Flame the wire loop again before putting it down on the bench.
- Within 10-30 seconds: A positive reaction is indicated by the development of a blue-purple colour, while a negative reaction is indicated by a lack of colour change or a pink colour.
I have conducted this test before, however the procedure is very simple and I think that even without previous experience I would have had no problems conducting it. I did sometimes find it difficult to interpret the results, as some samples produced a very light blue-purple colour. For these samples, I repeated the test and noted down my uncertainty.
As demonstrated here, is it fairly difficult to see but the sample on the right has a faint blue colour, while the sample on the left is pink/colourless
Wednesday 6th November
As with previous days, I began work by making up more MacConkey agar and getting it on to autoclave. I then collected my streak plates and motility test tubes out of the incubator. I observe my second batch of streak plates, again making detailed notes and taking photos of both the whole plate and individual colonies.
After I had finished my agar was cool enough to pour, so I poured my plates aseptically and left them to set. I then observed my API motility test tubes, making a note of the results for each sample, and taking photos. Although I haven’t used API motility tubes before I found it fairly easy to determine the results of the test and had no major problems.
I then decided to begin streak plating the third group/batch: Lactose negative mixed organisms (Suspected to be mixed samples of lactose positive organisms (Escherichia coli, Klebsiella, Citrobacter/Enterobacter) and lactose negative organisms (Salmonella, Proteus, Shigella/Serratia)). I selected these samples from the freezer, warmed them to room temperature, vortexed and then streak plated them onto MacConkey agar plates. Again, incubating them overnight at 37ºC.
Once I had finished my observations I began setting up to conduct both biochemical tests on my new batch of streak plates. As before, I conducted both oxidase and motility tests on my streak plated samples. Making sure to make up fresh oxidase reagent, as contact with oxygen causes it to become oxidised and unreliable. Again, I incubated the API motility test tubes overnight at 37ºC.
Thursday 7th November
Unlike earlier on in the week I decided not to make up anymore MacConkey agar, as I would only be processing plates for tomorrow and then it would be Friday. Instead I collected my streak plates and motility test tubes out of the incubator. I observe my third batch of streak plates, again making detailed notes and taking photos of both the whole plate and individual colonies.
Selection of plates from third batch of streak plates, suspected to be mixed samples of lactose positive organisms (Escherichia coli, Klebsiella, Citrobacter/Enterobacter) and lactose negative organisms (Salmonella, Proteus, Shigella/Serratia)
After this I observed my API motility test tubes, making a note of the results for each sample, and taking photos. I then decided to begin streak plating the fourth group/batch: Lactose negative organisms (Most likely Salmonella, Proteus, Shigella/Serratia). I selected these samples from the freezer, warmed to room temperature, vortexed and then streak plated them onto MacConkey plates. Incubating them overnight at 37ºC.
Once I had finished this I began setting up to conduct both biochemical tests on my new batch of streak plates. Again, I conducted both oxidase and motility tests on my streak plated samples. Making up fresh oxidase reagent and then incubating the API motility test tubes overnight at 37ºC.
Biochemical testing: Indole
The third biochemical test I am interested in conducting on my samples is the indole test, as it is often used to differentiate between members of the Enterobacteriaceae family, which of course is very relevant to my collection. Organisms such as Salmonella, Klebsiella and Proteus are indole negative, while Escherichia coli is indole positive. The indole test is used to investigate whether an organism can degrade the amino acid tryptophan and produce indole. However, for this test I require Kovac’s reagent with the lab currently doesn’t have. I asked some other postgraduate students and obtained an ordering form, which I filled out and sent to the Science Building’s administrator. Unfortunately I don’t know how long the reagent will take to get here, so I decided to just begin by preparing tryptone broth which is required to incubate the sample that you wish to test in.
How to make tryptone broth for indole testing
- Carefully weigh out your required amount of tryptone and sodium chloride into a flask (This will depend on how tryptone broth you need and how many indole tests you wish to conduct).
- Combine this with your required amount of distilled water and mix until homogeneous.
- Pipette 5ml of the broth into universal glass jars (Again, the amount will be based on how much broth you choose to make and how many indole tests you wish to conduct).
- Autoclave the broth at 121ºC for 15 minutes.
- Store the universals at 4ºC-10ºC .
Although I have never made this particular broth before, I found the process very simple and easy to conduct and I had no problems.
Friday 8th November
As it was a Friday I didn’t begin my work by making agar, but instead just collected my streak plates and motility test tubes out of the incubator. I observe my fourth batch of streak plates, again making detailed notes and taking photos of both the whole plate and individual colonies. After this I observed my API motility test tubes, making a note of the results for each sample, and taking photos.
I decided to just conduct oxidase tests on my newest streak plated samples, as it was the end of the week, and finish up my work on the bacterial collection next Monday. I conducted the oxidase tests as before, making up fresh oxidase reagent and taking note of the results.
This week has been fairly busy, however I am nearing the end of identifying the bacterial collection – which is really exciting! Next week I hope to finish off the last part of the collection, conduct indole testing, complete a detailed of list of my results so far and then begin preparatory work for the second part of my project.
A Year in the Life…