A Year in the Lab: Indole Testing

After spending last week out of the lab to work on literature work, I was really glad to find that my Kovac’s reagent had been delivered! So I headed back into the lab on Monday morning to get started on re growing my bacterial samples so I could conduct Indole testing.

Monday 18th November

While I was inputting data into my database spreadsheet last week I also made a note of those samples which needed to be Indole tested, and those which for what ever reason hadn’t been Gram stained, Oxidase or Motility tested yet. So I began the week by getting these samples out of the freezer, letting them reach room temperature before vortexing them.

IMG_3575Aseptic set up for spread plating samples onto MacConkey agar

I had a batch of MacConkey agar plates already made so I then spread plated my samples as before and incubated them overnight at 30ºC. As I will need more MacConkey agar plates I decided to make, autoclave and pour another batch.

Tuesday 19th November

I started the day by making more MacConkey agar and getting it onto autoclave. I also decided to make up some fresh Tryptone broth, decanting it out into universal jars (5ml each) before autoclaving.

While these were autoclaving I got my spread plates out of the incubator and viewed them using the colony counter, as before. I made detailed notes and took close up photographs of the colonies on each plate.

IMG_3591 IMG_3584 Selection of MacConkey spread plates showing colonies observed

I then heat fixed slides and Gram stained those samples which hadn’t been viewed yet. I observed these under light microscopy, making notes on whether the samples were Gram negative or positive and the cell morphology.

IMG_3627Gram staining heat fixed slides

I also conducted oxidase testing on those samples which hadn’t been tested yet. I took notes on whether the samples were oxidase positive or negative.

As my MacConkey agar and Tryptone broth had finished autoclaving, I removed them both from the autoclave and allowed them to cool. Once cool enough I poured my MacConkey agar plates aseptically, allowing them to set before inverting them.

Once my Tryptone broth had cooled, I labelled each bottle with the sample number and aseptically inoculated each.

How to inoculate Tryptone Broth

  • Begin by setting up an aseptic work space.

IMG_3625

  • Ensure you have a wire loop, spread plated samples and labelled tryptone broth in universal jars (4ml each).
  • 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.
  • Allow to cool slightly, and then pick up one colony from the overnight agar plate, making sure not to put the wire loop down or touch anything.
  • Carefully open the labelled tryptone broth universal jar, flaming the neck of the jar (Remembering not to put the lid on the bench) before putting the wire loop into the broth and swirling it around gently.
  • Remove the wire loop, flaming again before putting it down on the bench.
  • Re flame the neck of the universal jar and replace the lid.
  • Incubate for 24-48 hours at 35ºC.

IMG_3618

  • After incubation: Add 5 drops of Kovac’s reagent and read colour change. A positive result is indicated by the development of a cherry-red coloured ring. A negative result is indicated by no colour change.

I have inoculated other broths before, but not tryptone broth, however I found this procedure pretty simple and problem free.

Wednesday 20th November

I started the day by removing my inoculated tryptone broth samples from the incubator, then added 5 drops of Kovac’s reagent and observed any colour changes. I made notes of the results for each sample and took photographs.

IMG_3647Adding Kovac’s reagent to incubated inoculated tryptone broth

Although I haven’t performed Indole testing before, I found it really easy to differentiate between those which were Indole positive, and displayed a cherry-red ring and those which were Indole negative and displayed no colour change.

Thursday 21st  – Friday 22nd November

I had full and very busy days on Thursday and Friday (Lab demonstrating and other work) so I didn’t visit the lab again this week. Instead I inputted my new data into my database spreadsheet and carried on identifying my samples based on this data.

IMG_3615Collection of MacConkey agar spread plates, in stacks of suspected organism

Next week…

I’m very glad to have been able to start Indole testing, as well as completing any other tests that hadn’t been finished yet. I will be carrying on with Indole testing next week, as I ran out of time this week. Hopefully all testing will be completed by next week and I may even get chance to finally start working with the Spiral plater!

A Year in the Life…

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