Nutrient agar autoclaved failed to give colonies

Discussion in 'Supporting Biology' started by JHRoss, Feb 14, 2020.

  1. Greetings to everyone,

    I have used an 2,8% w/v nutrient agar autoclaved solution to prepare 10 petri dishes of agar,

    Before I autoclaved the agar I just heated it at 50 Celsius for a while with a magnetic stirrer just to dilute it,

    Then I autoclaved it and then put in in the fridge,

    What went wrong?

  2. What did you incolulate it with?
    How long did you incubate it for after inoculation?
  3. I wasn't present during the inoculation phase, I incubated it for 48 hours (actually the incubator doesn't work properly but is still dark and warm)

    I see now in wiki how site this:

    but don't use less than 1.2 grams (½ teaspoon) of agar powder for every 10 centimetres (3.9 in) Petri dish you wish to use.

    1,2 grams per petri dish?!?!? Could it be this?
  4. Nutrient agar I always use 28g/L - 2.8% as you did
    Incubation could be a little short depending on what you've put on the plates - some can take a few days more

    Other than that how were they sterilising their spreaders and loops?

    We usually have blank plates as a result of students putting immensely hot inoculation loops into their bacterial samples and killing all the bacteria...

    Doesn't seem like you've done much if anything wrong at your end tbh
  5. Well, they did use Bunsen burners to sterilize the metal incubating loops,

    But as I said I wasn't present in order to observe what they did in details
  6. It's likely that there was never any organism in the first place or that your colleague when flaming the loop did not let it cool suffiently before using it to innoculate the plates. In other words the orgamism was fried.:)
    Jaytee likes this.
  7. True, I was taught to 'tap' loop onto agar plate first to cool it.
  8. I find it hard to think that all the students had such a wonderful aseptic technique that they managed to kill all their stock and fail to inoculate with anything without contamination. This tends to suggest that the agar is at fault as some would have been contaminated.
    If there are no colonies on any of the plates, try inoculating one yourself to prove the plate is OK - in fact just take the top off for half and hour then incubate.
  9. Just last week, I send out a set of agar plates - the students inoculated half the plates and did clean and dirty finger dabs with the other half. The inoculated half were completely clear after 5 days in the incubator whilst the finger dabs showed some lovely bacteria. My guess is either the broths I gave them were dead (unlikely since they were nice and cloudy) or the students did something stupid like flaming their loops AFTER they had dipped them in the broths. From experience, as long as the agar is made according to the instructions and sets, its unlikely to be the plates at fault.
  10. agreed
  11. Yes as I've said, this is the obvious answer. The question that begs to be asked is the Teacher's skills up to scratch?:)
  12. not usually, the microbiology is complex and even the biology teachers wont have done much before.
    GeorgetheScienceTech and ClaireS like this.
  13. Exactly what I thought Paul.:)