Australia's Science Forum

Hi All

I have been having problems with my agar plates. The recipe I use is 15g agar 1L distilled water. I have sterile petri dishes. The agar sets perfectly the problem is that nothing grows, its like the agar is sterile. The students picked up germs from everywhere eg. coughing into plate etc. we incubated for 3 days at 35C but nothing grew. My procedure is get the water to nearly boiling point, add agar bit by bit until dissolved and continue to near boil for 15 mins. What am I doing wrong this is the 3rd lot that hasn't grown anything. Please help.

Nicky

Does your agar have nurtient broth in it? It sound like you only have the agar which is only a setting agent, bot a food source for the bacteria.

On another point, letting students cough onto the plate is not advisable as it is not allowed due to WH&S for Qld.

There are several different previous post about plate on site, check them for more information.

I also beleive that you are not allowed to incubate plates over 30c, to avoid the growth of human pathogens.

Nicky,

When making agar plates you normally add some sort of nutrient to encourage the bacteria to grow. I don't think agar is enough.
When I make my plates I add either beef stock powder or peptone.

The recipe is: 15g Agar, 5g peptone OR 3g alternative nutrient eg. beef cube (I use powder), Bonox, Vegemite.

1000ml H2O, add agar and nutrient, bring to boil stirring constantly( I use a magnetic stirring hotplate)

I then place this in a pressure cooker in a conical flask seal with cottonwool and foil and cook for 15 mins.

Allow the agar to cool to 50 - 55deg bgefore pouring into sterile pertie dishes

Incubate exposed plates at 30deg (this generally stops any nasties( Pathogen from growing)

This method has worked wll for me in the past.

Good Luck

Lorrae

Hi Nicky,

I use basically the same method as Lorrae but the ingredients are:

750mls distilled water
15 grams agar
1 gram sucrose
3.7 grams sodium chloride
3.7 grams peptone
2.5 grams beef extract (Vegemite)

We always get plenty of growth with this food source.

Best of luck,

Robbie

I lovely lady from Holsworthy High, sent her Organism to ICPMR Institute of Clinical Pathology and Medical research. In 2004, they identified the Pepper corn MIDI system as Bacillus subtilis. A safe Bacteria for class use in NSW.

Peppercorn Infusion
Simple & Safe way to grow bacteria for class use.

* Soak approximately 20g peppercorn in 200ml water in a conical flask. Stopper with cotton wool.

* Stand at room temperature for several days or place in incubator overnight.

* Cultures need to be a known bacteria as required in CSIS 3.2.6.2.P57
This peppercorn infusion has been analyzed ( as I put above)

* Bacillus subtilis a gram-positive rod.

* To simulate anti biotic discs and growth of bacteria, soak small disc of filter paper in "Home Brand" Disinfectant. Place on agar plates that have been streaked evenly with Peppercorn infusion, Incubate overnight, Observe.

* To dispose of peppercorn infusion place open plates in a container (eg Bucket or tray) pour "Home Brand" disinfectant over plates. Soak for several days, drain and discard to the garbage.

We have been useing the Safe way since 2004, when this lovely lady let us have a copy at a network meeting. I have rung her, and she is happy for this to go onto this site.

Regards

Thanks everyone for your input. I have used the peptone and beef extract before without any luck. Can anyone tell me if the peptone is supposed to be rock hard, I have to chisel it out of the bottle. If so what is the trick to getting it out.

Thanks Nicky

The best solution is nutrient agar from southern biological and I think Serrata has it as well. Dissolve 3g into 1L of water, boil, autoclave and pour into petri dishes. Easy and you can grow almost anything. Incubate at around 30-32oC.
Good luck Lada

Sorry, the amount of nutrient agar is 3g to 100ml water(around 3% soln)

No Nicky, peptone is not meant to be rock hard, it should be a powder. Being an organic substance, it has quite a short shelf life.

Diana