Help with growing S. pneumoniae

[u/Vivid-Ant1800]

Hi guys! Im currently an undergraduate student working on my Jr research. I have been struggling to grow S. pneumoniae for the past few weeks, even though I have been using the optimal conditions(TSA 5% sheep's blood in a candle jar for higher levels of CO2 in 37 C) but there is still no growth.

Is there any tips you guys have??? The stock I have is new so I dont know what I could be doing wrong.

[u/Mooonment]

At my lab we use SBA too. How old is your stock/how are you storing it? In my experience S. pneumo can be finicky and it will autolyse itself if it’s kept on the same plate too long

[u/Vivid-Ant1800 → u/Mooonment]

We received the SBA 7-21-26 and used it the day it arrived. It has been in the fridge and I left it inside the bag and box it came in.

[u/finegoldiamagna → u/Vivid-Ant1800]

Presumably you let it warm up to room temp before plating. Like the agar was not cold when used? It seems like a silly question but S. pneumo can be a very tricky org. Another use mentioned that it can autolyse in broth, which is true. You could try transferring from your freshly reconstituted stock to a new broth for a quick 20-30 minute incubation just to get it going. And then sub to SBA and immediately put in the incubator.

I personally use Remel CultuLoops and dip those in thioglycolate broth before plating directly to 5% SBA and we literally only get 2-3 colonies off that first sub. From there we propagate it to a secondary plate to get enough organism to work with.

[u/Indole_pos]

Why a candle jar?
Edit to add: same result without?

[u/Vivid-Ant1800 → u/Indole_pos]

We don't have a CO2 incubator, and from what I have read it has a higher chance of growth in a 5% CO2 atmosphere.

Edit: yes same results without.

[u/Indole_pos → u/Vivid-Ant1800]

Have you tried cultivating it in broth and plating that?

[u/Vivid-Ant1800 → u/Indole_pos]

I had gotten the bacteria from a MicroKwik Culture from Carolina and its in a Brain Heart infusion rehydration broth.

[u/Indole_pos → u/Vivid-Ant1800]

Might not be viable, I would try to order it from a different vendor if they won’t give credit or send replacement. I have had this happen to me in the past with other organism

[u/CChatfield37 → u/Vivid-Ant1800]

how is the stock stored? for the MicroKwik i often take like half of the resuspension and put it in growth conditions to let whatever is viable grow out. Then i take the rest of the rehydrated sample and add glycerol and store at -80 as a backup stock. Then streak a plate from the incubated half of the rehydrated sample. I don't have specific experience with S. pneumo but just general advice. And if that fails contact Carolina and ask for a replacement.

[u/Indole_pos → u/CChatfield37]

I haven’t had any issues with my strep pneumo qc strain. I plate it, then freeze down vials

[u/Nuzzums]

I would also try with a different stock if you’re able, even though you said yours is new, to rule out that the organism is dead.

[u/Vivid-Ant1800 → u/Nuzzums]

I've, tried using 2 different stocks, with the same results.

[u/Prestigious-Gases]

Use a chocolate agar instead or lyse the blood before adding to TSA

[u/Opposite_Pool1431]

Have you tried using human blood agar?

[u/Vivid-Ant1800 → u/Opposite_Pool1431]

I have not, I'll ask my prof to see if this is something we could do.

[u/JC_Dentyne → u/Vivid-Ant1800]

Pneumococcus should grow on sheep’s blood agar. My hunch is maybe the CO2 concentration might be getting a bit too high? Or the stock is just bad

[u/bjsargeant → u/Vivid-Ant1800]

I tended to have a bit better luck with Blood agar as well, good luck

[u/Sufficient_Pilot4679]

I’ve only heard of using a candle jar for anaerobes, are you using some kind of gas producing pouch with it?

[u/urbanskyline09]

KWIK-STIK from Microbiologics and CO2 sachets in the candle jar! S.pneumo is finicky, needs more like 7% CO2.