Can bacteria hide in liquid culture....

[DR.NNOO]

Hello, I hope I've come to the right place. I am a aspiring mycologist searching for some expert advice.

 

I intend on culturing pure mushroom mycelium in liquid media. Sometimes the liquid culture can get contaminated. It is a very controversial topic of whether or not this is always detectable by the naked eye.

 

Does anyone have any foresight or wisdom on this matter? I have a microscope that I've used to scope some liquid culture but I am not sure if this will suffice. 400x maginification is what I used.

 

Normally if a liquid culture is contaminated with bacteria, it turns cloudy, or there is obvious growth on the side of the container or top of the liquid media that is obviously not the intended organism meant to be propagated.

 

The LC's get stirred quite frequently, so I would imagine that the mix would be pretty homogeneous and with the growth rate of bacteria that its presence would be obvious but I could be mistaken. Maybe the bacteria interacts with the mycelium in a way I do not understand.

[CharonY]

Bacteria only turn the broth cloudy if there is a lot of them. Low-level contaminations are much harder to spot. A microscope with good optics could find bacteria at 400x, but it is easier if you stain them.

[DR.NNOO]

I'm pretty sure I am finding them. Using the iris diaphragm condenser I am able to make them visible, I believe. I just don't understand why the bacteria would remain at a low population if the food source is plentiful, like with liquid media. The mushroom mycelium only spreads so much, leaving a lot space uncolonized. Seems like they would proliferate as bacteria in sterile media tend to do.

Edited March 10, 2016 by DR.NNOO

[Robittybob1]

Do these bacteria affect the fungi mycelium in any way? What is the problem with bacterial contaminants?

[CharonY]

I'm pretty sure I am finding them. Using the iris diaphragm condenser I am able to make them visible, I believe. I just don't understand why the bacteria would remain at a low population of the found source is plentiful, like with liquid media. The mushroom mycelium only spreads so much, leaving a lot space uncolonized. Seems like they would proliferate as bacteria in sterile media tend to do.

 

Depends on the medium, the type of contamination and whether the fungus does something to the bacteria. Sometimes contamination are yeasts or other fungi (if they look like large cells) and there may be competition going etc.

[DR.NNOO]

The goal is to create a pure culture of mushroom mycelium in liquid that is used to inoculate sterilized grain. Because of the sterilized state of all the media, there is no competition upon inoculation. When you have a liquid culture that is contaminated, when you try to spread to other sterilized media, the bacteria tend to either take over, or impede growth of the mushroom mycelium. A lot of stuff in the mycology field is not exactly organized. There is a lot of how to but the mechanics behind why or why not things work are not always covered in grave detail. Contaminate fungi can be detected under scope. Yeast as well. Bacteria too, I need to purchase a 100x objective otherwise I cannot be as thorough in my screening.

 

Most contaminations can be detected visual in liquid culture. It is hearsay that a LC can be contaminated with bacteria but not give tell visually. I don't think I believe it. bacteria grows much faster than most organism, and should have no problem beating out competition in liquid culture. That is my opinion.....I've been wrong before lol.

 

I use malt extract at 3% to water for the media. Malt agar allows better inspection of growth. I've heard that bacteria can hide

 

 

Once again, bacterial contamination is detectable by the naked eye in most cases. Some argue that a LC can be contaminated with bacteria but not obvious to the naked eye. My argument is that liquid culture, especially malt, is too potent of a food source, and that liquid culture is too efficient of a growth media and that any bacteria present would dominate the media, as well be easily detectable. This is in the case of a bacteria vs mushroom mycelium, I believe. I am seeking information to prove me otherwise.

 

It is possible that the mycelium might produce metabolites that might retard the growth of bacteria but that would be more likely to happen with contamination after the mycelium culture was matured and not when newly inoculated.

Edited March 10, 2016 by DR.NNOO

[Robittybob1]

I have listened to many videos on growing mushrooms but you are the first to worry about bacterial competition. The usual emphasis is on eliminating other strains of mushrooms.

[DR.NNOO]

Please stay on topic.

[Robittybob1]

In #6 you seem to be talking about bacterial competition. So I feel I'm on topic.

[DR.NNOO]

Isolating single strains is done on agar and not liquid culture. They are two different beast. I actually grow mushrooms and I know for a fact that liquid culture and bacteria are a common topic. Bacteria and mold are all a factor during each level of expansion of mushroom mycelium and is the main challenge of growing. People have bacterial contamination all the time with liquid culture, grains, and bulk substrate, for all sorts of different reasons. If you watched a lot of videos on growing and never heard mention of bacteria I suggest you seek a more qualified source.

I don't want to come off impolite but I super excited to get this solved if possible.

 

I am here to talk about bacteria's ability or lack of ability to not proliferate a liquid culture.

Edited March 10, 2016 by DR.NNOO

[CharonY]

 

Most contaminations can be detected visual in liquid culture. It is hearsay that a LC can be contaminated with bacteria but not give tell visually. I don't think I believe it. bacteria grows much faster than most organism, and should have no problem beating out competition in liquid culture. That is my opinion.....I've been wrong before lol.

 

 

It really depends on the contamination, although I will agree that the most common ones are from the experimentator and many grow rather fast. However, you mentioned you are using malt broth. If memory serves the pH is usually low, somewhere around 5.5. This an intentional measure to limit bacterial growth and many, including fast-growers such as Escherichia grow very slowly under these conditions assuming temps of 20C or higher.

So it is quite reasonably to expect only modest bacterial growth under these conditions, of you do not have acidophilic contamination. Likewise temperature could also be an issue, if you incubate them around RT. But again, it depends with what you have contaminated the cultures.

[DR.NNOO]

Thank you for the the explanation.

 

Does anyone think there is a way to create a liquid culture that is sugar based that would always allow bacteria to be detectable by visual inspection?

Maybe if I use 1% malt instead of 4%? PH will be closer to neutral.

In the case of a contaminated LC that is used to inoculate grain spawn, the mycelium growth is significantly stunted, not usable. Can I create a liquid culture that won't grow my mushroom mycelium unless it is a pure culture?

You did a good job of putting that in perspective. Thank you for your help!

 

I still believe their are some other visual indicators that an LC is contaminated but they are somewhat hypothetical and based off limited personal experience. I had 5 LC's that were contaminated from their inoculation material, they all deceived me, but now that I know they are contaminated I remember several things not the same from previous, uncontaminated LC's.

Any how, is there a way to remedy this? I mean what you have said is true for all substrates but in the case of liquid culture the liquid disguises the bacteria where as on agar it would grow on top of the solid surface. I need for any contamination to grow very well, so it will not hide. Can this be created with liquid culture?

Edited March 12, 2016 by DR.NNOO