MadSci Network: Microbiology

Re: Why is it so hard to culture most bacteria?

Date: Sat Mar 17 08:57:52 2001
Posted By: Jim Caryl, Grad student, PhD Plasmid Molecular Biology, University of Leeds
Area of science: Microbiology
ID: 979409727.Mi

Hi there

Culturing bacteria is "feeding" them, such that culture media provides not only a nutritional framework, but a support media on/in which they can survive (whether this be solid or liquid phase).

Many bacteria will grow on general high nutrient media, otherwise known as undefined media. This is media that contains excessive amounts of all the basic nutritional requirements such as salts, and amino acids and vitamins and most importantly a carbon source; all at a physiologically compatible pH.

Such complex media may include casein digests, soybeans or sheep/horse blood. Perhaps the most common ones are yeast extract based media such as LB (Luria-Bertani) and 2x YT, which basically consist of a little sodium chloride, bacto-tryptone yeast extract and water - in varying proportions.

These media are highly nutritious but chemically undefined. Generally these are the first approach to culturing unknown organisms as most m/organism will grow on them. These include relatively easy to grow organisms such as Esherichia coli to the extremely fastidious organisms such as Leuconostoc mesenteroides. A fastidious organism is one with highly complex nutritional requirements.

The type of culture media one uses is defined by knowing the exact nutritonal reqirements of the organism you wish to culture. If these are known, it is possible to plate out swabs of microbial biofilm and only those organisms (9 times out of 10) that you want to grow will grow. (I say 9/10 as there is always some unidentified bacterium that can survive anywhere and is hard to get rid of!). These are called nutritionally defined media, or minimal medias. Unlike LB or 2YT, these medias have very specific amounts of essential salts, sugar and trace elements added e.g.

Potassium phosphate, potassium hydrogen phosphate, ammonium sulphate, magnesium sulphate, calcium chloride, glucose, trace elements (Fe, Co, Mn, Zn, Cu, Ni, Mo), distilled water and pH specified. Obviously some of these will have a greater presence than others, but between them the specific species / genus you wish to culture has an iso-osmotic medium with all the salts, sugar (carbon-source), and elements (required for enzymatic reactions, cofactors and protein constituents) they require.

Now, many fastidious organisms are called that because a) they have only recently been dentified and don't fall into any predefined genera, and as such noone has a clue what their specific nutritional requirements are; or b) are some form of extremophilic bacteria, which admitedly grow in amazingly diverse environments such as halophiles growing in 5+ Molar salt, or thermophiles growing at 68+ degrees celsius.....but take them out of their environments and one discovers that unless you recreate it perfectly, they don't survive. Microbiologists need to work for years to identify just what it is in that environment that allows them to survive.

In the whole scheme of things, pathogenic bacteria will often look no different to a non-pathogenic bacterium on an LB or 2YT type media (this is why care must be excercised working with unknown samples on these plates).

Generally, when you are referring to human pathogens, based on the parts of the body they invade, it is possible to deduce what their basic nutritional requirements are. This allows selective media to be created (also discovered by trial-and-error), which enables biomedical scientists/microbiologists in hospital laboratories or CDC to identify/confirm the causative organism of a bacterial disease.

There are many medias out there, some that make your bacterial species of interest appear as black, or blue, green, pink or red colonies. These are achieved by identifying certain sugars and chromogenic (colour producing) substrates that the pathogen of interest can ferment, but others can't.

It is a methodical, stepwise process where you start with a sample and initially you many wish to distinguish between fermenting bacteria and non- fermenting bacteria. As your pathogen is a fermenter, you take all the colonies that change the colour of the media (eg. MacConkey Media) when they ferment a specific sugar. These fermenters are known as enterobacteriaceae, which is a large taxon of enteric bacteria, some of which are pathogens, including yours.

Without going into too much detail of diagnostic microbiology, eventually you will be in a position where you have a media on which only your pathogen will grow. One that I use is Sorbitol MacConkey with is used to identify E.coli 0157 from a "refined" clinical sample. Obviously there are many srains of E.coli out there that won't do you any harm, but E.coli 0157 (if it is a certain serotype, i.e. H7) is a deadly haemorrhagic strain, but wouldn't look unlike a harmless strain on an LB plate.

Finally, there are of course a number of pathogens that we still are unable to culture, such pathogens include Mycobacterium leprae. This is because it is an intracellular pathogen, i.e. it lives inside of certain human cells called macrophages, which is why it is also quite hard to treat. For this reason it is not possible just to plate them out onto LB media as they won't grow. Currently they use special plates with confluent tissue cultures from the paw pads of an animal you will be familair with - Armadillos...which is why these animals have become very important in researching this disease that still affects millions worldwide.

So in answer, both general environmental organisms and human pathogens can be as easy or hard as each other to isolate, it very much depends on the organism. In general however, we tend to have more control over human pathogens as we understand better what nutrients are available to the bacteria in our bodies; however the environment is a big place and there could be any number of factors required for their survival. Remember of course that many human pathogens exist primarily 'out in the environment' to name a few such as Clostridium botulinum, Clostridium tetani and Clostridium perfringens, which cause botulism, tetanus and gas gangrene, respectively.

I would be enclined to say the easier to culture organisms are those which reside on humans, other animals and plants for which we have defined biochemistry; everything else is just a lot of hard work :o)

Hope that huge monologue helps....and sorry it's late - busy trying to grow fastidious organisms I guess!

Jim Caryl
MAD Scientist

A good starting point for microbiology (including culturing) is:

Madigan, M.T., Martinko, J.M., and Parker, J. (1997). Brock: Biology of Microorganisms. (8th ed). Prentice-Hall, New Jersey.

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