Culture Barriers in Bacteria

Why are so many organisms “unculturable,” that is, what makes them difficult to grow in the laboratory? In the first place, one needs to know something about the nutritional and environmental requirements of a strain to culture it. Macro- and micronutrients must be provided in a Petri dish; the oxygen tension must be correct, and any other factors required for growth must be present. Culturing bacteria in the laboratory is a good way to look for “knowns” but much less efficient for finding “unknowns”. Many unculturable organisms might ultimately be able to be cultured, but we need to understand their biochemistry first in order to develop protocols to grow them.

Second, some organisms live in symbiotic consortia in nature and so cannot be grown isolated in pure culture. Since pure culture methods are the mainstay of microbiological practice, it becomes impossible to grow them in the laboratory by conventional means. Some investigators have had success growing consortia of organisms. However, it is often necessary to have some sense of which species are required for co-survival in order to set up a multi-species culture.

In spite of all the pitfalls and challenges associated with culture, microbiologists will continue to attempt to culture new species. It is easier to test hypotheses in simple laboratory cultures than to do so in situ in ecosystems. Moreover, sequence data obtained during metagenomic studies are more easily ordered when reference genomes from known cultured organisms are available for comparison. Even though most of the published data on the marine cyanobacterium Prochlorococcus have been obtained using culture-independent methods, our understanding of this organism and its ecology is dependent on experiments performed on a handful of cultured strains, and their genome sequences. But Prochlorococcus still illustrates the importance of in situ metagenomic research—it is only by studying the ecology of this organism in the ocean that seawater-based media have been devised to grow it in the lab.

It is important to emphasize that, although culture-independent methods tend to capture more species than do culturebased methods, some species are missed by culture-independent techniques as well. Methods that amplify and then clone 16S rRNA genes and use these clone libraries as the basis for a species catalog will miss species that have 16S rRNA genes that are not as close a match to the oligonucleotide primers used as other species in the community.

One such study compared such culture-independent and culture-dependent methods to catalog bacteria residing on apple leaves and found only partial overlap between the two methods. Twelve OTUs were found by both methods. The culture-independent method identified 21 OTUs that were not found in the culture-dependent method, while the culture-dependent method detected 11 OTUs of the order Actinomycetales that were missed by the culture-independent methods. Whole shotgun sequencing of metagenomes (rather than targeted amplification of specific genes by PCR) further increases the species that can be identified, as illustrated by the example in Box 17.3 on ancient metagenomes. In addition, whole genome sequencing does not

require any prior knowledge of the community being studied, which is an advantage. In reality, the power of whole metagenome sequencing improves when some of the species identified have been previously characterized, since this will increase the number of DNA fragments that can be assigned taxonomically.