A pure culture is a laboratory culture containing a single species of organism. Isolation typically involves separating individual microbial cells from an environmental sample so that each cell can multiply to form a visible colony composed only of its descendants. Despite this technique’s long history, only about one percent of all known bacterial species have been successfully grown in pure culture under standard laboratory conditions. This means over 99% of the microbial world remains uncultivated because their complex natural habitats are difficult to replicate in a laboratory setting.
The Great Plate Count Anomaly
The realization that most microbes resist cultivation emerged from the Great Plate Count Anomaly. This discrepancy describes the significant difference between the number of microbial cells observed under a microscope and the number of colonies that grow on a nutrient agar plate from the same environmental sample. Historically, researchers would examine a sample directly to count the total number of cells, and then spread the same sample onto a Petri dish containing growth media to count the number of Colony Forming Units (CFUs).
The count from the agar plates is consistently and drastically lower than the direct microscopic observation. The difference is often by several orders of magnitude, meaning scientists observe thousands of cells for every one that successfully forms a colony. This observation demonstrated that traditional cultivation methods were only capturing a tiny fraction of the total microbial population.
Why Most Microbes Resist Cultivation
Many microbes resist cultivation because their growth depends on highly specific conditions. Each microbial species has evolved to thrive in a precise physicochemical niche, requiring a delicate balance of temperature, pH, osmotic balance, and pressure. For example, extremophiles living in hydrothermal vents require conditions of high heat and pressure that are challenging to reproduce in a conventional incubator.
Nutritional requirements represent another major barrier, as many microbes need complex nutrients or growth factors that are not contained in standard media. Specialized organic compounds, metal ions, or specific vitamins that are abundant in their natural environment may be entirely absent from the artificial medium. In some cases, preparing the media, such as autoclaving agar, can inadvertently create inhibitory compounds that prevent growth.
A significant reason for resistance is the interdependence between species, often referred to as syntrophy. In their natural environments, microbes rarely exist in isolation; they form intricate communities where the waste product of one organism serves as the food source for another. Isolating one species in a pure culture removes its necessary partner, starving it of an essential nutrient or allowing toxic metabolic by-products to accumulate.
Exploring the Unseen Majority
Because the majority of microbes cannot be grown in a laboratory, modern microbiology relies heavily on culture-independent methods to study the unseen majority. The field of metagenomics has revolutionized this effort by bypassing cultivation. Instead of growing the organisms, researchers extract the total DNA directly from an environmental sample, such as soil, ocean water, or a human gut sample.
This extracted genetic material, known as the metagenome, contains the collective genomes of all the microbes present in the community. High-throughput sequencing technology is then used to read the millions of DNA fragments in the sample. By analyzing these sequences, scientists can identify which species are present, estimate their relative abundance, and predict the metabolic functions they are capable of performing.
This approach allows researchers to understand the diversity and function of microbial communities without ever having isolated a single species in a Petri dish. Techniques like sequencing the 16S rRNA gene, a universal marker for bacteria, can reveal the taxonomic makeup of a sample. A related method, single-cell genomics, sequences the DNA from an individual, uncultivated cell, providing a high-resolution view of its genetic potential.