Two samples may have the same relative abundance of bacteria – for example, 50% bacterial species A and 50% bacterial species B – but if one sample has a total of 2 million bacterial cells and the other has 20 million bacterial cells, they have very different microbial loads. Thus, absolute abundance complements information on relative abundance in a sample. ¹ ²
In the context of human or animal health, relative abundance may be directly relevant when assessing bacterial overgrowth or the success of an antimicrobial treatment.
Three common methods are used for quantifying microbes in a sample: flow cytometry (cell counting), qPCR, or spike-in of defined numbers of microbial species absent from the samples to allow statistical calculation of both relative and absolute abundances. We provide the two most efficient and accurate methods – qPCR and spike-in.
With the qPCR method, calculation of absolute abundance is affected by DNA extraction biases. Extraction efficiency is not precisely linear, so extraction of twice as many microbes does not necessarily yield twice as much DNA.
In the spike-in method of microbial quantification, the same laboratory processing steps are used for calculating both relative and absolute abundance, as the spiked-in species are already included in the DNA extraction step. Our team can advise on the suggested number of spike-in species that will yield robust microbial loads without sacrificing too many reads.
Contact us for advice on whether QMP will add value to your study, and on the best method for assessing absolute abundance for your specific sample type.