ZymoBIOMICS Spike-in Control II (Low Microbial Load) consists of three bacteria strains, Truepera radiovictrix, Imtechella halotolerans and Allobacillus halotolerans. When spiked into a microbial sample, this product will serve as an in situ positive control for DNA-sequencing-based microbiome measurements. Of the three chosen bacteria, Imtechella halotolerans is Gram-negative and Allobacillus halotolerans is Gram-positive. Truepera radiovictrix is resistant to lysozyme lysis and has very high GC content. They represent different challenges in NGS-based analysis. Moreover, with accurately quantified cell number and a log abundance distribution, this standard enables absolute cell number quantification in cases such as pathogen load detection.