Protocol for the collection, packaging, and transportation of preterm newborn stool samples

•The steps prior to the fecal metagenomic analysis of premature infants are detailed.•Surgical tissue added to the diaper prevents absorption and preserves the microbead.•Hygienize the neonate after collection, pay attention to its comfort and vital signs.•The protocol guaranteed the preservation bacterial DNA in 78 % of the samples. The newborn's intestinal microbiota can vary with gestational age. Reliable analyses of stool samples require establishing the steps of collection, packaging, transportation, and storage. To describe the development of a protocol and test an algorithm for the sequence of actions and procedures for the collection, packaging, transportation, and storage of stool samples from Preterm Newborn (PTNB) admitted to a Neonatal Intensive Care Unit (NICU). This is a descriptive, observational study linked to a non-randomized controlled clinical trial conducted in the NICU of two public hospitals covenanted to the Unified Health System of a large city in inland northeastern Brazil. The methodology adopted in its development followed the Brazilian Ministry of Health Preparation Guide recommendations. The flow of actions and procedures was consistent with scientific evidence. The sequence of work process steps to implement the proposed protocol was tested and resulted in a graphical representation of an algorithm compatible with the reality of Brazilian public hospitals. Following the protocol steps with the description of the rules of conduct and recommendations regarding the collection, packaging, transportation, and storage of stool samples from PTNBs ensured the preservation and integrity of the bacterial DNA in the stools sample. The details of the recommendations will allow their reproducibility and improvement by professionals and researchers with similar study objects. World Health Organization (WHO) under Universal Trial Number (UTN) code U1111–1266–2295, under register RBR-3mm7cs in the Brazilian Registry of Clinical Trials (REBEC).