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Human milk is considered the optimum feeding regime for newborns and is a source of bacteria for the developing infant gastrointestinal tract. However, as with all low biomass samples, standardization across variabilities such as sample collection, storage, and extraction methods is needed to eliminate discrepancies in microbial composition across studies. The aim of this study was to investigate how different storage methods, temperatures, preservatives, and extraction kits influence the human milk microbiome, compared to fresh samples. Breast milk samples were processed via six different methods: fresh (Method 1), frozen at −80°C (Method 2), treated with RNAlater and stored at 4°C or −80°C (Methods 3 and 4), and treated with Milk Preservation Solution at room temperature (Methods 5 and 6). Methods 1‐5 were extracted using PowerFoodTM Microbial DNA Isolation kit (Mobio), and Method 6 was extracted using Milk DNA Preservation and Isolation kit (Norgen BioTek). At genus level, the most abundant genera were shared across Methods 1‐5. Samples frozen at −80°C had fewest significant changes while samples treated and extracted using Milk Preservation and Isolation kit had the most significant changes when compared to fresh samples. Diversity analysis indicated that variation in microbiota composition was related to the method and extraction kit used. This study highlighted that, when extraction from fresh milk samples is not an option, freezing at −80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrate that choice of extraction kit had a profound impact on the microbiota populations detected in milk., The aim of this study was to investigate how different storage methods, temperatures, preservatives, and extraction kits influence the human milk microbiome, compared to fresh samples. This study highlights that when extraction from fresh milk samples is not an option, freezing at −80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrate that choice of extraction kit had a profound impact on the microbiota populations detected in milk.

The composition of the microbiota associated with the human ileum and colon in the early weeks of life of two preterm infants was examined, with particular emphasis on the Lactobacillus and Bifidobacterium members. Culturing work showed that bifidobacteria and lactobacilli in the ileostomy changed over time, compared with the colostomy effluent where there was far less variation. The colostomy infant was dominated by two phyla, Actinobacteria and Firmicutes, while in the ileostomy samples, Proteobacteria emerged at the expense of Actinobacteria. Bacteroidetes were only detected following the reversal of the ileostomy in the final fecal sample and were not detected in any colonic fluid samples. Clostridia levels were unstable in the colostomy fluid, suggesting that the ileostomy/colostomy itself influenced the gut microbiota, in particular the strict anaerobes. Pyrosequencing analysis of microbiota composition indicated that bifidobacteria and lactobacilli are among the dominant genera in both the ileal and colonic fluids. Bifidobacteria and lactobacilli levels were unstable in the ileostomy fluid, with large reductions in numbers and relative proportions of both observed. These decreases were characterized by an increase in proportions of Streptococcus and Enterobacteriaceae. Clostridium was detected only in the colonic effluent, with large changes in the relative proportions over time.