4. Culture conditions for the dominant members of the bee gut microbiota
Culture-independent studies show that the gut microbiota of the honey bee is dominated by only 8 or so groups of bacteria (Martinson et al., 2011; Ahn et al., 2012; Moran et al., 2012). Given the difficulty of drawing delineations between species when it comes to bacteria, each “group” may actually represent one or more species. Nonetheless, these groups are valid in that they comprise distinct phylogenetic clades, and the bacteria within each clade are found in association with bees and not with other animals or environmental sources (Martinson et al., 2011; Ahn et al., 2012; McFrederick et al., 2012).
Bacteria from each of these groups can be cultured on standard microbiological media if supplied with the correct growth conditions. Recent culture-based studies have begun to support the findings of culture-independent surveys, giving due recognition to the dominant members of the microbiota (Olofsson and Vásquez, 2008; Engel et al., 2012; Tian et al., 2012; Vásquez et al., 2012; Kwong and Moran, 2013). By combining the low-bias culture-independent analysis of bacterial communities and the powerful molecular tools available in culture-based approaches, a rigorous study of the microbial associates of bees can be realized.
The following is a brief overview of the current state of knowledge regarding the cultivation of the bee gut microbiota for each of the dominant groups. All bacteria described here can be cultured by plating homogenized guts (aseptically removed from the bee using methods described in Section 2.1.1.) onto a suitable medium (often a standard nutrient-enriched agar that can be purchased from commercial suppliers). The plates are then incubated for several days, typically at 35-37°C, to allow growth of bacterial colonies.
Most of the bacteria associated with honey bee guts require a low-O2 atmosphere for optimal growth. To supply these conditions, plates may be incubated in dedicated CO2 incubators or sealed in pouches or jars with CO2-generating packets. Bacteria that require anaerobic conditions can be grown in dedicated anaerobic chambers by displacing air with nitrogen gas, or in sealed pouches or jars with the appropriate packets for generating anaerobic atmospheres. Packets for both CO2 and anaerobic atmospheres are available from commercial suppliers such as BD Biosciences’s GasPak system (Franklin Lakes, NJ, USA) and Oxoid’s CO2Gen and AneroGen (Basingstoke, Hampshire, UK). Identification of bacterial isolates should be carried out by DNA sequencing (as described in section 2.1.2. and 2.1.3.) rather than by phenotypic observations, as traits such as colony morphology and biochemical activities may be heterogeneous among members of the same species.
A word of caution, culturing methods will always lead to bias in what is recovered, and this bias is affected by the culture conditions you select. What is presented here is a general guide to methods that have successfully cultured some members of each dominant group, but it is important to recognize that these techniques may not always capture the full strain diversity within each group.