Abstract
A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.
Keywords: Biofilm, Cyanobacteria, Synechococcus elongatus, Chlorophyll measurement, Sessile, Planktonic
Background
Several recently published studies indicate an emerging interest in the mechanisms that underlie cell-aggregation and biofilm development in cyanobacteria (Fisher et al., 2013; Jittawuttipoka et al., 2013; Schatz et al., 2013; Enomoto et al., 2014; Schwarzkopf et al., 2014; Enomoto et al., 2015; Oliveira et al., 2015; Agostoni et al., 2016; Parnasa et al., 2016). We recently reported a self-biofilm-inhibitory mechanism that dictates planktonic growth of the model unicellular cyanobacterium Synechococcus elongatus PCC 7942 (Schatz et al., 2013; Nagar and Schwarz, 2015). Abrogation of the biofilm-inhibitory process by inactivation of particular genes results in robust biofilm development in this otherwise planktonic strain (Schatz et al., 2013; Nagar and Schwarz, 2015). These studies required quantification of the extent of biofilm development in various strains and under different conditions. Crystal violet is commonly used for quantification of biofilms in heterotrophic bacteria (O’Toole and Kolter, 1998). This staining procedure, however, quantifies only the sessile fraction of cells. Here we provide a detailed protocol for culture growth and quantification of cyanobacterial biofilms using chlorophyll measurement as a proxy for biomass accumulation in sessile as well as in planktonic cells. These measurements allow estimation of the total biomass accumulated and representation of the relative fraction of chlorophyll in sessile or in planktonic cells.
Materials and Reagents
Equipment
Procedure
Data analysis
The percentage of chlorophyll in suspension is calculated from the amount of chlorophyll in the planktonic and biofilm fractions. These values are calculated based on measured chlorophyll absorbances, sample volumes, and dilution factors, as described below. An example calculation is provided in Table 1. Table 1. An example of calculations of chlorophyll (Chl) in planktonic and biofilm cells
Notes
Recipes
Acknowledgments
This protocol is modified from previous studies (Schatz et al., 2013; Parnasa et al., 2016). Growth medium is based on the protocol by (Stanier et al., 1971). Chlorophyll measurements are based on previous protocols (Arnon, 1949; Ritchie, 2006). Rakefet Schwarz and Susan Golden are supported by the program of the National Science Foundation and the US-Israel Binational Science Foundation (NSF-BSF 2012823). This study was also supported by a grant from the Israel Science Foundation (ISF 1406/14) to Rakefet Schwarz.
References
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