Journal of Nature and Science, Vol.1, No.6, e116, 2015 ISSN 2377-2700 | www.jnsci.org/content/116 1 Jun 2015, Vol.1, No.6, e116 Microbiology Effect of Water Washing Purification on the Surface Morphology of Bacillus cereus Spores Jessica M. Goss 1 , Eric J. McCullough 2 , Cristina Stanciu 1 , Vamsi K. Yadavalli 2 , Christopher J. Ehrhardt 1,* 1 Department of Forensic Science, Virginia Commonwealth University, Richmond, VA 23284, USA. 2 Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA The architecture of spore surfaces plays an important role in the pathogenesis and natural ecology of many Bacillus organisms. However, changes to the laboratory preparation method for Bacillus spores may be a source of phenotypic variability in cell surfaces that can affect our understanding of in situ morphology and make cross-study comparisons more difficult. In this work, we examined how variation in the number of water washing steps during spore purification influenced the three-dimensional morphology of Bacillus cereus spore surfaces. Two strains of B. cereus, str. 14579 and T-strain, were cultured in liquid medium and purified using 0, 1, 3, or 5 water washing steps. Results showed that the nanoscale morphology of the spore surface is affected by the number of water washes such that early wash step samples had higher levels of roughness, quantified with a Root Mean Square (RMS) algorithm, which decreased with successive washes. The presence of large (>200 nm) surficial features, representing cell fragments and/or debris from the culture, was also inversely correlated with the number of washes. The presence of cellular debris in unwashed spore samples also changed the nanomechanical signatures of the outermost surface but did not affect the properties of the underlying cell. This suggests that variation in purification method can affect the nanoscale morphology and mechanics and should be considered when analyzing micro- to nano-scale surface properties of Bacillus cereus spores. Journal of Nature and Science, 1(6):e116, 2015 Bacillus cereus | spore | atomic force microscopy | nanomechanical Phenotypic profiling of organisms within the Bacillus ACT group (anthracis, cereus, thuringiensis) can be a valuable tool for investigating the natural ecology and pathogenic properties of virulent species/strains. Specific morphological features on Bacillus spore surfaces correlate well with taxonomic divisions and can differentiate closely related species (1, 2), an important goal for public health and biodefense agencies. Surface phenotypes may also indicate key aspects of the production method for Bacillus spores relevant to a microbial forensics investigation (3). Although many of the surface characteristics reported in previous studies (Table 1) are linked to intrinsic properties of the cell, spore surfaces are also dynamic features that can be affected by culturing environment and/or preparation method in the laboratory (2, 4, 5). Since the culturing protocol for spores often differs between studies and across laboratories, this may create artificial variability in surface textures that is unrelated to the underlying biology of the cell. This may complicate interpretation of surface phenotypes and make cross-study comparisons more difficult. One particular aspect of spore preparation that varies across studies is the purification method. After culturing Bacillus in the appropriate sporulation medium, cells are typically harvested and subjected to procedures designed to remove cell debris, vegetative cells, and residual growth medium from the intact spores (6, 7). There are many established purification methods and one of the most common is washing the spores several times in ultrapure water (e.g., Table 1 and references therein). The number of successive water wash steps can range anywhere from one to more than ten. Because the overall ‘purity’ of the spore culture (i.e., the proportion of vegetative cells and/or cell debris in solution or physically associated with cell surface) changes with each wash step (6, 8), the number of water wash steps has the potential to influence surface textures of individual spore cells. While previous work has indicated a correlation between the extent of purification and the nanoscale texture of individual Bacillus spores (9), this has not been explicitly tested across multiple strains and a relevant range of wash steps. Therefore, the goal of this study is to investigate how the surface morphology of Bacillus cereus spore surfaces is affected by the number of water washing steps used to clean the spore culture after harvesting. We use atomic force microscopy (AFM) as a tool to characterize the nano- and microscale topography of spores that have undergone different processing histories after laboratory culturing. AFM is particularly well suited for analyzing spore surfaces because it allows for high precision imaging and real-time measurement of cells without complex or destructive sample preparation steps (10, 11). Additionally, in comparison to other types of high resolution microscopic techniques (e.g., electron microscopy), morphological imaging with the AFM can be performed in conjunction with nanomechanical and biochemical analysis of the cell surface (12, 13). In this study, the nanomechanical properties of single spore cells were characterized before and after washing to further elucidate physical changes that occur with water washing purification of Bacillus spore cultures. Experimental Spore culture and preparation Two different strains of Bacillus cereus were used for all experiments: Bacillus cereus T-strain (BcT) which was donated by the FBI Laboratory’s Counterterrorism and Forensic Science Research Unit (Quantico, VA) and Bacillus cereus 14579 (ATCC#14579, Manassas, VA). B. cereus was chosen because it is biochemically and structurally similar to Bacillus anthracis (14, 15) and can be manipulated at Biosafety Level 1. All cultures of Bacillus cereus were maintained at 30°C on Trypticase Soy Agar (TSA) (30 g Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ), 15 g agar (American BioAnalytical, Natick, MA)). Broth starter cultures were made by picking single colonies of Bacillus cereus from TSA medium and inoculating into 125 mL of Trypticase Soy Broth (TSB). Starter cultures were incubated for ~16-18 hours at 30°C and 225 rpm. To induce sporulation, either 1 mL or 20 mL of starter culture (Bc14579 and BcT, respectively) were added to 250 mL of sporulation medium. Preliminary experiments were initially conducted with Bacillus cereus spores prepared in four different medium recipes, ‘G’ Medium (16), ‘GPep’ (G Medium supplemented with 8g -1 of meat Peptone), GTryp (G Medium supplemented with 8gL -1 Tryptone), and ‘GBHI’ (G Medium supplemented with 8 gL -1 Brain Heart Infusions). AFM analysis of various individual spores from each medium showed that variation in the complex nutrient source (peptone, tryptone, brain-heart, etc.) had no discernible effect on the surface morphology of the spore (data not shown). Therefore, only GTryp medium formulation was used for this study. Sporulation cultures were incubated at 30 °C and 300 rpm in an orbital shaker. The cultures were monitored throughout the incubation period and were harvested when the proportion of spores in the cell population reached ≥ 90 %. Cells were typically harvested between 24 and 48 hours depending on the strain. _________ Conflict of interest: No conflicts declared. *Corresponding Author. Department of Forensic Science, Virginia Commonwealth University, 1015 Harris Hall South, Richmond, VA 23284, USA. (804)828-8420. Email: cehrhardt@vcu.edu © 2015 by the Journal of Nature and Science (JNSCI).