Brewing Bokashi: Strengthening Student Skills in Dilution Theory through Fermentation Analysis†

Abstract

INTRODUCTION One of the fundamental techniques of microbiology is the use of dilution theory: the mathematical calculation of colony-forming units per millimeter (CFU/mL) of a fluid as determined by spread plate data and consideration of the involved dilution factors (7). In spite of student practice in the laboratory with spread plating and with calculating CFU/mL of samples from their plate counts, my students consistently perform poorly on examinations when asked to apply dilution theory. For example, from 2008 to 2013, only 20%, on average, of the students tested on dilution theory were able to correctly apply dilution theory to problem solving on a quiz. Addressing this deficiency has been an ongoing challenge. My students initially are introduced to dilution theory with biosafety level 1 (BSL-1) organisms to help familiarize them with the process of pipetting, aseptic technique, and spread plating. After further development of their skills in culturing and manipulating bacteria, I engage the students in a fermentation-based laboratory experience where students repeatedly perform serial dilutions and spread plating and apply dilution theory to their data, in an attempt to improve student understanding of and practice with dilution theory. Other educators have developed student activities related to plate counts to foster active learning. March et al. (3) found increased student learning about dilution theory and plate counts when students engaged in analysis of a provided data set. Serial dilution for the enumeration of bacteriophages has been used to strengthen student understanding of phage biology (1). Although the materials to perform this fermentation are simple and widely available, this laboratory project also contains elements to assess changes in microbial composition in a fermentation mixture commonly referred to as bokashi (Japanese for “fermented organic matter”). Bokashi is a mixture of microbes resulting from fermentation (often referred to as effective microorganisms, or EM) which is then used to inoculate an organic substrate such as rice straw or bran (6). EM and the resulting bokashi have been used to enhance growth of a variety of crops including peanut and pigweed (4, 5). PROCEDURE Note that the microbes cultivated in this experiment fall within the BSL-2 safety guidelines. In light of this, students must adopt the following practices during the entirety of the experiment: the use of personal protection such as gloves and goggles, decontamination of all microbially contaminated materials and workspace (via chemical disinfectants or autoclaving), and proper hand hygiene. Total time for the protocol is three weeks comprising four sessions for microbiological techniques. Working in pairs students shake 6 g of white rice and 6 g of brown rice (both non-instant generic brands obtained at a local grocery store) in 60 mL of distilled water for 2 minutes. The fluid is decanted into another bottle and a 1-mL portion is serially diluted in 0.1% peptone and spread plated in triplicate (0.1 mL/plate) onto nutrient agar (NA) and MRS agar. MRS agar is a selective media for lactobacilli (2). All spread plates are incubated at 30°C until colonial growth is present (typically 2 days). Specific population levels of bacteria in the fermenting fluid can vary from student group to student group so I recommend diluting the samples after each of the three 7-day incubations, up to 106-fold, to ensure countable numbers of colonies on the spread plates (30–300 CFU/plate is the target). Expect very few colonies on MRS media from the initial spread plating of the rice water, but these numbers will increase around 104-fold after the first 7-day incubation. The pH of the fluid is measured using a pH electrode when the spread plates are set up and the remaining fluid is incubated at 30°C for 7 days. Over the course of the experiment, the pH should drop approximately 2 pH units. As the pH drops over the course of incubation, the relative populations of bacteria cultured on NA versus MRS shifts, albeit in inconsistent ways. In some student groups the populations drop 100-fold while other student groups find an increase in bacterial levels, so I recommend plating from all dilution tubes in order to get reliable plate counts. After incubation of the fluid for 7 days, the previously mentioned methods of pH determination, serial dilutions, and spread plating are repeated as described above. Cells in the fluid are then simple stained using crystal violet to determine the frequency of cell morphologies, and 10 mL of fluid is centrifuged at top speed in a tabletop centrifuge to pellet bacterial cells. This pellet is suspended in 10 mL of sterile 0.85% sodium chloride and used to inoculate GEN III microplates (Biolog, Hayward, California). The GEN III microplate tests bacteria for the utilization of 71 different carbon sources and sensitivity to 23 different inhibitors and pH conditions. Absorbance at 750 nm in each well of the microplates is then determined via a plate reader after 24 hours of incubation at 30°C. The remaining rice water is added to 400 mL of whole milk (obtained from a local grocery store), the pH determined, and the mixture incubated for 7 days at 30°C. After incubation, curd formation has occurred and the supernatant of the mixture is decanted into a bottle for simple staining, pH determination, serial dilutions with spread plating, and microplate inoculation as described above. One mL of unsulfured molasses (Grandma’s brand) is added to this supernatant and the mixture incubated for 7 days at 30°C. After incubation, simple staining, pH determination, serial dilutions and spread plating, and microplate inoculation are performed as described above for the final time. The resulting fluid in the bottle is bokashi. At the end of making bokashi, students are required to calculate CFU/mL fluid at each step of the process based on the spread plate data collected and averaging the values obtained from triplicate plating. This data can be correlated with changes in pH, dominant cell and colony morphologies, and sensitivity/carbon utilization patterns from the GEN III microplates. In addition to the enumerative emphasis of the methods described above, subculturing of dominant colony morphologies can give students practice in streak plating and bacterial identification techniques using standard techniques not given in this exercise. Simple staining of the fermenting fluid gives the students opportunity to practice microscopy skills. Although the use of GEN III microplates adds additional cost, it allows for examination of the collective phenotypes of the shifting bacterial communities over time in the fermentation fluid which can be related to the decrease in pH of the fluid (which is typically found to decrease from 6.5 to 4.7). SAFETY ISSUES Nutrient agar grows a wide range of microbes, so plates and all bacteria-containing materials should be treated with the appropriate guidelines for laboratory biosafety level 2 (www.asm.org/images/asm_biosafety_guidelines-FINAL.pdf). Students should be instructed in aseptic technique and proper disposal of bacterially contaminated materials. Due to the selective nature of MRS agar, students handling these plates will most likely be working with lactic acid bacteria. Students should be informed that the bokashi product is not for human consumption (the strong odor should be a deterrent for those tempted!). CONCLUSION The total time to produce bokashi is three weeks. The schedule allows for four weekly serial dilutions and spread plating. I have the students spread plate in triplicate to assess the variability of their pipetting technique. The use of both NA and MRS agar shows the difference between nonselective and selective media on colony number and morphology (see Fig. 1). Appendix 1 includes some questions that students can address in their analysis of the data. All students should calculate the CFU/mL fermentation mixture throughout the experiment, but the instructor typically assigns different data exploration questions to different student pairs for analysis. FIGURE 1 Spread plates (0.1 mL fluid/plate) from a bokashi fermentation mixture. (A) Colonies on nutrient agar from the initial fermentation of rice water for 7 days. (B) Colonies on MRS agar from the fermentation of milk/rice water for 7 days. SUPPLEMENTAL MATERIALS Appendix 1: Media and reagent preparation, materials needed, and questions for students