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A soy-based alternative to traditional bacterial nutrient media.

A vegan is an individual who does not use or consume animal products of any kind (Vegan Society, accessed June 15, 2008). The term "animal product" in a vegan context refers to any material derived from animals for human use (Stepaniak, 2000), such as meat, poultry, seafood, eggs, dairy products, honey, fur, leather, wool, and silk (Vegan Society, accessed June 15, 2008). Common animal by-products include gelatin, lanolin, whey, casein, and beeswax (Vegan Society, accessed June 15, 2008). Reasons cited for choosing a vegan/vegetarian lifestyle include ethical or moral convictions concerning animal rights, concerns for the environment, human health, and religious concerns (Time/CNN, July 7, 2002; Social Survey Division of the Office for National Statistics, 2002; Stepaniak, 2000). Various polls have reported that vegans make up anywhere from 0.2% to 1.4% of the adult U.S. population, a group that seems to be growing (Time/CNN, July 7, 2002; Stahler, 2006). Further, most people in that group fall between the ages of 16 and 34 (The Vegan Research panel, accessed June 15, 2008, an age range typical of most college students.

Most complex nutrient media used in a general microbiology lab (Nutrient Broth, Luria-Bertani Broth, or Trypticase Soy Broth) contain animal-derived ingredients, such as casein, tryptone, or meat extracts. Our goal was to produce a medium that is free of all animal products, yet supports the growth of most non-fastidious bacteria in a manner comparable to commercially available animal-based media. To provide the growing vegan population (and anyone else uncomfortable with using animal products) a viable alternative in microbiology lab, we wanted to develop an equally-effective, inexpensive, animal-free substitute to conventional media.

We initially tested several combinations of soy flour and other additives on eight species of bacteria, and qualitatively compared the amount of growth on these plates against standard animal-based nutrient agar plates. From these trials, we found that a mixture of toasted soy flour (SoyQuik[R]) and yeast extract powder yielded the best, most cost-effective results. Quantitative and qualitative analyses were performed to compare growth of bacterial cultures in our soy-based media to growth in the standard animal-based media. As a final assessment, we tested the soy-based medium side-by-side against animal-based media typically recommended by experimental protocol during standard microbiology labs.

* Methods

Nutrient Media Preparation

Prospective animal-free additives were purchased in health food sections of local grocery stores or ordered online at the EnerGFoods Web site (Ener-G.com). These products (including toasted soy flour, potato flour, and rice bran) were incorporated in varying ratios into agar plates. Commercially-available Yeast Extract and Nutrient Broth (Difco[R]) powders were also used and prepared according to the manufacturer's instructions. All ingredients were dissolved in distilled water and sterilized as is typical in nutrient media preparation. When solid media was required, the preparation included 1.5% agar. Media recipes and their corresponding designations include:

* SF = 3% soy flour

* SP = 3% soy flour + 2% potato flour

* SR = 3% soy flour + 2% rice bran

* SY = 3% soy flour + 2% yeast extract

* SPY = 3% soy flour + 2% potato flour + 2% yeast extract

* SRY = 3% soy flour + 2% rice bran + 2% yeast extract

* NB = nutrient broth (made according to manufacturer's instructions)

* NBY = nutrient broth + 2% yeast extract

* YE = 2% yeast extract

* 2SF = 2% soy flour

* MSY (modified soy media) = 2% soy flour + 0.5% yeast extract

Qualitative Growth Analyses

Fresh, single colonies (< 40 hours old) of all microbes tested were streaked with a wire loop onto agar plates with various ingredients. Plates were incubated for 48 hours at 37[degrees]C before visual comparisons of growth. We recorded the amount of growth on the plates by assigning a number (0-4), where "0" indicated no growth and "4" indicated heaviest growth. Each experiment was performed no less than three times, and the rankings were averaged, rounding to the nearest whole number.

Quantitative Growth Analysis

An equal volume of an overnight liquid bacterial culture (Bacillus subtilis or E. colt) was added in a 30:1 dilution to either nutrient broth (NB) or to the modified soy media (MSY). At hourly intervals (for six hours), one ml of each culture was removed and serially diluted into a sterile saline solution. Equal portions of those dilutions were plated onto nutrient broth agar plates and grown overnight at 37[degrees]C. After 24 hours, colonies were counted and the number of cells per ml was calculated for each hourly interval (see Figure 1). Experiments were performed no less than three times with averaged cell counts shown.

* Results

Initial Qualitative Growth

We tested the effectiveness of different nutrient additives by growing several species of bacteria commonly used in a microbiology lab on agar plates containing various combinations of ingredients. As shown in Table 1, soy flour (SF) alone only moderately supported growth of most microbes tested, and the addition of rice bran or potato flour (SP and SR) only slightly increased growth. Not until we added yeast extract (a commonly used additive to bacterial media) did we observe good (3) to excellent (4) growth of most bacterial strains tested (SY, SPY, and SRY). The combination of soy flour, potato flour, and yeast extract (SPY) exhibited the best overall growth. In fact, SPY more efficiently supported growth than the animal-based nutrient broth (NB). (Compare "Total" numbers in Table 1.) Therefore, we chose SPY as our initial soy flour medium to be tested in typical experiments in Microbiology class.

[FIGURE 1 OMITTED]

Refining the Media

Though the initial soy flour medium (SPY) was effective in supporting growth, we found that the medium was opaque and quite grainy, as some of the ingredients did not completely dissolve during the sterilization process. This opaque nature of the media would make it virtually impossible to count colonies from the underside of a plate, or to use a spectrophotometer to measure growth of a culture. To clarify the medium, we altered the initial recipe. Since there was only a slight performance difference between media SY and SPY (Table 1), the potato flour was completely omitted. In addition, lesser concentrations of soy flour and yeast extract did not significantly affect growth. Table 2 shows qualitative growth comparisons of five common species of bacteria grown on various media. We found no significant change in overall growth when comparing the modified soy medium (MSY) to the initial soy medium (SPY), and MSY still remains slightly more effective in supporting growth when compared to the traditional nutrient broth (NB) (compare "Total" numbers in Table 2).

Though the modified medium (MSY) was less grainy in nature, additional techniques were used to filter the medium. The medium was initially filtered with cheesecloth or via vacuum filtration. However, this procedure led to a significant loss of volume to the medium, was quite laborious, and resulted in a less effective growth medium (data not shown). Since our goal was to generate a medium that was a comparable substitute to a traditional medium, including the time and effort to prepare it, we decided that filtering the media was an unnecessary step for intended uses of the media in a basic microbiology lab setting. The media was still opaque, but we found this to be a minor limitation for its intended uses. Therefore, our modified soy-based medium (MSY) contained unfiltered 2% soy flour and 0.5% yeast extract.

Quantitative Growth Comparison

Since qualitative analyses can be quite subjective, often leading to varying results from experiment to experiment, a quantitative growth comparison of two common bacterial species (Escherichia coli and Bacillus subtilis) was performed and growth was monitored on an hourly basis. Results shown in Figure 1 demonstrate that both media types (MSY and NB) supported the growth of B. subtilis and E. coli nearly equally well (within a factor of six) during the six-hour monitoring period, and E. coli may even prefer growth in the soy-based media to that of the nutrient broth. These results agree with those of the qualitative analyses shown in Table 2, that the modified soy flour medium (MSY) supports the growth of bacteria as well as the conventional animal-based medium.

Using MSY for Typical Classroom Experiments

To test the adaptability of the modified soy flour medium (MSY) in a microbiology laboratory, we performed side-by-side comparisons with conventional media by having a student use MSY instead of the called for conventional animal-based medium for several basic experiments performed throughout the semester. Experimental protocols followed from the course lab manual (Bey, 2001) included The Effects of Oxygen on Microbial Growth, Enumeration of Bacteria (serial dilution technique), Bacterial Transformation, and Viruses of Bacteria (plaque counts). In all experiments, MSY was equally effective in supporting bacterial growth as the conventional animal-based medium called for in the lab manual (data not shown).

Benefits of the Medium

Ethical Concerns

The initial motivation for this research stemmed from the ethical and environmental concerns of using animal-based products. Our soy flour medium would alleviate objections by students or researchers who may be opposed to using animal-based media for experiments.

Cost-effectiveness

Not only is our MSY medium effective in promoting microbial growth, it is also more cost-effective than conventional media. SoyQuik[R] soy flour can be purchased at most large supermarkets or online at $2.75 per 465 g (a 16 oz. package) (EnerG.com). Adding this cost to that of the 0.5% yeast extract, the final price per liter is $0.98. Comparing that cost to the higher prices of some commonly-used media listed in Table 3 demonstrates the cost benefits realized by having investigators use this medium for common experiments in the lab. There are times of course, when Trypticase Soy (TSB) or Luria-Bertani (LB) broths may be the better media choice for a particular experiment. But we have found for most basic microbiology lab experiments involving the growth of non-fastidious bacteria, the less expensive NB medium will sufficiently support bacterial growth. And results reported in this manuscript show that our soy media is a comparable, cost-effective vegan substitute to NB.

Limitations of the Medium

The main limitation of the soy-based media is that it was opaque and somewhat grainy in texture. While this did not affect growth or the results of the lab experiments we performed, it does make some experiments involving colony counts slightly more labor intensive than if the media were fully dissolved and transparent. Typically, colonies growing on agar plates are counted using a marker to touch the underside of the plate wherever there is a colony; transparent media easily allows this. Since our media doesn't allow visual inspection of the colonies through the agar, we were unable to use this method. However, this can be easily remedied by taping the lid of the Petri plate to the bottom plate, then counting colonies through the lid. Taping prevents spinning of the lid during the counting process, which could result in inaccurate numbers. We found that students in lab who counted colonies in this manner had no more trouble doing it than those counting from the underside of the plate.

We also found that since the media was not homogeneous (the particulates settle to the bottom of a tube), measuring light transmittance through a spectrophotometer was not reproducible, and therefore an unreliable means of quickly measuring growth of a culture. This limitation however, has little consequence for introductory microbiology experiments.

* Conclusions

We have described the development of a soy based nutrient medium and have demonstrated that its usefulness in typical microbiology experiments is comparable to that of conventional, animal-based media. The medium supports growth of many common microbes, can be used for most microbiology experiments, and is less expensive than conventional animal-based media. Despite a few minor limitations, the soy flour medium is a viable option for microbiology students who may be uncomfortable using animal-derived ingredients and laboratories concerned with the environmental impact of the animal-based media. We are continuing experiments to improve the clarity of the media, and plan to test other soy powders, vegetable stocks or broths, and other non-animal based ingredients.

* Acknowledgments

We would like to thank Dr. Andrew Lukowiak and Dr. Tom Klubertanz for their critical review of this manuscript.

References

Bey, R.F. (2001). Microbiology Lab Manual. Belmont, CA: Wadsworth/Thomson Learning.

Ener-GFoods. Food for restrictive diets. Accessed June 15, 2008. Available online at: http://www.ener-g.com/.

Fisher Scientific. (2008). Available online at: www.fishersci.com.

Social Survey Division of the Office for National Statistics in accordance with the Official Statistics Code of Practice. (2002). The National Diet & Nutrition Survey: adults aged 19 to 64 years, Vol. 1. Norwich, England: St. Clement's House. Available online at: http://www.food.gov.uk/multimedia/pdfs/ndnsprintedreport.pdf.

Stahler, C. Now many adults are vegetarian? Vegetarian Journal, 4. Available online at: http://www.vrg.org/journal/vj2006issue4/vj2006issue4.poll.htm.

Stepaniak, J. (2000). Being Vegan, pp. 2,5,17,148-150. U.K.: McGraw Hill Contemporary.

The Vegan Research Panel. Factfile. Accessed June 15, 2008. Available online at: http://www.imaner.net/panel/profile.htm.

Vegan Society. Criteria for vegan food. Accessed June 15, 2008. Available online at: http://www.vegansociety.com/html/food/criteria.php.

Vegan Society. Time/CNN Poll: Do you consider yourself a vegetarian? Time Magazine, July 7, 2002. Available online at: http://www.time.com/time/covers/1101020715/poll/.

BIO

Rachel Mosher and Kyler Crawford are students at the University of Wisconsin in Madison, WI 53705. Andrea Lukowiak is Assistant Professor of Biology, University of Wisconsin-Rock County, Janesville, WI 53546; e-mail: lukowiaks@charter.net.
Table 1. Microbial growth on various media. Bacteria
were incubated for 48 hours at which time their growth was
assessed. The amount of growth was ranked from 0-4; 4
denotes heavy growth, 0 denotes no growth. Gray shading
highlights the most effective growth conditions. The last row
indicates the sum of the rankings of an individual medium.
Media abbreviations are defined in the Methods section.

 MEDIA TYPE

Organism SF SP SR SY SPY SRY NB NBY YE

Escherichia coli 2 3 2 4 4 1 4 4 2
Serratia marcescens 2 3 1 4 4 3 3 3 2
Bacillus subtilis 2 3 2 3 4 4 3 3 3
Micrococcus luteus 1 1 1 4 3 3 3 4 3
Staphylococcus aureus 0 1 2 4 4 4 3 3 3
Pseudomonas aeruginosa 2 2 3 4 3 4 2 3 3
Proteus mirabilis 2 2 3 3 4 4 1 3 3
Bacillus megaterium 2 3 3 3 4 4 3 3 3
TOTAL (out of 36) 13 18 17 29 30 27 22 26 22

Table 2. Modifying the media.
Bacteria were incubated for 48
hours at which time their growth
was assessed. The amount of
growth was ranked from 0-4; 4
denotes heavy growth, 0 denotes
no growth. Gray shading highlights
the most efficient growth condi
tions. The last row indicates the
sum of the rankings of an individual
medium. Media abbreviations are
defined in the Methods section.

Organism NB SPY MSY

Escherichia coli 3 3 3
Serratia marcescens 3 4 4
Bacillus subtilis 3 4 4
Staphylococcus aureus 4 4 3
Proteus mirabilis 1 3 3
TOTAL (out of 20) 14 18 17

Table 3. Media cost comparison.

Nutrient Medium Cost per liter

MSY $0.98
NB $1.07 *
Luria-Bertani (LB) Broth $2.33 *
Trypticase Soy Broth (TSB) $2.58 *

* All prices for the commercially available media
were taken from the 2007-08 Fisher Scientific
Catalog (fishersci.com).
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Title Annotation:HOW-TO-DO-IT
Author:Mosher, Rachel; Crawford, Kyler; Lukowiak, Andrea
Publication:The American Biology Teacher
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2009
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