Microbial Diversity in Oklahoma Soils: A CFU/mL Study

Introduction

Microbes are present everywhere in day to day life. The soil present in the world in one area where microbes play one of the biggest is soil. Microbes present in soil assist in decomposing residues residing in the soil, creating and providing nutrients for plants present within the soil, convert certain elements into energy and by products, assist in nutrient cycle within the environment many other processes(1). The most common types of microbes found within soil include bacteria, fungi, Algae , and many others(2).

The factors that can influence which type of microorganism that are found within certain soils include temperature, pH, moisture ,vegetation, and light(3). The soil sample collected for the experiment varied as soil sample 1 was dark brown, organic rich, and had earthworms present.

The first 3 inches of the soil was sampled on Jan 23,2019 with recent precipitation and temperature of 43°F.

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Soil sample 2 was a dry red clay with no vegetation within the sampling area ,and rocky environment. The first two inches were sampled on Jan 23,2019 with recent precipitation and temperature of 43°F. The variety and quantity of microorganism in soil sample 1 will be greater than soil sample 2 based on the characteristics of the soil. The hypothesis will be tested based on a viable colony count and colony morphology conducted using a serial dilution and three varieties of media plates which include nutrient agar for bacteria, glycerol yeast extract agar for actinobacteria, and sabouraud dextrose agar with antibiotics for fungi growth.

Abstract

In the following experiment two variations of soil will be examined and quantified for variations and quantity of microorganism existing within each respective sample.

Soil sample 1 should contain more variety and quantity of microorganism based on characteristic compared to soil sample 2.The procedure for the following experiment will consist of a serial dilution and viable colony count of each sample within a variation of media consisting of nutrient agar, glycerol yeast extract agar, and sabouraud dextrose agar with antibiotics .Soil sample 1 contained higher quantity and variation of microorganism based on analysis. The findings supported the hypothesis to an extent as the variation and quantity were not as expected.

Methods and Materials: The following methods was presented in Exercise 8-11 “Soil Microbial Count” pg. 642 Microbiology Laboratory Theory and Application 4th Edition, Leboffe and Pierce was preformed and adapted with modifications. Plates were labeled according to dilutions factor instead of alphabetically .Two soil samples were obtained and utilized instead of a single sample. Media plates were prepared ahead of time ,and plates were incubated at 37°C instead of 25°C for 7 days.

The following experiment will require per soil sample: 8 Nutrient agar plates used to grow simple bacteria within the sample ,8 glycerol yeast extract agar plates used to grow Actinobacteria within the sample, and 8 Sabourad dextrose agar plates with Abx used to grow Fungi within the sample. Additionally, a 20-200 µL micropipette and corresponding autoclaved tips,1.5mL autoclaved microcentrifuge tubes,10 mL of sterile saline, Bunsen burner, ethanol, metal spreading utensil (hockey stick).

Once everything necessary has been obtained begin by preforming a serial dilution. When preforming the dilution to ensure sterile practice change micropipette tips after each dilution and keep caps of microcentrifuge tubes closed when not in use. The dilution will range from 10-1 to 10-7 with one control each microcentrifuge tube will contain 900 µL of sterile saline and 100 µL of diluted sample, and the control only containing 900 µL of sterile saline. Once the soil sample has been prepared with 10g of sample soil and 100 mL of sterile H2O in a 1.5mL autoclaved microcentrifuge tube begin the 10-1 dilution using 900 µL and 100 µL of the prepared soil sample and continue until a 10-7 dilution has been completed making sure that 100 µL has been taken from each previous dilution and distributed in order.

Once the dilution is complete obtain your media plates and label according for each dilution and control starting at 10-2. Once labeled obtain 100 µL of the diluted sample and transfer to the media plate and spread evenly across the plate using the spreading device. When spreading to ensure sterile practice dip the utensil in ethanol and flame between each spread and keep the lid of the media plate closed as much as possible to avoid any unnecessary contamination. Once the dilution has settled within the media flip over to avoid condensation and incubate for 1 week at room temperature. Repeat for remaining soil sample and media plates .Once the plates have completed incubating record results(1).

Results

The results displayed were obtained after the incubation period was completed. Viable colony count, three most predominant colony morphology, and colony forming unit per mL were obtained, calculated and organized in the figures and sample calculation below for each soil sample and corresponding variation of media plate.

Table 1: Viable Count

Table 1: Viable count of colonies per each sample and specific media .(TMTC=To many to count),(*quantity selected for number of colonies in CFU/mL calculations)

Table 2: Colony Morphology

Table 2: Colony morphology of each sample and specific media. Morphology order follows color, form, elevation, margin and any additional characteristics strongly present within colony.

Table 3: CFU/mL Calculations

Table 3: CFU/mL quantities per sample and specific media formula and calculations seen below. Viable count of 20/30-200/300 colonies per plate selected for calculations based

Calculations

Viable Colony count: Hand counted

CFU/mL: ((# of colonies))/((Dilution Factor)(mL plated))=X CFU/mL

CFU/mL: ((144))/((1*(10)^(-1) )(.1 mL plated))=1.44*(10)^(4 )CFU/mL

Discussion/Conclusion

The results from the following experiment contained empathize of three main microorganism within the soil samples. The three main microorganism that were searched for were bacteria, actinobacteria and fungi as the nutrient, glycerol yeast extract, and sabourad dextrose with antibiotics media were designed to grow these three microorganisms specifically. The colony forming unit per mL quantities for soil sample 1 in comparison to soil sample 2 were largely but not by massive margins. The colony morphology for each sample within different medias were similar but did differentiate to an extent .Variations of bacteria and fungi were present on the plates during analysis. The results were as expected to an extent as the quantity and variation of microorganism within soil samples.

Soil sample 1 did result in higher colony forming units per mL per each specific media ,but not to the extent predicted. Colony morphology did not vary much either when analyzed after incubation(1). Soil sample 2 with sabourad dextrose agar with antibiotics did not grow enough colonies to provide a sufficient CFU/mL quantity this could be due to possible error in dilution technique or spreading with use of hockey stick as the dilution could have been made incorrectly or the hockey stick was to hot and may have caused microorganism to die when spreading on the media. Other possible errors could include contamination during dilution and transfer methods of the experiment. Improvements and consideration for this experiment would include using more diverse soil sample, multiple sets of selective medias for more microorganisms, and possible higher dilution factors.

References

1. Savage-Ashlock K. 2019. Week of January 28 Module, Ex 8-11 Soil Microbial count [Powerpoint]. Retrieved from University of Oklahoma MBIO 3812 Course website https://canvas.ou.edu/courses/109155/files/14026162?module_item_id=1907856
2. Nationwide, S. A. R. E. Soil Microorganisms. SARE: Sustainable Agriculture Research and Education. SARE: Sustainable Agriculture Research and Education. https://www.sare.org/Learning-Center/Books/Building-Soils-for-Better-Crops-3rd-Edition/Text-Version/The-Living-Soil/Soil-Microorganisms. Accessed 15 February 2019
3. AGP - Physical factors affecting soil organisms. International Rice Commission Newsletter Vol. 48. FAO of the UN. http://www.fao.org/agriculture/crops/thematic-sitemap/theme/spi/soil-biodiversity/soil-organisms/physical-factors-affecting-soil-organisms/en/. Accessed 15 February 2019