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Microbiology is the branch of biology dealing with the structure, function, uses, and modes of existence of microscopic organisms. Soil microbiology is study of microorganisms in soil and their functions. The functions these microbes perform range from nitrification, nitrogen fixation, denitrification, and decomposition. Nitrification is the process of changing nitrogen in the form of ammonia (NH4) to nitrites or nitrates in the forms of NO2 and NO3 which are used by growing plants. Actinobacteria are responsible for the breakdown of organic matter into ammonia which is used during nitrification to make nitrates. Some free living bacteria are able to constantly put nitrogen into circulation for biological uses; such as azotobacter. These types of bacteria perform what is called nitrogen fixation and are able to convert nitrogen from the atmosphere into substances that contain nitrogen. Denitrification is the opposite of nitrification. Instead of transforming ammonia into nitrates, nitrates are changed into ammonia. This is undergone by changing NO3 -> NO2 -> NH3 and is important for the returning of nitrogen to the atmosphere. These functions are all very important in the nitrogen cycles of the environment as demonstrated in Figure 1. The biotas that undergo these processes do so by using oxygen in different ways such as Aerobes(1) which strictly use oxygen in their processes, Anaerobes(2) which strictly cannot use oxygen, Facultative anaerobes(3) which prefer to use oxygen but can switch to fermentation if oxygen is not present. Microaerophiles(4) which need oxygen to survive but perform optimally in areas with less oxygen than found in the atmosphere, and Aerotolerant bacteria(5) which do not require oxygen for growth. These different oxygen uses can be viewed in figure 2 which shows the optimal growth places for the bacteria.
- Materials and Methods
For the experiment to take place two soil samples were collected and used to isolate bacteria from; Forest soil and Agricultural soil. Each soil sample was first diluted by putting 1 gram of soil into a beaker containing 99ml of DH2O. 1 ml of this was then pipetted into a test tube containing 9 ml of deionized H2O. 1ml of this solution was then taken and placed into another test tube. This was done to make solution with the concentrations of 10-2 to 10-7. Using aseptic technique each soil from the 10-2 was used to make slants, broths, deeps, and streak plates, and spread plates. One for each of the soil types. Eight pout plates were created using four samples of each soil ranging from 10-4 to 10-7. Observations were then taken in the next week and recorded. As well in the 2nd week four different looking bacteria colonies were taken and put on their own TSA plate and slant as to separate the colonies from each other for further study. The colonies were grown for a week and the visual observations were taken and recorded. Each of the 4 different bacteria types were then used in the experiment setup for week 3 to test for nutrient cycling. Two starch plates were split in half and each bacteria was spread over one half to test for starch hydrolysis. Each bacteria was tested for H2S and motility using four SIM deeps. Ammonification was tested using four peptone (4%) broth tubes. Using four ammonium sulphate broth tubes and four nitrite broth tubes, nitrification was tested for each bacterium and denitrification was tested on all four bacterium using four nitrate broth tubes. As well oxygen tolerance was tested using four thioglycollate medium tubes and catalase was tested using four microscope slides. The next week each test underwent testing using different reagents to test for different color changes and their meanings. Starch hydrolysis was used with iodine to see if the iodine reacts clear or turns blue/black. H2S and motility was tested using Kovac's reagent to try and obtain a red color. Nessler's reagent was used with the peptone broths to see if a yellow color was produced and ammonification was undergone. Nitrification was tested using both different broths. The ammonium sulphate broth was mixed with Nessler's reagent and Trommsdorf's reagent to test for a yellow color and then a dark purple/blue/black color in the transformation of NH4->NO2. The Nitrite broth was tested using Trommsdorf's reagent and H2SO4 to test for a brown color and then with phenylamine to test for a blue color for the transformation of NO2->NO3. Denitrification used reagents A +B for a pink color change and reagent C if no color change was observed in A+B. As well as using the different reagents in week 4 on the week 3 broths/plates, week 4 was used to set up the experiment to test environmental factors on the bacterium growth. Four TSA plates were used to test the effects of different temperature on the bacterium, sixteen TSB tubes were used to test each bacterium in four different pH ranges, and four TSA plates were used with different salt concentrations to test the growth during different osmotic pressures.
- Results -
The observations for bacterium 1 were recorded and placed into a table summarizing the different results of each test and their meanings.
Cicular form; raised elevation; shinny appearance; opaque; Orange
Rods - Singular arrangement
Clear with iodine reaction - positive
Tan color of media around bacteria - negative for H2S production
Yellow color of kovac's reagent - Positive for H2S motility
Yellow color of kovac's reagent - Positive for H2S motility
Yellow color of peptone broth with Nessler's reagent - positive for ammonification
(NO3 to NO2)
Clear color with reagent A and B - Negative for denitrification
(NO3 to NH4 or N2)
Grey color with Reagent C - negative for denitrification
(NH4 to NO2)
Yellow color using nessler's reagent - Negative for ammonia
Purple color using Trommdorf's reagent and H2SO4 - positive for nitrite.
Positive for nitrification NH4 to NO2
(NO2 to NO3)
Light Blue/clear color using diphenylamine reagent and H2SO4 - Negative for Nitrate
Negative for nitrification NO2 to NO3
Bubbles when H2O2 added - positive
Microaerophile - no growth near surface nor at bottom
Negative for oxidase
Minimum is 4C; Maximum is 37C; Optimal is 22C
Optimal Salt Concentration
Minimum N/A; Maximum 5%; Optimal 0-0.5%
Minimum pH 3; Maximum pH >9; Optimal pH 9
Table 1. Summary of Results and Observations for Bacterium 1.
Bacterium 1 was observed firstly by its appearance. The colonies of the bacterium were circularly shaped, with a slight elevation from the agar. The outside was slightly shiny and the colony color was tinted a strong orange. The bacterium underwent a gram stain which came out indicating a positive stain. Under the microscope bacterium 1 was found to be made of rods in a singular arrangement. In week 3 nutrient cycling was tested. Iodine was added to the colony which reacted with it and came out with a clear color indicating a presence of starch. Kovac's reagent was added to a colony and reacted yellow indicating a H2S motility but the tan color around the media indicates H2S was not reduced. The peptone broth was used with Novac's reagent which reacted yellow indicating Ammonification. Denitrification was tested using reagent A and reagent B together. The bacterium reacted clear with the two reagent indicating a negative response of the transformation of NO3 -> NO2. Reagent C was then added to the solution which did not react pink indicating that NO2 was not transformed to NH4 or N2. Nitrification was first tested using Nessler's reagent which reacted yellow indicating no presence of ammonia. Tromsdorf's reagent and H2SO4 were added, which turned purple in the presence of nitrite. The colony was also tested using H2SO4 and diphenylamine which did not turn a blacky color but turned a light blue color indicating no presence of nitrate. Nitrification tested positive in the transformation of NH4 -> NO2 but negative for NO2 -> NO3. Week 4 tested the bacterium in different solutions containing different pH, salt concentration, and temperature. It was found that the optimal pH for bacterium 1 was 9, the optimal salt concentration was 0-0.5%, and optimal temperature for growth was 22C. The bacterium would not grow at a higher salt concentration that 5% and temperatures over 37C, as well as under 4C. The growth for higher pH than 9 was not determined.
- Discussion -
Using these findings and the table of common soil bacterium found on blackboard under the Microbiology section, it was determined that bacterium 1 was either Rhodococcus or Flavobacterium. These two choices matched the most effectively with the results of bacterium 1 but were still not perfect matches. Rhodococcus was found to have a positive gram stain, and orange color, made of rods, positive for starch hydrolysis, negative for H2S production, negative for nitrification NO2 ->NO3, negative for oxidase and positive for catalase, and temperature in common with bacterium 1. Although it differed in the denitrification, nitrification NH4 -> NO2, Salt concentrations; it had 9 categories in common making it a possible match. Flavobacterium had 8 things in common with baceterium 1 including the orange color, make up of rods, negative for H2S production, positive for ammonification, negative for the denitrifications, negative for nitrification NO2 -> NO3, positive for catalase, and undetermined optimal pH. Each of the choices had 9 categories in common with the tested bacterium indicating the bacterium could possibly be either choice. Tests with higher pH could help distinguish the bacterium from the choices as well as repeating the tests could help eliminate human error during the gram stain and complex tests of that nature. Through the tests it was found that the bacterium was a slight nitrifcator which indicates that the bacterium is important in the environment in its ability to take ammonia (NH4) from the decaying matter around it and transform it into NO2, which is usable for growth by plants.
- Refrences -
Dictionary.com. (2010). Microbiology. Retrieved February 19 from
Borneman, J., Et al. (1996). Molecular microbial diversity of an agricultural soil in Wisconson.
Applied and Environmental Microbiology, Vol 62. Retrieved February 24 from Google
Robie Vestal, J and White, David C. (1989). Lipid Analysis in Microbial Ecology. BioScience, Vol 39(8),
Soil life. (2010). Retrieved February 19 from http://en.wikipedia.org/wiki/Soil_life
Facultative Anaerobic Organism. (2010). Retrieved February 20 from