Isolation And Phenotypic Characterization Of An Unknown Bacterium Biology Essay

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The balance of soil ecosystems rely on living organisms such as vegetation, animals, and bacteria (Smith and Smith, 2001). Microbial communities that live in soils are largely responsible for supporting plant growth through the formation of organic matter, which is formed from inorganic materials (Willey et al., 2008) like the nitrogen that is released through human activities (Vitousek et al., 1997). Many microbes also help break down plant materials, like cellulose (Willey et al., 2008; Smith and Smith, 2001; Paul and Clark, 1996).

The types of microbes that are found in soil depend on the physical and chemical properties of the environment in which they are living (Smith and Smith, 2001). Climatic factors have a strong effect on microorganism diversity and distribution. Some of these influencing factors include temperature, pH, available oxygen, and nutrient supply (Agnelli et al., 2004; Newton et al., 2006). However, Adl describes that agricultural practices can largely effect distributions of microbes in the soils of cultivable lands (2003) meaning that there would be differences between forest and garden soil.

Mixed populations of unknown microorganisms from samples of forest soil and garden soil were collected. Four pure cultures of soil bacterial isolates were obtained. The objective of this experiment was to find a possible identity for one of the unknown soil bacterial isolates.


All of the procedures were conducted using aseptic technique. Two soil samples were weighed, 1 g of garden soil and 1 g of forest soil, and mixed into separate beakers; each contained 99 mL of sterile deionized water. These 10-2 solutions were then used to make serial dilutions of 10-3, 10-4, 10-5, 10-6, and 10-7. As described in Microbiology Lab Manual, broths, slants and deeps were inoculated for each of the two soil samples (10-2 dilutions) and streak plates (for 10-4 to 10-7), pour plates (10-2 to 10-5), and spread plates (10-2) were made as well. After incubation, the pour plates were examined and the numbers of Colony-Forming Units (CFUs) were counted, with those with 20 or more than 200 exempt. These data were then converted into # of CFUs/g soil by multiplying the number of colonies by the dilution factor of each plate. One plate for each bacterium was chosen and the colony morphology was described. These plates were then used for sub-culturing procedures (Robertson and Egger, 2009).

A gram stain was prepared for each of the bacteria isolates as outlined by Robertson and Egger (2009), where crystal violet was used as the primary stain and safranin as the counter stain. Prior to staining, each isolate was viewed used the oil immersion lens and the cellular shape, size, and arrangement was recorded. Each bacterial isolate was tested for starch hydrolysis, hydrogen sulfide production, and motility. They were also tested for ammonification, nitrification (NH3/NH4+ to NO2- or to NO3-), denitrification (NO3- to NH3/N2 or NO2-), and catalase production. The bacteria were incubated at four different temperatures, 13-15oC, 26oC, 37oC, and 50oC, four different salt concentrations, 0%, 0.5%, 2%, and 5% NaCl, and four different pH levels, 3, 5, 7, and 9. One week later, the TSA plates were visually examined and the extent of bacterial growth for each isolate at each temperature and osmotic concentration was recorded. The TSB tubes were tested using a spectrophotometer set to a wavelength of 580 nm (Robertson and Egger, 2009).


The colonies of the unknown bacteria were circular in form and ranged in elevation from flat to slightly convex. They also ranged in diameter, with a sample measurement of 6.2 mm. With an entire margin, the colonies were also smooth, shiny, and opaque with off-white colouring. Staining the bacterium showed that it was gram positive. With the oil immersion lens (1000x magnification) the bacterium appeared to be rod-shaped and arranged in chains, determining that it was streptobacillus with dimensions of 3.0 μm by 1.2 μm.

Table 1 shows that the unknown bacterium was capable of hydrolyzing starch, and therefore used it as a source of carbon. As the test for hydrogen sulfide was negative, the bacterium was unable to reduce inorganic compounds which contain sulfur. After the motility test, there was evident bacterial growth throughout the tube. This showed that the bacteria was able to travel and, therefore, must have been equipped with flagella. The unknown bacterium did not perform ammonification, but it did undergo denirtification of nitrate to nitrite as well as nitrification of ammonia/ammonium to nitrite (Table 1). Table 1 also shows that the unknown produces catalase.

The environmental test results showing the optimal temperature, pH, and osmotic pressure indicated that the unknown bacterium was a mesophile with an optimal temperature of 26oC, a neutrophile with a pH optimum of 7, and a nonhalophile which had grown best in 0% NaCl (Table 1).



Colony morphology

Circular, flat of slightly convex, with an entire margin. Shiny, smooth, opaque with off-white colouring. Measured diameter of 6.2mm.

Cell morphology

1000x magnification, streptobacillus, 3.0μm long and 1.2μm wide.

Gram stain

Positive (Purple)

Starch hydrolysis


H2S reduction






Denitrification (NO3- to NO2-)


Denitrification ( NO3- to NH3 or N2)


Nitrification ( NH3/NH4+ to NO2-)


Nitrification ( NH3/NH4+ to NO3-)

N/A (Negative)



Optimal temperature


Optimal pH

pH 7

Optimal salt concentration

0%Table 1. Results of a variety of tests for an unknown bacterium


The genus Cellulomonas shares nearly all of the characteristics with the unknown bacterial isolate in this experiment. It was evident that the unknown was a gram-positive bacterium which either required oxygen, being aerobic, or prefer it, being a facultative anaerobe. Cellulomonas sp. satisfied those needs as they were gram-positive and faculatively anaerobic. Corresponding with the tests, they had an optimum temperature of approximately 30oC, grow best at a neutral pH, are able to hydrolyze starch, produced catalase, and reduced nitrate to nitrite (Sneath et al. 1986). Colonies were described as being convex (Sneath et al. 1986; Winn et al., 2006) which also matched up to part of the description of the unknown. While sources state that at the genus level, colonies were typically yellow (Sneath et al. 2008; Winn et al., 2006; Brown et al., 2005), colouring at the species level varied from yellow, to cream (Sneath et al. 1986; Malekzadeh et al., 1992), to white (Sneath et al. 1986).

A very important defining characteristic of both the unknown bacterium isolate and Cellulomonas sp. was their motility. Along with hydrolyzing starch, members of the genus Cellulomonas also processed cellulose and gelatine (Winn et al. 2006). They were said to decolourize quite quickly during the staining process (Sneath et al. 1986), which would explain why the unknown bacterium wad stained numerous times before the colour was identifiable. They were also described as microbes that do not produce endospores (Sneath et al., 1986) or capsules (Brown et al., 2005). These points caused increased assurance that the unknown bacterium isolate was Cellulomonas sp..

Members of the genus Cellulomonas were principally considered to be environmental bacteria (Winn et al., 2006) and, among other bacteria, play a very important role in the process of decomposition (Paul and Clark, 1996). They help to break down cellulose which is very abundant in plant residue and have particles in their cell walls to affect the efficiency of this process (Paul and Clark, 1996). They are also said to work in symbiosis with nitrogen fixing bacteria; they provide glucose to the nitrogen fixers, and in turn, receive nitrogen (Willey et al., 2008). Consequently, the majority of isolated cultures are collected from soil samples (Sneath et al., 1986).

To confirm that the unknown bacterial isolate is Cellulomonas sp., a few more tests would be desirable. Firstly, a test for acid production would have provided helpful results. Also, it would have been preferable to test for gelatinase production. Finally, the most ideal piece of information to identify the bacterial isolate would have been the results from the DNA extraction test from Lab Exercise 5 of the Microbiology Lab Manual (Robertson and Egger, 2009).

There are a number of limitations that arise when culturing and indentifying unknown bacteria. As noted by Kirk et al. (2004) only 1% of the bacteria found in soil can be cultured by every-day laboratory techniques and plate counts tend to favour faster-growing microorganisms. Similarly, unculturable microbes are not detected during plate counts and results are, therefore, inaccurate (Snyder, 1947). On top of that, the process used to identify the bacterium in this experiment was both labour intensive and time consuming, as multiple tests were needed to narrow down possible identities of an unknown bacterium. Although it was very educational, it would not be ideal for efficient culturing.

Each test had various sources of error, when describing colony morphology, the descriptions could have been subjective to the point where they do not match up to those described by Sneath et al. (1986). During gram staining, some gram-positive bacteria, such as Cellulomonas sp. decolourize quite quickly, which may have caused confusion. Visual errors may have occurred when measuring the cells and using old cultures could have also caused some error in results. Contamination would have affected any of the biochemical tests that were conducted.