Isolation By Dilution And Matabolic Testing Biology Essay


The variety of bacteria even in one gram of common soil could be teeming with as many as 20 000 of species (Brady and Weil 2002), and 400-5000kg of bacterial biomass can be found per hectare. This high density of species has forced the evolution of a variety metabolic functions pathways that allow this co-existence observed. Bacteria usually inhabit the top 15 cm of soil Brady and Weil 2002, aerobic bacteria near the top and anaerobes near the bottom based on their oxygen tolerance. Certain bacteria have also developed specified metabolic pathways in the event of extreme environmental conditions or to take advantage of a newly opened niche. Such as metabolizing starch or amino acids for energy; or using organic nitrogen, inorganic sulfur, or oxygen as their final electron acceptor; or preferring different environmental optima; like pH, temperature, or osmotic pressure. These optima could be at the extremes to these conditions, or possibly in the middle ground.

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In this experiment a bacterial colony will be isolated from a forest soil sample by dilution techniques. The colony will then be identified by subjecting it to various metabolic and environment tests and comparing the results to know data.


First (Robertson and Egger 2010), a 10-2 dilution of the bacteria was made by adding 1g of forest soil sample to 100mL of distilled water (dH2O). Then five serial dilutions were preformed to obtain solutions of 10-3, 10-4, 10-5, 10-6, and 10-7. The previous methods were repeated for an agriculture soil sample as well. A broth, slant, and deeps tubes, additionally a streak and spread plates, were aseptically inoculated with the 10-2 solution for each soil. Pour plate were prepared from the 10-3 to 10-7 dilutions of each soil. All plates and tubes were allowed to incubate for 48 hours at 22°C.

A bacterial colony from the 10-7 dilution of forest soil was chosen for further testing. The morphology of the colony was recorded. Then streak plate and slant tubes were prepared for the bacteria. Then the bacterium was gram stained and the cell morphology was observed through at microscope (X1000). Theses plates and tubes were allowed to incubate for 48 hours at 22°C.

Then the following tests for metabolic pathways were conducted. A starch agar plate, incubated 48 hours at 25°C, and Iodine solution was used to test for starch hydrolysis. A deeps tube of Sulfide, Indole, and Motility (SMI), incubated for 48 hours at 25°C, was used to test for H2S, motility and Kovac's reagent for indole. A peptone broth, incubated for 7 days at 25°C, and Nessler's reagent were used to test for ammonification. A nitrite broth, incubated 7 days at 25°C, was tested with Trommsdorf's reagent and dilute H2SO4 for NO2- nitrification; and phenylamine and concentrated H2SO4 for NO3- nitrification. A nitrate broth, incubated 7 days at 22°C, and sulfanilic acid and N,N-dimethyl-1-1-naphthylamine used to test for NO2- denitrification; and zinc for NH4+ or N2 denitrification. Thioglycollate broth, incubated 7 days at 22°C, used to test for oxygen tolerance. Lastly, the addition of H2O2 to a sample to bacteria was conducted to test for catalase.

Then the environmental optima and tolerance of the bacteria were tested. pH was tests in pH broths of 3, 5, 7 and 9; optimum measured by absorbance of the solution. Temperature was tested by incubating bacteria at 4, 22, 37 and 50°C; and osmotic pressure tested by incubating bacteria in salt concentrations of 0, 0.5, 2 and 5 % NaCl. All treatments were incubated for 36 hour period. The treatment with maximal growth was determined the optima. The previous tests were also conducted to three other bacterial isolations. The outcomes of these tests were used to make a possible identification of one of the chosen bacteria down to the Taxa of Genus.


A single bacterial strain was successfully isolated from the 10-7 dilution of forest soil. The isolated bacteria had colony had a circular raised form, and a slightly undulate margin. The colonies were also a rusty-orange colour, with a smooth and shiny optical property. The cells were gram negative, bacillus shaped found in small clusters, and approximately 1.96 μm in length. The cells also lacked an endospore; the existence of a capsule could not be ascertained.

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This bacterium was found to be a facultative anaerobe, able to carry out starch hydrolysis, tryptophan metabolism to indole, denitrification, and catalase reactions (Table1). The bacterium was also noted to be incapable of using inorganic sulfur or organic nitrogen as a final electron acceptor, or of motility (Table 1). The bacteria is possibly capable to nitrify NO2, but this is a questionable result (Egger pers. comm.). Environmentally, the bacterium is indicated to be a mesophile, neutralphile, and non-halophile (Table 1).

Table 1. Results of various metabolic and environmental tests on unknown bacteria



Gram Stain


Starch Hydrolysis


H2S reduction






Indole Product



(NO3-to NO2-, NH4+or N2)

Positive to NO2-, possibly to NH4+or N2

Nitrification (NH4+to NO2-)



(NO2-to NO3-)

Positive, but negligible results (Egger pers. comm.)



Oxygen Tolerance

Facultative Anaerobe

Optimal Temperature

Mesophile, optimum at 22°C

Optimal pH

Neutralphile, optimum at pH 7

Optimal [NaCl]

Non-halophile, optimum at 0-0.5%


The morphology of the cell, red-orange pigment, gram-negativity, cell size and shape, lack of endospore, along with the bacteria's environmental preferences (Table 1.) the Family of the bacterium was determined to be Enterobacteriaceae (Egger 2010). Then utilizing the metabolic nature of the bacterium, starch hydrolysis, Indole Product, catalase reaction as well as pigment and colony motility (Table 1.); the most likely identification of the bacterium was to the Genus Morganella (Probabilistic Databases). Morganella is a small Genus consisting of one species morganii, which is broken into two sub-species morganii and sibonii (Zaas 2008). This makes the most probable identification of the bacteria, Morganella morganii.

M. morganii cells are gram negative rods that lack endospores; they are facultative anaerobes capable of respiration and fermentation of certain sugars (Krieg 1984). M. morganii is capable of indole production, denitrification, and catalase reactions; they are motile, and incapable of H2S production (Krieg 1984). Also has an optimal growth temperature at 28-29°C, making them mesophiles (Krieg 1984). Though common in soil, M. morganii is greatly studied for its pathogenic properties. M. morganii is in the same Family as Escherichia coli, and as such is commonly found in the intestinal track of humans and many other mammals (Miller and Emmons 2009), as well as their feces (Krieg 1984). The pathogenic effects only occur if the bacteria are able to penetrate the host dermal or intestinal tract, usually through lacerations or ulcers. Once infected M. morganii can possibly cause urinary tract infections, sepsis, pneumonia, wound infections, musculoskeletal infections, CNS infections, pericarditis, chorioamnionitis, endophthalmitis, empyema, and spontaneous bacterial peritonitis (Miller and Emmons 2009). The fact that is gram negative does pose the specific challenges of circumventing the extra cell membrane. Most infections are smaller non-life-threatening, when properly treated with Carbapenems such as -lactamase inhibitors (Wang et als. 2005; Zaas 2008).

The greatest limitation of this study was the fact that so few tests were conducted to really specify the type of bacteria isolated. The limited amount of data that the tests provided was only sufficient for a poor identification, meaning minimal matching of metabolic, environmental, and morphological factors. Specifically the oxygen tolerance could have been further tested by testing for the presence oxygenase. Or metabolically the bacterium could have been cultured on a variety of sugar mediums, as well as aerobic and anaerobic condition. This could have more accurately identified the possible nutrient sources that those bacteria can glycolysize, and a test for facultative pathways. The bacterium could also have been cultured in a media that were subjected to a greater variety of environmental factors, such at more incubation temperatures, to more accurately pin point the environmental optima.

The validity of the majority of these tests was strong. The most questionable though is undoubtedly the most important, the gram staining test. The problem arises with the strength of the chemicals needed to perform the stain. Firstly if the bacteria could have been over heated during fixation; or over decolourized with ethanol; over washing with water; or the fact that different strains have variable affinity to the Iodine-complex (Robertson and Egger 2010).

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Every test indicated that the bacterium was M. morganii. The motility test the only erroneous tests, which indicated that the bacterium was immobile, while M. morganii was mobile (Krieg 1984). Though it is possible for M. morganii to be immobile at low temperatures, and the SIM deep was incubated below optimal temperatures. It is also possible that the SIM was not allowed to incubate for a sufficient time period to observe the colonies migrating horizontally out from the stab line.

A singular strain of bacteria was isolated from a forest soil sample. That isolate was determined to most likely be M. Morganii. The isolation and identification of bacteria are essential tools in continuing the expansion of understanding of bacterial species, new medications, and ecological roles. This all adds to our cumulative knowledge of the natural world.


Brady, N.C. and Weil, R.R. 2002. The Nature and Properties of Soils, 13th edition. Prentice Hall, N.J.

Egger, K.N. 2010. Common Soil Bacteria Key. UNBC.

Egger, KN. 2010. Professor of Microbiology at UNBC.

Krieg, N.R. (Ed.). 1984. Bergey's Manual of Systematic Bacteriology (Vols. 1). New Jersey: Lippincott Williams & Wilkins.

Probabilistic Databases for The Identification of Bacteria. Probabilistic Identification of Bacteria: Enterobacteriacae.

Miller, J.R, Emmons, W.W. 2009. Morganella Infections. Emedicine, vol. 5 number. Retrieved February 24, 2010, from < 222443->overview

Robertson, S and Egger, K.N. 2010. BIOL 203 Microbiology Laboratory Manual. UNBC.

Wang, T.J., Huang, J.S., and Hsueh P.R. 2005. Acute postoperative Morganella morganii panophthalmitis. Eye, vol. 19: 713-715

Zaas, A. 2008. Morganella. Johns Hopkins, POC-IT. Retrieved February 24, 2010, from contentInstanceId=255875.