Bacterias were some of the first signifiers of life that we know of and have continued to boom throughout the legion mass extinctions and changes the Earth has undergone. They are found in about every known environment on Earth, from the deep ocean deepnesss to the north-polar tundra. One of the countries where bacteriums are most outstanding is in dirt environments where the figure of species found here are legion. Bacterias populating these dirt environments provide a figure of services to the ecosystem. Decomposers by and large break down disintegrating organic affair in the dirt and thereby return to the dirt organic affair utile for other beings ( Ingham 2010 ) . Others are nitrogen-fixing and are associated with works roots. Here they take N from the air and supply workss with N in functional signifiers. Other services include chemoautotrophs which use non-carbon compounds for energy and are of import in N cycling and debasement of pollutants ( Ingham 2010 ) .
With so many bacteriums found in these dirt environments it is of import to separate the type of bacteriums you have isolated. By analyzing the stray civilization and executing several biochemical and environmental factor trials, one can contract down possible individualities of the stray civilization. When analyzing the civilization one can depict the civilization macroscopically by its pigment, signifier, lift, border, visual aspect, and texture ( Egger 2010 ) . Microscopically one can qualify the cells by their size, form, and gm staining. Some biochemical trials that can be performed include trials for H2S production, nitrification, ammonification, denitrification, presence of oxidase, presence of catalase, and the optimum temperature, pH, and, salt concentration for growing ( Egger 2010 ) . Using these consequences one can find the civilizations individuality utilizing known belongingss of bacterial species.
This experiment pursued the involvement of insulating a bacterial settlement grown from forest dirt and placing this isolate through a figure of biochemical and environmental factor trials. Through these trials and by depicting the cell and civilization morphology, the bacterial isolate was able to be narrowed down to a individual genus. The isolate was determined to be of genus enterobacter due to the similarities of negative gm staining, both being mesophiles, facultative anaerobes, non-halophiles, and alkaliphiles, positive ammonification, and deficiency of H2S production. The methods used in this designation procedure can be used to find the individuality of other bacterial civilizations.
The undermentioned methods were adapted from Biology 203 Lab Manual 2010 ( Egger 2010 ) . Dilutions of 10-2, 103, 10-4, 10-5, 10-6, 10-7 ( w/v ) of agricultural and forest dirt were made. Broths, angles, deeps, spread, and streak home bases were made utilizing 10-2 dilutions of agricultural and forest dirt utilizing sterile technique. Four pour home bases were besides made utilizing 10-4 to 10-7 for both the agricultural and forest dirt samples. These were grown over a hebdomad and the bacterial growing was recorded. A specific civilization was chosen and a streak home base and angle were prepared to insulate the bacterial civilization. The civilization was observed under a microscope for signifier, visual aspect, coloring material, texture, and other observations. The cells were so gram stained and observed.
The stray run home base was so used to fix a figure of home bases and tubings to find biochemical belongingss. A amylum agar home base was prepared to prove for amylum hydrolysis, a sulfide, indole, and motility ( SIM ) deep to prove for motility and H sulfide production, a peptone stock tubing to prove for ammonification, an ammonium sulfate and nitrate stock tubing to prove for nitrification, a nitrate stock to prove for denitrification, a thioglycollate stock tubing to prove for O tolerance, and a TSA run home base to prove for presence of oxidase and catalase. Four TSA run home bases were made to be grown at 4, 22, 37, and 50oC to prove for optimum temperature. Four TSB tubings at pH 3, 5, 7, and 9 were grown to prove for optimum pH. Four Transportation security administration home bases at 0, 0.5, 2, and 5 % NaCl ( w/v ) concentration were prepared to prove for optimum salt concentration.
After the first home bases were made from the wood and agricultural dirts, a ecru, smooth settlement from the forest dirt was chosen, isolated, and used for the balance of the survey. After sing the settlement macroscopically it was found to hold round signifier, umbonate lift, full border, glistening visual aspect, and smooth texture ( Table 1 ) .
Table 1. Summary of qualitative observations, biochemical trials, and environmental factors to place unknown bacteriums
Beige/Brown, Circular, Umbonate, Shiny, Translucent, Smooth, 10mm Diameter
Hydrogen Sulphide Reduction
Positive aa‚¬ ” High sums of ammonium hydroxide
Denitrification ( NO3- to NO2- )
Denitrification ( NO3- to NH4+ or N2 )
Nitrification ( NH4+ to NO2- )
Nitrification ( NO2- to NO3- )
22oC – Mesophile
9 – Alkaliphile
Optimal Salt Concentration
0 % ( w/v ) – Non-halophile
Microscopically it was found that the cells were rod shaped and had dimensions of about 1.25um ( Table 1 ) . The cells were found to be gram negative, hydrolyze amylum, have positive ammonification, and denitrification from NO3- to NO2- ( Table 1 ) . The cells were besides found to incorporate catalase, no oxidase, and grew optimally at 22oC, pH of 9, and 0 % NaCl ( w/v ) concentration ( Table 1 ) . The isolate had a temperature scope of growing from 4oC to 37oC, growing at pH from 7 to 9, and growing at salt concentrations from 0 to 0.5 % NaCl ( w/v ) .
The consequences of the biochemical and environmental factor trials were used to give a possible individuality for the bacterium. The negative gm staining was detected due to the pink coloring material under a microscope and positive amylum hydrolysis was detected due to the absence of a dark coloring material when I was added ( Table 1 ) ( Egger 2010 ) . The bacterium was found to be negative for H sulfide decrease because of the deficiency of black precipitate and positive for ammonification because of a brown coloring material nowadays when Nessleraa‚¬a„?s reagent was added ( Table 1 ) ( Egger 2010 ) . It was negative for motility as the growing was limited to the entry point of the vaccinating needle found in the deep and positive for denitrification from NO3- to NO2- as after the add-on of reagents A and B, the sample turned ruddy ( Table 1 ) ( Leboffe and Pierce 2005 ) . The bacterium showed positive nitrification from NO2- to NO3- as deep blue coloring material was observed when diphenylamine and H2SO4 were added, but no nitrification from NH4+ to NO2- as no coloring material alteration was seen when Trommdorfaa‚¬a„?s reagent and H2SO4 were added ( Table 1 ) ( Egger 2010 ) . The bacterium besides showed presence of catalase as a bubbles were seen when H2O2 was added, but no presence of oxidase as no coloring material alteration was seen ( Table 1 ) ( Leboffe and Pierce 2005 ) . The bacterium was determined to be a facultative anaerobe as there was growing throughout the thioglycollate tubing, but best growing near the top ( Table 1 ) ( Leboffe and Pierce 2005 ) . It was besides found to turn optimally at 22oC doing it a mesophile, pH of 9 doing it an alkaliphile, and at 0 % NaCl ( w/v ) concentration doing it a non-halophile ( Table 1 ) ( Egger 2010 ) .
Using these consequences I was able to come up with a possible individuality of Family Enterobacteriaceae and a possible individuality of genus enterobacter. This household of bacteria has some strong distinguishing features similar to the unknown isolate, those being both found in dirt, length being between 1.2 and 3.0 um, being facultative anaerobes, H2S non being produced, both being mesophiles ( turning best between 20 and 30oC ) , and similar cell morphology ( Holt 1984 ) . This peculiar genus from this household is chosen as it had the most similar consequences with neither it nor the unknown being motile ( Holt 1984 ) . The positive nitrification did non compare with many suspected bacteriums genus. This may be a consequence of heterotrophic nitrifiers in the dirt supplying a positive consequence.
The genus enterobacter consists of many bacteriums which are pathogens, impacting the urinary and respiratory piece of lands. Members of this genus are found worldwide throughout a figure of environments including H2O, dirt, workss, insects, and worlds ( Pontes et al. 2007 ) . Recently members of this genus have become progressively popular due to their clinical importance ( Pontes et al. 2007 ) .
To assist corroborate the individuality of the unknown bacterial isolate many other trials could be performed, some specifically utile for Enterobacteriaceae. These include trials for urease, lysine decarboxylase, and gluconate which would assist separate from other gram negative facultative anaerobes ( Holt 1984 ) . Some restrictions of the trials performed include the deficiency of precise measuring for add-on of substrates for trials and possible taint of bacterial civilizations ensuing in hapless trial consequences.
Through the progressive isolation of a individual bacterial settlement from a mixture of settlements from dirt, a individual bacterial settlement was able to be isolated for word picture. Through a assortment of biochemical and environmental factor trials, every bit good as qualitative observations, this bacterial isolate was able to be characterized to a peculiar genus ( Table 1 ) . The consequences of these experiments showed that this unknown bacterial isolate was most likely to be a portion of the genus enterobacter ( Holt 1984 ) . Members of the genus enterobacter are known as pathogens and are found in legion environments including dirt, another suggestion that this is the right bacteriums ( Pontes et al. 2007 ) . Other trials including the sensing of urea, lysine decarboxylase, and gluconate could be used to further corroborate the individuality of this unknown isolate ( Holt 2984 ) . The methods and trials used in this experiment can be used in other research to assist find the individuality of an unknown bacterial isolate.