Identification Of Nocardia In An Agricultural Soil Biology Essay

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Microbes play an important role in peoples everyday lives. They are responsible for diseases such as the common cold, and are important in the food industry in items like yoghurt and bread (Willey et al. 2008). Another place that microbes live is in soil. Microbes that inhabit the soil are less obvious to people, but not any less important. These microbes play critical roles in nutrient cycling and decomposition (Whitman et al. 1998). Without these processes, plants, and therefore animals and humans, would not be able to survive (Whitman et al. 1998). The products of some microbe's metabolic activities release molecules that are necessary for the survival and growth of plants and other microbes (Whitman et al. 1998). Some soil microbes fix nitrogen from the air, which makes Nitrogen available in a usable form for plants and other microbes (Willey et al. 2008). Other microbes hydrolyze starch, which converts unusable starch to usable glucose and can be digested and used as an energy source (Robertson and Egger 2010). Different environmental factors such as temperature, oxygen concentration, salt concentration and pH can determine where microbes live in the soil. Different soils will have different distributions of microbes, depending on the environmental factors in the soil (Murray et al. 1989).

In this experiment, a microbe colony was isolated from an agricultural soil sample, and the identity of the microbe was determined after performing multiple tests that helped identify properties that were specific to that microbe.

A sample of agricultural soil was obtained and diluted in distilled water to make a 10-2 dilution. A spread plate of this dilution was prepared by using aseptic techniques to spread the colony on a petri dish containing Agar (Robertson and Egger 2010). The petri dish was incubated at room temperature for 48 hours. One colony was selected from the plate, and a streak-plate subculture was prepared, by using an aseptic technique to streak another sterile petri dish containing Agar (Robertson and Egger 2010). This petri dish was also incubated at room temperature for 48 hours.

The colony was tested with a gram-stain and viewed under the microscope. Iodine was used to test for starch hydrolysis, and Kovac's reagent was used to test for the production of H2S. The colony grew in a peptone broth, and then was tested with Nesslar's reagent for the presence of ammonia. The colony was grown in an ammonium sulfate broth, and tested with Nesslar's reagent for the presence of ammonia, and tested with Trommdorf's reagent/H2SO4 for the presence of nitrite. The colony was grown in a nitrite broth and was tested with Trommdorf's reagent /H2SO4 to see if nitrite was oxidized, and was also tested with diphenylamine reagent/H2SO4 to test for nitrate production. The colony was grown in a nitrate broth, and tested for denitrification. The colony was grown in a thioglycollate medium to test the oxygen tolerance of the microbe. H2O2 was used to test for catalase activity, and aminodimethylamine was used to test for oxidation. The colony was also grown at different temperatures, pH and salt concentrations to determine what kind of environment the microbe could live in.

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RESULTS

The microbe colony first appeared on the Agar spread plate, and Table 1 shows that it had a circular shape, with a 7mm diameter. It had no colour, and was translucent and dull (Table 1). The colony was flat and had a rough texture with many visible tiny filaments (Table 1). A Gram stain was done on a sample of cells from the colony, and the light pink colour indicated that the colony was Gram negative. The cells were found to be single rods, and measured to be about 3μm (Table 1). Iodine turned a light yellow when placed on the colony, indicating that the colony could hydrolyze starch (Table 1). The colony did not produce H2S , or Indole (Table 1). The microbe also did not show any motility (Table 1). Nessler's reagent turned the peptone broth with the colony in it a light yellow, which indicates that ammonia is being produced in small amounts (Robertson and Egger, 2010). The microbe was found to produce nitrate, but not nitrite through the process of nitrification (Table 1). The microbe was found to produce nitrite from nitrate through the process of denitrification , although the nitrite was not further reduced to ammonia or nitrogen gas (Table 1). The microbe colony was also found to have the enzyme Catalase, because bubbles were formed when H2O2 was put on the colony. The colony did not turn a dark black colour when a small amount of aminodimethylamine was added and so the microbe does not carry out oxidation.

The microbe was found to be aerobic because all of the microbe growth occurred at the very top of the thioglycollate medium, (Table 1). The microbe was found to be mesophilic, because the most growth occurred at 37°C, although a little

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occurred at 22°C (Table 1). The microbe was found to be neutrophilic , because the most growth occurred at pH 7 (Table 1). Finally, the microbe was found to be nonhalophilic to halotolerant, because the most growth occurred from 0% to 2% NaCl, and some of the microbe grew at 5% (Table 1).

Table 1: Summary of characteristics and results of biochemical and environmental tests carried out on a singular microbe colony isolated from a sample of agricultural soil.

Test

Results

Test

Results

Colony Morphology

Circular flat colonies, with visible filaments, no colour, dull and translucent. Rough texture, approx 7mm diameter.

Denitrification

(NO3-to NH4+or N2)

Negative

Cell Morphology

Single rods, approx. 3μm

Nitrification

(NH4+ to NO2-)

Negative

Gram Stain

Negative

Nitrification

(NO2-to NO3-)

Positive

Starch Hydrolysis

Positive

Catalase Activity

Positive

H2S Reduction

Negative

Oxygen Tolerance

Aerobic

Motility

Negative

Optimal Temperature

37°C - Mesophile

Ammonification

Positive

Optimal pH

7- Neutrophile

Indole production

Negative

Optimal salt concentration

0%-2% , some at 5% Nonhalophile, Halotolerant

Denitrification

(NO3- to NO2-)

Positive

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DISCUSSION

The isolated colony from the agricultural soil sample was classified as being part of the genus Nocardia. The morphology of the colony aided in the identification

of the microbe. Valero-Guillen and Martin-Luengo (1984) and Robertson and Egger

(2010) also found the Nocardia colonies to contain no colour, tiny hyphae filaments and to be circular. The microbe was found to be aerobic and mesophilic, which is consistent with the findings of Bergey's Manual, (1986). The microbe was also found to be nonhalophilic, and halotolerant which was consistent with Robertson and Egger(2010). Bergey's Manual, (1986) found that the hyphae of Nocardia break off into small rods, which would explain the single rods found in the microbe sample. The characteristics that set this Nocardia microbe apart from the other filamentous bacteria was the ammonification test, Gram stain and the aerobic nature of the microbe. This microbe was strictly aerobic (Table 1), while other genera like Actinomyces are preferentially anaerobic (Bergey's Manual, 1986) The Rhodococcus genus does not produce ammonia through ammonification (Robertson and Egger 2010), and this microbe did produce ammonia (Table 1). Also the Bergey's Manual (1986) found the Nocardia to be Gram positive to Gram variable, while other filamentous bacteria were found to be Gram positive. This microbe was found to be Gram negative, which could be due to the fact that the Nocardia is acid-fast, and so it does not retain stains very well (Bergey's Manual 1986). Rinsing for too long with ethanol could have made the gram positive bacteria appear gram negative. A possible error that might have skewed the results could have been contamination of the colony when transferring to perform tests. Other microbes might have given

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different results, and could have skewed the data.

This experiment classified the microbe as a Nocardia primarily based on morphology, and a few subtle differences in biochemical and environmental tests between Nocardia and other filamentous bacteria. Other tests that would help identify the Nocardia genus, would be to test the chemical components of the cell wall. Nocardia have very distinct mycolic acids on the cell wall, and any bacteria containing these acids are classified as a Nocardia (Bergey's Manual, 1986 and Valero-Guillen and Martin-Luengo, 1984). These characteristics were not examined in this experiment, and future studies would include these tests to help classify the microbe as a Nocardia.

The Nocardia inhabits many different soil types, and since it is chemoorganotrophic, the more organic matter in the soil, the more Nocardia are present (Valero-Guillen and Martin-Luengo, 1984). The role of Nocardia in nature is not well studied, but the Nocardia could be important in the Nitrogen cycle, because they require the presence of Nitrogen to grow, and they produce ammonia and nitrite (Table 1 and Bergey's Manual, 1986). Some Nocardia are also an opportunistic human and animal pathogen (Bergey's Manual, 1986).

The colony isolated from the agricultural soil sample was successfully classified as a Nocardia bacteria primarily based on morphology, and a few biochemical differences between Nocardia and other filamentous bacteria. The classification could be more definite if further tests were carried out to see if the bacteria contained mycolic acids on their cell walls.

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