In a soil environment, microorganisms live in mixed communities (Robertson and Egger 2010). They are composed of prokaryotic Archaea and bacteria and eukaryotic fungi, protozoa and algae. Soil microorganisms are essential for decomposition and the recycling of essential nutrients that are in organic macromolecule material.
To help study microorganisms in a soil environment, isolating bacteria from a sample is needed. Growth and survival depends on nutrient supply and a favourable growth environment. A sterile medium is needed to ensure that it is free of contaminating microorganisms. Culture mediums can come from culture tubes including broths, slants, and stabs (deeps) as well as agar plates. These plates allow large surface area to study microorganisms with a number of inoculating methods including spread plates, pour plates and steak plates.
After successfully obtaining a pure culture, observations of the morphological appearance have to be noted to help identify the bacteria being studied such as colony morphology, cell morphology, cell size. Additional studies and tests are required to help determine characteristics that can further narrow the cultured bacteria including staining, adding salts, gram staining, etc. Tests are needed to determine specific biochemical activities that the microorganism possesses to classify the microorganism. To determine tolerated and optimal growth condition changes in temperature, pH, saline concentrations, and other tests are needed to help classify the microorganism. Techniques used as above can be used to categorize to help identify the microorganism and can find favourable traits that can be used for agriculture, medicine and other industrial applications. The objective is this paper is to be able to isolate and culture an individual soil microorganism and to identify the bacteria cultured with techniques described below.
Methods and Materials
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Place 1 g of Agricultural soil in a beaker containing 99mL of sterile deionized water (dH2O), mix well and allow settling. In this 10-2 dilution, extract 1mL from the solution and place in a labelled 10-3 test tube that has 9mL sterile H2O. Continue sterilely diluting soil until you reach 10-7 dilution.
Using the 10-2 soil dilution, use the aseptic technique described in Bio 203 Microbiology Lab Manual 2010 material and method section 3. Place a loopful of inoculum into the broth, stir briefly, and withdraw loop. Using the same aseptic technique, streak the tip of the loop in a wiggle shape across the surface of the slant with the inoculating loop. With the aseptic technique, dip the inoculating needle the solution in TSA deeps about Â¾ of the way down into the agar of the deep.
Divide streak plates into 4 quadrants by drawing lines on bottom of plate, with the aseptic technique, drag the loop back and forth 2-3 times across the surface in quadrant 1. Flame the loop and place in ethanol after cooled. Turn the plate clockwise Â¼ turn. Heat the loop again and cool the hot loop on the agar at the side of the plate. Drag the loop through the first streak towards quadrant 2 then streak back and forth without further touching of the original streak. Replace the lid, sterilize the loop, and turn plate another quarter in the clockwise direction and repeat until all four quadrants are done.
Prepare pour plates with melted TSA medium by placing 1mL of soil dilution 10-4 diluted soil sample and add cooled melted TSA. Gently swirl until solidify in a figure 8 motion. Place the lid halfway to prevent condensation build-up until cooled. Repeat with 10-5-10-7 soil solutions.
Prepare spread plates by pipetting 0.1mL of 10-3 soil dilution onto TSA plate agar surface. Using aseptic technique, place L-shaped rod onto agar and turn plate with other hand distributing evenly solution until no liquid is longed seen. Repeat spread plate technique on a Brewer's anaerobic spread plate medium with 10-3 soil dilution.
Place all petri dishes inverted to prevent condensation in an incubator at room temperature for 48 hours.
Identify a colony from a plate above. Prepare a streak plate as mentioned earlier.
Place a drop of water on a clean microscope slide, with aseptic technique place a small amount of colony into water and spread evenly and allow drying. With forceps, pass slide through Bunsen burner 3 or 4 times to heat-fix. Use gram staining technique described in Bio 203 Microbiology Lab Manual 2010 and record results in Table 1.
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Place a single streak on a starch agar plate of bacterial isolate with aseptic technique, invert and place in incubation at 25 degrees for 48 hours. Add iodine at the edge of the culture, a clear zone will be present if hydrolyzed around the bacterial growth and a blue/black color if not. Record results in Table 1.
With the aseptic technique and inoculating need, stab the SIM deep test tube and place in incubation at 25 degrees for 48 hours. Look for black precipitate indicating hydrogen sulphide production and growth around original stab indicating motility. Add three drops of Kovac's reagent to determine if using the aseptic technique, place an inoculum of bacteria in the peptone broth and place in an incubator for 7 days at 25 degrees. Repeat steps with an ammonium sulphate, nitrite broth tube, nitrate broth tube, thioglycollate medium. After the incubation, add one drop of Nessler's reagent to a spot plate and add a loopful of peptone broth culture and record results in Table 1. Repeat test with ammonium sulphate. As well place 3 drops of Trommsdorf's Reagent and 1 drop dilute H2SO4 and add a loopful of ammonium sulphate broth. Record results in Table 1. Repeat Trommsdorf's reagent with the Nitrite. To see whether nitrate was produced, add on drop phenylalanine and 2 drops of concentrated H2SO4 and add a loopful of nitrite. Add reagents A and B to nitrate broth tube to see if nitrate reduction. Add reagent C if no color is produced after two reagents added and record results. Note location of growth in tube. Place loopful bacteria on glass microscope and add a few drops of 3% hydrogen peroxide to observe with a microscope if the enzyme catalase is present.
Place a single wavy line on the agar surface of a TSA plate in four separate plates and incubate at temperatures 4, 22, 37, and 50 degrees Celsius. Repeat on TSA plates with salt concentrations: 0, 0.5, 2, & 5% NaCl. Inoculate the broth test tubes with the aseptic technique with pH of 3, 5, 7, 9. Allow to incubate for 36 hours in respective conditions. Record level of growth in each condition in table 1.
Table 1 - Experimental result
Creamy White, smooth, translucent
Temperature (degrees Celcius)
Linear chain bacillus
Color of iodine reaction
Color of media around bacteria
Color of Kovac's Reagent
Color Peptone broth w/ Nessler's reagent
Color of ammonium sulfate w/ Nessler's Reagent
color ammonium sulfate w/ Trommdorf's reagent and H2SO4
color nitrite broth w/ Trommdorf's reagent and H2SO4
Color of nitrite broth w/ diphenylalamine reagent and H2SO4
Color broth w/Reagents A+B
Color broth w/Reagents C
Osmotic pressure (%NaCl)
Growth in Thiogycollate
Growth throughout but better growth near top
Bubbles when H2S2 added?
Osmotic pressure Classification
A summary of the important facts from Table 1 is that the bacteria isolated is the colony morphology has a circular, creamy white appearance. The cellular morphology is a bacillus shaped in linear chains with a positive gram stain indicating that the cell wall has a thick homogenous layer of peptidoglycan. Further testing indicated that starch hydrolysis is positive and H2S production is negative. Ammonification is also positive, Denitrification is converted from NO3- to NO2- and not all the way to NH4+ or N2. Nitrification is positive. The growth in the thioglycollate indicates that it can grow in the aerobic and anaerobic conditions but were optimal in aerobic conditions indicating that it is a facultative anaerobe. Catalase activity was also positive and a negative indole production result. The ranges on conditions the bacteria grew under and optimal conditions indicated that it was a Mesophile, Neutrophile and Halotolerant.
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The genus could most likely be determined as Actinomyces. The bacteria's characteristics determined from the experiment indicate 16/18 similar with the common soil bacteria key (Egger 2010). The results nitrification was suppose to be negative and not positive to be an Actinomycetes. The size is 10 um which is a bit bigger as well than the 2.0-4.0um x 0.5um.
Actinomycetes play an important role in creating soil structure and cycling carbon and nitrogen via decomposition and mineralization of plant and animal remains. They are also capable of degrading substrates such as lignocelluloses, keratin and chitin (Lederberg 2000). The also play an important role in bioremediation of hydrocarbon contaminated sites. Other environments include Rhizospheres, composts and mouldy fodders, aquatic environments and a variety of other environments. An interesting point is that Actinomycetes-derived antibiotics have been in agriculture for the past 40 years as feed additives for growth promotion in farm animals which role is not known but thought to cause weight gain by affecting the animal's gut microflora. This might be a possible route for transmission of antibiotic resistance to humans (Lederberg 2000). Actinomycetes have been valuable in biotransformation in the alteration of hormones and steroids for the pharmaceutical industry (Lederberg 2000).
Two-dimensional Electrophoresis to sequence the 16 s rRNA can be used to determine the genus. Examinations of the genome with reveal a length of approximately 8Mb with a characteristically high G+C (mol %) content between 69-73% with a linear, circular structure (Lederberg 2000). Use of a more powerful microscope would review a closer look and a 3-dimensional look at the bacteria to help determine cell morphology characteristics which can help determine the isolated bacteria. A limitation in the test was a 1000x microscope could determine the general shape but more could have been done with a stronger microscope.
Errors in the test could have been made error in observations, with contaminating microorganisms that result in false characteristics. The bacteria identified could be another bacteria as the denitrification is suppose to be negative unless this was misread, recorded incorrectly. The size of the bacteria is 10um recorded which is a bit bigger than the size 2.0-4.0 um x 0.5um as well the length was not accounted for so a miscalculation in the correction factor is most likely and/or recording error.
The objective was met as a bacterium was successfully isolated from a soil sample. This undergone further culturing to determine cellular and colony morphology observations, as well determined cell wall composition, biochemical pathways the bacteria possess and determining optimal condition and ranges the bacteria could survive in all to classify the genus. This was done as a gene Actinomycetes was found with a high probability that it is the correct bacteria from the tests preformed. Further tests may be needed to confirm results.