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Identification of Bacillus Coagulans from Agricultural Soil

Introduction Soil is a common substance that is found almost everywhere in the world and is home to a diverse community of microorganisms, especially for bacteria. Bacteria are so common in soil that surface soil can be home to 108 to 109 bacterial cells per gram of dry soil (Prescott et al., 1999). This quantity of bacterium in soil, however, decreases deeper into the subsurface of the soil. There are small variations in bacterial quantity in subsurface layers, but the surface layer is always the most numerous in quantity (Weaver et al., 1994). This is because these bacteria prefer the smaller soil pores (2 to 6 ?m in diameter) that are more commonly found in surface soil. This is likely a method to avoid being eaten by protozoa (Prescott et al., 1999). As small soil pores are common habitats for bacterium, and soils contain higher concentrations of CO2 and CO, with lower concentrations of O2, many of these bacteria have developed the ability to grow in microaerobic or anaerobic conditions (Prescott et al., 1999).
Although bacterial quantity is plentiful in the surface layers, it is common to find relatively few bacteria of the same species. Rather, there is much variation in the species of bacterium cohabitating in these soil layers (Weaver et al., 1994). As a diversity of bacteria brings a diversity of metabolic pathways and nutrient fixation, this proves beneficial for plants and insects that live in the soil and rely upon the nutrients produced by these bacteria (Weaver et al., 1994).
Through isolation, culturing, and testing of agricultural soil bacterium, this lab attempted to isolate and identify a single species of bacterium from agricultural soil.
Methods A 10-2 dilution in distilled water was created using 1 g of agricultural soil. From this dilution, a TSA streak plate was made and incubated at 22°C for 48 hours and then chilled at 4°C until further analysis could be preformed (Egger 2010).
From this streak plate, a single culture was chosen to be sub-cultured on a TSA streak plate and was incubated as above. This culture was also gram stained and observed under microscope. The bacterial cell dimensions were calculated and cell shape and arrangement were recorded.
The bacterial subculture was then observed for colony morphology. Individual cells were then tested for starch hydrolysis, H2S reduction and motility, ammonification, nitrification, denitrification, oxygen tolerance, and for catalase production (Egger 2010).
Further testing was then performed to determine optimal growth temperature, osmotic pressure, and pH. Temperature testing was done at 4, 22, 37, and 50°C; osmotic pressure testing done at 0, 0.5, 2, and 5% NaCl; and pH testing was done at pH 3, 5, 7, and 9 (Egger 2010).
Data from tests were pooled and used to determine the possible identity of the unknown culture.
Results The streak plate of dilute agricultural soil revealed a large quantity of different bacterial colonies varying in colour, shape, size, texture, and elevation. After isolation of a single bacterial culture, there was little to no variation in these characteristics.
This single bacterial culture revealed a circular form with a flat elevation and a rough texture. The colour was observed to be clear to white in colour and translucent in appearance.
Further observation under microscope revealed staphylobacillus approximately 40 ?m in length.
Gram staining and testing for starch hydrolysis, and nitrification all turned out positive, while testing for H2S reduction, motility, ammonification, and denitrification all proved negative, which is further outlined in Table 1.
This bacterium was then determined to be a facultative anaerobe with an optimal temperature of 37°C, optimal pH of 7, and optimal osmotic pressure of 0.5% NaCl. It was also observed that there was little growth at 5% NaCl and reasonable growth at pH 5.

Discussion As the bacterium isolated in this experiment was rod shaped, gram positive, and catalase producing, it is likely that this specific bacterium belongs to the genus Bacillus (Butler 1986). Determination of the specific species of Bacillus proved more difficult.
As this bacterium was able to hydrolyse starch, was a facultative anaerobe, had an optimal growth temperature around 37°C, and an optimal pH around 7, the possibilities were narrowed to either Bacillus coagulans or Bacillus licheniformis (Butler 1986). Although B. licheniformis is common to most ground soils, there proved to be a few common factors that supported the greater plausibility of this specific bacterium to be B. coagulans. The isolated bacterium was unable to reduce nitrate to nitrite and was unable to grow significantly in a NaCl medium of 5%. As B. licheniformis is capable of reduction of nitrate to nitrite and growth in 5% NaCl medium, it is unlikely that this unknown bacterium was B. licheniformis (Butler 1986). It was also noted that the unknown bacterium was capable of growth at pH 5, which is a characteristic of B. coagulans (Butler 1986). Specifically, B. coagulans has been recorded as having an optimal pH of 6 and a minimum of 4.0-5.0 depending on specific strains (Butler 1986).
In order to further verify that the unknown bacterium was B. coagulans, it would have been beneficial to test the bacterium’s ability to hydrolyse casein and gelatine as B. coagulans cannot hydrolyse casein and gelatine while B. licheniformis can. It would also have been beneficial to further investigate growth in 7% and 10% NaCl to further verify the identity of B. coagulans if growth did not occur.
Unfortunately, not all tests that were performed were useful in the identification of the specific species of the unknown bacterium. The tests were useful, however, in determining genus. As the genus Bacillus was reasonably simple to identify based on the rod shaped bacterium and catalase production, some of the tests, such as the test for H2S reduction were not as useful as others, such as the test for catalase production.
If the unknown bacterium did prove to be B. coagulans, this would be an uncommon isolation as B. coagulans is not common in soil (Butler 1986). For this reason, there is little information recorded on B. coagulans ecological role in soil. It would be advised that further investigation into the ecological role of B. coagulans should be performed as, although uncommon, B. coagulans is still a soil bacterium and must therefore play an ecological role in soil. It may also be beneficial to further investigate B. coagulans as it is a common additive in medicated creams and antacids (Butler 1986). This is because of the possible pro-biotic benefits of B. coagulans in the gastrointestinal tract, although these benefit claims have been questioned as to their validity in recent years (De Vecchi and Drago 2006). There has also been a recent study that suggests that application of B. coagulans to arthritic joints may cause a decrease in pain and a reduction in disability of these joints. Although, these are preliminary studies, and the function of this pain relief is unknown, there is significant reason to peruse B. coagulans as a treatment for rheumatoid arthritis (Mandel et al. 2010).
Although the unknown bacterium is likely B. coagulans there were possible sources of error. A major source of error could have come from the gram staining process. There was a possibility that the stain was not rinsed with ethanol for long enough, which would have caused retention of the dye in the cell wall of the unknown bacterium. This would have lead to a false positive for gram staining. If this was true and the unknown bacterium was gram negative, this would indicate a possibly different genus and definitely different species.
Although errors could have lead to misidentification of the unknown bacterium, based on the data collected, it is most likely that the unknown bacterium isolated was B. coagulans. There was no reason to believe that the isolated colony was contaminated with different species of bacteria, and testing lead to a conclusive identification of the unknown bacterium. From this it is reasonable to say that the objectives of this experiment were met.

Effects and Importance of Osmosis

Osmosis is the movement (natural) of a solvent, in the case of living organisms (water) selectively through a semi-permeable membrane down a water potential gradient. In other words it is the movement of water across a selectively semi- permeable membrane from an area of high water potential (low solute concentration) to an area of low water potential (high solute concentration) (Bowen, 2000).
Semi-permeable membrane
A membrane is partially (semi) permeable, if it will let in water molecules but not the molecules or ions dissolved in water (the solutes such as sugar molecules). Many cell membranes function in this manner. Osmosis is there for an important mechanism in the transport of fluids in living organisms (Bowen, 2000).
Osmosis: Movement of water across a selectively permeable membrane from an area of low solute concentration to an area of high solute concentration.
Key: Water, o – Solute
Osmosis is important in biological systems, as many biological membranes are semi-permeable. In general, these membranes are impermeable to organic solutes with large molecules, such as polysaccharides, while permeable to water and small, uncharged solutes. Permeability may depend on:
solubility properties,
charge, or chemistry, as well as
Solute size.
Water molecules travel through the plasma cell wall, tonoplast (vacuole) or protoplast in two ways, either by diffusing across the phospholipids’ bilayer directly, or via small transmembrane proteins similar to those in facilitated diffusion and in creating ion channels). Osmosis provides the primary means by which water is transported into and out of cells. The turgor pressure of a cell is largely maintained by osmosis, across the cell membrane, between the cell interior and its relatively hypotonic environment (Maton et al., 1997).
The process of osmosis accounts for many functions that maintain life. In relation to blood cells, blood cells placed in pure distilled water will swell and burst. If these cells are placed in a Hyper osmotic (hypertonic) solution, i.e., the solution has more dissolved particles, salts, sugar, etc., than is in the cells, they will shrivel up (a process called crenation in the case of blood cells).
The energy that drives the process of osmosis is called osmotic pressure.
In animal (human beings included), the red blood cells are very important to the survival of the organism because they transport oxygen from the gills, skin, or lungs to the cells of the various tissues (muscles, nerves, etc.). These blood cells are transported in a fluid (serum) that has approximately the same salt content as sea water.
Effects of Osmosis on Red Blood Cells
Red blood cells as in all animal cells don’t have cell walls. In cases of hypotonic solutions, red blood cells will swell up and burst (explode) .when the cell is in danger of bursting due to accumulation of too much water in it, contractile vacuoles will pump out the water out of the cell to prevent it from bursting. In hypertonic solutions, water will diffuse out of the cell due to osmosis and the cell shrinks. For the red blood cell to stay in its normal condition, it’s always surrounded by isotonic solution.
If the concentration of the cells cytoplasm is lower then medium (the medium is hypotonic) surrounding the cell, then osmosis will result by the cell gaining water, hence the cell will swell up and burst.
If the concentration of the water inside the cell is the same as that outside the cell (the medium is isotonic solution), there exists a dynamic equilibrium, meaning the number of cells getting in and leaving the cells is the same hence the cell will retain its original size. The red blood cell retains its shape because of the isotonic nature of the plasma.
If the water concentration inside the cell is higher than that of the medium (the media is a hypertonic solution), hence the number of molecules diffusing out will be more than that entering, and the cell will shrink.
The kidneys in the human body provide the necessary regulatory mechanism for the blood plasma and the concentration of water and salt removed from the blood by the kidneys, which is controlled by the hypothalamus. This process of regulating the salty and the mineral salts in the blood is called osmoregulation.
Osmosis and diffusion a have related concepts: Both processes involve the movement of materials from an area of high concentration to an area of low concentration. Diffusion involves the movement of chemical molecules from a low concentration to a higher concentration whereas osmosis involves the movement of water molecules from a high to low concentration via a semi permeable membrane.
Importance of Osmosis in the animal/human body
Salts and minerals are transferred from water through osmosis. Osmosis transfers water through the plasma membrane (which is selective and semi permeable) of the cell. It manages the mixing of water, glucose and salts in the body cells, this is important, otherwise the cells would loose too much water and eventually die. Hence osmosis plays an important role in keeping the cells alive.
Osmosis plays an important role in the functioning of the kidneys, it is also important in the helping to transfer water and various nutrients between the blood and fluid of the cells.
People who suffer from kidney diseases depend in kidney machines to remove waste substances (products from their blood, such machines use a process called dialysis, which is similar to the process of osmosis.
Salt water fish constantly consume a lot of water, which is released to the environment through osmosis, while fresh water fish don’t drink water because their skin is responsible to absorbing water.
Diffusion of Potassium Permanganate (KMnO4)
Diffusion is the movement of molecules from an area of high concentration to an area of low concentration across a permeable membrane as a result of kinetic energy of random motion. It is a random movement of molecules which is directional from an area of high concentration to an area of low concentration until equilibrium is achieved.
Molecules are in a constant state of motion. For example, if you dissolve KMnO4 in water so that the concentration is initially higher in one part of the water that another, diffusion will occur so that there is a net movement of KMnO4 from area if high concentration to an area of lower concentration. However, if the KMnO4 molecules have a complete even and random distribution through out the water, there will be no movement of KMnO4 in any direction.
The rate of diffusion will be affected by properties of:
The cell
The diffusing molecule
Surrounding solution

The rate of diffusion increases as the concentration gradient increases. When the concentration of molecules outside the cells is very high, relative to the internal concentration, the rate of diffusion will also be high. If the internal concentration are the same (low concentration gradient), the rate of diffusion will be low.

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