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Sporosarcina Bacteria Identification

Microbial communities are an immensely important part of soil structure and function. Microbes cycle nutrients, such as carbon and nitrogen, in the soil, making them available for use by plants (Rogers and Tate III 2001, Wu et al. 2008), and they affect the physical structure of soil by forming microaggregates of soil particles (Wu et al. 2008). The microbial community is also influenced by the soil; the soil type and its properties (Wu et al. 2008) and the litter type (Rogers and Tate III 2001) can affect the size and activity of the microbes. The flora growing in the soil can also have a major impact on microbial communities; forest and agricultural soils having very different community compositions (Macdonald 2009).
There are several ways to identify the microbes composing a soil community, including soil enzyme activity, microbial community enumeration, and metabolic diversity patterns (Rogers and Tate III 2001). In this study, bacteria were cultivated from agricultural and forest soils, and a single bacteria was identified by characteristics such as cell morphology and biochemical cycling and metabolism.
Methods The methods were adapted from Roberston and Egger (2010). Cultures were prepared from 10-2 dilutions of agricultural and forest soils in slants, broths, deeps, streak plates, spread plates, and anaerobic spread plates, and from dilutions of 10-4-10-7 pour plates were prepared. From these, four distinct colonies were subcultured on streak plates and slants, and gram staining was applied to the original cultures. The subcultures were then cultured in TSA plates, starch agar plates, SIM (Sulfide, Indole, and Motility) deeps, peptone, ammonium sulfate, nitrite, nitrate, and thioglycollate broths to be tested for nutrient cycling. TSA subcultures were tested with H2O2 and p-Aminodimethylaniline oxalate, an additional test not included in Robertson and Egger (2010), starch plates tested with iodine, peptone and ammonium sulfate broths with Nessler’s reagent, ammonium sulfate and nitrite broths with Trommsdorf’s reagent and dilute H2SO4, nitrite broths with diphenylamine and concentrated H2SO4, nitrate broths with Reagents A,B, and C, SIM deeps observed for motility, H2S production, and tested with Kovac’s reagent, and thioglycollate broths observed for oxygen preference. The subcultures were then cultured at 4, 22, 37, and 50°C on TSA plates to test for temperature preference, in broths at pH 3, 5, 7, and 9, and in NaCl concentrations of 0, 0.5, 2, and 5%. The temperature and NaCl plates were observed for growth extent, and the absorbance at 580nm was measured for the pH tubes. From the information collected, a single bacterial genus from the forest soil was identified.
Results The isolated bacteria was a gram-positive coccus of diameter 1.46um with cells that did not aggregate together and were found to be motile (Table 1). Colonies were round and shiny, with a pale pink to orange cream color ranging to non-pigmented (Table 1). The bacteria did not hydrolyze starch, reduce hydrogen sulfide, or produce indole (Table 1). Nitrate was not denitrified, but tests indicated that the bacteria did nitrify nitrite to nitrate, and produced ammonia from nitrogen compounds (Table 1). Tests for oxygen tolerance indicated an aerobic or microaerophilic bacteria, although there was no oxidase or catalase activity (Table 1). Growth occurred between 4 and 22°C, optimally at neutral pH, and in up to 2% NaCl (Table 1). From the observations and test results, it was determined that the bacteria was most likely a member of the genus Sporosarcina (Egger 2010).
Discussion Using colony morphology, cell morphology and gram-stain, the possible identity of the bacteria was first narrowed to the genera Micrococcus, Sporosarcina, Staphylococcus, Sarcina, and Arthrobacter (Egger 2010). Motility, ammonification, colony morphology, and cell morphology indicated that of these, Sporosarcina was the most probable identity (Egger 2010). Lack of denitrification, starch hydrolysis, indole production, and H2S production also supported the identity, as did oxygen tolerance (Egger 2010). These characteristics were further supported by Claus and Fahmy (1986), Kocur and Martinec (1963), and Yoon et al. (2001). Although Egger (2010) indicated that Sporosarcina was both oxidase and catalase positive, some species in the genus are oxidase negative (Kocur and Martinec 1963). Egger (2010) also indicated that Sporosarcina cells aggregated as diplococci or in tetrads, but Kocur and Martinec (1963) and Claus and Fahmy (1986) have shown that there are species in the genus occuring as single coccus, as the isolated bacteria did. Several species of Sporosarcina are psychrotrophic or psychrophlic (Yoon et al. 2001, Reddy et al. 2003) as the isolated bacteria was. The isolated bacteria did not reduce nitrates to nitrites as some species in the genus do, but it has been shown that this trait can be variable (Kocur and Martinec 1963, Yoon et al. 2001).
An erroneous characteristic the indication that the isolated bacteria nitrified ammonium ions to nitrate. This is typically a characteristic of heterotrophic nitrifying bacteria like Nitrosomonas, Nitrosococcus and Nitrobacter, aerobic gram-negative bacteria (Egger 2010). The indicative test results were odd, as Nessler’s reagent indicated no ammonia and Trommsdorf’s reagent indicated no nitrite in the ammonium sulfate broth, and diphenylamine reagent indicated the presence of nitrate in the nitrite broth (Table 1). The test of the nitrite broth with Trommsdorf’s reagent, however, produced a brown product for all four of the bacteria tested; a negative result should have produced no reaction, leaving the liquid clear, and a positive result should have yielded a blue-black colour (Robertson and Egger 2010). No explanation was found for this phenomenon.
The negative result of the catalase test is also problematic. All indications are that Sporosarcina are catalase positive (Kocur and Martinec 1963, Claus and Fahmy 1986, Yoon et al. 2001, Reddy et al. 2003, Egger 2010). The only catalase negative bacteria provided by Egger (2010) were Sarcina, Actinomycetes, and Nitrobacter, all of which did not match the other characteristics of the bacteria. No explanation was found for this indication, though Busse et al. (1996) stated that test for catalase can be difficult to standardize and interpret.
The reliability and success of the tests used must be taken into consideration when identifying a bacteria such as Sporosarcina. Many of the tests used to identify Sporosarcina indicated the presence of biochemical pathways and phenotypes which can be variable between species of the same genus (Busse et al. 1996). For example, Egger (2010) and Kocur and Martinec (1963) show that Sporosarcina are oxidase negative, while Clause and Fahmy (1986) indicate that the genus is oxidase positive. Some results for these tests, such as the nitrite broth reaction with Trommsdorf’s reagent, cannot be explained if they do not give one of the expected results. Busse et al. (1996) also point out that many tests, for example those for indole, catalase, and oxidase, are difficult to standardize and interpret, which may explain the negative results for the catalase test. They also point out that newly isolated strains of a culture may react differently than those which have been stored or subcultured over a longer period of time and that many identification systems were designed for specific taxa and may not work the same way with other organisms (Busse et al. 1996). It must also be considered that some techniques and tests used, such as gram-staining, take practice to be done properly and well. All the tests undertaken in the lab were being performed for the first time by someone with no previous experience. In order to ensure accurate results, it is suggested that multiple cultures be made in test media and the tests themselves be performed in duplicate.
There are also a variety of other tests that could be used to confirm the identity of Sporosarcina. As Sporosarcina are endospore forming bacteria (Kocur and Martinec 1963, Claus and Fahmy 1986, Yoon et al. 2001), testing for endospore formation using terbium diplocolinate photoluminescence (Pellegrino et al. 1998) or total luminescence spectroscopy (Smith et al. 2004) could substantiate the identification. Some species have the ability to decompose casein, gelatin, and tyrosin (Claus and Fahmy 1986), so testing for removal of these substance from medium may indicate Sporosarcina. It has also been found that Sporosarcina possess menaquinone systems (Claus and Fahmy 1986) so identification of the these could contribute to a positive identification. 16S rRNA analysis is an extremely useful tool for identifying phylogenetic relationships between bacteria (Busse et al. 1996) which has been successfully used to distinguish species by their similarities to Sporosarcina sequences in Antarctic soil (Aislabie et al. 2006, Aislabie et al. 2008), and so could be used identify the isolated bacteria as a member of the genus.
Like the bacteria in Antarctica, Sporosarcina are predominantly found in soil (Claus et al. 1986, Acha et al. 2009), as well as some species such as S. macmurdoensis found in ponds. One of the large roles of Sporosarcina in the soil is the production of urease, which catalyzes the production of CO2 and ammonia, lowering the pH of surrounding soils and causing the precipitation of mineral ions (Acha et al. 2009). The addition of urea to the soil can have a detrimental effect on germinating seedlings and young plants, as well as populations of microorganisms (Omar and Ismail 1999), and species like S. ureae play an active role in the decomposition of urea (Claus and Fahmy 1986).
To the extent possible with the given biochemical and nutrient tests and observations, the bacteria isolated from the forest soil was identified as a member of the genus Sporosarcina. Not all tests, such as the catalase test, matched to Sporosarcina, but the similarities between Sporosarcina and the isolated bacteria were enough to provide an identification as a member of the genus. Without more biochemical testing, or even DNA or RNA analysis, it cannot be said for certain that the bacteria is a member of Sporosarcina but the study was successful in providing a very probable identity for the bacteria as Sporosarcina.

Positive and Negative Contrast Media

In the field of radiology, some investigation or procedure on a patient in need of contrast into the patient’s body through a vein, artery, mouth or anus, which is located in the body. Since the invention of the era of the 1950s, iodine contrast media (ICM) is apparently more secure have been used extensively, almost daily use and is a widely used drug in medical history. Various forms and types of contrast media was used for the development of medical imaging. Most current diagnostic imaging contrast agents depends on this. Such as pharmaceutical drugs most others, this contrast agent was almost no negative effects even with a good safety record and proven medicine/drug is safe for use.
If seen on x-ray film, there are usually pictures of the anatomy such as bone, and there also appears a white colored gas that surrounds around the organs. The real aim is the use of contrast media to increase the density of the organ is usually not visible on radiographs, such as passage of urine or blood vessels, which originally did not appear at regular chest x-ray contrast media are not used. After application of contrast media, the resulting image will be white showing a hidden organ. Contrast media is composed of two types of ionic and non ionic contrast media is devided into two categories
Positive contrast media
Negative contrast media
Positive contrast media Positive contrast agents are agents that have the atomic mass and a high rate of obstruction (attenuation) high. So these agents are indeed radiopaque material. Positive contrast media are as iodine, bromine, and barium.
Typically, these materials will be incorporated into the body (according to appropriate procedures) and contrast agents will meet the target organ. Effects resulting radiopaque would explain the situation and structure of the organ / tissue targeted at radiographic images. Generally, a positive contrast agent is the key to ensuring that the patient has allergies or not for the selection of the contrast media can be tailored to the patient to avoid the effects of anaphylactic shock.
Negative contrast media One of x-ray absorption is the weight of the atomic mass of a substance. If the atomic mass is low, the effect of obstacles (attenuate) is low and the reverse occurs when the atomic mass increases. When the mass of the atom is high, it will produce an effect of density / contrast radiography of the image because the image will become more permeable and dark. This feature is useful when an anatomical structure is shown with a background of very permeable and the large difference in contrast between the image and the background (without producing blur the boundaries between image and background).
Contrast media such as air (oxygen, carbon dioxide, etc.) are good examples as negative contrast agents. Negative contrast agents often used in conjunction with contrast agents positf and one common procedures often incorporate the use of these agents were negative double barium enema.
Examples of gas used to produce negative contrast in radiographic images: Carbon dioxide
Produced in the gastrointestinal tract (GIT) with barium sulfate to demonstrate mucosal pattern. Double contrast barium meal for example. Carbon dioxide can also be produced by the colon when given double contrast media, as it reduces pain and reducing stomach discomfort immediately.
Produced in the body cavities such as in the knee when examining arteriogram to demonstrate the knee joint.
Produced by the patient during the radiographic examination, for example, took a deep breath and hold during chest examination.
Basic iodine contrast media Several investigations in the radiology needs of this contrast into the patient’s body through a artery or a vein. A frequent example is the intravenous urogram. Usually only the bones and the wind will be visible on x-ray images. To distinguish the path of blood in the urine or blood vessels, contrast media containing iodine to be added to increase the intensity of blood or urine. Then, this will cause the structure to look ‘white’ in films such as the bones of patients in x-ray images.
Classification of Contrast Media There are two parts: the ionic and non ionic.
Ionic compound is divided into two parts; high level of osmolar contrast media(hocm), and low level of osmolar contrast media(locm). This classification is based on the iodine concentration of particles (generally as elements of contrast media is frequently used in the procedure IVU) absorbed / dissolved into the blood. If the rate of absorption of iodine is too high, it can slow the blood flow into the central nervous system (hyperosmolality). This situation is a major factor many negative reactions to patients due to contrast media that are not appropriate.
Agents Rate of osmolality(mOsm/kg) High-osmolar contrast media
low-osmolar contrast media
Non-ionic compounds
Cerebrospinal Fluids
Ionic: Content molecules tightly to the anion and cation (positive and negative) in the blood causing high osmolality – toxic / reaction high.Properties of molecules containing benzene rings, for example – salt compound meglumine diatrizoate and diatrizoate sodium (Renografin 60 and Hypaque 76 – osmolaliti high)
Characteristics of iodine contrast agents; i. Osmolality (no liquid particles in 1 kg of water)
ii. viscosity – the size of the rejection of (resistant) to the flow of fluids (viscosity / concentration)
iii. Toxicity – (poisoning / injury), it is based on the molecular configuration.
Non-ionic: Molecules are not resolved in the fluid / blood.For example, a compound of sodium diatrizoate (iopamidol, isovue, iohexol / omnipaque)
– Measures of care; i. Can not exceed the dose.
ii. Stay away from the sun.
iii. Stored at room temperature.
Reactions to contrast agents – iodine: Use of Iodine contrast media (ICM) can cause unwanted side effects and manifestations of anaphylaxis. Symptoms include nausea, vomiting, erythema area, generally a hot sensation, headache, coryza or larynx edema, fever, sweating, astenia, dizziness, paleness, dyspnoea and moderate low blood pressure. more acute reactions in such as vasodilatation cardiovasluar system with pronounced peripheral hypotension, tachycardia, dyspnoea, action, bluish skin condition(cynosis) and lack of consciousness, needs emergency treatment. On behalf of this reason the use of contrast media should be limited in cases of diagnostic procedures must be shown. Side effects associated with use of intravascular contrast medium is constant iodinated of natural light to moderate and temporary, and less often by non-ionic than ionic preparations. Side effects to ICM are classified as special and nonidiosyncratic. The pathogenesis of these adverse reactions may involve direct effects of mobile phone; enzyme induction, and activation of quinine, fibrinolytic complement, and other systems.
ATTENTION AND CONTRAST AGENTS CONTROL MEASURES. Firstly, check the history of the patient.Make sure the reading of the BUN-blood urea nitrogen (8-25mg/dl kretinin) – (0.6-1.5 mg / dl).Actions taking steroid / anti-histamine before contrast examination. Check patient heart condition.If the patient have Multiple myeloma or myelo dysplastic syndrome or severe diabetes (preferred drink a lot of water).
Anaphylactic shock. The other name is Vasogenic shock. Histamine is released in response to an antigen (foreign substance). They give effect to;smooth muscle – causing bronchial spasm (bronchospasm) respiratory (sneezing, wheezing, a pilot). Increased vascular permeability – (urticaria, edema of the mucosa, red spots (rash). Increased secretion of mucous glands (nausea, vomiting).
Sign of the symptom(anaphylactic shock) are patients will begin to be Agitated. Rate of pulse will be increased dramatically.The patient appeared pale and weak and lifeless body.They do not have the ability to think too long.The patient feels cold, sticky and itchy of the patient will be reddish and spotted (urticaria). Patient’s neck like a choking / strangulation and Trouble breathing.
Mild symptoms Weak symptoms include the following disease: dispersed urticaria, which is the very commonly stated as adverse reaction; sickness of pruritus; rhinorrhea; brief retching or nausea or vomiting; dizziness, diaphoresis; coughing. Patients with mild symptoms should be noticed about the progression or development of a more severe effect, which needs treament.
Moderate symptoms Medium symptoms include the following: constant vomiting; diffuse urticaria; pain in head; facial oedema; laryngeal oedema; weak bronchospasm or difficulty in breathing; palpitations, rapid heart beat(tachycardia), or slowness of heart beat (bradycardia);high blood pressure; and abdominal spasms or cramps.
Severe symptoms Acute symptoms include the following: life-threatening arrhythmias (in example; ventricular tachycardia), laryngeal oedema, faint, over bronchospasm, pulmonary oedema, spasm, low blood pressure, and death.
Comparison of contrast agents: Ionic agent Non-ionic agent Toxicity – high risk of allergy
Toxicity – less / low risk
Osmolality high
low Osmolality
High viscosity
low viscosity
Contrast media needed for Radiological examination: To distinguish the path of blood in the urine or blood vessel, usually just the bones and the air will be visible on x-ray and to improve the density of urine or blood.
Advantages of contrast media: easily dissolved.
Provide better mucosal detail
There is no osmotic effect, so the radio density is long-lasting.
Relatively delicious
Disadvantages of contrast media: Aspiration pneumonia if inhaled.
If got leakage into the cavity or organs of the body. It can’t survive indefinitely and may cause granulomatous reactions.
Contrast media management Reactions that occur while the procedure of contrast media are not predictable, and the patient should be observed carefully during the procedure. The following is general advice to be given by an x-ray contrast media as well as introduced to the patient.
explain to the patient about the procedure in detail
the patient and medical history
approved by completing the patient consent form
check the serial number or code and expiry date of contrast material
free virus check on each appliance and packaging that will be used
ensure that emergency equipment used are provided
patients are encouraged to drink plenty of water and make sure the patient’s body dehydration. Most of the contrast media will be out through the urine after four hours
make sure the contrast agent used was based on body temperature
need to take special protection and be alert of additional risks that can be derived as severe cardiovascular disease, severe renal impairment, asthma or a history of allergies, suspected a high sensitivity of iodine, a very old (> 65 years) and very young (<6 months) , severe respiratory diseases, diabetes and had suffered a reaction to the medication.