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Evaluating Post-irradiation Tissue Alterations

Towards evaluating post-irradiation tissue alterations Abstract
We seek to call to attention the paucity of data concerning irradiation effects on the extracellular matrix and of organised tissues. Examples of such research are cited as are some of the limiting factors towards obtaining meaningful results. We further seek to engender a range of research towards further improving the quality of life, most pointedly of those receiving radiotherapy.
Keywords: Irradiation, Extra Cellular Matrix, Tissues
Introduction Not long after the discovery of X-rays it was recognised that tissue effects can occur as a result of irradiation. Most notable of the early as opposed to late changes in tissue properties was the observation of skin erythema (the reddening of skin), first recognised at relatively high doses received from soft x-rays, the severity of reddening increasing with dose. Indeed, the observation of the occurrence of skin erythema was soon to be followed by if not the first, then at least one of the earliest means of measuring dose, with the degree of reddening equated with dose through the so-called skin-erythema dose (SED). By the very early 1920s it had become more than apparent that x-ray exposures were producing a range of effects, including opacifiation of the lens of the eye (cataract formation) and of even greater alarm, malignancy; see for instance (1) concerning the widely recorded developments leading to the X-Ray Martyrs and to the first controls on radiation exposures. It would of course be unacceptable nowadays and indeed proscribed within any modern radiation controls legislation to knowingly produce such deterministic effects in an individual, other than for the purpose of producing net benefit for the individual, as for instance would typically be determined in medical practice in which the net benefit far outweighs the risk.
The safe, optimal use of radiation for the benefit of humankind has advanced considerably since those first very tentative steps, and with widening exploitation of radiation methods there now exist a wide range of fundamental studies aimed at providing strong underpinning knowledge of radiation effects in living tissues. These include both studies at the intracellular level (with which this article will not concern itself, not least because of the massive range of studies already devoted to the areas of endeavour) as well as of extracellular effects, the latter including the extracellular matrix (ECM). As an example of the latter, interest has been shown in changes in protein structure, starting with investigations using high doses of radiation up to about 1 MGy, such as the work of Cassel in 1959, reported by Bailey (2). While at such high doses it is of no surprise that one could detect these extracellular changes even given the instrumentation capabilities available at that time, the sensitivity of instrumentation for ECM and organised tissue changes occurring at doses of clinical interest is rather more challenging, confronting this challenge is one of the prime themes of present review. This interest relates to the continuing desire to provide the most effective outcome for the patient while suppressing untoward side-effects. Here note is made that while the aim of radiotherapy is to deliver the maximum radiation damage to tumour cells, damage can occur to organs at risk (OAR), such as for instance the heart in breast radiotherapy. Epidemiological studies of patients receiving radiotherapy for the left breast have shown a significant increase in cardiovascular death (3), albeit there being well-established practices for limiting dose to the tissues of the heart. In what is to follow, we review efforts aimed at studying changes in the pericardium, the intention being that such efforts be regarded as a model system, on the basis of which other organised tissue effects can also be studied. We similarly review radiation induced effects in hyaluronic acid, important throughout the body, not least in terms of lubractive role.
Pericardium is a part of the heart, mainly made of collagen fibres and can show changes in its structure at doses as low as a few Gy (4). In our own studies, we have used hyaluronic acid (HA) and collagen as models for effects of radiation on tissues. HA is prevalent between the two layers of the pericardium, offering smooth motion between the serous and visceral layers. It is also of course prevalent in the skin and in the space between the articulating bones of skeletal joints, offering similar lubricating roles. The HA and the framework offered by the collagenous systems provide for support and stability of motion and changes in the mechanical properties of these can result from insults that include irradiation as well as disease processes. Using well controlled radiation doses allows us to study both of these tissue components as models for investigation of biomechanical changes in a variety of tissues, not only directly as a result of irradiation but also in examining associated functionality changes from disease-based deformations. It should be mentioned that most of the work cited herein concerns doses at radiotherapy levels, an exception being food studies wherein the work also concerns viscosity changes, albeit at much higher doses (up to 10 kGy). Our work has attracted a favourable degree of citation, Table 1 indicating the prime drivers of those referencing present studies.
Rheological studies
Given irradiation to a sufficient dose, changes can be observed in the viscosity of the HA that forms the basis of synovial fluid (being one example of ECM). As an aside it can be noted that HA is a non-Newtonian fluid, in particular showing dependency upon shear rate. Changes in the viscosity of HA can be explained in terms of depolymerisation and polymerisation, as investigated by our group (15), use being made of rotating viscometers to alter shear rate. The rotating viscometer as well as the falling sphere method represent particular forms of tool whose use enable measurement of the effects of irradiation on the structure of HA and of other proteins. The importance of this work is also clearly seen in measuring the side effects of irradiation of sensitive structures such as the rectum in prostate radiotherapy, pericardium effects in regard to left breast radiotherapy or indeed even in studies of the viscosity of food sauces, as seen in Table 1. HA is found to be in all body fluids and organs, as in for instance, in the vitreous humour, synovial fluid of the joints, umbilical cord, and skin (16). Table 2 shows the concentration of HA in some tissues and tissue fluids. One of the distinctive properties of HA is its high-molecular weight and therefore high viscosity, making it a crucial element of the extracellular matrix (2).
Effect of radiation on HA Out own work on HA has concerned the synovial fluid of articulating joints, with HA forming a major component of this fluid, playing the dominant role in joint lubrication. In particular, studies have shown HA to be the major determinant of viscoelastic behaviour in synovial fluid (18, 19). It has been established that after typically the third decade of life the body will begin to lose the ability to produce HA, making it all the more important that irradiation changes in HA attract attention, particularly in regard to radiotherapy of the joints, with loss in viscosity and hence of wear resistance being expected to impact upon the quality of life.
In more detail, irradiation of HA will result in ionization and excitation of the atoms of HA and surrounding ECM, to the extent that this may lead to changes in the physical and chemical nature of the polymeric HA. Alterations can be a result of several effects, including chain scissions and cross-linking (2, 20) and bond deformation (21). Chain scissions will result in reduction of the molecular weight and the associated viscosity (2). Conversely, cross-linking will result in increasing viscosity, a reflection of increasing molecular weight. Since viscosity is an important property of the HA polymer, giving rise to its viscoelastic behaviour in the synovial joint, it is important to investigate this in regard to any concomitant radiation cosmesis effects i.e. effects impacting on the quality of life of the individual following radiotherapy. While chemical degradation of HA induced by reactive oxygen-derived species (ROS) has attracted considerable attention in regard to HA depolymerisation, irradiation induced depolymerisation or bond deformations at low levels of dose has been largely neglected. Thus, it has been the intention of the studies previously reported by this group to make measurements of viscosity and shear stresses on HA solutions, conducted at different shear rates, use being made of various types of viscometer for different concentrations (0.01% – 1%w/v) of HA.
Collagen studies
In regard to irradiation effects upon collagen, it first needs to be mentioned that collagen forms an essential framework that not only provides biomechanical support to tissues but also moderates nutrient flow within the body, to be altered by both physical insult such as irradiation and also from the presence of disease, as in infiltrating ductal carcinoma. Collagen fibres are fundamentally fibrous proteins, made up of long filaments arranged side by side, sometimes forming networks and also sometimes forming annuli. Indeed, there exists a rather extensive range of types of collagen, approaching 30 variations, such that between them they can accommodate a range of biophysical needs. Thus it is of no surprise to learn that among the particular challenges in irradiation studies is the wide variation in collagen fibre types and widths, not least given that collagen fibre width represents one of the most important physical properties of collagen fibres, potentially altering as result of irradiation. It is clear that studies of for instance biomechanical stress-strain are particularly prone to misinterpretation.
Effect of irradiation on pericardium (fibrous ECM)
Before discussing the radiation effects on pericardium it is first worth briefly discussing the effects on both proteins and collagen. Irradiation is one of the mechanisms (physical or chemical mechanisms) that can provide for modification of the structure of proteins. In general, irradiation of proteins can cause ionization and excitation of the atomic constituents, leading to changes in the physical and chemical nature of peptide chain. This can take place as a result of three different actions, bond deformation, chain scissions and cross-linking of the hydrogen bonds (2). One or more of these effects can take place in both dry and aqueous states of proteins. It should be noticed that in dry state, the radiation effects are predominantly direct action on the amino acids as a result of disruption of the secondary structure of proteins. Conversely, irradiation in aqueous state also involves action of free radicals, where and radicals interact with the surrounding molecules in the solution, mainly hydrogen atoms.
In the early 1960s a number of studies investigated the effects of irradiation on collagen, a major component of the extracellular matrix (2, 22-24). The studies were conducted at particularly elevated doses, from a few tens of kGy, through to extremely high doses of ~ 1 MGy, as in the work of Cassel in 1959, as reported by Bailey (2). Unsurprisingly, at such doses detectable changes were observed. Changes on collagen structure as a result of irradiation are similar to those occurring for other fibrous proteins. Irradiation can be connected to changes to hydrogen bonds that make up the backbone of the triple helix and might lead to chain scission or/and cross linking within the basic structure which can affect its mechanical strength.
In regard to pericardial alterations, with potential mortality from cardiovascular disease, a particular concern is that of ultrastructural changes of the heart following therapeutic thoracic irradiation. This was first documented by Burch and his group in work published in 1968 (25). The changes of the heart tissues were found to differ from those resulting from ischemia or infarction (25). Further such early work was published on the effect of radiation on the heart, changes being investigated through variations in electrocardiography (ECG) readings (26). Studies of tissue morphology started in the late 1960s, the heart being considered at the time to be of low sensitivity to radiation at radiotherapeutic doses (27) until in rabbits Fajardo and Stewart demonstrated radiation induced damage at low single doses (up to 40 Gy) (28).
Radiation induced changes to the heart, resulting from radiotherapy of cancers of organs close to the heart have been reported in a number of studies (see below). These changes have also been observed in different animal species, including rats (29), rabbits (27, 28) and monkeys (30). Effects in different organs in the chest, such as breast, oesophagus and lymphoma were subsequently documented for humans (31-33). In regard to the incidence of heart disease following breast radiotherapy, this has been estimated to be 3.4 per cent, compared to 5.8 per cent for lymphomas cancer patients (34). Mechanisms for radiation-induced changes in the heart can be broadly summarised to be a result of direct interactions with cell nuclei, with subsequent degradation of DNA, together with compromised vascular, extracellular matrix, neural damage and affects upon the heart mediated by the viscous and mechanical alterations. Radiation-induced damage on the heart can be observed in the pericardium, myocardium, valves and coronary arteries or interstitial cellular medium (ECM) (3, 26, 31, 35). Although the aforementioned structures of the heart can be potentially damaged by irradiation, pericardium has been found to be more frequently affected than other structures, in particular the parietal part (36). It has also been noticed in the myocardium that damage is more frequent in the anterior wall of the left ventricle than the right ventricle. This may explain the reported reduction in heart output after irradiation (37, 38). It should also be mentioned her that the incidence of damage is dose related, depending on the volume of the heart irradiated (32).
Issues in AFM studies of pericardium with fat
In study of bovine pericardium samples by the present group, use was made of the tapping mode and a hard cantilever. In doing so, although some images were initially found to be of acceptable quality, there was observed to be a progressive reduction in spatial resolution. Guidance showed this to be a result f a contaminated tip, the hard cantilever applying excessive force on the sample surface. Remnant fat on the sample added to such difficulties. Since it would be impractical to change the tip for each new scan, technical advice lead to use of reduced hardness cantilevers for soft samples.Although as an alternative, use could be made of chemical treatment to remove fat deposits, described in the literature as a means of stabilizing pericardium, it has nevertheless been reported that the fibre width and d-spacing will change as result of use of those chemicals (39-42). Such chemical treatment might also affect the mechanical properties of samples, possibly increasing stiffness. Glutaraldehyde (GA) and dimethyl suberimidate (DMS) are examples of such chemical agents that could be applied in pericardium processing in order to produce prostheses. It should be mentioned that these chemicals affect pericardium, mainly through a cross-linking mechanisms (39-42). In considering making use of chemical treatment, specifically as applied in treating food products, no evidence has been found to negate change in the properties of such samples. Therefore, in our own work we have consistently avoided such treatment. A summary table showing some of these chemicals is shown in table 3, with their possible effects on collagen fibres, observed using AFM scanning.
Table 3 Summary of chemicals and effect on topography/other properties of collagen fibres.
Concluding Remarks In this commentary piece, we have sought to stimulate further research in this important area of endeavour. It would seem to bea matter of curiosity that while remarkable efforts have been made in regard to intracellular research and quite deservedly so, very limited efforts have been made in regard to the ECM and at the organised tissue level. We have pointed out a number of drivers of such research, also indicating some of the challenges towards obtaining meaningful results when confronted by multifactorial dependencies, failure to take these into careful account limiting the quality of results. An important fundamental limitation of such irradiation effect studies is that these are typically conducted in silico and with the lack of vitality of life, one is unaware of the possible effect of recovery of tissues post-irradiation.
Table 1 Prime drivers of research citing work reviewed herein.
Field of interest
Study purpose
Breast radiotherapy
Pericardial stress-strain
Prostate radiotherapy
HA degradation
Rectal dose as a result of prostate radiotherapy
Dose reduction through collagen injection
Rectal dose as a result of prostate radiotherapy
HA sensitivity
Rectal dose as a result of prostate radiotherapy
HA degradation
Pericardium dose
HA degradation
Effect of radiation on HA expression
HA degradation
Biomedical applications reviewed: hot topics areas cited twice
HA and collagen changes
Biotechnology/Effect of radiation on different sauces
Viscosity measurements
Aortic valve
Viscoelastic properties
Prostate radiotherapy
HA degradation
Table 2 Concentration of hyaluronan in some tissues and tissue fluids.
Tissue or fluid
Concentration, mg/l
Human umbilical cord
4100
Human synovial fluid
1420-3600
Bovine nasal cartilage
1200
Human vitreous body
140-338
Human dermis
200
Human thoracic lymph
8.5-18
Human urine
0.1-0.5
Human serum
0.01-0.1
Table 3 Summary of chemicals and effect on topography/other properties of collagen fibres.
Chemical used in prep
Effect seen using AFM
Tannic acid
Considerable change in surface topography

Gram Negative Unknown Lab Report

Jennifer Mbanasor
The primary motivation behind this experiment is to uncover the distinctive bacteria organisms placed in my unknown broth. The issue is that this obliges various tests with the end goal that I should have the capacity to identify the microorganisms. My intention in this trial is to acquire the right names for these subjects with a specific end goal to progressively compose a fitting report. My methodology to this test will be an experimentation that will inevitably acquire the correct results to getting the name of my cultures. I then eventually did the analysis by leading my tests and crossed out the bacteria that did not meet the specifications from my experiment. My results will be uncovered after my clarification of what I did and the tests I performed to figure out the bacteria’s nomenclatures.
My unknown culture accompanied two types of species of bacteria that had been inoculated previously. The information I got from class is that one of them ought to be a gram negative. The other ought to be a gram positive and conceivably have an alternate morphology. To recognize the two organic entities I first needed to go for the TSA streaking strategy. This technique obliged that I streak the blended culture on a TSA plate and incubate it for 24 hours. From that point, I needed to inspect the plate for two separate shapes of the colonies. This is a piece of my experimentation approach on the grounds that after I analyzed the plate I didn’t see a colossal contrast on the plate. I expected that either the test didn’t work or I simply just streaked the plate in the wrong way. The move down to the blended culture of the TSA streaking system was the selective media technique. With the selective media and differential media I will have the capacity to clarify what the cultures I got from my TSA plate are. There is a distinction between selective media and differential media. Selective medium sort segment is planned to support the development of one gathering group of organisms, yet repress the development of another. These media contain antimicrobials, dyes, or alcohol to hinder the development of the organisms not took a gander at or not focused for study. Selective medium sorts include EMB agar, Mannitol Salt agar, MacConkey agar, and Streptococcus Faecalis (SF) agar (Highlands 2013).Then again, differential medium sorts are those that recognize microorganisms from each other focused around development attributes that are present when they are grown on particular medium sorts. Organisms with varying development attributes essentially indicate obvious growth in development when set on differential media. Illustrations incorporate blood agar, Eosin Methylene Blue (EMB) agar, Mannitol Salt agar, and MacConkey agar (Highlands 2013). The Eosin Methylene blue agar or (EMB) contains dyes like eosin and methylene blue. The name clarifies it itself. This media is picked particularly for gram negative species. Lactose-fermenting creatures, for example, E. coli or Enterobacter will create a precipitate on the EMB. The colonies will either be black or have dull focuses with clear rings. At that point the non-lactose fermenters like Salmonella will seem red or pink or even uncolored. The MacConkey agar has its closeness to EMB on the grounds that it excessively likewise chooses for gram negative species. MacConkey is both selective and differential in light of the fact that it chooses for gram negative and separates the lactose-fermenting bacteria by uncovering a red or pink color for the lactose aging microbes while seeming colorless for non-fermenters. Mannitol salt agar is produced using 7.5% NaCl (Highlands). It is selective for staphylococci and is differential regarding mannitol fermentation. No one but halophiles can become on this high centralization of salt in the medium. This media is then motioned by the generation of acidic items heading phenol red in the media to transform from a neutral red-orange to bright yellow. The SFA plate is utilized for the separation of Enterococcus species from the Streptococcus bovis group and other streptococci. Particularly, the EMB and Mac plates were everything I needed to inspect the distinction in the gram positive and gram negative. After I had cultured those plates, I still felt free to streak two TSA plates simply so the bacterium would not have dye on it when I assess it through the microscope.
The gram staining procedure is the most widely recognized method utilized today to have the capacity to distinguish the diverse types of bacteria. The thing is gram negative and gram positive stain distinctively with this procedure for a couple of reasons. Gram positive have thick multilayered peptidoglycan which traps a color called crystal violet better than how gram negatives can hold the dye. Gram negatives have a slim layered lipopolysaccharide on its peripheral covering that doesn’t hold the stain well while the ethanol alcohol wash. This is the reason that after the gram stain transform, that the negatives uncover a reddish to pink color under the microscope and the positives will remain a purple shade.
I needed to do construct a harder biochemical test first so I continued to perform the biochemical test needed for gram negatives first. One of the tests would be the triple sugar iron agar test or TSIA which tests the capacity of the bacteria to deliver sugars like glucose, and to create hydrogen sulfide. The methyl red and Voges-Proskauer test includes the testing of acid fermenters. All the more particularly it tests to see the oxidation of NADH to NAD and glycolysis (Weber, 2009). The Vogues-Proskauer test permits me to examine the organisms capacity to kill acid results of glucose fermentation utilizing 2, 3 butandiol. The citrate test uncovers if the life form can create a catalyst called citrate-permease. From that point, citrate is transported into the cell and changed over into pyruvate which is simpler for the cell to change over into items. The urease test “an intracellular enzyme test”, focuses on the bacteria species digestion system and the way it breaks down urea into ammonia and carbon dioxide. The gelatin test is a test for gelatinase which hydrolyzes proteins that are gotten from collagen. The SIM test looks at hydrogen sulfide production, indole generation, and motility. The triple sugar iron agar test (TSIA) is a media test that gives three results which incorporate sugar usage, gas creation, and sulfur reduction.
The main test I started was the citrate test. I clarified prior that it includes the testing of catalyst called citrate-permease. I was required to figure out if my unknown can utilize citrate as a wellspring of carbon for its energy. Bacteria that are able to do this can likewise change over ammonium phosphate into NH3 NH4OH creating the media to turn into a soluble substance (Badon). I utilized a whole alternate method for inoculating other than how I did with the past tests and cultured a needle. I cleaned it and touched the most superficial layer of the citrate with the refined secured needle. I then incubated the citrate tube for 24 hours. Bromthymol blue color is the thing that I added after the overnight incubation to focus the pH scope of my culture. A green shade demonstrates a pH of 6.9, and blue color shows a pH of 7.6 or more (lab slides).
The following test I began was the VP test to check whether my unknown produces 2, 3 butanediol rather than acids from glucose aging (Badon, 2013). Since it was hard to test the occurrence of 2, 3 butanediol specifically, the test really shows the occurrence of acetoin which is a forerunner of 2, 3 butanediol. This test obliges Barrits reagent A and Barrits reagent B to uncover a color change. A red or pink shade change implies it is sure for acetoin creation which likewise shows that it is a butanediol fermenter. Before I included the reagents, I needed to inoculate my unknown culture by disinfecting the loop and after that adding it to the tube from my plate. I then needed to incubate it for three days.
The next test I performed was the MR-VP test. Which I clarified in the previous sentence that it includes the testing of acidic fermenters and glycolysis. This test obliges methyl red marker in order to figure out if it is certain for the occurrence of the acid or not. Red significance it is positive, orange implies that the test is negative and no change of the color implies a negative result. I disinfected my loop and vaccinated my unknown culture from the plate and swirled the bacteria into the MR-VP test tube, then to be inoculated and then put it in the hot room overnight for 24 hours.
With a specific end goal to figure out whether my unknown contains the chemical enzyme urease, I was required to do a urease hydrolysis test for it. I then started to inoculate my culture into the urea broth produced using yeast concentrate and urea. It likewise contains phenol red which will change the color of the juices from an orange-yellow to pink if the pH climbs up. This test was obliged 8 days of review so I essentially held up an entire week before I returned to record my data.
The gelatinase test was also exceptionally straightforward and straight to the point just as was the citrate test. The reason for this test is to form whether the gelatin (got protein from collagen) gets to be hydrolyzed by a catalyst known as gelatinase. The gelatin medium is made out of gelatin, peptone and also a beef extract. This test likewise also requires an incubation time of 8 days so I inoculated my culture then put away the gelatin filled tube into the hot room in a test rack with the other test tubes.
The SIM test was my next in my biochemical tests which records for three disclosures. It tests for sulfur reduction, Tryptophanase action, and motility (Badon). The SIM medium contains supplements, for example, iron and sodium thiosulfate. It likewise also incorporates amino acids, for example, tryptophan (ACC, 2000). For the sulfur reduction parcel, I needed to experiment on if my unknown could diminish sulfide by utilizing both of the two catalysts: cysteine desulfurase, or thiosulfate reductase. For the indole bit I needed to watch whether my microscopic organisms could create the catalyst tryptophanase which hydrolyzes tryptophan to pyruvate, ammonia, and indole. I needed to utilize a substance known as Kovac’s reagent so as to focus my result whether it is red for positive indole creation, or brown for negative indole production response. The motility allotment is a third test to test if my microbes had motile flagellum. This is the motivation behind why I needed to immunize my bacteria by stabbing my loop in the SIM semi-solid media. On the off chance that I were to see cloudiness around my wound line, then it would be sure for motility. This test will take me 24 hours to watch, so I likewise put away it in the hot room with whatever is left of my test tubes.
The following test I did was for the TSIA slant. Like the SIM test it additionally gives three test outcomes which incorporate sugar use, gas generation, and sulfur reduction. The segments of this semi-solid media is produced using three sugars: lactose, sucrose, and glucose. It likewise is made out of phenol red to demonstrate the aging of sugars. In the event that there is no color change, then that means that there is no sugar aging. On the off chance that there is a detectable shade change, then it shows that my unknown is either a glucose fermenter, lactose fermenter, or both. A dark black color would then uncover that it delivers hydrogen sulfide. This is one of the reason I needed to return to the lab in the next 6 hours after I inoculate this media. The dark color may toss of my results a little bit for inspecting it for sugar fermentation. In the event that there are any indications of any type of bubbles at the lowest part of the tube then it demonstrates that gas has been delivered. After I noted my results after 6 hours, I set the TSIA plate back into the hot room for another 20 hours.
The EMB test was next in my rundown of biochemical media tests. I likewise did the T-streak method for this media the same way I did the streak for my MSA. This will only be able to choose for most gram negative bacteria that are able to ferment lactose.
To begin my gram positive testing, I decided to choose the few differential and selective plates required to create a few outcomes about that permit me to take out the conceivable unknown decisions. The MSA media is the plate I wanted to T-streaked first. The MSA plate will help me to figure out if my unknown will be able to survive the high centralizations of salt and can be able to ferment mannitol as well (ICT/MHRD, NME 2013). I streaked my MSA plate, and then put away it in the hot room for incubation and then proceeded with the following test.
The coagulase test was an alternate test I did for my gram positive unknown. This test will uncover if my unknown is either positive for the protein coagulase or negative. I utilized my time to inoculate my loop and then swabbed some of my unknown onto one of my slides. I then started to blend the coagulase onto the slide as well. I then was able to see a couple of white big clumps as I blended the substance. I recorded the results of what I saw and proceeded to do the following test.
The bile esculin test which contains ferric citrate and esculin tests for the results of 6, 7-dehydroxy-coumarin. For this test I basically got my loop and was able to stab the media and find out my gram positive unknown. I put away this test in the hot room to be incubated for 2 days.
The SFA test is utilized to focus the contrast amongst enterococci and streptococci. The peptone and dextrose are what deliveries the enterococci it supplements. A positive response will uncover a yellow-brownish shade because of the fermentation of dextrose. So as to check whether my positive unknown can ferment dextrose, I then did the T-streak method on the plate to be inoculated for 24 hours.
After all the information I gathered from my biochemical tests, I at last accumulated all my results. My results turned out uniquely in contrast to what I had anticipated. For the morphology of my unknown bacteria, I anticipated that it will be round formed staphylococcus. In reality my unknown was a gram negative grouped bacillus. Keeping in mind the end goal to truly separate my unknown microbes, I needed to record my results and contrast it with a spreadsheet agenda on my lab manual. When I investigated my SIM test, I perceived that there weren’t any discernible changes whatsoever. A slight development of my unknown bacteria, there was no indole generation as per the Kovac’s reagent, and without a doubt there was no sulfur creation in light of the fact that the shade did not change to dark. My VP results hinted at some change however. It demonstrated to me that it was sure for the occurrence of acetoin. So yes the creation of 2, 3 butanediol is available. I recognized a negative turn out about my citrate test. I recollected that when the semi-solid used to be green yet it then ended up being blue after 30 hours. This implies my unknown cannot utilize citrate as a carbon source and the pH hopped up the whole time. My urea test additionally hinted at an antagonism as it didn’t change shades and stayed orange. I then realized that it didn’t contain the protein urease and pH continued as before subsequently. For my TSIA slant test, which I was able to recognize that the shade of the media was red at the top and yellow at the bottom which shows glucose maturation. That was the main thing that was distinctive on the grounds that there were no rises at the lowest part. My microorganisms likewise brought about a negative test for MR-VP. For the MR test I can infer that creation of blended acids were missing. For the gram positive segment of my biochemical test I saw from the MSA plate that it was sure for mannitol aging. It doubtlessly finishes the coagulase test in view of the recognizable bunches I saw. My bile esculin turned to a dark substance and the SFA continued as before significance there were no enterococci present. In conclusion from my gram staining I recognized that my gram positive microbes were formed as cocci groups.
So what is the importance of my results? In the wake of inquiring about and utilizing my procedure of disposal, I inferred that my unknown bacteria number 31 was nothing other than Escherichia coli for my gram negative. The outline for it matches precisely the same with the revelations I brought up for the unknown bacteria number 31. I made a couple of arrangement to my procedure of disposal to acquire my data. In the meantime the Escherichia coli was negative for the citrate, VP, urease, and gelatin and positive for the. The EMB and the MacConkey had a green color shade. From these results I can infer that my unknown in fact is E.coli.
References
ICT/MHRD, NME. “Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab.”Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab. Amrita, 2013. Web. 21 Nov. 2013
Edu, Highlands. “Use of Selective and Differential Media.”Use of Selective and Differential Media. Highlands Acedmics, 2013. Web. 23 Nov. 2013.
Weber, Dereck. “Unknown Bacterium Identification Project.” Unknown Bacterium Identification Project. Softchalk.com, 09 Jan. 2010. Web. 02 Nov. 2013.
“Welcome to Microbugz – SIM Medium.” Welcome to Microbugz – SIM Medium. Ed. Linda Beaver. ACC, Apr. 2000. Web. 03 Nov. 2013.

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