The Risks Of Exposure To Radiation Biology Essay

Ionising radiation is commonly utilized in our daily lives in small amounts e.g. X-rays. However, radiation exposure results in harmful effects on DNA structure, leading to base modifications (oxidation, alkylation), cross-link formation or bulky lesions. Repair mechanisms exist to correct these modifications, however, misrepairs may lead to increases in mutations such as base substitutions and single and double-stranded breaks, the latter being the most important.

At present, mouse models are used to measure the effects of exposure to radiation. From this, the risk of gaining harmful mutations is calculated (Bouffler et al, 2006) .This is performed by identifying markers in the mouse genome (Neel, 1990, Sankaranarayanan, 2001). Phenotypical changes can be seen with the different doses of radiation exposure, e.g. fur colour Is dose-dependent. Unfortunately, the frequency of these mutations is very low (about 1 in 100 000) and hence large amounts of radiation needs to be used. Despite this, these experiments were useful as they showed that the mutations occurred at or near the sites of exposure to ionising radiation.

Subsequently, it was discovered that the mouse and human genomes contain tandem repeat DNA loci (TRDLs) which have a higher mutation frequency than the mouse genome markers (Bois 1999), which can be used to induce mutations. The higher mutation frequency of these genes in mice are evidence that these mutations are “untargeted”, that is, the mutations are not limited to the sites of exposure to ionising radiation. Further experiments have shown that exposure to ionising radiation can induce mutations in cells many years after exposure (Little, 1994). Although the exact mechanism of untargeted and delayed mutations is not fully understood, it is thought that radiation exposure results in genome instability. The regions of higher mutation in the human genome are known as “minisatellites”, and in mice, “expanded single tandem repeats (ESTRs)”. The difference in nomenclature is due to the different structures of these regions in mice and humans (Bouffler et al, 2006).

Mammalian TDRLs consist of microsatellites which are 500 base pairs long and repeat sizes of 1-4 base pairs. ESTRs are 0.5-16 kilobases long with repeat sizes of 4-9 base pairs. Human minisatellites are 0.5-10 kilobases long and the repeat sizes are 9 – 60 base pairs (Bois 1999, Ellegren, 2004, Vergnaud, 2000). TDRLs can make up about 10% of human genes. The research significance of these regions of the genome is that any additions or subtractions occur in the repeat units as a whole, and hence are useful to assess the risks of exposure to radiation.

Minisatellites are highly variable sequences (Vergnaud,2000). In humans, they are located in the sub-telomeric locations of the gene (Bouffler, 2006). Not only is this less so in pigs, rats and mice (Jeffreys, 1999), but these genomes also have higher mutation rates. Two approaches can be used to assess mutation rates in human minisatellites. These are pedigrees, where mutations are identified in complete pedigree trees. and the small pool PCR (SP-PCR) method (Tamaki, 1999). The DNA is obtained from lymphocytes and locus-specific probes are used to identify the minisatellite regions. A mutation is a new segment of DNA which does not arise from the parents’ genes and this is easily ascertained because of the high mutation frequencies of these minisatellite loci. For the pedigree method, it is important to avoid errors from non-paternity and other human errors. In the PCR technique, DNA from sperm are diluted and amplified, so that mutations can be detected. Therefore, the pedigree method identifies mutations in the maternal germline and the PCR technique, in the paternal germline. The mutation rate per generation is calculated as follows:

In the case of human minisatellites, a greater number (approximately four fold more) of paternal mutations than maternal mutations have been detected, (Bouffler, 2006). This difference has also been fairly consistent with human microsatellites (Yauk, 2004). Why this occurs is not understood. Studies involving minisatellites have also revealed that mutation rates vary between somatic and germline DNA, with the high frequencies only being seen in the germline DNA (Buard, 2000). A possible explanation could be the “gene conversion-like” processes which occur in the germline DNA, which cause many of the mutations. (Boufler 2006 ). Many additional mutations also occur in germline DNA e.g. DNA double strand breaks, recombination from meiosis at genetic hotspots. (Buard, Shone, Jeffreys, 2000). Nearly half of the minisatellites in the human genome (Denoeud F, 2003) occur in the coding sequences, therefore resulting in alterations to protein structure. The HRAS gene codes for the HRAS protein, a GTPase, which controls cell division in the presence of growth factors. Hence minisatellites within this gene will alter gene expression and thus increase the risk of familial cancer. In addition, minisatellites in the introns can alter gene splicing, due to sequence overlap between the minisatellite repeat and the splice regions. The actual purpose of minisatellites is not known, however the multiple minisatellite loci, heritability and their existence in the genes of higher mammals propose that they do serve an important purpose.

WWII Hiroshima and Nagasaki atomic bomb

Mutations in minisatellite loci were studied in the genomes of families that were exposed to ionising radiation during the WWII Hiroshima and Nagasaki atomic bomb explosions (Kodaira, 2004). The sample size studied consisted of the children of 30 fathers and 32 mothers who were exposed to the ionising radiation, and 60 children of unexposed parents. The majority of the 62 children from exposed parents were born a decade after the explosions. The Dosimetry System 86 (DS86) was utilized to process the radiation exposed by the parents. The average parental exposure was 1.9Gy and this was seen mostly in the cases where only the mother was exposed (Bouffler, 2006). The initial experiments identified low mutation rate sequences in the genomes of the exposed group (Kodaira, 1995). Subsequently, single-locus probes identified very high mutation rate minisatellite sequences (Kodaira, 2004). However, dramatically-changed mutation rates were not found. Possible reasons could be the insufficient number of exposed individuals, the large number of exposed groups consisting of exposed maternal lines and unexposed paternal lines, possible correction of any mutations in the genome before conception, or finally, a genuinely low mutation rate caused by the exposure to ionising radiation.

Chernobyl Disaster

Mutation frequencies in minisatellite loci were studied in families who were exposed to ionising radiation during the Chernobyl disaster. These families were located in the countryside of Belarus and the Ukraine (Dubrova, Grant, 2002). The sample size consisted of 127 children from exposed parents in Mogilev in Belarus. The control individuals were 120 Caucasian children of unexposed parents in the UK. High mutation frequencies were found within minisatellite sequences, through the use of 2 multilocus and 8 single-locus probes. This showed that the mutation frequency was higher in the exposed group. The mutation frequency also directly correlated to increased exposure to cesium-137, although the individuals were also exposed to other forms of ionising radiation. Another discrepancy is that the control individuals were of a different ethnicity to the exposed individuals, therefore, it cannot be proven that the increase in mutation rate is purely due to radiation exposure (Bouffler, 2006). Hence, to validate these conclusions, mutation frequencies at minisatellite loci were studied in exposed and unexposed individuals who were born in same countryside districts of the Ukraine (Dubrova, Grant, 2002). To ensure the highest degree of validity possible, the exposed and unexposed groups were made sure to have the same ethnicity, lifestyles and maternal age. Single-locus probes was used to identify any germline mutations. However, a significant increase in mutation frequency was only found in the paternal germline. This could be due to the greater early exposure immediately after the Chernobyl incident. In terms of the analogous maternal mutation rates, this could be a result of non-irradiation of the mothers during the meiotic phase of pregnancy in order to result in mutation at the minisatellite loci. These studies support the hypothesis that increased exposure to ionising radiation results in raised germline mutation frequency (Boufller, 2006).

Nuclear Weapons Testing

Semipalatinsk was an area where nuclear weapons testing was executed by the Soviet Union from 1949-1989 . As in the other studies, the frequency of mutations at minisatellite loci were analysed in forty families resident near this area (Dubrova 2002). The main contact with the ionising radiation occurred via the surface explosions executed from 1949-1956. At present, the level of radioactivity in this region is minimal. The control sample comprised 28 families resident in the Taldy Kurgan region in Kazakhstan, where there was no exposure to ionising radiation. It was ensured that all the individuals being analysed were of similar ethnicity, maternal age, occupation and smoking. The eight single-locus probes were used once again for this study. The experiment concluded that exposure to ionising radiation nearly doubled the minisatellite mutation frequency in the irradiated families. Also, the consequence of the reduced exposure during the lack of surface explosions post-1950s resulted in minisatellite mutation frequency not linking with the year that the parents were born. Hence, there is the possibility of confirming that the initiation of minisatellite mutation frequency is dose-dependent, and that exposure to ionising radiation is directly responsible for the raised minisatellite mutation frequency in the exposed group (Bouffler, 2006).

Chemotherapy and Radiotherapy

The very methods used to treat cancer patients, could ironically and potentially have the ability to induce harmful mutations that could also affect neighbouring non-cancerous cells. Hence, genetic experimentation is vital in assessing the level of mutation initiation to the germline caused by chemotherapy and radiotherapy. Once again, minisatellite mutation frequency was analysed in male cancer patients. This was performed using the SP-PCR method (May, 2000). DNA from sperm were diluted and amplified to identify novel mutations within one male patient (Jeffreys, 1994), and therefore requires a much smaller sample size than the pedigree method. However, the disadvantage of the SP-PCR method is that there is a high variation frequency within each locus (Tamaki, 1999). This makes it unsuitable for assessing the differences in mutation frequency between the exposed cohort and controls. As such, this method is used specifically to assess mutation frequencies before and after chemotherapy or radiotherapy in only one man. Another disadvantage of SP-PCR, is that only minisatellite alleles that are less than 5kb can be amplified. Initial experiments concluded that cyclophosphamide, etopside and vincristine did not alter the mutation frequency of the MS205 minisatellite (Armour, 1999). However, cyclophosphamide was shown to initiate alterations to germ cell mutations specifically during post-meiosis. On the other hand, etoposide induces mutations specifically to germ cells during meiosis (Vilarino-Guell, 2003), which creates a narrow window for assessment. Hence, these drugs are meosis stage-specific. A further study involved sperm from ten individuals post-chemotherapy for Hodgkin’s disease (Zheng, 2000). No raised mutation frequency was detected in the patients treated with vinblastine or a combination of adriamycin and bleomycin, which correlates with similar findings with these drugs on mouse models. However, procarbazine, which was shown to affect cells in the pre-meiotic stage in mouse models, raised the genome mutation rate majorly in humans (Zheng, 2000).

In the case of radiotherapy, again, no changes in mutation frequency was seen in the B6.7 and CEB1 minisatellite loci (May, 2000). The dosage delivered to the patients were 15 courses of X ray exposure (0.4-0.8Gy). This is near the doubling dose exposure for mouse models (UN. And Sanakaranarayanan, 2000). However, the results obtained could be due to the fact that the exposure to radiation in this case is fractionated, as opposed to one large dose.

In conclusion, the results of these different studies have not all been consistent with each other. Possible improvements could be to increase the sample sizes. Perhaps a novel method of calculating the effect of ionising radiation could be explored. In addition, transgenerational mutations have been detected in mice, but not in humans. This reflects the possibility that the mechanism of ESTR mutation initiation in mouse models could be different from that for human minisatellite loci. However, the mutations in mice were only seen after high-dose exposure. Research is currently taking-place to understand the possibility of a stress-like response that needs to be activated before transgenerational mutations are seen in mice. Results from these studies could be useful in understanding this process in humans, and hence our understanding of disease processes related to exposure to ionising radiation.

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The Processes Leading To Skeletal Muscle Contraction Biology Essay

Biology » The Processes Leading To Skeletal Muscle Contraction Biology Essay

Essay title Give an overview of the processes that lead to skeletal muscle contraction (50%). Discuss how these processes are disturbed by muscular dystrophies and discuss one particular dystrophy in detail (50%).

Muscle contraction is when a tension is made from an action potential to cause a movement in of the muscle, this requires interactions between actin filaments and myosin. The Skeletal muscles contract in a sliding filament model: Firstly an action potential that comes from the CNS reaches an alpha motor neuron. This then transmits an action potential down its axon. The action potential then spreads by activating the sodium dependent channels near to the synaptic cleft along the axon. The action potential them reaches the motor neuron terminal and so causes a calcium ion influx through the calcium dependent channels.

The influx of the Ca2+ causes exocitosis of the vesicles containing acetylcholine so it fuses with the plasma membrane and releases acetylcholine into the extracellular space. The acetylcholine then diffuses across the synapse and binds to the nicotinic receptors on the motor end of the muscle cell. When the nicotinic receptor is activated, the sodium/potassium channels open and cause sodium ions to enter in a surge and potassium ions go out. The overall charge on the muscle fibre becomes positive due to the difference in potential (voltage) from sodium in the muscle fibre and potassium outside the muscle fibre. This causes an action potential. As the action potential spreads across the muscle fibre it becomes depolarized.

The depolarization of the muscle fibre activates the voltage dependent calcium channels in the T tubule membrane. The Sarcoplasmic reticulum then releases calcium due to the activated calcium release channels. The calcium then binds to the troponin C which is on the actin containing thin filaments of the myofibrils. This causes the troponin to modulate the tropomyosin. Normally the tropomyosin blocks the myosin binding sites on the thin filament but, the calcium binding to the troponin C causes unblocking of the binding sites.

Myosin can then bind to the newly uncovered binding sites on the thin filament. They are bound to the actin in the strong binding state. When the ATP binds the myosin it allows the release of actin becomes a weaker binding state. The myosin then hydrolyzes the ATP and uses the energy to move into the correct conformation. Calcium is actively pumped back into the sarcoplasmic reticulum and when calcium is no longer present on the thin filament, the tropomyosin changes conformation back to its previous state so it blocks the binding sites again. The myosin stops binding to the thin filament, and the contractions stop.

There are many types of dystrophies that can affect the processes involved in skeletal muscle contraction. One such dystrophy

is the becker muscular dystrophy is a less severe variant of Duchenne muscular dystrophy and is caused by the production of a partially functional form of dystrophin.

Another dystrophy is the congenital muscular dystrophy which includes several disorders with a range of symptoms. Muscle degeneration can be severe as problems may be restricted to skeletal muscle, or muscle degeneration could be paired with the effects on the brain and other organ systems. There are a number of the forms of the congenital muscular dystrophies that are caused by defects in proteins and are thought to have some link to the dystrophin glycoprotein complex. Some types of congenital muscular dystrophy show severe brain malformations.

Emery-Dreifuss Muscular Dystrophy is one that people normally show signs of in childhood and the early teenage years. Clinical signs can include weakness and wasting of the muscles, this can start in the distal limb muscles and progress to the limb-girdle muscles. Most of the patients usually also suffer from arrhythmias or cardiac conduction defects. If they’re left untreated, there is an increase in the risk of stroke and possible death.

Facioscapulohumeral muscular dystrophy is a dystrophy that at first affects the muscles in the face, shoulders, and upper arms as they become weaker. The Symptoms are shown in the teenage years and some people who are affected can become severely disabled. The pattern of inheritance is autosomal dominant, but the underlying genetic defect is inadequately understood.

The Limb-girdle muscular dystrophy shows a similar distribution of muscle weakness, affecting both the upper arms and legs. Many forms of this dystrophy have been identified through varying patterns of inheritance. In the autosomal recessive pattern of inheritance, an person would have two copies of the defective gene, one from each parent. The recessive ones are more common than the dominant forms and can usually have childhood or teenage onset. The dominant genes usually show adult onset. There are some of the recessive forms that have been linked to defects in proteins that make up the dystrophin-glycoprotein complex.

Myotonic muscular dystrophy is the most common adult form of muscular dystrophy. It is known for muscle wasting and weakness. Myotonic dystrophy varies in seriousness and affects many body systems in addition to skeletal muscles, which include the heart, gastrointestinal tract and the endocrine organs. Myotonic dystrophy follows an autosomal dominant pattern of inheritance. While the exact mechanism of action is not known, the molecular change could interfere with the production of vital muscle proteins.

One particular dystrophy known as the Duchenne muscular dystrophy, is a neuromuscular disorder. It’s an inherited disorder with an incidence of 1 in 3300 live male births.

Duchenne muscular dystrophy is a devastating inherited neuromuscular disorder with an incidence of 1:3,300 live male births.

In patients with Duchenne muscular dystrophy, muscle biopsy characteristically demonstrates necrotic or degenerating muscle fibres, often observed in clusters.

Such necrotic fibres are surrounded by macrophages and CD4+ lymphocytes.

In the early stages of the disease, one also finds small immature centrally nucleated fibres which represent muscle regeneration from myoblasts. This indicates that there is balance between necrotic and regenerative processes.

Later, the regenerative capacity of the muscles appears to be exhausted and muscle fibres are gradually replaced by connective and adipose tissue

The gene responsible for this defect codes for the protein dystrophin.

It is the absence of dystrophin that leads to Duchenne muscular dystrophy. However, the comprehensive understanding as to how the absence of this protein leads to muscular degeneration is still not fully understood.

Dystrophin is found in the vicinity of many other proteins and these all form a large complex.

The best studied roles for the dystrophin-associated complex involves structural stabilization of the sarcolemma.

Mutations of other dystrophin-associated protein complex components also cause muscular dystrophy (see later) by disassembling the complex and compromising the linkage between the extracellular matrix of the fibres to the cytoskeleton.

The various pathophysiologic hypotheses for Duchenne muscular dystrophy include:

(i) mechanical hypothesis;

(ii) impaired calcium hypothesis;

Much old data had indicated that there appeared to be an excessive fragility of the muscle fibres in this disease.

The discovery of the dystrophin-associated protein complex scaffolding supported the view that the absence of one of these proteins could compromise the muscle membrane integrity of the fibres.

This could particular be so after sustained contractions, as the ability to sustain contraction with forced lengthening appears to be dramatically reduced in Duchenne muscular dystrophy.

The absence of dystrophin results in a striking alteration in membrane structure related to delocalization of the dystrophin-associated proteins from the membrane. The dystrophin-associated complex together with additional proteins (e.g. vinculin, desmin, spectrin) normally form rib-like lattices on the cytoplasmic faces of the sarcolemma (these regions are called costameres): these anchor the cytoskeleton to the extracellular matrix.

Costameres act as mechanical couplers to distribute contractile forces generated in the sarcomere laterally through the sarcolemma to the basal lamina and thereby maintain uniform sarcomere length along the fibre.

Absence of dystrophin, leads to the loss of the dystrophin-associated protein complex and disruption of the costameric lattice, and it is this that is thought to underlie membrane fragility.

Evidence for membrane fragility in patients is shown by cytoplasmic accumulation of proteins that are not normally present in muscle fibres, such as albumin and immunoglobulins. This indicates that the permeability of the muscle membrane has increased.

Other research would appear to indicate that exercise could provoke greater damage in dystrophin-deficient muscles than in controls.

These ideas clearly have some bearing on the possible management of the disease because whilst physical therapy appears to improve or stabilize muscle functions, too much exercise could lead to further muscle damage.

There is documentation of calcium accumulation and of hypercontracted fibres in muscle biopsies of Duchenne muscular dystrophy patients.

In dystrophin-deficient membrane there is an increased flux of calcium which appears to occur through a voltage-independent mechanosensitive calcium channel.

However, measurements of the basal [Ca2+]i are normal and this could suggests that there may be only abnormal calcium concentrations at localized submembranous compartments.

If mechanical stress induces microlesions in the muscle fibres then this could lead to high influx of extracellular calcium and this could overide the cells capacity to maintain a physiological cytosolic concentration of calcium.

Clearly, higher [Ca2+]i can lead to the activation of proteases (e.g. calpains) and this can lead to further damage and even lead to apoptosis and cell death.

Muscles of patients with Duchenne muscular dystrophy exhibit inflammatory changes.

It would seem that selective chemokine up-regulation may be a key determinant in the inflammatory response. However, no studies have provided any direct insights into the mechanisms implicated in cell death.

Nevertheless, corticosteroids, which have potent anti-inflammatory effects, are the most common used drugs in Duchenne muscular dystrophy.

e.g. prednisolone treated patients experience significant delay in the disease progression, they are able to move about freely for longer and there is prevention of curvature of the spine.

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The First Case In Phytopathogenic Bacteria Ralstonia Solancearum Biology Essay

Biology » The First Case In Phytopathogenic Bacteria Ralstonia Solancearum Biology Essay

Filamentous phages belong to the genus Inovirus, members of which infect almost exclusively Gram-negative bacteria. The virions of these phages are slender filaments usually about 6 nm in diameter and 800–2000 nm long, comprising a helical capsid of several thousand copies of the major coat protein (pVIII) surrounding a single-stranded circular DNA genome about of 5–8 kb, encoding around 10 genes (Marvin, 1998). These phages usually use type IV pili as receptors to infect bacterial cells (Marvin, 1998).

Infection of bacteriophage to the host cells causes many change in morphology, physiology and genetics. Webbs et al. (2004) found that Pseudomonas aeruginosa, a human lung pathogen, to be form small colony variant (SCV) caused by Pf4 phage infection. Infection of Pf5 phage was reported to be increase the virulence of P. aeruginosa (). Moreover, virulence factor of pathogenic bacterias have been shown to be encoded by prophage (Brussow et al., 2004). CTX phage was reported to be increase the virulence of host bacteria, Vibrio cholera, by promoting CT toxin that affect to the development cholera disease (Davis and Waldor, 2003). Indeed, the define exotoxin from pathogenic bacteria, Shiga toxin by Escherichia coli (Johannes and Römer, 2010); botulism toxin by; diptheria toxin by , have been show to be expressed from phage-encoded gene.

In Phytopathogenic bacteria, phages have been reporting to infects and affects the virulence of host bacteria. Filamentous phage Xf and Xf2 were reported to infect Xanthomonas campestris pv oryzae NP5850 and caused increasing virulence due to overproduction of extracellular polysaccharides (Kamiunten and Wakitomo, 1982). Moreover, Tseng et al. (1990) reported that filamentous phage Lf which infect X. campestris pv campestri increased virulence by promoting EPS production.

Recently, Yamada et al. (2007) have been succeed to isolate various bacteriophage that infect phytopathogen, Ralstonia solanacearum. Moreover, Kawasaki et al. (2008) characterized the genome of two kind of Ff-like phage (inoviridae),RSS1 and RSM1, were have host genome integration activity. Askora et al. (2009) also found a Ff-like phage, RSM3 and RSM4, that are enhance bacterial cell aggregation and reduce the bacterial host virulence. However, detailed information of cell aggregation and reducing virulence is unclear.

R. solanacearum, a widely distributed soil-borne pathogen belonging to the ß subdivision of Proteobacteria, causes a lethal wilting disease of more than 200 plants species including economically important crops (Hayward, 2000). During infection, bacterial express virulence and pathogenicity factors resulting in typical wilting symptoms. Virulence and pathogenicity of R. solanacearum was known controlled by T2SS and T3SS (Poueymiro and Genin, 2009). As virulence factors, R. solanacearum produce consortium of plant cell wall-degrading enzymes (CWDEs), which are secreted via the type II secretion system (T2SS), such as ß-1,4-endoglucanase (Egl), endopolygalacturonase (PehA), exopolygalacuturonases (PehB and PehC), ß -1,4-cellobiohydrolase (CbhA) and a pectin methyl esterase (Pme) (Denny et al., 1990; González and Allen 2003; Huang and Allen 2000; Tans-Kersten et al., 1998). Moreover, secretion of protein (T3Es) via Type III Secretion System (T3SS) was known as important factor of bacteria pathogenicity. Loss of these secretions causes the loss ability to infect host-plant (Genin et al., 2005). During pathogen infection into plant host, plant will enhance and alter some physiological chance to challenge pathogen infection such as expression of pathogenesis-related gene that encode pathogenesis related protein (PR-protein). Hase et al. (2006) showed that Tomato pathogen infection, Pythium oligandrum, was shown high expression of PR-2b, PR-3b and PR-5b gene of plant which related to the expression of PR-protein. Moreover, Aime et al. (2008) compared infection tomato plant with virulence and avirulent strain of Fusaium oxysporum which showed high accumulation of 5 PR-protein in root and leaves of tomato inoculated with virulent strain compared with avirulent strain.

In this study, we showed an evidance of avirulent formation of R. solanacearum after infection by filamentous phage RSM3.This study was conducted both in bacterial cell and in the interaction of host-pathogen.

To observe the culture color, a single colony of Twenty four house cultures in CPG Agar of Ralstonia solanacearum was picked-up and transfer to CPG broth and was incubated in 28OC with shaking 250-300 rpm until 3 days. Changing of culture color was recorded every day. Culture characteristic on CPG agar was determined by streak plate of OD600 1.0 of 24 hours bacterial culture. To obtain a single colony size, a twenty four hours culture was recovered by centrifugation and was washed two times with ddH2O and was adjusted OD600 of 0.1 and was streaked onto CPG and MM agar. Observation was done after 48 hour of incubation in 28OC.

Twenty four hours cultures in CPG of R. solanacearum was adjusted to OD600 of 0.01 as initial OD with CPG Broth. The culture was incubated in 28OC with shaking 250-300 rpm and was measured the OD600 every 1 hour grown for 1 day at 28OC. Curve of bacterial growth was calculated using absorbance on OD600 and time of incubation. To observe the change of characteristic of liquid culture, bacteria was cultured in liquid CPG for 3 days and observed for color change of liquid CPG medium.

Aggregation assay was performed from Bacterial cell from Twenty four hours cultures in CPG bya harvested cells by centrifugation at 15,000 x rpm for 2 min at 4°C, washed three times with ddH2O and adjusted the OD660 of 1.0 with 1 x PBS pH 7.0. Suspension was incubated without agitation in 28OC. absorbance at 660 nm of upper suspension (100 µL) was measured after 0, 0.5, 1, 2, 4, and 6 h of incubation. Percentage of aggregation curves were then constructed following the formula (Eboigbodin et al, 2005).

% Aggregation= OD0- ODtOD0 x 100

where OD0 is the optical density at 660 nm of bacteria immediately after resuspended with 1 x PBS and ODt is the optical density after particular time.

Cultures of R. solanacearum were grown for 1 day at 28OC in CPG broth. Bacterial cells were obtain by centrifugation at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with ddH2O and was adjusted the OD600 to 1.0. Fifty microliter of suspension was dropped into test media for each assay (MM for twitiching motility, SWM for swimming motility and SRM for swarming motility). Motility was observed by measuring the diameter of dropped-culture up to 6 days.

To test the movement in tomato stem, GFP-expressing strain was used in the experiment. Both strains, MAFF106603 contain pRSS12 and its infected cell by FRSM3 was grown in CPG media for 24 hours. Bacterial cells were obtain by centrifugation at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with ddH2O and was adjusted the OD600 to 1.0. One microliter of suspension was injected into tomato stem between cotyledon and the first leaf and incubated at 28OC. After 1 week incubation, plant stem was cut 20 µm in thickness with microtome and then observed using a Leica MZ16 microscope with GFP3 filter.

Total endoglucanase activity was quantified by measuring the reduction sugar (Nelson, 1944) released during incubation at 50OC for 4 hours with 4 mg/ml of carboxyl metylcellulose in 120 mM phosphate buffer (pH 7.0) In soluble material was removed by low speed centrifugation before absorbency readings were taken on 540 nm. One unit of enzyme activity was defined as releasing 1 nmole/minute of glucose.

Cultures of R. solanacearum were grown for 3 days at 28OC in EG broth (Denny et al., 1990). To recover EPS, culture supernatants were adjusted to 0.1 M NaCl and 4 volume of acetone were added. After incubating overnight at 4OC, precipitated material was recovered by centrifugation (15.000 rpm, 10 minutes, 4OC), dissolved in 200 µl of ddH2O, heated at 65OC for 10 minutes and centrifuged for 5 minutes to remove in soluble material. The concentration of hexosamine in culture supernatants was estimated with a modified Elson and Morgan reaction. Appropriately diluted samples (0.45 ml) were mixed with 0.15 ml of concentrated HCl, hydrolyzed at boiled water (110OC) for 30 minutes in sealed tube and the colorimetric assay performed. The results were read at OD530, the background due to residual media components substraced, and the hexosamine concentration determined from N-acetyl D-glucosamine standard curve. N-acetyl D-glucosamine standards were subjected to the entire analysis procedure beginning with hydrolysis step.

Cells of R. solanacearum strains were streaked heavily on minimal medium plates (Clough et al., 1994) and incubated for about 22-24 hours. The colonies were washed off in a small volume of 10 mM Tris-HCL buffer pH 8, and the cell suspension was forced five times through a 25-gauge hypodermic needle. Bacterial cells were removed by centrifugation with a R12A2 rotor in a Hitachi himac CR21E centrifuge at 8,000× rpm for 20 min at 4°C. The bacterial surface appendages were collected by ultracentrifugation with a R12A2 rotor in a Hitachi himac CR21E centrifuge at 136,000×g for 60min. Precipitated exostructure were subjected to Tris-Tricine PAGE according to Schagger and von Jagow (1987).

All strains, R. solanacearum MAFF106603 and its infected cell by FRSM3, were grown in Minimal Medium (MM) at 28OC for 24 hours. The new sub culture were made by adjusting the OD600 to 0.02-0.03 in new MM and continued growing to reach OD600 of 0.1.

Total RNA was prepared from 3 ml of the exponential phase culture with RNeasy Mini columns (Qiagen), and was eluted in 60 µl of DEPC-treated water. The total RNA was treated with Recombinant DNase I (Takara) to remove any genomic DNA contamination by incubation with 10 U of RNase-free DNase I for 30 min at 37°C. The DNaseI was inactivated by phenol/chloroform extraction method. To confirm the presence of DNA contaminant, a 30 PCR cycles was performed using a gene specific primer (Table 1.) with DNA genome of MAFF106603 as a positive control.

The cDNA of target genes was synthezed by using M-MLV Reverse Transcriptase RNase H- (ReverTra Ace, Toyobo, Osaka). Specific primers were designed using the Primer3 (v.0.4.0) software (http://frodo.wi.mit.edu/primer3/#PRIMER_MAX_TEMPLATE_MISPRIMING) and were checked for gene specificity using DDBJ/Blast (v.2.2.18). Breafly, 20 µl of Reaction Mixture (5 µl of 0.1-1 µg total RNA, 1 µl of 5 pmoles of gene spesific primer, 4 µl of 5X ReverTra Ace Buffer, 2 µl of 10 mM dNTPs, 1 µl of 100 units ReverTra Ace and 7 µl of DEPC-treated water) was incubated for 60 minutes at 42OC and was inactivated by incubation at 99OC for 5 minutes. Negative control reactions to eliminate the possibility that residual DNA was amplified were performed in the same way, except that the RT was omitted from the reaction mixtures.

Real-time PCR was performed using a Line Gene Fluorescence quantitative detection system (BioFlux, Tokyo) with cDNAs prepared from R. solanacearum MAFF106603 strains. A PCR mixture containing SYBR-green (SYBR premix ExTaq, Takara Shuzo, Kyoto) was used. PCR reactions in a final volume of 10 µl reaction mixture containing 5 µl PCR mixture, 1 µl diluted cDNA and 0.5 µM each primer (Table 1.) were carried out under the following conditions: 3 min at 95°C and 45 cycles at 95°C for 10 s, 62°C for 10 s, and 72°C for 15 s. At the end of the program, the specificity of the primer set was confirmed by melting curve analysis (65-95°C with a heating rate of 0.5°C/min). The copy numbers of spesific target gene and 16s rRNA were estimated by comparing the results of real-time PCR with several dilutions (102, 103, 104, 105 copies/µl) of each gene. The mRNA level of 16s rRNA was used to normalize the expression ratio of each gene.

Table 1. Primers used in real-time qRT-PCR study

Target

Forward primer 5'–3'

Reverse primer 5'–3'

Size (bp)

egl

CAG CGC GAC CTA CTA CAA GA

299

hrpB

TTC TCG ATG ATG TAG CGA TAG G

238

phcB

CTA CCA GAT CGT CGT CAA TGA A

172

pehC

AGT CAA ACG ATT GCC TGA ACT A

227

16srRNA

CTA GAG TGT GTC AGA GGG AGG TAG A

349

Note. Bolded characters of oligo primer sequence refers to the Primer used in cDNA synthesis.

Table 2. The tested genes and their putative functions

Gene

Function

egl

Endoglucanase, enzyme which involved in degradation of plant cell wall glucans. (Roberts et al. 1988)

hrpB

Regulator the expression of T3SS biosynthesis genes, and probably >60 effector substrates, and T3SS-dependent export pathway (Occhialini et al., 2005).

phcB

Requiring for production of an extracellular factor (EF), identified as the fatty acid derivative 3-hydroxypalmitic acid methyl ester (3-OH PAME), which function as autoinducer (quorum sensing signal) in R. solanacearum (Flavier et al., 1997a, Flavier et al., 1997b)

pehC

Exopolygalacturonase (Gonzalez and Allen., 2003).

16srRNA

Encode 16S ribosomal RNA.

Virulence assay and HR test

Tomato plants were cultivated in 5-cm pots filled with a soil–peat mixture. Plants (4 leaves) were selected for inoculation. Twenty four hours bacterial culture was centrifuged at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with d ddH2O and was adjusted the OD600 to 1.0. One microliter of suspension was injected into 6 weeks old tomato stem between cotyledon and the first leaf and incubated at 28OC. As a control, distilled water was injected in the same manner. Disease index was observed following Poueymiro et al. (2009) up to 1 week after inoculation with scale ranging from 0 to 4, according to the percentage of wilted plants (0= no wilt; 1 = 1 to 25%; 2= 26-50%; 3=51-75%; 4=>75%).

HR-eliciting ability was tested by infiltrating a serial dilution of R. solanacearum strain solution into the intercellular space of tobacco leaves (Nicotiana tabacum) (Kim et al., 1997). Plants were incubated in 25OC and hypersensitive response was observed 24-48 hours after infiltration.

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The Development Of Stem Cell Technology Biology Essay

 

The research of embryonic stem cell is classified under the study of biotechnology, which is one of the most recognized areas of research by the public, as well as one that have the potentials of benefited from the completion of the research7. The embryonic stem cells are the absolute base form of all cells that is within a human, they are frequently known for two of their main characteristics. One is the ability to self-renew though a long period of time while remain non-specialised, and the other main characteristic is the stem cell’s ability to differentiate and give rise to any specialised cell3. Because of these characteristics, it brings hope to cure diseases that were not possible before, by replacing the damaged cell with a new one that is being derive from a stem cell. Neurodegenerative diseases are a prime example where scientists believe that stem cells are the key to treating the disease. However, the research of embryonic stem cells has always being surrounded with controversy, due to the pro-life movement around the world. This report will be mainly focused on the use of stem cell on treating neurodegenerative diseases, and a general overview of the progress in the research of stem cells. (202 Words)

Review 667 words

The majority of the research done on embryonic stem cells is directed mostly toward medical treatment such as bone narrow transplant, regrowing a new nerve cell to heal Neurodegenerative diseases and more. The researches of stem cell started in the mid eighteen hundreds, when scientists discovered that some cells have the ability to generate into others. But the application of stem cell only start to boom from 1960’s, when the human understanding on natural biological systems had increased to the point that it allowed scientists and doctors to do bone marrow transfers between two humans which resulted in curing a patient with severe combined immunodeficiency in 1968.

This is the reason that stem cell research attracted attention from the public, due to the possibilities of curing several different types of diseases or healing damages that were beyond humans’ natural healing ability. Stem cells are the fundamental cells of all the different types of cells that are in a human body. It has the ability to renew itself, under the correct environment and performs obligatory asymmetric replication (see diagram 1 at appendix 1). This, combined with their ability to transform into any specific cell type that can be found in a human body, makes it possible to create new cells to replace those that were damaged by disease or treatment processes. However, the current understanding of the behaviour of stem cells is not enough for scientists and doctors to use them to completely cure a certain disease. Nevertheless, we currently have enough understanding through research, to show that stem cells do have the ability to lessen the symptoms such ailments as Alzheimer’s disease.

The research into stem cells has potential of curing most of the long-term diseases known to doctors; however the cost of this research is the main reason of controversy. The reason of the controversy around stem cells is focused mainly because embryonic stem cells can only be gathered when an egg is being fertilized.

The process of gathering a sample of stem cells started by triggering a human egg that has been left in cryo for more than the legal limit, to grow into an embryo like the normal growth of a child. Afterward, the egg started to grow for five to seven days where the blastocyst started to form along with the inner cell mass. The whole blastocyst is then moved to a specially prepared culturing plate that is covered in a feeder layer, that is designed to support the growth of the stem cell, and chemicals are added to break the blastocyst to allow the inner cell mass to be exposed. From this point on it is merely waiting for the inner cell mass to grow and divide into stem cells that the researcher can use to test in a controlled environment by using different growth factors that will hopefully allow the scientist to map out the reaction of different types of proteins and the chemical environment that would decide the growth path of these stem cells (see diagram at appendix 2).

By this point, most biotechnologists require assistance from people in another field, such as chemical engineer, protein specialist and doctors to help culturing or to understand how this stem cell works. These specialist from other field of science, help scientists to understand the growth factor that is involved in the differentiation of the cells. A prime example would be the use of growth factors.

Unfortunately this is also where the controversy starts; a lot of people around the world that support pro-life organisations believe that the research of stem cells is immoral; since every embryo that is being used to extract the stem cells have the possibility of becoming a full child, thus the process of extracting the stem cells would destroy the possibility of life. They believe that the same research can be conducted through adult stem cells, or known as somatic stem cells, that can be isolated from bone narrow, or any other cells that have the ability to self repair, without the need of creating a full embryo. While it may be possible to do so, the concentrations of somatic stem cells are extremely hard to isolate and researchers are currently unable to grow them in cultures for further study. However, most scientists believe that while each embryo has the possibility of developing into a child, the long period of cryo that is used to store the egg, is damaging it, to a point that the death rate of the infant will be higher, along with the fact that these cells were originally scheduled to be destroyed, thus it does not make much difference. Therefore, until either the somatic stem cells can be cultured and proven to be as potent as embryonic stem cells, or the latest induced pluripotent stem cells that are created by “reprogramming” the adult stem cell back into the pluripotent stem cell stage of development. Scientists are stuck on using embryonic stem cells for the majority of the study. (792)

Application Case study 800 word

The advancement in the field of biotechnology’s research into stem cells has already started to show results. In the past, diseases such as Alzheimer’s, which are diseases that cause the degradation of neurons on nerve cells, which were not treatable by doctors before. The most that doctors were able to do was to relieve the pain caused by the disease, they were not able to locate the source of the disease, or how they affected patients.

However, since the development of stem cell technology, scientists have regain hope of finding cure or treatment process that can permanent treat these diseases. Using the same system that was originally designed to house the stem cells, they were able to isolate the infected neuron samples that were affected by the diseases, and therefore, were able to devise a treatment or cure for the diseases as they now have a sample with which they can work.

Alzheimer’s disease is one of the most common neuron degradation diseases seen in the world. In America alone, there are at least 5.3 million people that have this disease. Alzheimer’s disease attacks the nerves and neurons that are in the brain over a long period of time, which slowly causes memory loss, problem solving skills, thinking, to the point that the brain can no longer function properly, leading to death. Alzheimer’s disease currently has no cure, as the rate that the human body regenerates the brain cells are much slower than the rate of infection. Therefore, theoretically it will be possible to cure Alzheimer’s if all infected nerve cells could be removed, and replaced with new nerve cells that were derived from the adult stem cell of the patient. This same application can also be used on other diseases such as Parkinson’s disease or sport injury that normally requires years of recovery.

Based on a published journal in 2009, scientists were able to use bone marrow derived the mesenchymal stem cell to relieve symptoms of Alzheimer’s on a mice model that have been purposely induced. According to the research journal, when a patient (or mice in their case) that is presented with the disease, the concentration of amyloid-b (Ab) peptides in the form of amyloid plaques in the brain parenchyma increases, which causes neural loss to the patient. Therefore, in a direct treatment the research team injected bone-marrow-derived-mesenchymal stem cells into the brain of the induced mice, the result showed that there is a diminution of amyloid-b by the reactivation of microglia that were re-stimulated by the injection of the stem cells. Thus the research team arrived at the conclusion that BMMSCs can promote reduction of the amyloid-b through the microglial activation in the induced Alzheimer’s brain, suggesting a potential therapeutic agent against Alzheimer’s disease. (453) however, in the journal the research team fail to considered the other cause of Alzheimer the neruofibrillary tangles

Conclusion 267 words (135)

In conclusion, it is clear that the development of stem cells is one field of biotechnology that is emerging quickly as the understanding of natural biological systems increase, along with the fact that the diversity of biotechnology is potentially limitless in the field of medical research. For example, during the process of creating a suitable environment for the embryonic stem cells to grow, researchers have accidentally created a solution that can keep donated organs for longer periods of time, allowing a higher chance of success to the transplant surgery after a long period of travel. In the future, once more is understood about stem cells, the whole technology may be advanced enough to reconstruct an entire limb. There remains much controversy around this field, due to the researchers’ ethical practices, notably, obtaining new stem cells.

Referencing

Kil Lee, Jong, Hee Kyung Jin, and Jae-sung Bae. "Bone marrow-derived mesenchymal stem cells reduce brain...." Neuroscience Letter 450.2 (2009): 136-41. Web. 31 May 2010. .

S. Shihabuddin, Lamya, and Isabelle Aubert. "Stem cell transplantation for neurometabolic and neurodegenerative diseases." Neuropharmacology 58. (2010): 845-54. Web. 12 May 2010. .

Stem Cell Basics. In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Wednesday, May 12, 2010] Available at

Unknown, Unknown. "What is the history of stem cell research?" History of Stem Cell Research. AllAboutPopularIssues.org, 2010. Web. 12 May 2010. .

Unknown, Unknown. "What is Alzheimer?" Alzheimer's Association. Alzheimer's Association, 06 April 2010. Web. 28 May 2010. .

Unknown, Unknown. "Stem cell." Stem Cell. AlphaMed Press, 28 May 2010. Web. 12 May 2010. .

Keener, Kevin, Thomas Hoban, and Rekha Balasubramanian. "BIOTECHNOLOGY and its APPLICATIONS." North Carolina State University. North Carolina State University, 06/03/2010. Web. 12 May 2010. .

Lindvall O (2003). "Stem cells for cell therapy in Parkinson's disease". Pharmacol Res 47 (4): 279–87. Doi: 10.1016/S1043-6618(03)00037-9. PMID 12644384. 

Goldman S, Windrem M (2006). "Cell replacement therapy in neurological disease". Philos Trans R Soc Lond B Biol Sci 361 (1473): 1463–75. Doi: 10.1098/rstb.2006.1886. PMID 16939969.

Unknown, Unknown. "Stem Cells and self-Renewal." isscr. ISSCR, 2010. Web. 28 May 2010. www.isscr.org/public/SC_self-renewal.pdf

Unknown, Unknown. "Neurofibrillary Tangles." Department of Pathology. University of Oklahoma, 2010. Web. 30 May 2010. .

Word distribution

Point/total point*word limit

Introduction

10/75*2000=266

Review

25/75*2000=667

Case Study

30/75*2000=800

Conclusion

10/75*2000=267

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The Problem And Possible Treatments Of Tuberculosis Biology Essay

Biology » The Problem And Possible Treatments Of Tuberculosis Biology Essay

Tuberculosis or simply called TB is an infectious disease caused by the bacteria called Mycobacterium tuberculosis which usually affects the lungs but it can also involve almost any part of the body. This airborne disease is a killer for people mostly those with HIV. In 1993, World Health Organisation (WHO) declared TB a global emergency as it has murdered approximately 2-3 million people per annum. One third of World’s population infected with 5-6% ends fatally. (1) Tuberculosis is an airborne disease which means it is spread through air. When an untreated pulmonary TB patient sneezes or coughs, the air containing pathogen released by patient is breathed in by person nearby causing him or her get infected. (3, 11) About 90% of the cases show that the host’s natural defences prevent any further progression of the infection where the Host-Parasite equilibrium is reached in which many of the mycobacteria are destroyed but some lie dormant in tubercle foci. Upon activation of the dormant bacteria, the patients will suffer chronic cough with sputum-containing-blood, chest pains, fever, night sweats, weakness and weight loss. (2, 11) Since tuberculosis is fatal, what is possible treatment to cure that disease?

Self-administered Therapy (SAT)

Before treatment, the patients must be stressed of the importance of compliance in order to prevent development of resistance of the bacteria towards the drugs used. The duration of drug therapy will take about 6 months or longer. The Full Blood Count (FBC), liver, and renal function have to be checked. The drug therapy usually done by the patients themselves based on prescription given by physicians. (2)

Drug therapy is an effective way in providing chances of fully recovery. The therapy is divided into two phases which are initial phase and continuation phase.

Initial phase:

This phase takes about 8 weeks. 4 drugs are given depending on susceptibilities. The drugs are:

Rifampicin 600-900mg (child 15mg/kg) Per Oral (PO) three times/week

Isoniazid 15mg/kg PO three times/week + pyridoxine 10 mg/24h

Pyrazimide 2.5g PO three times/week

Ethambutol 30mg/kg PO three times/week for 2 months

Continuation phase:

For this phase, it takes about 16 weeks on two types of drugs, rifampicin and isoniazid at same doses. The most common medicine used is Rifinah 300® which contains a combination of 300mg rifampicin and 150mg isoniazid. The patients are advised about the compliance from time to time. The pyridoxine is given throughout the treatment.

Isoniazid is one of the most common drugs used to treat TB. It is effective, inexpensive and easy to take. When it is used with other types of drugs, it can prevent and even cure TB. Thus, isoniazid preventive treatment is recommended to person that:

Have a close contact with person who have infectious TB

Show a positive tuberculin skin test reaction (2, 7)

The drug therapy may need a longer period if the TB patients also suffer another disease like meningitis and osteomyelitis. The HIV positive patients may need to be treated with extra-drugs and longer time. (2)

Why SAT?

The drug therapy works very efficiently when TB patient takes medicine according to doctor’s advice. This is because the mycobacterium tuberculosis will be destroyed from body. The graph below shows how the drug therapy reduces cases of tuberculosis in US.

We can see that the rate of TB cases declined from 1981 to 1984. It has reduced at about 6-7 % annually. The drugs used during the therapy had efficiently recovering many TB patients. This reduces further spreading of that disease to other person as the number of active TB patients keeps reducing. There is fluctuation from 1985 to 1991. This happens due to some reasons like poverty and substance abuse. These reasons raise the rate of infection of TB. However, the drug therapy still able to control the number of TB cases. (9)

Social

Economic

Some people might say that the cost for the initial therapy is relatively affordable that is about $1,206 or RM 3,789.98 but this only include the direct costs of the initial therapy neglecting the further spending required if there is failure in the treatment. (15) This will require more money for treatment. The money that should be used for other necessities have to be spent for the treatment. This becomes a problem in Malaysia as most Malaysians only have a moderate income. Thus, the patients will have a heavy burden to pay for the bills. Besides, as the drugs used for the treatment have side effects, more money will be needed to cure the effects caused by the drugs. This indicates that a lot of money required for the treatment.

During drug therapy treatment, combination of different drugs is used to treat TB. Different drugs will react differently. This improves to rate of survival of the patients. The duration of this treatment which is usually takes about 6-9 months provides sufficient time for antibiotics to destroy the TB bacteria. The drugs also may be combined together as a single tablet. For instance, Rifater contains isoniazid, rifampin and pyrazinamide. The patients can take the medicine effectively and easily. (17)

However, TB drugs can cause very serious side effects. They can cause jaundice (a yellow colour to skin), blurred vision, tenderness in abdomen, loss of appetite and vomiting. They also can be highly toxic to liver. (17) Besides, the treatment of tuberculosis by drug therapy may lead the emerging of drug resistance bacteria. This happens as the patients are allowed to have a self-administrated therapy. They take the medication completely unsupervised. (8) Drug resistance happen when the patients fail to complete drug therapy that lasting about six months or longer. The TB bacteria that manage survive will reproduce and multiply. They become more drug resistance. (16) Consequently, the treatment requires a longer period and a higher cost in order to cure the airborne disease. The number of people get infected will keep increasing (18) Therefore, alternative way shall be taken to prevent this happening.

Directly Observed Therapy (DOT)

It is a treatment where the doses of drugs used are increased for rifampicin, ethambutol and isoniazid from 1-4 days but not with thiacetazone, ethionamide or streptomysin. The process is same as the SAT but it is fully conducted and observed by hospital. (4, 7) This provides better result rather than the patients take the drug without being monitored. In Hospital Albert Schweitzer, 138 TB patients were treated with DOT and 143 TB patients were treated without observation. This gives a higher successive rate for DOT which is 87% while self-administered drug therapy only has 57% successive rate. (5)

Besides, the treatment failure can be reduced and less drug resistance will be acquired. (6) This is because the patients are give doses on schedule. However, the program the programs of directly observed therapy are not easy to conduct. They require a flexible administration.

Immunisation

Prevention is better than cure. With the development of science, the BCG (Bacillus Calmetta-Guerin) vaccine has been invented. BCG is made up of living attenuated strain of Mycobacterium Bovis (M. Bovis) that produces a localised and self-limiting infection. This will stimulate cell-mediated immunity. (12) The young babies and person that have high risk of infection are given BCG injection. BCG will protect infants against tuberculosis. (10) The vaccine is given intradermally by injection to upper left arm. Putting hand on hip so the arm is at 45° will make it easier for the nurse to administer it. After three weeks, a small red spot appears. The site must be kept clean and dry. This eventually heals to form a round flat scar after 3-4 months. (12, 13) The usage of this vaccine is much more money-savings than treatment.

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The Introduction And Spread Of Japanese Beetles Biology Essay

Japanese Beetles (Popillia japonica) are a destructive pest of turf, landscape, agriculture, and ornamental plants. They were introduced into the United States sometime in the early 1900s on a shipment of iris bulbs. They were first identified in a nursery near Riverton, New Jersey in 1916 during a routine inspection. They are also found in their native Japan, China, Russia, Portugal, and Canada. They have become an important pest as both larvae and adults destroy roots and defoliate plants. The Japanese beetle easily established in the U.S. after its introduction. As with a lot of introduced pest they were lacking in natural enemies and found an abundant food source and favorable climate. Efforts have been made to control these invasive pests. In 1918, the USDA and New Jersey authorities attempted to eradicate the infestation. But after only two years the populations were so well established the efforts were deemed unproductive and were stopped. The best strategy for eradication is early detection, isolation and the total elimination of populations. The annual cost of control in the U.S. exceeds $460 Million (Shetlar 2008).

What are the implications of the introduction of Japanese beetles? And how can their spread across the United States be stopped in their tracks? This paper will be a thorough look into how Japanese beetles have spread across the U.S. and how to cope with this pesky pest.

Japanese beetles are polyphagous generalists and have a wide range of over 300 hosts. They are important pests of fruit, vegetables and field crops. Japanese beetles have one generation per year. A percent of the grubs may take two years to mature based on the soil conditions if they are cold and wet. The larvae are white colored grubs and are typically C Shaped around 1 inch in length. The third instar larvae overwinter in the soil below the frost line. The grubs develop in the soil feeding on the roots of plants. They can destroy lawns and turf grass. They are considered the most destructive pest of turf-grass in the United States. The adults are gregarious and collect together on a single plant. Single plants and trees may be completely defoliated. The adults are ½ inch in length and are metallic green with bronze-colored elytra. The males are typically smaller in size than the females. The adults skeletonize the leaves of their hosts by eating the material between the veins. The veins are often left untouched after feeding and the leaves will crumple and die. They feed on the upper and outermost parts of the plants and work their way down the plant (Cook and Gray 2010).

Japanese beetles are found throughout the Midwest and eastern United States and are spreading west ward. The furthest west they are expected to reach on their own is central Nebraska because of the unfavorable climatic conditions. There are areas in Western states that are suitable for their survival if they hitchhike to the area. The movement of these pests is a major concern to horticulture and farmers. They have been introduced into California three times and successfully eradicated. Material such as nursery commodities, sod, soil, compost and manure from infected states are now prohibited without a PPQ inspection. The USDA closely monitors the movement of cargo planes into the unaffected areas. The beetles are known to fly onto the cargo plane when the doors are open during times of adult beetle flight (Hamilton et al. 2007).

Airports are potential vectors for Japanese beetle infestation. A State Plant Health Director which is an APHIS employee arranges a monitoring survey to determine the risk status of an airport. Inspectors survey the airport to determine the potential risk. Monitoring surveys will determine the threat of entry into the aircraft and the population level in surrounding areas. The Indianapolis Airport is one of many airports where Japanese beetles pose a threat. Traps around the airport over a 4 year period showed that the relative distribution of adult beetles along the trap line were stable and at the same level in the same areas. Conclusions were drawn that the environment around the airport has considerable influence on the distribution of beetles and potential threat of a hijacker flying on board. The soil type, land use, location of host and nonhost plants, soil moisture all effected the movement of the Japanese beetles. A change in the environment such as land use and irrigation could affect the Japanese beetle distribution and be a viable practice to decrease the Japanese beetles’ opportunities to fly onto open airplanes (Hamilton et al. 2007). By manipulating the land around the airport the risk could be minimized.

In 1917 the former U.S. Bureau of Entomology established the Japanese Beetle Laboratory in 1917. They studied the biology of the pest in its new environment and worked on developing methods for control (Potter and Held 2002). They studied the pest-host interaction attempting to gain an understanding of this invasive pest. Entomologists realized this introduction had the potential of becoming a devastating pest that would lead to high economic costs.

In 1998, the National Plant Board initiated the first version of the U.S. Domestic Japanese Beetle Harmonization Plan. This established a plan for the movement of commodities that host Japanese beetles such as nursery stock and other regulated commodities. This program has two objectives to establish a framework that encourages states to assess the Japanese beetle risk and infestations status. The second objective is to provide a more uniform adoption and implementation of pest risk improvement measure to reduce the risk of pest introduction to receiving states. Nursery stock will requires a phytosanitary certificate for moving to states that consider Japanese beetle a quarantine pest (National Plant Board 2011). Movement is restricted from quarantined states, all but Florida, Mississippi, and Louisiana in the east are restricted. Infestations west of the Mississippi River are eradicated before they become established, although partial infestation occurs in Arkansas, Iowa, Kansas, Minnesota, Missouri, and Oklahoma.

Areas that are at risk for introduction have implemented protocols to eradicate this pest if they are introduced. If the Japanese beetle becomes established in Oregon and generally disperses throughout the state, the economic impact to all crops, commodities, and other related businesses could be over $34 million (Hamilton et al. 2007). The absence of natural enemies has allowed the Jap beetles to establish and spread in areas with little predator control. The indigenous host plants that Japanese beetles use as foods source are highly susceptible due to the lack of coevolutionary history with the beetle leaving the plants defenseless.

Japanese beetles are classified as an A1 quarantine organism for EPPO and of quarantine significance for CPPC, JUNAC, and OIRSA. They are at low risk of establishing in the Mediterranean area, and the risk is also low in the UK, Ireland and continental Europe north of 53N, because of the naturally cool summers. The area of concern is between latitudes 43N and 53N east to longitude 30E. If they are introduced and established they could cause massive damage and have significant economic loss. The EPPO makes a suggestion that countries prohibit the importation of plants with roots from countries where P. Japonica is established. If material is imported it should be planted in inorganic growing medium or in treated medium and kept under conditions which prevent infestation (Potter and Held 2002). The removal of soil from the roots of nursery plants will remove the larvae, in some cases the soil should be treated. The Japanese beetle was established in Terceira Island where in 1970 it escaped from a U.S. air base (National Plant Board 2011).

Japanese beetles release an aggregation pheromone and often are found in mating clusters. Invasive pests can benefit native populations through the plant stress-mediated interactions. Grapes are among their preferred hosts. This is an interesting interaction between an invasive species benefiting a native species. The Japanese beetle facilitates the green June bug feeding on grapes by biting through the skin providing access to the GJB and by eliciting yeast-mediated fermentation volatiles that the GJB uses in host-finding and aggregation. The GJB are usually unable to break through the tough skin of grapes so they benefit from the Japanese beetle feeding. This allows the GJB to expand its range into the fruit-growing region. Green June bugs were able to feed on grape plants before but the Japanese beetle injury leads to an additive increase in feeding and aggregation by breaking apart the fruit and attracting the GJB to the plants. In this case Japanese beetles have elevated the economic impact of an indigenous species (Hammonds et al. 2009). If these beetles are introduced and established into California this could potentially threaten the grape industry.

Japanese beetles have favored hosts that they prefer to feed on but will feed successfully on a list of non-preferred hosts. The preferred hosts successfully attract the beetle to feed. Since they attract other beetles to food sources, killing beetles or hand picking them off plants will reduce the populations. Some commercial traps bated with pheromones will also attract them. Grubs have trouble surviving in dry soils, so irrigation should not be used if an infestation is suspected. Bacillus papillae, a bacterial milky spore disease can kill the buried grubs. The bacteria can be applied to the soil and the spores will remain in the soil to infect further inhabitants (Shetlar 2008).

The insect-plant system is a complex interaction that understanding will benefit management strategies. Japanese beetles are generalists that feed on over 300 species making them an important pest of horticultural crops. They are attracted to hosts by green leaf odors and color instead of specific host stimuli. These generalists avoid nonhost plants because they sense deterrents in these plants such as cucurbitacins, a bitter triterpenes characteristic of cucurbits (Potter and Held 2002).

Although, corn and soybeans plants are not a preferred host of the Japanese beetle, they can do considerable damage defoliating soybeans and feeding on silks of corn plants which can reduce the pollination and kernel set. Both the grubs and adults can cause injury in corn. The grubs are able to feed on the root hairs of the plant which interferes with the plant taking up water and nutrients. If fields are heavily infested they can experience reduced plant stands. The adults beetle feed on the silks and this interferes with pollination. In soybean fields, they can also cause damage in both the larvae and adult stage. The grubs also feed on the root hairs of the soybean plants, this usually and economic important issue. The adults can cause extensive damage to the soybean plants, if this happens during the reproductive stages during the plant life a loss in yield will occur. They will feed on the flowers and defoliate the leaves. Management can be difficult. Adults are very mobile and can quickly move from fields. Fields should be scouted to determine the percent defoliation and to correctly identify the species. If the standard economic thresholds are met for corn or soybeans the fields should be sprayed with insecticide (Cook and Gray 2010).

The larvae have limited mobility so the oviposition site is very important for survival of the offspring. They have a strong preference to oviposit in grass the chemical cues stimulate females to lay eggs in the soil (Szendrei and Issacs 2005). Japanese beetles have an interesting mating behavior. The egg load and body size may influence their mating frequency. They have a promiscuous mating system in which both males and females mate repeatedly during their lives during this type the females moves to the soil to lay her eggs. The males are attracted to the virgin females by sex pheromones released (Tigreros and Switzer 2010). They seek out non-virgin females on food plants where they tend to aggregate. There is a last male advantage for fertilization in sperm competition. The males will mount unpaired females or try to remove the male from the paired female. Males may stay on the female for a couple hours “guarding” the female. Males will choose larger females over smaller females and guard these longer. This could be because larger females have more eggs and may oviposit more frequently (Switzer et al. 2008).

Management systems are multifaceted and can be complicated when trying to treat both the grub and adult stage. They both damage plants through different modes of feeding. The adults are mobile and move from place to place while the grubs are restricted to the area where the female laid her egg. They need to be laid by a suitable host for feeding if they are to survive.

Cultural controls can help decrease the larval populations in grasses. Avoiding irrigation during peak beetle flights this can make it difficult for the beetles to oviposit. One of the interesting discoveries was concerning geraniums. Beetles that feed on petals of geraniums roll over on their backs and remain paralyzed for 24 hours. They will typically recover but under field conditions are often preyed on by predators. Some type of substance in these plants paralyzes the beetle. The plants do not deter feeding and could potentially be used as a control agent for Japanese beetles if the volatile could be identified (Flores 2010).

The Japanese beetle problem should be treated using an IPM strategy to fight the infestation. A couple of management tips to follow are to avoid irrigation in areas where grubs are feeding and to avoid planting plants that are preferred hosts of Japanese beetles. Spot treat areas with insecticide if infestation is heavy, treat lawn with pesticides when the soil is wet for better absorption. Being aware of the life cycle of this pest and ways to manage them can help decrease expanding populations. Infestations are usually a threat in areas where there is a history of feeding, so scout and be aware of the potential threat.

Since the accidental introduction of the Japanese beetle into the United States they have slowly spread throughout the country threatening indigenous plants and ecosystems. The Japanese beetles are here to stay. They are voracious generalists that aggregate and can quickly defoliate plants and trees. Their grubs can destroy turf grass leaving irritating bald areas across your lawn. Comprehensive strategies restricting the movement of these mobile pests are in place and should be stringently followed. Stopping the spread of these beetles is very important for areas in the West such as Oregon and California where preferred host plants would be ravaged by their feeding. The combination of their mobility and reproductive capabilities makes Japanese beetles an invasive pest that will need to be closely monitored and quarantined in the present and future.

Cook, K.A. and M.E. Gray. 2010. Japanese Beetle Popillia japonica Newman. University of Illinois Extension. http://ipm.illinois.edu/fieldcrops/insects/japanese_beetles/

Flores, Alfred. 2010. Geraniums could help control devastating Japanese beetles. U.S. Department of Agriculture.

Hamilton, R.M., R.E. Foster, T.J. Gibbs, C.S. Sadof, J.D. Holland, and B.A. Engel. 2007. Distribution and Dynamics of Japanese Beetles along the Indianapolis Airport Perimeter and the Influence of Land Use on Trap Catch. Environment Entomology 36 (2): 287-296.

Hammons, D.L., S.K. Kurtural, M.C. Newman, & D.A. Potter. 2009. Invasive Japanese beetles facilitate aggregation and injury by a native scarab pest of ripening fruit. PNAS, 106 (10): 3686-3691.

Krischik, Vera and Maser, Doree. 2010. Japanese Beetle Management in Minnesota. University of Minnesota Extension. http://www.extension.umn.edu/distribution/horticulture/dg7664.html

National Plant Board. 2011. U.S. Domestic Japanese beetle Harmonization Plan. http://nationalplantboard.org/docs/jbcolumn.pdf

O’Neill, B.F. A.R. Zangerl, E.H. DeLucia, and M.R. Berenbaum. 2008. Longevity and Fecundity of Japanese Beetle (Popillia japonica) on Foliage Grown under Elevated Carbon Dioxide. Environmental Entomology 37(2): 601-607.

Popillia japonica. Data Sheets on Quarantine Pests. 2010. Prepared by CABI and EPPO for the EU.

Potter DA, Held DW. 2002. Biology and management of Japanese. Annual Review of

Entomology 47:175-205.

Shetlar, David. 2008. Japanese Beetle Fact Sheet. The Ohio State University Extension. http://ohioline.osu.edu/hyg-fact/2000/2504.html

Switzer, P.V., Enstrom, P.C. and C.A. Schoenick. 2009. Behavioral Explanations Underlying the Lack of Trap Effectiveness for Small-Scale management of Japanese Beetles (Coleoptera: Scarabaeidae). J.of Ec. Entom. 102(3): 934-940.

Switzer, P.V., Enstrom, P.C. and C.A. Schoenick. 2008. Environmental Conditions Affect Sperm Competition Risk in Japanese Beetles (Coleoptera: Scarabaeidae). Ann. Entomol. Soc. 101(6): 1154-1161.

Szendrei, Zsofia & Rufus Isaacs. 2005. Do plants cues influence the oviposition behavior of Japanese beetles? The Netherlands Entomological Society 117: 165-174.

Tigreros, N. & Switzer, P.V. 2008. Effects of food deprivation, body size, and egg load on the mating behavior of female Japanese beetles. Ethology Ecology & Evolution 20: 89-99.

Tigreros, Natasha, R. Jadhav, K.A. Kowles, B.P. Nathan and P.V. Switzer. 2010. Physiological Status of male and Female Popillia japonica (Coleoptera: Scarabaeidae) Affects mating and Grouping Behavior. Environmental Entomology 39 (3): 892-897.

USDA. Department of Agriculture. 2010. Japanese Beetle Program Manual for Airports.

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The Experiemental Haematological Index Of Rats Biology Essay

As demonstrated in (Table 1.)the experimental haematological index of rat is greater than both the standard human male and human female haematological indices, 6.25 x 1012 erythrocytes per litre. The RCC gives the oxygen carrying capacity of the blood. The higher the RCC, is the greater the oxygen carrying capacity of blood. This higher capacity is a requirement for rats as they are highly active organisms, and therefore their cells have a high rate of metabolism. Hence the haematological index of rat needs to be higher than that of humans. Since the MCV of rat erythrocytes is smaller than humans, relatively high number of erythrocytes is required to support the higher haematocrit and haemoglobin content which have ranges almost similar to human ranges.

The experimental rat haematological index is slightly below the standard rat haematological index. The cells need to be distributed homogeneously in the diluted blood solution in order to obtain accurate results.

The erroneous results may be due to insufficient mixing of blood with the preserving fluid before microscopic examination or due to rapid mixing, resulting in rupture of some red blood cells. The value is almost within the range.

There may have been miscount of cells.

As demonstrated in (Table 1.)The experimental haematocrit or the packed cell volume of the rat is within the range of the haematocrit for both standard human male and female haematocrit and the standard rat haematocrit.

The haematocrit is the proportion of blood volume occupied by red blood cells.

The haematocrit depends mainly on the non-cellular component; plasma and the number of erythrocytes in blood. The size of erythrocytes (MCV) also has an effect to a lesser extent. The experimental MCV obtained for the rat are within normal ranges for rat. (as shown on Table 2.) Even though the RCC estimated was slightly below the expected range, the haematocrit falling within the normal range explains that there may have been miscounts during the Neubauer count.

As demonstrated in (Table 1.) The experimental haemoglobin content of rat is slightly above the standard human female haemoglobin content range but within the range of standard human male haemoglobin content.

The haemoglobin content varies mainly depending on the RCC and to some extent the MCH. The standard normal ranges for haemoglobin content varies extensively in healthy organisms, with age, sex ,physiological variations such as pregnancy, ethnic origin and even method used for measuring, including the position of the model organism(that is upright or lying down), when the blood was obtained to be tested. (Ravel,1995). Therefore haemoglobin content of the rat falling slightly off range does not necessarily mean it is ill.

Generally the haemoglobin content ranges of human females is lower than that of human males of the same age. These variation are due to physiological changes in women. . (Ravel,1995; Rushton, et al.,2001). This explains why the higher experimental haemoglobin content of the rat went above the haemoglobin content range of human females.

The higher experimental haemoglobin content of rat 167g/l is within the relatively high standard range of haemoglobin content of human males. This may be due to either a blood disorder of the experimental rat or an adaptation of the rat’s blood to survive in enclosed places with low oxygen levels.

Increased haemoglobin content could reflect the rat to have polycythaemia, but not due to an increase in the RCC but decrease in the plasma volume. This is indicated by the haematocrit reading, 50%, suggesting there’s relatively high amount of erythrocytes relative to the rest of the blood, even though the value is just within the normal range.

This could be caused due to various reasons including dehydration of rat. Dehydration may be due to chemicals that were used to kill the rat, or due to warming the rat’s tail; where blood is usually obtained, thereby increasing blood circulation, easing the extraction of blood from its tail. (Diehl et al.,2001). Dehydration causes the blood to become more concentrated, showing an increase in the haemoglobin content in blood relative to the blood volume.( apparent polycythaemia) (Bain,2004).

There may have been carboxyhaemoglobin in the rat’s blood. The presence of carboxyhaemoglobin in blood has an effect on the aberrant haemoglobinometer reading. Carboxyhaemoglobin conversion into azide-methemoglobin is low, resulting in erroneous readings. (Shephard et.al.,2000).

The Wintrobe indices, MCV, MCH and MCHC are calculated using the haematocrit, haemoglobin content, and the RCC, which are within normal ranges or very close to the normal ranges. Thus values calculated using them are also within normal ranges..

The white blood cells demonstration shows that the cell sizes and numbers are within normal range without any signs of abnormality in them.

To avoid errors that could occur during microscopic cell counts, modern medicine includes more accurate, automated apparatus’ to perform cell counts; which involves the generation of an electrical impulse by each cell passing a sensing zone, and these signals correspond the number of cells. By using these methods the human errors that could occur during microscopic cell counts may be avoided. (Bain,2004).

There are automated apparatus for the measurement of the haematocrit, after the blood sample is centrifuged. (Bain,2004).

The method and area in which blood is obtained, also could be standardised. For instance, rat blood could be drawn from the sublingual vein; with repeated anaesthesia. This way dehydration of the rat is avoided, providing more accurate results. (Diehl et al.,2001).

It is advisable to take into account all the haematological blood tests into (even the ones that are not mentioned) before diagnosis of a disease and to repeat tests in order confirm the accuracy of their results.

Variations of the Wintrobe indices, blood counts, haemoglobin content and/or haematocrit off normal ranges may indicate different blood disorders as types of polycythaemia or anaemia, caused due to varied reasons, as iron deficiency (iron deficiency anaemia), change in shape of erythrocytes (sickle cell anaemia), vitamin deficiencies (pernicious anaemia), anaemia due to haemorrhage (haemorrhage anaemia), due to lysis of erythrocytes as a result of bacterial infections (haemolytic anaemia), and destruction/depression of bone marrow (aplastic anaemia),etc. and thereby aid the diagnosis of diseases which are associated with these blood disorders. (Bain,2004).

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Water Quality Dissolved Oxygen Biology Essay

Figure 4.1 show the temperature and DO concentration in EM treated water sample and control water sample. The reading has been taken once in 2 days by using YSI 52 CE DO meter (S.N 06L1446) for 26 days in a row in unit mg/L (ppm). The optimum range of DO concentrations for tilapia culture is between 5.00 ppm - 8.30 ppm. Lower than 5.00 ppm, the fish will distress and if the concentration of DO less than 2.00 ppm the potency of fish dies is high.

Figure 4.1: Concentration of DO in EM Tank and Control Tank

Base on figure 4.1, the concentration of DO starting from day 0 till day 26th not shown an obvious changed in both water samples. At day 0, the DO concentration for EM treated water sample is 5.06 ppm and at the end of the study which is at day 26th, the concentration of DO in EM treated water sample is 5.15 ppm. The highest concentration of DO in was appeared at day 24th which is 5.92 ppm. In contrast, the lowest concentration of DO in EM treated water sample is 4.79 ppm which at day 10th. For EM treated water, the DO concentration at day 8th (4.86 ppm) and at day 10th (4.79 ppm) were recorded below the optimum concentration.

Conversely, the highest concentration of DO in Control water sample is 5.83 ppm which at day 8th. On the contrary, the lowest concentration was recorded at day 20th which is 4.88 ppm. Furthermore, the DO concentration at day 4th (4.97 ppm) and day 20th (4.88 ppm) appeared at low then optimum concentration. Even though the concentrations of DO at both days are slightly low then optimum concentration, this was not affected the Tilapia culture.

Theoretically, the temperature of water sample will affect the concentration of DO. Oxygen becomes less soluble in water as the temperature increases (Geer and Kamila, 2005). Meaning that, warm water is less capable in dissolving the gases like oxygen while cold water has greater ability in holding oxygen. How ever, those days which the DO concentration were shown not in the optimum range, the temperature was lower (24°C) compared to others (Figure 4.1). However, this fact was assumed to show negative result due to the deepness of the probe when DO concentration reading. More deep of water from the surface, the temperature and DO are lower compare to the temperature and DO at the surface. Therefore, the more deep the probe of DO meter goes from the surface during reading, the lower the temperature and the DO concentration.

In conclusion, the concentrations of DO for both tanks are almost the same. No significantly changed in DO concentrations due to the aeration system that has been used to supply the oxygen. Therefore, the hypothesis can be made as the used of EM are not affecting the levels of DO in Tilapia culture. The level of DO is mainly control by aeration system. Even the surrounding temperature can affect the DO concentration, but it is not indicate an obvious change to the DO concentration.

Temperature also acts as an important parameter which needs to be checked in fish culture. The temperature of EM treated water samples were in range of 24-27°C and in control water samples were in the range of 24-26 °C. From figure 4.2, the data had shown there was no significant change through out the whole study. More over, the temperature of the water for both tanks was not away from the optimum range which is between 20 and 35 °C.

Figure 4.2: Temperature in EM Tank and Control Tank

For EM treated water sample, the lowest temperature recorded was 24°C, while the highest temperature was 27°C. In contrast, the lowest temperature for control water sample was same with EM treated water sample which is 24 °C but the highest temperature recorded in control water sample is 26 °C.

In general, the temperature for both tanks was control by the environment. Due to the location of the culture tanks which was under the roof, the heat from the sun are not directly affect the temperature. Moreover, in rainy day the temperature reading is not dropped significantly because the rain water does not drop into the culture tank and the volume of water in culture tanks remain the same.

Figure 4.3 show the concentration of Total Ammonia Nitrogen (TAN) in EM treated water sample and control water sample. Starting from day 0 to day 10th, the TAN concentration in both water samples increased quite in the same way. At day 0, the TAN concentration in EM treated water sample is 1.64 ppm and rose to 4.85 ppm at day 10th. On the other hand, the TAN concentration in control water sample rose from 1.65 ppm at day 0 to 4.90 ppm at day 10th. However, the concentrations of TAN in both water samples show major differences starting from day 12th to day 26th. The TAN concentration in control water sample started to acumulate at higher rate compared to EM treated water sample from 4.92 ppm at day 12th to 5.25 ppm at day 26th. On the contrary, TAN concentration in EM treated water sample show in decreased (33.70%) from 4.57 ppm at day 12th to 3.03 ppm in day 26th.

Figure 4.3: Concentrations of Total Ammonia NH3-N

The waste from fish pallet was proven to be the main contributor to the TAN concentration in water. For that reason, the concentration of TAN in control water sample was significantly increased by time. The ammonia cycle in control tank was not effective enough to reduce the concentration of TAN. This fact is mainly due to the lack of bacteria that utilized ammonia. In contrast, the decreased concentration of TAN starting from day 12th to day 26th in EM treated water sample was mainly due to the used of EM treatment. The microorganisms in EM were proven to be effective in reducing the TAN concentration. Moreover, the microorganisms such as Nitrosomonas and Nitrobacter that can make use of ammonia as energy source may be include in the consortium of microorganisms in EM.

Besides, the concentration of TAN will affect the pH of the water (refer chapter 4.1.4) and the concentration of Un-ionized ammonia (refer chapter 4.1.5). Since ammonia is alkaline, it will indicate higher pH value when its concentration increases. Moreover, the greater the concentration of TAN, the higher concentration of Un-ionized ammonia will produce in the water system (Ruth and Craig, 2005)

The value of the pH has an effect toward the toxicity of ammonia and increasing in pH will increase the toxicity of ammonia. The recommended value for Tilapia culturing is between 6 - 8 ppm (Tilapia culturing technique, Lembaga Kemajuan Pertanian MADA). If pH readings are beyond this range, fish growth is reduced and at values below 4 or above 10, mortalities will occur.

Figure 4.4: The pH Values in EM Tank and Control Tank

Figure 4.4 shows the pH value for EM treated water sample and control water sample. The pH values for both tanks were increased for the first 10 days. At day 10th where the EM solution was introduced to the EM tank, the pH values for EM treated water samples were started to decrease. On the contrary, the pH values for control water sample still show an increase.

For the first 10 days, the pH value for EM treated water sample increase from 7.13 ppm to 7.46 ppm. While, pH value in control water sample had been increase from 6.98 ppm to 7.81 ppm. After 2.5 L cultured of EM was added to the EM tank at day 10th, the pH value in EM treated water sample has slightly decreased from 7.46 ppm to 7.27 ppm. While, the pH values in control water sample still increased until 8.31 ppm toward the end of the study (day 26th). For EM treated water sample, the value of pH remain in the recommended pH value and still suitable for the culture of Tilapia. Whereas, the pH values in control tank at day 14th, 16th, 18th, 22nd, 24th and 26th (figure 4.4) has go beyond the recommended range of pH value for Tilapia culture (6-8 ppm).

These results proved that the use of EM in aquaculture will reduce the pH level to suitable pH range for the Tilapia culture. Since the pH value is related with concentration of total ammonia in water, the used of EM will solve both problems. Hence the production cost will decrease and the productivity of Tilapia will increase by using EM.

Figure 4.5 shows the concentration of un-ionized ammonia (UIA) in both EM treated water sample and control water sample. UIA is a toxic form and the toxicity begins as low as 0.05 ppm. If the UIA is higher than 0.05 ppm, the fish gill is being damaged. As the concentration rises above 0.05 ppm it causes more and more damage and at 2.0 ppm fish will die. The UIA can be calculated from the concentration of TAN multiply by the Fraction Factor (Appendix 7). Prior to the calculation of the UIA, the pH and temperature of the water sample need to be determined.

Figure 4.5: Concentrations of Un-ionized Ammonia (UIA)

It was shown that the concentration of UIA in EM treated water sample shown an increase from day 0, 0.0098 ppm to 0.0686 ppm at day 12th. The concentration of UIA is related to the concentration of TAN. Since there was an increasing in TAN concentration in EM treated water sample for the first 10 days (Figure 4.3), therefore, the UIA in EM treated water sample was increased from day 0 to day 12th. However, after EM has been introduced, the concentration of UIA was slightly decreased (57.58%) from 0.0686 ppm at day 12th to 0.0291 ppm at day 26th. This happened because the decreasing of TAN (33.70%) in EM treated water sample from day 12th to day 26th (Figure 4.3).

On the other hand, the concentration of UIA in control water sample was increased with time. The concentration of UIA at day 0 is 0.0099 ppm and rose significantly to 0.4620 ppm at day 26th. This observable fact was due to the increasing of TAN in control water sample. In general, the higher the concentration of TAN, the grater the concentration of UIA in water sample.

Figure 4.6 show the concentration of nitrite in EM treated water sample and control water sample. Started from day 0 to day 10th, the nitrite concentration in both water samples increased in the similar manner. At day 0, the nitrite concentration in EM treated water sample is 0.036 ppm and rose to 0.427 ppm at day 10th. On the other hand, the nitrite concentration in control water sample rose from 0.042 ppm at day 0 to 0.453 ppm at day 10th. But after addition of EM into EM treated tank, the concentration of nitrite in both water sample show noticeably differences starting from day 12th to day 26th. The nitrite concentration in control water sample started to increase at higher rate compared to EM treated water sample from 4.92 ppm at day 12th to 5.25 ppm at day 26th. In contrast, nitrite concentration in EM treated water sample show in decreased from 0.299 ppm at day 12th to 0.193 ppm in day 26th (35.45%).

Figure 4.6: Concentration of Nitrite NO2-N in EM Treated Water Sample and Control Water Sample

Since nitrite is the product of the ammonia metabolisms in nitrogen cycle, the concentration of TAN will affect the concentration of nitrite. In conclusion, EM was proven in reducing the concentration of nitrite due to the reducing of TAN concentration.

Gas Chromatography Mass Selective (GC-MS) detector should be used in this analysis. Due to some technical problem that cannot be accounted, alternatively, Gas Chromatography with Flame Ionized Detector (GC-FID) has been used. However, the chromatograph did not show the peak of interest but show a lot of unknown peaks.

This problem was believed due to the GC type that has been used. The GC-MS is more sensitive in detection because it detection is based on the mass of the compounds. In contrast, GC-FID detection is based on ionization. More over, GC-FID that being used is equipped with ultra-5 column which is semi-polar instead of ultra-1 column which is non-polar that needs to be used. Since GEO and MIB is semi volatile polar compound, the used of ultra-5 as a column is not good for separation of these compounds. Further more, the method used is well-matched with GC-MS but the same method has been used when running GC FID.

Conversely, sample preparation also one of the factor that contributed to this problem. Methanol has been used as a solvent in dilution of the standard samples which are GEO and MIB. Even though the solvent has been filtered with 0.45 µm and 0.2 µm filter membrane, but there were still impurities or contaminant occurs in the standard samples. This impurities and contaminant has shown in the chromatograph as unknown peak (Appendix 14-16). How ever, after methanol (solvent) or blank has been injected in GC, all the unknown peaks have been identified from the solvent itself (Appendix 16).

Therefore, as an alternative, the sensory evaluation method has been used to investigate the effect of EM in elimination of off-flavor in Red Tilapia.

Figure 4.7 and figure 4.8 show the graph of Average Score of Sensory Evaluation for EM treated fish sample and control fish sample. The four attributes that has been judged for 5 evaluation session were texture of the fish fillet, earthy taste of the fish, moisture and acceptability. The major attribute focused in this study was earthy taste in fish.

The evaluation session was carried out once in 3 days. It was started with first session that was held on day 13th followed by second session on day 17th, third session on day 21st, fourth session on day 25th, and last session on day 29th. At each session, six fish samples have been introduced to the panelist consisted of EM treated fish samples and control fish samples.

Figure 4.7: Average Score of Sensory Evaluation in EM Treated Fish Sample

Figure 4.8: Average Score of Sensory Evaluation in Control Fish Sample

The first attribute that has been study is texture of fish sample. The score vary from 1 for hard to 5, soft. Based on figure 4.9, the average score for EM treated fish sample and control fish sample not show significant differences. For the 1st session till the 3rd session, the average score given by the panelist for EM treated fish sample and control fish sample are almost the same. It was found that the 4th session of evaluation, the average score for EM treated fish sample is 4.2 while for Control fish sample, the average score is 3.5. On the other hand, at 5th session, the average score for EM treated fish sample is 4.1 compared to control sample which is 3.6.

Figure 4.9: Average Score in texture for EM treated fish sample and control fish sample According to Evaluation Session

In general, the fish samples that had treated with EM have higher quality of texture compare to control sample (Zulkafli A. R. Pemahaman asas-asas mutuair: panduan mudah untuk penternak. Unpublished note, Pusat Penyelidikan Perikanan Airtawar). Further study need to carry out to confirm this fact because the effect of EM on fish texture is time consuming process and need longer study period to see the result. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 16).

Figure 4.10 show the distribution of earthy taste in EM treated fish sample and Control fish samples. The score vary from 1 (very dislike), 2 (dislike), 3 (neither dislike nor like), 4 (like) and 5 (very like). From the figure 4.12, the earthy tastes in EM treated fish sample show drastically increase in quality. In 1st session, the average score is 2.7 which mean the panelist not sure whether they like or dislike. But at the end of the study, at 5th session almost the entire panelist agreed that the earthy taste in EM treated fish sample has decreased and give an average score 4.5 (almost very like).

Figure 4.10: Average Score in Earthy Taste for EM treated Fish Sample and Control fish Sample According to Evaluation Session

On the other hand, the control fish samples did not show in drastic improvement on earthy taste. The average score are varying from 2.1 (dislike) for 1st session to 2.8 (neither dislike nor like) at the last session. The panelist agreed that the earthy taste is still in the fish sample after 5 session of evaluation.

As a hypothesis, the used of EM will reduce the earthy taste in the fish tissues. The earthy taste had change from dislike to almost like very much after treatment of EM to the fish sample. Based on this study, 16 to 20 days after treatment with EM was enough to reduce the earthy taste in fish sample. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 17).

Figure 4.11 show the average score for moisture in EM treated fish samples and control fish samples. The scores vary from 1 (dry) to 5 (wet). For the 1st session, the average score for EM treated fish sample is 3.6 while for control fish sample, the average score is 3.3. Moreover, the average score for control fish samples at 2nd and 3rd session are similar which is 3.2. Towards the end of the evaluation session, the average score for EM treated fish sample is 4.1 compared to control fish sample 3.9.

Figure 4.11: Average Score in Moisture for EM treated Fish Sample and Control fish Sample According to Evaluation Session

In conclusion, the effects of using EM toward the moisture content in fish sample are not clearly defined in this study. Since there is no significant different in moisture content between EM treated fish sample and control fish sample, the hypothesis can be made as EM was not affect the moisture contain in fish tissue. May be other factors such as the genetic of the fish, aging or the way of sample preparation will affect the moisture contain in fish. Therefore, further study must be carry on by using different methods to determine the moisture content in fish. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 18).

Figure 4.12 show the average score for acceptability in EM treated fish sample and control fish sample. For this attribute, the score vary from 1 (worse), 2 (bad), 3 (fair), 4 (good) and 5 (best). These attributes were judged to know the level of satisfactoriness toward the fish samples. In other word, this attribute was indicated that either the fish sample is satisfied to eat or not.

Figure 4.12: Average Score in Acceptability and Earthy Taste for EM treated Fish Sample and Control fish Sample According to Evaluation Session

Figure 4.12, shown that the average score for EM treated fish sample always higher compared to control fish sample. In 1st session, average score for EM treated fish sample was 3.2 whereas control fish sample was 2.3. Towards the last session of the evaluation session, the average score for EM treated fish sample also higher than control fish sample. Overall, the average score for EM treated fish sample has change from Fair (3.2) to Good (4.3) toward the end. On the other hand, for control fish sample not show drastically change. For the 1st session the average score was bad (2.3) and at the lass session of evaluation the score is still at the same score (2.7) but a bit higher toward Fair score.

Moreover, figure 4.12 shows the relationship involving acceptability and earthy taste. The change in average score for acceptability for both sample were affected by the earthy taste in the fish sample. The less of earthy taste in the both fish samples or in other word the higher the average score in earthy taste indicate higher average score in acceptability.

Therefore, the earthy taste was the main problem contributed to the rejection of Tilapia in the local and global market. Even though there are other attribute that has been judged in this study, but the main attribute contributed to the negative response of tilapia was earthy taste. The used of EM has changed the panelist tolerability toward fish sample from fair to good (figure 4.12). From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 19).

Table 4.1 shows the comparison of the average CFU between the EM treated water sample and the control water sample. From the spread plate results, control soil sample recorded an average 6.0 x 107 CFU/mL at day 14th, 1.6 x 108 CFU/mL at day 19th, and 1.2 x 108 CFU/mL at day 24th. In general, the average microorganism colonies for EM treated water sample are 2.7 x 108 CFU/mL at day 14th, 5.7 x 108 CFU/mL at day 19th, and 6.5 x 108 CFU/mL at day 24th. Furthermore, by combining all these results, the average microorganism colonies for control water sample is 1.13 x 108 CFU/mL while the average microorganism colonies for EM treated water sample is 4.97 x 108 CFU/mL which is 4.398 times (339.82%) higher. In simple word, the application of EM has vitally increased the number of beneficial microorganisms in the water.

Table 4.1: Comparison of CFU

2.7 x 108

6.0 x 107

5.7 x 108

1.6 x 108

6.5 x 108

1.2 x 108

4.97 x 108

1.13 x 108

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