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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The Recent Advances In Gene Expression Profiling Technologies Biology Essay

Biology » The Recent Advances In Gene Expression Profiling Technologies Biology Essay

Breast cancer starts from breast tissue, usually in the inner lining of the milk ducts or lobules which supply ducts with milk (Sariego J., 2010). It is becoming increasingly difficult to ignore the effect of 1/8 women in the world. So far, breast cancer has been known as one of most famous cancer disease, with an incidence rate higher than twice that of colorectal cancer or cervical cancer, and its incidence rate just lower than lung cancer. (Norazizah Shafee, et al, 2008) Like other cancer disease, the pathogenic mechanism of breast cancer usually associates with environment and genetics risk factors. It is a very complex cancer disease, but still keeps poorly understood about its pathogenesis. Several studies have produced some evidences shown that it is about 100 times between the incidences rates of women than men, but survival rates are equal in both sexes (American Cancer Society, 2008).Nobody knew why women could get breast cancer. However, there is increasing concern that women have a large number of risk factors to get it.

The treatment of breast cancer is usually determined by the size, stage, rate of growth and characteristics of cancer. The multistep process of breast cancer development could be obvious defined as the sequence of pathologically stages. According to the patients’ clinical behavior, the breast cancer is really difficult to accurately classify by these predictors for metastases, such as lymph node status and histological grade (McGuire, 1991, Goldhirsch A, 1998 and Eifel P et al, 2001). As the recently research, people thought the primary breast cancer usually starts from the premalignant stage of atypical ductal hyperplasia (ADH), develops into ductal carcinoma in situ (DCIS) which is the preinvasive stage of breast cancer, and terminates in the potentially lethal stage which is invasive ductal carcinoma (IDC). This linear model of breast cancer progression has been used for diagnosing and treating breast cancer at earlier clinical stages (Tabar, L et al., 2000). However, the breast cancer in stages of DCIS and IDC are related to heterogeneous with mitotic activity and cellular differentiation. Therefore, breast cancer can be divided into three cancer grades in which grade I, II, and III lesions according to subtype the stages of DCIS and IDC with well, moderately, and poorly differentiated breast cancer (Dalton, L. W, 2000 and Holland, R., 1994). However, the cancer genesis of breast cancer is still understood poorly, such as limited identified the biologically relevant genes related to the different pathological stages. Chemotherapy or hormonal therapy usually could reduce approximately 1/3 risk distant metastases, but 70–80% of patients by this treatment would not have the chance of survival (Hedenfalk I, 2001 and Ahr A, 2001). Therefore, the research of breast cancer need technology to outperform all currently used various clinical and pathological factors in predicting outcome of cancer and adjuvant therapy methods. Gene expression profiling technologies may be provided researchers with a very good opportunity to carry out exhaustive genetic profiling of breast cancer and establish a molecular profile for breast cancers (Ahr A,2001) .

Over the last 20 years, gene expression profiling technologies have the advances lead to the ability to examine plenty of genes expression at the same time and provide good methods to analysis of multiple markers quickly. To recognize the trails of gene expression is also very useful to identify tumor markers. Until now, gene expression profiling technologies have developed to identify hundreds and even thousands of genes expression at the same time. Gene expression array, such as DNA microarray, tissue microarray, is one of most widely applied gene expression profiling technologies for genome-wide gene-expression studies which received a big deal of attention on the global level.

The gene expression array based on oligonucleotide-microarrays is popular (.Hardiman, G, 2006 and Perou, C. M, 2000). There are two major methods to construct oligonucleotide arrays: 1) microarrays are composed of 25 bases short oligonucleotides which are synthesized directly onto a solid matrix with photolithographic technology (Affymetrix); 2) microarrays are composed of 55–70 bases long oligonucleotides which located on an ink-jet printing process (Agilent) or spotted by a robotic printing persist in a solid medium, such as the fibrous mesh membranes (CodeLink) or glass slides ‘‘chip’’. Through DNA microarray technique, thousands of DNA spots which located single gene onto the single slide or chip (Eisen MB, 1998 and Khan J, 1998). The Affymetrix microarray hybridizes one sample per chip by a single-color detection scheme, but Agilent technology could hybridize the same array with two different samples by a two-color scheme.

High-throughput screening techniques have been fundamentally developing biomedical research now. It is one of best option to prioritize and select the best targets from thousands of candidate genes and proteins. Analysis of the molecular targets in situ at the cellular level, assessment of the molecular targets expression could be analyzed in all tissues and diseases and be used to evaluate their clinical significance with important to target selection through additional information. Compared with the high-throughput techniques of genomics and proteomics, most tissue-based molecular analyses are slow, cumbersome and require extensive manual interaction.

Opposed to the classical northern-blotting analysis, the target is hybridized in parallel to a great number of DNA sequences, immobilized on a solid surface by the microarray experiment principle (Bast RC Jr, 2001). It can detect and quantify thousands of transcript species at the same time. Recently, the technology of gene expression array has been developed rapidly. More powerful robots for arraying, novel surface technology for glass slides, and novel protocol of labeling and dyes, could be advanced together. They can extend the quality and complexity of microarray experiments to increase different organisms’ genome-sequence information including human. The high-density cDNA microarray technology (LMC) offers a novel chance for high-throughput breast cancer genetic analysis (Schena, M., 1995). It has the capacity to develop monitoring of plenty of genes. Most current microarray studies have the ability to perform array-based expression analysis with in vivo-derived genetic material originating from morphologically distinct cellular subpopulations within neoplastic tissue. It can be used to analyze gene expression in a clinical cancer specimen.

Especially in the construction of tumor markers for purposes of diagnose, the single gene identification express in cancer cells with great pharmacological interest, but not in normal cells (EllisM, 2002). The protein can be used to design the target strategies for chemotherapy or immunotherapy, which could be identified on cancer cell surface (Paik S, 2004). The transcriptional profiling of solid tumor is very complex. In fact, they may include a variable great number of infiltrating tissue, for example endothelial, stroma and lymphoid cells. RNA isolated from purified cancer cells by laser-capture micro dissection needs amplification of substantial target and significant bias (Sauter G, 2002). Recent developments of gene expression profiling technologies prove that the identified potential tumor markers using whole solid tumors are very useful. Through this tissue microarray technique, cancer specimens which are up to 1,000, can be marked in correlative for fluorescence or immunohistochemistry to resemble gene expression with the rearrangement of chromosomal in situ hybridization (Sallinen, S.,2000). Gene expression profiling technologies also have direct contribution for the annotation of the human genome sequence by the regulation of gene transcription. (Sauter G, 2002)

It is also one of best prognostic or predictive context of prospective clinical method to assay novel gene factors (Hayes DF, 1996 and 1998). It can be directly designed as the objective of trial and the function of targets. However, these prognostic factors can only be considered as one possible therapy method in fact. If the prognosis of patients is poor, these factors will be useless. For this reason, the clinical utility of these prognostic factors is really complicated to identify without treatment factors, and these factors also need to layout severe clinical trial. However, the evidence of research on most tumor marker is not easy to confirm, and have more chance to advance novel clinical treatment hypotheses.

It is also used to identify the gene target or pathways with unknown function drug, and have the possibly chance to offer a quick way to the description of novel drugs. Loss of the gene encoded the desired drug target should stop all the gene expression changes which caused by the novel drug. It might result in some needless correlation with other protein, which called “off-target” side-effects (Buyse, M., 2006). Plenty of large DNA microarray data sets of gene expression profiling technologies were located in the public domain, such as specific pathways activation, treatment of novel drug, and cataloguing gene expression changes which regard to mitogens, etc. To a certain extent, these techniques could be able to analysis original investigated data with more significant sense of the expression trails compared with other data sets.

Several research reports revealed that the same RNA samples hybridized on distinct microarray programme might lead to contradictory results by application of gene expression profiling technologies (Shi, L. et al., 2006). Therefore some skepticism about the reproducibility and the reliability of the techniques appeared. However, in fact, these different results reflect that data analyses by high throughput technologies were very complex, without any significant meaning on the techniques are inferior or unreliable. Definitely, most the differences were considered as illogical sequence correctness and explication, low specific spotted DNA/RNA and tissue microarray, different isoforms lack of specific probe, or differences in hybridization and measurement of fluorescence (Kothapalli, R., 2002, Tan, P. K., 2003, Baum, M., 2003, Barczak, A., 2003 and Hardiman, G, 2006). Gene expression profiling technologies have also been used throughout on cancer-derived cell lines and to organize distinct cancer cell-line panels from cancer origin (Loi, S., 2007 and Dai, H., 2005).

Gene expression profiling technologies could be used to identify breast cancer into classification from different gene expression trails (Colozza, M.,2006). According to the analysis of cluster, breast cancer can be classified into different synthesized expression profiling which can be characterized by the upregulated expression of several genes targets, such as predominantly oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative and ERBB2-negative(Perou, C. M.,2000, Sorlie, T., 2001 and 2003, and Sotiriou, C., 2003). Even more important, the novel molecular subgroups have obvious clinical application outcomes and therapy responses. In fact, the PR-negative and ER-negative could be recognized by different characterization of breast cancer. The expression trail of gene targets revealed the active hormone-mediated transcription activity associated with the androgen receptor (AR) pathway. It points out the latent target therapeutic pathway in breast cancer. Breast cancer sensitive in luminal A tumor with low proliferation and grade (Doane, A. S., 2006 and Farmer, P., 2005).

Stupendously, the clinical regard parameters have dissimilar gene-expression trails, such as nodal status tumor size and menopausal status (Sorlie, T., 2003). On the contrary, these significant prognostic parameters gain more tumor stage, but not the intrinsic biological possession of the breast cancer. Therefore, it has already begun to change the clinical therapeutic method and identify standard clinico-pathological parameters. The main challenge on the world is to detect novel potential gene targets for new therapeutic application development by gene expression profiling technologies.

To sum up, data of gene expression profiling associated with analysis of genomic changes, such as the relative genomic hybridized microarray analysis, whole genomic DNA microarray hybridization. These technologies could be used to recognize gain or loss of gene expression which lead to advance breast cancer (Teschendorff, A. E., 2006). It is also expected to recognize gene expression signatures which suggest breast cancer .clinical behavior.

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The Effect Of Three Antiseptics On Staphylococcus Aureus Biology Essay

Biology » The Effect Of Three Antiseptics On Staphylococcus Aureus Biology Essay

With food and air- borne infections rising by the day, skin infections are the most commonly known infections. Staphylococcus aureus bacteria generally cause skin infections that lead to various diseases. Antiseptics are well known for preventing the bacteria from infecting the skin. Therefore in my experiment I would like to conduct a check on “The effect of three antiseptics on Staphylococcus aureus.”, keeping the time for the antiseptic to act on the surface constant but varying the dilutions to check the effect on the rate of bacterial growth on the surface. This experiment has been proved successful and is termed as the microbial challenge test.

Antiseptics and disinfectants are used extensively in hospitals and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections. A wide variety of active chemical agents (or “biocides”) are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection, and preservation. “Biocide” is a general term describing a chemical agent, usually broad spectrum that inactivates microorganisms. Because biocides range in antimicrobial activity, other terms may be more specific, including “-static,” referring to agents which inhibit growth (e.g., bacteriostatic, fungistatic, and sporistatic) and “-cidal,” referring to agents which kill the target organism (e.g., sporicidal, virucidal, and bactericidal).

Types of Antiseptics

Chlorhexidine Gluconate

A biguanidine derivative, used in concentrations of 0.5–4.0% alone or in lower concentrations in combination with other compounds, such as alcohols. Used as a skin antiseptic and to treat inflammation of the gums (gingivitis). The microbicidal action is somewhat slow, but remanent. It is a cationic surfactant, similar to Quats.

Alcohols

Most commonly used are ethanol (60–90%), 1-propanol (60–70%) and 2-propanol/isopropanol (70–80%) or mixtures of these alcohols. They are commonly referred to as "surgical alcohol". Used to disinfect the skin before injections are given, often along with iodine (tincture of iodine) or some cationic surfactants (benzalkonium chloride 0.05–0.5%, chlorhexidine 0.2–4.0% or octenidine dihydrochloride 0.1–2.0%).

Quaternary ammonium compounds

Also known as Quats or QAC's, include the chemicals benzalkonium chloride (BAC), cetyl trimethylammonium bromide (CTMB), cetylpyridinium chloride (Cetrim, CPC) and benzethonium chloride (BZT). Benzalkonium chloride is used in some pre-operative skin disinfectants (conc. 0.05–0.5%) and antiseptic towels. The antimicrobial activity of Quats is inactivated by anionic surfactants, include chlorhexidine andoctenidine.

Infection: Before a microbe or parasite can invade the host and cause infection, it must first attach to and penetrate the surface of the epithelial layers of the body. Organisms gain entrance into the body through active or passive means. That is they have to pass physical barriers such as the skin or epithelial cells which line the mucosal surfaces of the respiratory, gastro intestinal and genitourinary tracts. The active passage for these microbes and parasites are the dead layers of the skin.

Skin: The term ‘skin’ applies to the outer covering of vertebrate animals. The skin is the largest organ of the body and has many different functions. The structure of the skin varies in different vertebrate groups. The basic structure of human skin will be described here. The skin is composed of two main layers, the epidermis and dermis. These cover the underlying, or ‘subcutaneous’ tissue, which contains specialised fat- containing cell known as adipose tissue. The thickness of the epidermis and dermis varies according to the region of the body and from person to person.

Epidermis- The cells of this region are separated from the dermis by a basement membrane. The epidermis is composed of many layers of cells that form a stratified epithelium. The cells immediately above the basement membrane are cuboidal epithelial cells and form a region known as the Malpighian layer. The cells in this layer are constantly dividing by mitosis and replace all the cells of the epidermis as they wear away. The Malpighian layer forms the lower region of the stratum granulosum, which is composed of living cells that become flatter as they approach the outer region of the epidermis, the stratum corneum. Cells in this region become progressively flattened and synthesise keratin, which is a fibrous protein which makes the cells waterproof. As the keratin content of the cells increases, they are said to become ‘cornified’. Their nuclei disappear and the cells die. The thickness of the stratum corneum increases in part of the body where there is considerable friction, such as the ball of the foot and the bases of the fingers. The outer covering of the skin forms a semi- transparent, thin, tough, flexible, waterproof covering by the hair follicles and by pores, which are the pores, which are the openings of the sweat glands. The outermost squamos epithelial cells are continually being shed as a result of friction.

The stratum corneum has become modified in many vertebrates to produce nails, claws, hooves, horns, antlers, scales, feathers and hair. Keratin is the main component of all these structures.

Dermis- The dermis is a dense matrix composed of connective tissue rich in elastic fibres and contains blood capillaries, lymph vessels, muscle fibres, sensory cells, nerve fibres, pigment cells, sweat glands and hair follicles.

The skin also has other more specific immune functions. Langerhan cells, which are immunocompetent cells, are involved in recognizing and processing foreign substances. These decrease with age, altering immune function (Gilchrest, 1999).

Mucous membranes, covering our eyes, alimentary canal, and genito-urinary tracts, play a protective role in providing a barrier and differential absorption. Usually, the skin prevents invasion by microorganisms unless it is damaged—for example, by an injury, insect bite, or burn.

Staphylococcus Aureus: Out of the many bacteria entering the human body through the dead skin, Staphylococcus aureus, is the most potent organism infecting the skin.

Facultatively anaerobic - do not require oxygen for growth but do grow better in its presence

The most common species of staphylococcus is Staphylococcus aureus which is a pathogen of both humans and animals. It is characterized by its ability to clot blood plasma by action of the enzyme coagulase

a) Colony Characteristics of Staphylococcus aureus

Size - 1 micrometre

Gram nature - Gram positive

Appearance- Grape-like cluster but some occur as single or pair of cells

usually non-capsulate

Form - Colonies are circular, 2-3 nm in diameter with a smooth shiny surface when grown on nutrient agar, milk agar or blood agar for 24 h at 37 degrees

Pigmentation - Colonies are often pigmented, though a few strains are unpigmented

Halophile i.e.Salt-tolerant

b) Pathogenesis of Staphylococcus aureus

Present in the nose of 30% of healthy people and may be found on the skin, causes infection at sites of lowered host resistance.

Eg: - damaged skin or mucous membranes

c) Epidemiology of Staphylococcus aureus

i) Infected Lesions:

Large no. of staphylococci are disseminated (spread widely) in pus and dried exudates discharged from large infected wounds, burns, secondary infected skin lesions and in sputum coughed from the lung of a patient with bronchopneumonia. Direct contact is the most important mode of spread, but air-borne dissemination may also occur

ii) Healthy Carriers:

Spread into environment by the hands, handkerchiefs, clothing and dust consisting of skin squames? and cloth fibres of healthy carriers. Some carriers, called shedders, disseminate exceptionally large no. of cocci, comparable to the number disseminated by patients with large superficial lesions or lower respiratory tract infections

iii) Animals:

Domesticated and some wild species may disseminate Staphylococcus aureus from infected lesions or carriage sites and so cause infections in humans.

Mode of infection may be either exogenous (from an external source) or endogenous (from a carriage site or minor lesion, elsewhere in the patient's own body). Staphylococci do not grow outside the body except occasionally in moist nutrient materials such as meat, milk and dirty water. Although not spore-forming, they may remain alive in a dormant state for several months when dried in pus, sputum, bed clothes or dust

They are fairly readily killed by heat (65 degrees for 30 min), by exposure to light and by common antiseptics. Therefore I would like to study on the research question,

Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality from treatment failures and increased health care costs. Appropriate antimicrobial antiseptic use has unquestionable benefit, but physicians and the public frequently use these agents inappropriately.

The microbial challenge test would be most appropriate to study the effect of antiseptics on Staphylococcus bacteria.

Microbiological challenge testing has been and continues to be a useful tool for determining the ability of a product to prevent the growth of pathogens. Microbiological challenge tests also play an important role in the validation of processes that are intended to deliver some degree of lethality against a target organism or group of target organisms.

The design, implementation, and assessment of microbiological challenge studies is a complex task that depends on factors related to how the product is formulated, manufactured, packaged, distributed, prepared, and used.

When conducting a microbiological challenge study, a number of factors must be considered.

These include:

(1) the selection of appropriate pathogens,

(2) the level of challenge inoculum,

(3) the inoculum preparation and method of inoculation,

(4) the duration of the study,

(5) formulation factors and storage conditions,

(6) sample analyses.

The interpretation of the data and pass/fail criteria are critical in evaluating whether a product needs time/temperature control for safety.

It is also important to incubate and prepare the challenge suspension under standardized conditions and format. Shifts in the incubation temperature used to propagate the challenge organisms and the storage temperature of the product have been shown to change the length of the lag period of the challenge study itself.

The inoculum level used in the microbiological challenge study depends on whether the objective of the study is to determine product stability and shelf life or to validate a step in the process designed to reduce microbial numbers.

The preparation of the inoculum to be used in microbiological challenge testing is an important component of the overall protocol. Typically, for vegetative cells, 18 to 24 h cultures revived from refrigerated broth cultures or slants or from cultures frozen in glycerol are used. The challenge cultures should be grown in media and under conditions suitable for optimal growth of the specific challenge culture.

The method of inoculation is another extremely important consideration when conducting a microbiological challenge study. Every effort must be made not to change the critical parameters of the product formulation undergoing challenge. There are a variety of inoculation methods that can be used depending upon the type of product being challenged.

Standard suspensions of insoluble Barium sulphate precipitates have been described by Mc. Farland (1907) and Brown (1919-20). A series of standards of different BaSO4 concentrations correspond to suspensions of different numbers of bacteria/ml.

Preparation of Mc. Farland standards.

Std. Tube no.

BaCl2 1% (ml)

H­2SO4 1% (ml)

Corresponding bacterial concentrations (millions/ml)

0.5

0.05

9.95

150

1

0.1

9.9

300

2

0.2

9.8

600

3

0.3

9.7

900

4

0.4

9.6

1200

5

0.5

9.5

1500

Use:

In a tube of the same internal diameter as the standard, prepare a uniform suspension in saline of bacteria under test to a density greater than that required.

Select a standard opacity tube of the density required and shake it vigorously until all the deposit is raised into uniform suspension.

At once compare the bacterial suspension with the standard. View with oblique illumination against a dark background or over a printed page.

Adjust the bacterial suspension by diluting with saline until it matches the standard, re- shaking the standard before each comparison.

Total Viable Count (TVC) gives a quantitative idea about the presence of microorganisms such as bacteria, yeast and mold in a sample. To be specific, the count actually represents the number of colony forming units (cfu) per g ( or per ml) of the sample.

A surface viable count is made when the bacterium is best grown in surface culture or on an opaque medium. Ten fold dilutions of the bacterial suspension are made. A suitable volume of each dilution, e.g. 0.1ml, is pipetted on to the surface of each of the plates and at once spread widely with sterile glass spreader or sterile cotton swab. The plates are incubated at 37ºC for 24 hours. The viable count is calculated from the average colony count / plate. A viable count is essential to determine the level of the challenge inoculum.

Only plates (or replicate plates from the same dilution) with 30-300 colonies are counted. Plates with fewer than 30 colonies give statistically unreliable results, while plates with more than 300 colonies are too crowded to allow all the bacteria to form distinct colonies. Usually, more than one dilution in a series is plated, just to be sure that results in a countable range will be obtained. Ignore dilutions giving results outside of the countable range.

The concentration of bacteria in the original sample is calculated as:

 CFU ml-1 (or g-1) =

 colonies on plate

 final plate dilution

Frequently, volumes other than one ml are used to inoculate the plate. For example, 0.1 ml is often used when surface-plating, as larger volumes may not be absorbed by the agar.

For this reason, the size of the inoculum is usually incorporated with the dilution factor to give the "final plate dilution" (d x i). When 1.0 ml of a 10-4 dilution is plated, the final plate dilution is 10-4. When 0.1 ml of the same dilution is plated, the final plate dilution is 10-4 X 10-1 = 10-5.

The formula becomes:

 CFU ml-1 (or g-1) =

 colonies on plate

 d x i

Of the many media available, Muller Hinton agar is considered to be the best for routine susceptibility testing of nonfastidious bacteria for the following reasons.

It shows acceptable batch to batch reproducibility

It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.

It gives satisfactory growth of most nonfastidious pathogens

Meat infusion, beef extract, acid hydrolysate of casein in the medium provide with C, H, N and other trace element sources.

Starch in the medium acts as a protective colloid against toxic substances.

Hydrolysis of starch during autoclaving provides with small amounts of dextrose which acts as a source of energy.

Nutrient Agar consists of peptone, beef extract and agar. This relatively simple formulation provides the nutrients necessary for the replication of a large number of microorganisms that are not excessively fastidious.

The beef extract contains water soluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts.

Peptones are the principle sources of organic nitrogen, particularly amino acids and long chained peptides.

Agar is the solidifying agent.

Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms.

Composition: Chlorexdine Gluconate Solution

I.P 1.5%v/v

Cetrimide Solution (strong)

Specified working range: 1:15 dilution.

Diluent: St. Distilled water.

Composition: Chloroxylenole IP 4.8 %, Terpineol BP 9.0

IP 13.1 % v/v (Alcohol)

Specified working range: 1:20 dilution.

Diluent: St. Distilled water.

Composition: Povidone-iodine

IP 5%w/v. (0.5%w/v available iodine).

Purified water IP.

Media: Muller Hinton Agar (200ml)

Nutrient Agar (40ml)

Diluent: Autoclaved distilled water (50 ml)

Sterile Saline (100ml).

Organism:

Glass ware: Sterile assay tubes (15)

(10 ml) Sterile glass pipette (5 pipettes)

Petri plates (15)

Miscellaneous: Auto pipette (vol. 1000ul, 100ul)

Sterile tips (vol. 1000ul, 100ul)

Cotton Swabs.

Instruments: Incubator at 37ºC.

Votex.

Autoclave

Oven

Laminar Air Flow Unit.

HiMedia Laboratories Pvt. Ltd.

Ingredients: gms/litre

Peptic digest of animal tissue 5.00

Beef extract 1.50

Yeast extract 1.50

Sodium Chloride 1.50

Agar 15.00

Direction of preparation:

Suspend 28.00 gms in 1000ml of distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121ºC) for 15 minutes.

The mouth of the nutrient agar flask was flamed to sterilize it and sufficient nutrient agar was poured into the sterile plate (approximately 20ml). The plate was then covered with the lid and then placed on a flat surface and slightly rotated it so that its agar was equally spread on the surface on the plate. The agar solidified in a span on 10 minutes.

2. Mueller-Hinton (MH) agar:

HiMedia Laboratories Pvt. Ltd.

Ingredients: gms/litre

Beef infusion from 300.00

Casein acid hydrolysate 17.50

Starch 1.50

Agar 17.00

Direction of preparation:

38.0 gms in 1000ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121ºC) for 15 minutes. Mix well before pouring.

The mouth of the Mullar Hinton flask was flamed in order to sterilize it and poured sufficiently into the plate (approximately 20ml). The plate was then covered with the lid and placed on a flat surface and slightly rotated it so that its agar was equally spread on the surface on the plate. The agar solidified in a span on 10 minutes.

3. Sterile saline:

To prepare saline water 100 ml of distilled water was poured into the flask and gms of sodium chloride was added to the distilled water. It was then autoclaved at 15 lbs pressure (121ºC) for 15 minutes.

4. Inoculum preparation:

Using colonies taken directly from an overnight (20 to 24 hou r) nutrient agar culture plate, a suspension of the test organism ( S. aureus ) was prepared in sterile saline. The suspension was adjusted to a turbidity equivalent to a 0.5 McFarland standard. This suspension contained approximately 1 to 4 x 108 CFU/ml. Within 15 minutes after adjusting the turbidity of the inoculum suspension, it was used for plate inoculation.

To check the growth of Staphylococcus aureus bacteria on the Nutrient Agar plates it is necessary to check the viable count of the bacteria. The 10 fold dilutions were carried out and 10-1, 10-2, 10-3, 10-4and 10-5 dilutions were plated.

Using colonies taken directly from an overnight (20 to 24 hour) nutrient agar culture plate, a suspension of the test organism ( S. aureus ) was prepared in sterile salinein a sterile assay tube. The suspension was adjusted to a turbidity equivalent to a 0.5 McFarland standard.

5 Sterile assay tubes were labeled as follows - 10-1, 10-2, 10-3, 10-4, 10-5

Using a sterile 10 ml glass pipette 4.5 ml of sterile saline was added to each tube.

0.5 ml of S. aureus suspension was transferred to the first assay tube labeled 10-1 using a 100 – 1000 ul autopipette .

The tube was vortexed and 0.5 ml of culture from this tube was transferred to the second tube labeled 10-2

Again this tube was vortexed and 0.5 ml of culture from this tube was transferred to the third tube labeled 10-3

Similarly dilutions were carried out for the remaining tubes.

After all the dilutions were done, 0.1 ml of the culture (using a 20 – 200 ul autopipette) from each tube was spread plated on respective nutrient agar plates using a sterile cotton swab.

All the plates were incubated at 37ºC for 24 hours.

Microbial Challenge test, which is established by internation practice, is a most widely used to method to evaluate the efficacy of antiseptics. The test is performed over a period of time to simulate clinical scenarios in the process of manufacturing and handling. In this test, samples are inoculated with the test microorganisms and then inspected visually and determine if the antiseptic system is effective. The microbial challenge test method is the classical means to evaluate antiseptic efficacy because it closely mimicks the practical situation

Varying dilutions of the antiseptic were made in sterile assay tubes.

To each dilution add 0.1ml of the culture ( using 20 – 200 ul autopipette ) was added from the inoculum prepared for viable count.

Each tube was vortexed and allowed to stand for 1 minute.

After 1 minute, 0.1ml ( using 20- 200 ul autopipette) from each tube was taken and spread plated on the respective Muller Hinton agar plates using sterile cotton swabs.

All the plates were incubated at 37ºC for 24 hours

1. Viable count:

Dilution

No. Of colonies

10-1

Mat growth

10-2

Mat growth

10-3

Too many to count (TMTC )

10-4

101 X 4= 404

10-5

12

2. Microbial challenge test:

Sample

Dilution

No. of Colonies

A

1:5

0

1:16

1

1:60

0

B

1:10

0

1:20

1

1:50

0

C

1:5

0

1:15

0

Full strength

0

10-4 = 101 x 4 = 404

= 404 x 104 x 10 = 4.04 x 107

~ 4 x 107 cfu/ ml.

10-5 = 12 x 105 x 10

= 12 x 106

= 1.2 x 107

~ 1 x 107 cfu/ ml.

Average: 4 cfu/ ml.

From the viable count the cell density of the initial inoculum was 4 cfu/ml.

Microbial challenge test:

For sample A

In the 1: 15 dilution and the 1: 60 dilution there is no colony growth. Whereas in the 1:16 dilution there is presence of 1 colony of Staphylococcus aureus Bacteria.

For sample B:

In the 1: 10 and 1:50 dilutions there is no colony growth. However in the 1: 20 dilution there is growth of a single colony of the bacteria.

For sample C:

In the dilutions of 1: 5, 1: 15 and full strength dilutions do not have any bacterial growth and therefore no colonies are observed on the plate.

From the observations it can be concluded that the sample A of the antiseptic is effective on staphylococcus bacteria within and beyond its range.

The conclusions that can be drawn from the observations are that the sample B of the antiseptic agent is effective within and beyond its range.

The sample C is effective on the staphylococcus bacteria this conclusion can be drawn from the observation of no bacterial growth on the dilutions of sample C.

Therefore from the results obtained it can be concluded that the staphylococcus bacteria is killed with the help of every antiseptic with varying concentrations in the specific constant time. This proves the effectiveness of the antiseptic. However it is recommended in hospitals and laboratories to change the antiseptic monthly because the bacteria become immune to the antiseptic after a certain period. Therefore with the help of this experiment I have checked the effectiveness of the antiseptics on the Staphylococcus aureus bacteria.

From the Microbial Challenge Test conducted it is observed that all the three antiseptics are effective with and beyond their respective range. However comparing the antiseptics among each other, it can be concluded that the sample C is most effective, since the Sample A and Sample B have 1 colony growth in one of the dilutions. However Sample C does not have any colony growth. The third sample is most effective because its composition includes Povidone- iodine which is not included in the other two samples. However a further question for research has been probed in this experiment to identify if these antiseptics cause any harm to the human body. There is a suspect that these antiseptics are toxic to the human body. Therefore these antiseptics are on account of further research.

The Mechanism of Action and Efficacy Evaluation Methods for Preservatives

Hongyu Xue, Guiqi Yin, Evelyn G. Su

Nanjing Zhongshi Chemical Co., Ltd.

http://www.merck.com/mmhe/sec17/ch188/ch188d.html

http://agrc.ucsf.edu/supplements/immune/immune_system_06.html

http://www.bd.com

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Influence Human Being That Affecting The Shoppers Behaviour Marketing Essay

Marketing » Influence Human Being That Affecting The Shoppers Behaviour Marketing Essay

Hence, the expanding application of scent as a component of store atmosphere that led to increase the awareness in consumer marketing. To adopt scent marketing strategy into service process to increase shopper’s cognitive and behavioural responses by retail settings. Since retail atmospherics mediate the duration and frequency of the shopping trip, which has direct affection to spending, average sales transactions, even a lift of the sales significantly (PEOPO, 2008).

Therefore, to investigate the trend which use of scent as atmospheric cue as marketing strategy by retail stores in Hong Kong, researches will be conducted in relation to Hong Kong market because the market is still in infancy stage but high potential to grow (BRANDCFE, 2007).

By concluding the observation that scent marketing has been well established and widely use to improve service experience. "Our body needs us to breathe, so that scent marking prevails hard selling", which is reveal the effectiveness of scent marketing is fruitful because people may ignore the message that the posters conveys or they may neglect the salesmen’s persuading words (BOSS, 2010). As scent will be directly channeled to Limbic System. That can process the human's emotion and memorizing the effectiveness of the sub-conscience and consuming behavior which is passively affected by scent (NEWSTATE, 2010).

The purposes for this study are to provide information of human behaviour toward scent marketing in retail industry of Hong Kong. This study provides timely and meaningful to this industry. On the other hand, the aspect of this research towards retail industry as academic research which is to furnish a clearer picture how scent marketing can carry out in Hong Kong.

After determine the purpose of this study, the summarized research aim is as follow: “To study Consumer Perception as Mediators of the Ambient Scent on Purchasing Behavior in Retail Store (Duty-Free). The factors affecting the intention to actual purchase are effect of scent, perceptions of merchandise quality and global environment, as well as the amount of spending in a retail store with the mediating role of evaluating the shoppers’ purchasing behaviour and attitudes toward the moderating role of gender which applies to retail store.

As previously stated, this study is to examine the effect of ambient scent on consumer perceptions and purchasing behaviour in Hong Kong retail store. In order to undertake this research, Theoretical framework is necessary for analyzing the influences. The following section will explore the various theories and the available of literature findings which is relevant fields of interest that service environment, scent as environmental cues, effects on perceptions and behaviour, as well as those have potentially apropos of moderating variables of gender.

Ambient Scent and Marketing

Atmospherics are subset of widespread research streams on physical environment in service industry (Baker, 1989; Bitner, 1986; Booms and Bitner, 1982). Based on some extensive literature review that indicated the retail environment is undoubtedly influence shoppers’ behaviour (Turley and Milliman, 2000) and increase sales (Stanley and Sewall, 1976). In addition, retail atmosphere can mediate merchandise evaluation (Wheatly and Chiu, 1977; Rappoport, 1982; Bitner, 1986) and customer satisfaction (Bitner, 1990). Moreover, customer attitudes toward retail store environment as well as products (Darden and Erdem, 1983).

Atmospherics and Service Environment

The atmospherics concept defined the effort to create desirable environment of buying process is needed to induce consumer emotional responses and increase the probability of purchase ultimately. Hence, atmosphere form an undeniable competitive advantage (Kotler, 1973; Mehrabian and Russell, 1974; Baker, 1986; Bitner, 1992; Baker, Grewal and Parasuraman, 1994).

The Theoretical Models of Consumer Response to Atmosphere is based on the framework of Stimulus-Organism-Response (SOR) paradigm (Donovan and Rossiter, 1982; Spangenberg, Crowly and Henderson, 1996) as follow:

The pleasure-arousal-dominance (PAD) Model (1974)

Mehrabian and Russell proffered theoretical model in which emotional states are elicited by environment stimuli and act as mediators to acquired affective responses rather than approach-avoidance behaviour. Environmental cues coming out from the physical circumfluent can excite voluntary or involuntary emotional reactions.

Servicescape Model (1992)

As clientele and employees both perceived the objective environmental cues, Bitner designed the integrated framework about the effect of physical surrounding on consumers and service personnel interactions. In turn, the approach or avoidance reactions can be qualified toward surroundings (Mehrabian and Russell, 1974).

Perceptions toward environmental cues led to certain emotions, beliefs and physiological sensations that influence behaviors (Bitner, 1992). Service setting is notion of non-verbal communication that affected shoppers’ beliefs of organization, employees and merchandise by cognitive responses. Therefore, that can affect the shoppers with unrelated beliefs and emotions toward service setting (Bitner, 1992).

Model of Influence of Ambient Scent on Consumer Responses (1995)

Gulas and Bloch based illustration the effects of ambient scent toward emotions and behaviours. The ambient scent is objectively assumed that is dependent on different individuals and the seal acuity of individuals under the physical environment (Gulas and Bloch, 1995).

Scent awareness and emotions as potential moderators that comprise other atmospheric factors and congruity of scent can evaluate the products offering and assessments of the overall service setting (Gulas and Bloch, 1995).

Then, environmental psychology models are beneficial in determining the pertinent methods and measures required to examine and analyze the impact of atmospheric cues on consumer responses to retail setting (Spangenberg, Crowley and Henderson, 1996).

The following conceptual foundations proposed the current study is based and from which the research hypotheses were drawn. It presents comprehended and potential influences of the ambient scent as atmospheric cue on various consumer responses, which previously outlined in prior sections. Detailed methodology of information which to be collected for the research will be provided next section.

Conceptual Background and Model

The present research such as Stimulus-Organism-Response (SOR) paradigm (1974), as well as Bitner’s Servicescape conceptualization (1992) takes the foundation in environmental psychology. The proposed model is in addition consistent with Gulas and Bloch’s Model of Olfaction Effects (1995). Also, it is affiliated with Lazarus’s “cognition-emotion” theory (1991), which sustained the preeminence of cognitions with the effects of ambient stimuli on evaluation toward the retail setting and affective responses.

This study designed differs from those atmospheric studies as that will not assumed shoppers’ emotions will play a role in hypothesized relationships. It examines the paths of link from environmental cue to consumer perceptions and purchasing behaviour. The approach is in concurrence with the Elaboration Likelihood Model, which provided support the belief of attitudes can be differentiated from affect (Cacioppo and Petty, 1989). The conceptualization evidence has revealed that affective responses are not developed by ambient scent all the time (Yalch and Spangenberg, 1993; Spangenberg, Clowley and Henderson, 1996; Bone and Ellen, 1999).

Consequently, the proposed flow (Figure. 1) limited the research to study the effects of environmental stimuli, ambient scent. It not only assesses the impact of internal evaluations, perceptions of merchandise quality and global environment, but also assess on an approach-related measure, purchasing behaviour. Hence, the model predict that perceptions will have mediating affect to the spending related to the retail service setting and the gender will moderate the relationship among the ambient cue and consumers; evaluation and behavior reactions.

Fig.1 The proposed flow for hypotheses testing

Accordingly, based on conceptual model of the above, research objectives are as follows:

Provide comprehensive understanding of ambient scent as an environmental cue impact on shoppers’ perceptions regarding retail store in Hong Kong.

Justify the factors leading to the phenomenon in order to design the desire service environment of retail store to shoppers that affects the purchasing behaviour of actual expenditure.

Determine the assessment of whether male or female (gender) that affected by retail store atmospherics.

Evaluate the scent marketing strategy as differentiating tools for retail store can be improved.

Research Hypotheses

The existence and valid studies between retail settings, shoppers’ perceptions and intended behavioural responses and reactions have been established by numerous researchers (Bitner, 1990; Baker, Levy and Grewal, 1992). Hence, the research hypotheses on the impact of ambient scent are primarily in the following, which based on the literature related to the main stream, individual shopper interactive influenced by environmental stimuli on empirical evidence.

The conclusive findings about the influence of atmospheric stimuli on merchandise evaluations in the relevant studies (Gardner and Siomkos, 1986). The following hypotheses are derived through cognitive approach and processing to atmospheric cue (Shostack, 1977; Lazarus, 1991). That is concordance with the study that discovered the existence of pleasant ambient scent positively affect shoppers’ perceptions toward product quality and the retail setting (Spangenberg, Crowley and Henderson, 1996). Also, the assessment of global environment and retail density could be affected by similar fragrance that influences shoppers’ cognitive responses (Michon, Chebat and Turley, 2005). Moreover, many journal founded that merchandise and retail setting are inference concern with environmental cues (Gardner and Siomkos, 1986). Since atmosphere contains multidimensional concept, which assumed that impact of associated environmental factors will be greater than the sum of its parts (Gulas and Bloch, 1995). It is therefore hypothesized that:

The direct and positive relationship among the existence of pleasant ambient scents and intentions of purchase, with consumer spending of transaction have established in studies (Hirsch and Gay, 1991; Hirsch, 1995; Spangenberg, Crowley and Henderson, 1996). Nonetheless, presence of scent demonstrated may certainly affect shoppers’ shopping time and frequency, even chances of return to store to acquire the products (Bone and Ellen, 1999). Thus, it is hypothesized that:

As mentioned before, theory of “cognition-emotion” supported that emotional responses can be engendered without cognition occur (Lazarus, 1991), which presented as a base for subsequent behaviours (Cacioppo and Petty, 1989). The premise about emotions do not perform as mediators relationship among environmental stimuli, consumer appraisals and behavioral responses that supported by the empirical evidence (Chebat and Michon, 2003).

Various researches have observed that the comprehensive perceptions of physical retail environment and of product quality are affect the consumer behaviour, thus, revealed the shoppers’ evaluations is independently affect both approach-avoidance behaviors as well as the actual spending (Zeithaml, 1988; Baker, Levy and Grewal, 1992; Donovan et ai.,1994). The influence of shoppers’ perceptions on behaviour still has rooms to research, as to find out the importance of perceptions in service environment. In consequence, it is supposed that:

The findings showed that there is possible to hypothesize the perceptions may follow a path than inferences on merchandise quality (Baker, Grewal and Parasuraman, 1994). Therefore, the assessment of product quality served as antecedents toward the evaluations of service setting and performing as mediator of the relationship among environmental stimuli with shoppers’ perceptions of retail environment. Hence, it is hypothesized that:

To review the olfaction research found that women tend to enjoy shopping in a scented atmosphere (Robert and Kerry, 2007). In addition, female perceive superior emotional responses with affective and evaluative reactions than the counterpart of male (Bone and Ellen, 1999). The appearance of variance is the possible result of hormonal factors, together with women’s higher sense in acuity and verbal skills. That sensitivity have enhanced by olfactory cues and thus familiarity with smells (Levine and McBurney, 1986; Richardson and Zucco, 1989; Ellen and Bone, 1998; Bone and Ellen, 1999). Hence, it is assumed that:

To analysis the effects of scent on consumer perceptions and purchasing behaviour towards retail environment, both primary data and secondary data should be collected.

To start with secondary data, the data collected for purpose other than the problem at hand (Malhotra & Birks 2007). It includes internal and external data (Malhotra & Birks 2007). Internal data is information generated in –analysis of earlier studies such as customer files, like past customer transactions (Schiffman & Kanuk 2007). Company report like The Body Shop, Starbucks and Mrs. Fields will collected as internal data. This gives the examples and past information of the new trend of scent marketing.

Apart from internal data, external data is collected by an outside organization. Federal government is the main source which publishes information collected by scores of government agencies about the economy, business and virtually all demographics of the population (Schiffman & Kanuk 2007). For this dissertation, text book is used to learn about theory development and establish research approaches, as well as methods of data collection. Besides, academic journals and articles. will also collected as external data to provide up-to-date and rigorous reports on research of the study, which gives different ideas that to be summarized with respect to the study. Moreover, the industrial reports and news of retail operation and scent marketing strategy are collected to establish the background of this research and provide useful figure and picture to describe the trend of rising attention with emotional bonding towards retail environment. Nevertheless, the census data from Hong Kong Government and experimental research company to provide accurate figure toward the retail industry.

To mention about primary data, it is originated by the researcher specifically to address the research problem (Malhotra & Birks 2007). Primary data is original and unique element of this research. It can be divided into qualitative and quantitative information.

For this dissertation, focus group is used to collect qualitative information. This provides views of consumer behaviour on ambient scent. The increased understanding provided is a key to reduce negative aspects of consumer behaviour and scent marketing which so-called ‘dark side’. Research focused on understanding consumer experiences is called interpretivism (schiffman & Kanuk 2007). However, if there is any idea uncovered, quantitative studies should be tested. Therefore, quantitative information is collected. For this study, observation research is used to determine the research sample and face-to-face questionnaire survey with representative sample from the wider population are conducted in order to measure the behaviour, emotions and purchasing decision in terms of actual spending of potential consumers in Hong Kong. Result are used for making strategic marketing decision whether to implement scent marketing in Hong Kong retail industry ( Schiffman & Kanuk 2007).

Based on the information to be collected, the research designed for the study is as the follow:

First of all, secondary data is collected from online databases, which are ABI/INFORM of City University. Moreover, text books like “Marketing”, “Service Marketing”, “Scents” and “Consumer Behavior” are used to provide basic information to study. Besides, some of the academic journals and articles are searched through we'd searching engines such as Google, etc. Finally, Hong Kong Government website provides the census data and industrial reports.

To achieve triangulation between quantitative and qualitative information, the overall research design will be as follows:

The quantitative research is conducted to quantify data and apply forms of statistical analysis (Malhotra N. & Birks D. 2007). It is descriptive in nature and used to understand the effects of various promotional inputs on the consumer, thus enabling marketers to “predict” consumer behaviour (Schiffman & Kanuk 2007). For this study, observation and face-to-face questionnaire survey will be used.

Observational research is the recording and counting of behavioural patterns of people to obtain information about the phenomenon of interest (Malhotra N. & Birks D. 2007). And also, it is an important method of consumer research because marketers recognize that the best way to gain an in-depth understanding of the relationship among people towards products and environment is by watching through the process of customers get into the store and whether purchase the merchandise or not (Schiffman & Kanuk 2007). Therefore, it is the first step of conducting marketing research as the researcher observe and record the numbers of customer who have intention to visit the shop and make actual purchase. This is used to determine the appropriate time of the face-to-face questionnaire survey to be conducted. The structure observation will be conducted as the researcher clearly defines the behaviours to be observed and the techniques by which that will be measured.

Conducting survey in familiar and non-artificial surroundings, people tend to act more naturally that allows researchers to obtain data on subjects’ actual emotions, perceptions and behaviour. Thus, this study will take place at Nuance-Watson, the leading retail operator in Hong Kong, provides wide range of products including watches and jewellery, fashion and accessories, perfumes and cosmetics, electronic equipment and clothes, etc. The result and findings are consequently more representative to the situation. Additional consideration was given into selection process to ensure that the retail store would be a neutral environment. Hence, the potential for observer bias can be reduced and the reliability of the data can also be enhanced.

Then, before doing face-to-face questionnaire survey, sampling techniques should be carried out. For the quantitative research, large and probability samples which each element of the population has a fixed probabilistic chance of being selected for the sample is used (Malhotra N. & Birks D. 2007). The data collection instruments are valid and reliable, thus the results can be viewed as representative of the universe (Schiffman & Kanuk 2007). There are four probability sampling methods that are simple random, systematic, stratified and cluster sampling (Malhotra & Birks 2007). The cluster sampling technique is adopted this time. It is because the sample size is clustered into 3 groups by geographically, New Territories, Kowloon and Hong Kong Island that with different purchasing behaviour while under a "scented" retail store.

In the study, target group was retail consumers aged 15 or above who regularly purchased in retail store in Hong Kong. The reason for this selection was that during this type of consumers are in their working period that the teenagers may get the part-time jobs and adult normally have earning their own income. Therefore they have sufficient purchasing power to buy product. The sample size of this survey is set at 100, as the resource limitation. The manpower and time give the restriction to carry out larger sample size for the research. However, it is believed to be good enough for studying the potential consumers and for suggesting the recommendations.

After setting the sample size, the face-to-face questionnaire survey of quantitative research will be conducted. Respondents are intercepted while they shopping in the retail store (Malhotra & Birks. 2007). They may be questioned there and asked about their consumption experiences. Therefore, questions with predefined possible responses and open-ended questions that has to be code numerically. Most questionnaires include attitude scales and, the questions are not disguised (Schiffman & Kanuk 2007).

Once the questionnaire was finished off, face and content validity, pilot testing of 30 respondents will be conducted to improve questionnaire by identifying and eliminating potential problems (Malhotra N. & Birks D. 2007). All aspects of the questionnaire should be tested, including question content and instructions, wording, sequence, form and layout, question difficulty. More importantly, it ensures all answer options are provided. The respondents should be similar to those who will be targeted in the actual survey in terms of background characteristics, familiarity with topic, and attitudes and behaviours of interest.

Due to triangulation, besides the quantitative research that qualitative research is needed. It is an unstructured, primarily exploratory design based on small samples and intended to provide insight and understanding (Malhotra & Birks. 2007). However, the findings tend to be somewhat subjective since it is administrated by highly trained interviewer-analysts who also analyze the findings (Schiffman & Kanuk 2007).

For this study, focus group will be carried out which is a discussion conducted by a trained moderator among a small group of participants in an unstructured and natural manner (Malhotra & Birks. 2007). It is generally made up of 8-10 members. Due to group of fewer than 8 are unlikely to generate the momentum and group dynamics necessary for a successful session. Likewise, group of more than 10 may be too crowded and may not be conducive to a cohesive and discussion (Malhotra & Birks. 2007).

Method to organize focus group

The group discussion “focused” on the subject of research interest to gain insights by creating a forum where participants feel sufficiently relaxed to reflect and portray their feelings and behavior, at their pace and using their language and logic (Malhotra & Birks. 2007). However, since sample sizes are necessarily small, findings cannot be generalized to larger populations (Schiffman & Kanuk 2007). Hence, quantitative research is needed. The type of questions should be open-ended, unstructured questions and further probing by the interviewer.

Moreover, the sample size should be small with non-probability sampling methods as the findings are generally not representative of the universe under study (Schiffman & Kanuk 2007). The non-probability sampling techniques are convenience, judgmental, quota and snowball sampling. However, convenience sampling is applied as only the shoppers who have intention to walk around or consumed in the retail store are selected.

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Tile And Introduction Concept Of Physiological Properties Biology Essay

Biology » Tile And Introduction Concept Of Physiological Properties Biology Essay

Dissociation constant “pka” It shows the charge on the molecules. Partition coefficient shows the substance which is well partitioned in liquid and water both the parameters are required for understanding the behaviour of the drug, many substances would varied from its physical and chemical properties. So it’s important to determine ionic form of a present molecule is necessary.

Partition coefficient and PKa both in combination can be help full to determine distrubutation of drug compound in a system. Absorption execration and penetration. Affects both the parameters.

Dissociation constant

Ionisation of both acid and base

Pka is not dependent on the concentration for acid

Pka = -log 10 (ka) where ka = (H+) (A-) / (HA)

Concentration ionisation constant, measured by spectroscopy. It is temperature dependent constant

Log p and partition coefficient

It can be shown by

Partition coefficient P = (organic) / (aqueous)

Log P = log 10 (partition coefficient)

Where log D = distributation coefficient

{{54 Earll, Marks. 1999-2006 ;}}

There may be different choose of partition solvent

Octanol the most commonly used solvent

Through which partition or distrubutation ratio can be determined. It’s also determines the distrubutation or partition law. which shows inconsiderateness to concentration and temperature .Concentration studies shows that distribution ratio was clearly concentration dependent in case of” benzoic acid” between water and benzene

The octal water coefficient studies show the resemblance to the body system .so it is mostly used solvent for determination of partition coefficient. Its shows different quantitative structural activity relationship. It shows correlation of biological activity and chemical structure .its lead to high activity, application, implementation in the theory,

It can be shown by BR = a+Bb+cC+......

Where BR is Biological response, B, C are the molecular properties and log Kow, shows large fraction of QSARS relation BR = a+b log Kow

Biological response interferes with the large numbers of reaction to be undertaken in concerned with interaction of xenobiotic activity (in vivo), and in sense of chemical interaction in biological reaction (in vitro). Concepts of pka and partition coefficient studies is relatively important in drug discovery or formulation in pharmaceuticals .it is also showed in vivo chemical studies as Bioaccumulation and relates with the tissue partition coefficient.

{{55 Sangster. 1997 ;}}

Physiochemical properties serve to be the important factor in the drug penetration, solubility, permeability, ionisation, bioavailability, and also adsorption surface.

{{57 Pallasch, Thomas, J. January 22 1988 ;}}

Pka is an essential factor in determining solubility and lipophilicity of drug molecule for better solubility, drug need to be converted into suitable salt form. Traditional method for calculation of pka was potentiometric titration. It is available commercially, due to its high throughput (up to 30 compounds / day) with mini sample requirement method used was Yasuda Shedlovsky Method.

Spectral gradient analysis is recently used technique. UV absorption spectrum is used . Along the GLPKa Sirius Instrument.

Measurement of physiochemical properties can be done along with the properties

Solubility is an important factor in drug absorption. low drug solubility which is always associated with plasma binding, slow tissue distribution, drug-drug interaction .so overtaking factors to determine solubility of a particular drug .Methods such as Potentiometery Titration and Nephetrometric Assay can be implemented but this methods having high throughput, its solubility range is limited .so solubility in buffer solution is measured by LC/MS based solubility determination protocol

Partition coefficient and distrubitation coefficient are the two factors between two immussiable solvent phase (octonal and water) are mostly used to determine lipophilicity of organic compounds.

In a development of drug log D at PH 7.4 is widely used which shows an indication of lipophility of drug molecules at PH of blood plasma. It is necessary to determine the PH due to changing environment of biological system such as gastro instential tracks for proper permeability. Lipophility is one of the factors which are related to ADME properties. So drugs with low or high lipophilicity, shows drug with low lipophility have improper absorption through passive transport and with high lipophlic molecule may get fascinated inside the membrane. Other complication are also related to high lipophility is important. Indirect methods such as REVERSE PHASE HPLC have been proven to be high throughput method. It is an indirect method for lipophilicity determination but traditional methods such as conventional SHAKE AND FLASK method is still used to its simplicity and cost efficiency.

{{56 Yan, Zhengyin.Caldwell, Gary, W. 2004 ;}}

Fluconazole used in the form of semisolid formulation to be applied tropically. It is used as an antifungal agent. To determine partition coefficient is necessary for studies. Distribution of drugs, partition gives information on surface tension, hydrolysis, solubility, dissociation of substance along with structural formula. Partition coefficient of solute between octanol and water is widely used to predict drug pharmacokinetic properties ,techniques involved as traditional shake flask method, potentiometeric titration , liquid chromatography , counter current distribution ,

Drug shows toxic effect and also reduced systemic absorption due to the drug contacted with adsorption force which may be physical or chemical applied to them. Adsorption is important phenomenon in terms of interaction of drug and excipients. The is effected by many different factors such as ph, time of surface area, nature of adsorbent used. “Langmuir and freundlich” adsorption isotherm equation describes adsorption theoretically.

Fluconazole is a bis –triazole antifungal agents, it highly reactive against Cryptococcus neoforman and Candida species in vitro because of their higher infection of Candida species except C albuian they are highly susceptible to oral formulation so needs an alternative antifungal therapy. So partition coefficient of fluconazole is studied in different organic and phosphate buffer to identify the technique which is suitable for tropical use. physiochemical properties such as adsorption , partition coefficient are important to study for development of dosage form , so that behaviour of drug can be understood through which suitability and compatibility of drug can be predicated.{{59 Hajare,Ashok,A.Mali,Mahesh,N.Sarvagod,Sushil.Kurane,Sachin.Patwardhan,Shweta.and.Dange,Arun,S. April-June 2009;}}

Studies show that bioavailability of fluconzole is more concerned with IV and oral administration.

Pharmacokinetics of fluconazole

Forms of administration

1. Administration through intravenous injection by rapid bolus or infusion.

2. Oral administration through tablet, capsules or solution /suspension

Studies proven on voluntaries shows

Tissue penetration of fluconazole

Penetration in the tissue and fluid is important, fluconazole having low lipophility due to low affinity towards plasma proteins. It readily penetrates into body tissue by the means of passive diffusion. Long plasma life gives better distribution phase, which is necessary for fluconazole, available at the site of infection during implementing dosage. It has also properties to penetrate through CSF by the oral means.

Correlation to studied, fluconazole posses good pharmacologic properties due to rapid absorption due to water solubility, high oral bioavailability, and volume of distributation is high due to the relatively high plasma concentration along with high oral bioavailability in feeding and fasting state. In gastroinstenial disease it can be also admistred due to proper absorption, which shows extreme distbutation of fluconazole in body .Due to its low affinity for plasma proteins. {{58 Brammer, K.W.Farrow, P.Rand.Faulkner, J.K. March-April 1990 ;}}

Buffer Solutions

Method: Part one

First prepare four different solutions A, B, C and D in following concentration

Solution A, 30ml of sodium acetate (CH3COONA), with 0.1m was added with 30ml of acetic acid (CH3COOH) with 0.1m.

Solution B was also prepared by adding 20ml of sodium acetate (CH3COONA) with 50ml of acetic acid (CH3COOH) with 0.1m of both.

Solution C, prepared by adding 10ml of sodium acetate (CH3COONA) with 50ml of 0.1m acetic acid (CH3COOH) .

Lastly Solution D was prepared with 50ml of sodium acetate (CH3COONA) with 100ml of 0.1m, CH3COOH.

Next step was to measure the ph by PH meter of solution A, B and C along with its temperature, values are noted.

Part second:

It involves the titration of 50ml of solution D with HCL along the addition of 2ml of HCL each time and measurement of PH to be obtained until the total volume up to 90ml. Reading of PH was obtained by titertaion of solution D with 0.1m HCL.

TABLE

52ml

4.28

54ml

4.17

. . 56ml

4.08

58 ml

3.98

60 ml

3.83

62 ml

3.65

64 ml

3.35

66 ml

2.78

68 ml

2.33

70 ml

2.17

72 ml

1.93

74 ml

1.84

76 ml

1.78

78 ml

1.72

80 ml

1.65

82 ml

1.63

84 ml

1.65

86 ml

1.59

88 ml

1.55

90 ml

1.54

Part third

It involves the titration of 50ml of solution D with 0.1m of NAOH. Adding 2ml of 0.1m NAOH each time along with notification of PH value until the total 90ml volume, 20 different reading where obtained for Alkali.

TABLE

52ml

4.61

54 ml

4.45

56 ml

4.56

58 ml

4.62

60 ml

4.67

62 ml

4.75

64 ml

4.82

66 ml

4.84

68 ml

5.06

70 ml

5.01

72 ml

5.11

74 ml

5.16

76 ml

5.26

78 ml

5.39

80 ml

5.72

82 ml

5.78

84 ml

6.22

86 ml

9.88

88 ml

11.14

90 ml

11.47

Note: each time tip of PH meter is to be cleaned with distilled water in order not to vary the readings.

Readings of both acid and alkali were obtained to plot a graph and calculate the value of pKA and Ka along with the buffer capacity of both. Experiment was performed in the group of three.

Method

To determine partition coefficient of fluconazole .Two different solution where prepared, solution A and Solution B

Part first

Preparation of solution A

Phosphate buffer of PH 7.4, in which 0.1w/v fluconazole was dissolved for preparing stocking solution A.

Solution A was used to prepare different range of calibration of each 1ml containing 5,10,15,20,25,30,35,and 40 ug/ml of fluconazole in phosphate buffer of PH 7.4, seven readings where obtained each of standard lammad max 260nm, with the help of phosphate buffer PH7.4 , it was used as a blank reading to calibrate the absorbance for each time .Reading noted are shown as follows.

Concentration of fluconazole Absorbance of lammad max 260nm

5 0.007

10 0.021

15 0.06

20 0.043

25 0.075

30 0.083

35 0.103

40 0.092

Part second

Preparation solution B was prepared with 0.01% w/v of fluconazole dissolved in 1-octanol. Second part involves the preparation of partitioning solution in four different funnels. First separating funnel should be checked for the leakage, to fit the tap at its bottom, so that after loading the solution it should not be leaked. Four different samples were prepared as follows

First solution involves 5ml solution B with 20ml of 1- octane and 25ml of phosphate buffer.

Second solution, 10ml solution B with 15ml of 1- Octanol and 25ml of phosphate buffer.

Third solution , 15ml of solution B with 10ml of 1- octanol and 25ml of phosphate buffer.

Fourth solution, 20ml of solution B with 5ml of 1-octanol and 25ml of phosphate buffer.

Four solutions are needed to be shaken gently as soon as they are prepared. Shaking for about 30-60min is needed. After shaking check weather two clear layers are appeared in the funnel. Time taken was about 55min of shaking for getting the two different layers. After getting the layers separate attach funnel to the holder stand. Amongst the two different layers, lower layer was water and upper layer was octanol. This layer where separated in two different 50ml tubes as octanol and water. Total four pairs, each of octanol and water. Last part was to recoding the absorbance on UV visible spectrophotometer of lammad 260nm of water and octanol phase. This can be shown as follows

Reading

Organic Buffer

0.041 0.172

0.096 0.150

0.106 0.044

0.133 0.166

Lab Practical Results:

Ph of three solutions where noted as follows

Solution A ph 4.82.

Solution B ph 4.41.

Solution C ph 4.13.

Calculate pka=ph+log [base/acid] pka = -log ka , so Ka = antilog pka

Concentration of base = volume /total volume* molarity.

Solution A

Pka = 4.82

Ka = 1.5*105

Solution B

Pka = 4.01

Ka =1.0*105

Solution C

Pka = 3.42

Ka =2.6* 105

Plot graphs of pH against volume of 0.1M HCl and pH against volume of 0.1M NaOH on the same sheet of graph paper and calculate the buffer capacity with respect to both acid and alkali.

Graph

C:\Users\hp\Desktop\pras.png

Buffer capacity:

Buffer capacity of acid = 15.05ml

Buffer capacity of base =27.02ml

The concentration of fluconazole in the water and octanol phase, Cwat, Corg, respectively, for each composition of the partitioned mixture from the standard curve.

Graph

C:\Users\hp\Desktop\prashant cc.png

Calibration curve, slope is obtained as follows

Slope of the line is 0.0027

Now concentration of fluconazole in organic and buffer

For the solution 1

Concentration of fluconazole in buffer = 15.18

Concentration of fluconazole in octanolol =63.70

For the solution 2

Concentration of fluconazole in buffer = 35.55

Concentration of fluconazole in octanolol =55.55

For the solution 3

Concentration of fluconazole in buffer = 39.25

Concentration of fluconazole in octanolol =16.29

For the solution4

Concentration of fluconazole in buffer = 49.25

Concentration of fluconazole in octanolol =61.48

Calculate the partition coefficient Kow of fluconazole at each of the partitioned mixture,

Kow = Corg/Cwat

Kow =0.172/0.041 = 4.19

Kow =0.150/0.096 =1.56

Kow =0.044/0.106 =0.41

Kow =0.166/0.133 =1.24

Express the average value of the partition coefficient Kow from four experiments, and compare it with values from literatures.

Average value 1.85

, standered range shows the lipophilicity value in the range between -0.89 to 2.21.so average value falls in the range of standard value.

{{60 Alimuddin,Muhammad.Grant,Daniel,Bulloch,Daryl.Lee,Noelle.Peacock,Martin.and.Dahl,Russell. May 6 , 2008;}}

Calculate the distance from the active ser oxygen to the C- terminal carbonyl carbon of the peptide?

Ans: Distance between His 57 and Ser195: 8.30 A

Asp 102 and His 57 :6.47A

C:\Users\hp\Desktop\prashant 1.png

Calculate the distance from nitrogen to the peptide carbonyl oxyg5en, what do these imply?

Ans: 2.82A, shows hydrophobic properties

Peptide specification

C:\Users\hp\Desktop\manaoj 4.png

Identify the location of loops and amino acids type. Compare these to the equivalent residue in trypsin .pdb?

Ans: Amino acids: PRO, SER, GLY, ALA, VAL.

Location: AA- Amino Acid

A A ATOM X, Z, Y ATOM CORDINATE ATOM B FACTOR

PRO 225 C 26.376 9.625 45.862 9.51

SER 221 O 23.446 4.945 48.704 9.61

SER 214 CA 28.692 7.173 36.496 8.10

GLY 216 CA 28.971 5.629 43.557 12.57

ALA 185 CA 22.264 10.587 49.513 7.42

VAL 188 CA 17.455 6.480 46.705 7.05

C:\Users\hp\Desktop\prashant05.png

Calculated the surface in the context of residue 16-245.from chymotrypsin?

Ans : The surface context residue from 16 to 245 from chymotrypsin can be drawin as follows

Area : 8021

Volume:28018

C:\Users\hp\Desktop\prashant mol.png

(Due to some errors of software it shows two images)

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