Preparation Of Crude Extracts Using Methanol As Solvent Biology Essay

In this survey, the intervention of the sponge samples prior to extraction procedure is important as it affects the bioactive compounds that could be extracted from the samples. The fresh sponge samples were dried in an oven at a temperature non lower than 40oC to forestall the oxidization of active enzymes and non higher than 60oC to avoid the high temperature from damaging the biogenic compounds. In add-on, the drying procedure reduces the H2O content in tissues, therefore forestalling the debasement by enzyme and fungous infection ( Fennell et al. , 2004 ) . In this survey, Haliclona sp. samples were dried at 45oC in an oven until the weight become changeless. After that, the dried samples were ground into all right powdered signifier to supply a higher surface to volume ratio easing the incursion of the extraction dissolver.

In this survey, the dried samples were used in the extraction procedure to obtain the petroleum infusion of Haliclona sp. alternatively of fresh samples. This is due to the high H2O content in the fresh samples may interfere with the extraction procedure and cause improper separation during partitioning ( Wright, 1998 ) . Furthermore, clip is another critical factor impacting the extraction procedure. This was proved with the trouble in covering with the fresh samples if at that place happened to be any clip hold between the sample aggregation and processing ( Kueh, 2007 ) . The dried samples are more advantageous as they allow storage for a long period before analysis. To obtain the petroleum infusion, there are several traditional ways of pull outing the bioactive compounds including utilizing the Soxhlet extractor, magnetic scaremonger, boiling, maceration, crunching and heat reflux ( Yang et al. , 2008 ) . In this survey, the maceration method was used due to its low cost and easiness in pull outing the secondary metabolites from marine sponges.

In general, the extraction method depends mostly on the type of extraction dissolvers employed, energy input, and agitation to better the chemical solubility and efficiency of mass transportation ( Yang et al. , 2010 ) . The major factor impacting the extraction procedure is the type of organic dissolvers used. Harmonizing to Kandhasamy and Arunachalam ( 2008 ) , the pick of organic dissolvers used for extraction affects the bioactive compounds present in the petroleum infusion and its activity. Likewise, the solubility and mutual opposition of bioactive compounds affect the effectivity of dissolver used for extraction. In this research, methyl alcohol was chosen as the dissolver used for the extraction procedure. This is due to high mutual opposition belongingss of methyl alcohol which is a good dissolver for pull outing broad assortment of bioactive compounds from the Marine sponges. Harmonizing to Silva et Al. ( 1998 ) , the alcoholic dissolver can perforate the cell membranes efficaciously which permits the extraction of high sums of intracellular constituents. Probe of antimicrobic activity of Marine sponge Haliclona exigua revealed that the methanol infusion of the sponge samples showed promising fungicidal activity against several species of Fungi ( Lakshmi et al. , 2010 ) . This consequence indicates that methyl alcohol is a good dissolver for pull outing bioactive compounds from natural beginnings. Besides, several surveies sing the effectivity of extraction method revealed that the methanolic infusion exhibited greater antimicrobic activity as compared with other dissolvers such as n-hexane and benzine ( Rosell and Srivastava, 1987 ) . In this research, of the 20.0 g of dried Haliclona sp. , 1.298 g of petroleum infusion was acquired and has contributed to the high output per centum of methyl alcohol which is 6.4 % indicating that methyl alcohol is an effectual dissolver for extraction.

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5.2 Screening of antimicrobic activity of Haliclona sp. methanolic petroleum infusion

In this survey, the methanolic extract readying was used to prove on a list of trial beings including 12 bacteriums, 6 Fungis and 2 barms. The intent of this trial is to find the antimicrobic activity of Haliclona sp. and its possible to be recovered and developed as a new antimicrobic drug. Harmonizing to the old survey by Monks et Al. ( 2002 ) , the organic infusion from Haliclona aff tubifera demonstrated important antibacterial activity against B. subtilis whereby the strongest activity was exhibited and moderate antibacterial activity was observed on E. coli. However, this consequence opposed with the testing consequence of Haliclona sp. infusion from local Penang country whereby the methanolic petroleum infusion exhibited no antibacterial activity on E. coli. This fluctuation in the antibacterial activity could be due to several extrinsic factors. For case, method of extraction, pick of dissolver used, seasons of the sponge samples aggregation, geographical location of sponge samples, temperature, alimentary handiness are among the extrinsic factors lending to this fluctuation ( Kandhasamy and Arunachalam, 2008 ) .

The temperature has been proposed as the chief factor impacting this fluctuation. This is supported by the production of ecthyonine in Microciona prolifera which decreases in cold months. This compound plays function in the biochemical ecology of sponge, and its fluctuation could ensue from alterations in the environmental conditions ( Betancourt-Lozano et al. , 1998 ) . Bakus et Al. ( 1986 ) , in their surveies of sponges along the Mexican seashores suggested that there is a seasonal fluctuation in the activity degrees of the antimicrobic belongingss of sponges with supportive informations demoing that sponges exhibited antibiotic activity during the warm season as opposed in the cold season. Therefore, in the research of antimicrobic activity of Marine sponges, standardisation of the parametric quantities involved is critical in the findings of possible antimicrobic agents.

In this survey, the phonograph record diffusion method was employed to analyze and find the antimicrobic activity of Haliclona sp. This method is chosen as it is a comparatively robust and easy technique to execute and it gives rise to high duplicability ( Barker and Kehoe, 1995 ) . Besides, the phonograph record diffusion methodological analysis yields a quantitative consequence ( zone of suppression ) and a qualitative interpretative class ( susceptible or immune ) . It is a cost effectual method which normally provides qualitative consequences 24 hours sooner than the standard broth dilution methods ( Espinel-Ingroff, 2007 ) . Furthermore, missing the demands for specialised equipment is another advantage of this method ( Matar et al. , 2003 ) . In this survey, the spread home base method was employed. This is due to the cell denseness from this method was greater, taking in bend to comparatively smaller zone sizes ( Barker and Kehoe, 1995 ) . Initially, the inoculant was spread equally on the surface of the agar ( Acar and Goldstein, 1986 ) . After vaccination, the home bases were left to dry for 15 proceedingss at room temperature with the lid ajar. A multidisc was so placed onto the agar utilizing unfertile forceps and pressed down gently to guarantee contact. However, many factors such as agar beginning, inoculant size, phonograph record authority, incubation temperature and the length of incubation can act upon the diameter of zone size ( Acar and Goldstein, 1986 ) .

Based on the showing consequences, the methanolic petroleum infusion exhibited important antimicrobic activity. Besides, the consequences besides revealed that the bioactive compounds of Haliclona sp. exhibited antimicrobic activity merely on the unicellular procaryotes, but non on the eucaryotic beings. From the consequences, the methanolic infusion exhibited important antimicrobic activity by suppressing 6 species of bacteriums including 4 Gram-positive and 2 Gram-negative species. This consequence is in understanding with the old research done by Ely et Al. ( 2004 ) where the methanolic infusions of nine Marine sponges ( Porifera ) and two seaweeds collected from the south east seashore of India demonstrated important antimicrobic activity against one or more of the trial being. In this survey, methyl alcohol was used as the dissolver to fade out the petroleum extract paste. Therefore, a negative control with methyl alcohol was included so as to observe the solvent effects. However, the showing consequences revealed that methyl alcohol did non suppress the growing of all of the trial being. Hence, methyl alcohol is a suited organic dissolver to be used as the dissolver to fade out the petroleum infusion of Haliclona sp. in this survey.

The methanolic petroleum infusion of Haliclona sp. exhibited important antimicrobic activity against both the Gram-positive and Gram-negative bacteriums. This revealed that it possessed a wide spectrum of activity. From the showing consequences, the petroleum infusion of Haliclona sp. was observed to be exhibiting stronger antimicrobic activity on the Gram-positive bacterium as compared to the Gram-negative bacterium. This was due to the differences in the cell wall construction and composing between the Gram-positive and Gram-negative bacteriums. For the Gram-negative bacteriums, their cell wall possesses an outer membrane which constitutes the outer surface of their cell wall. This outer membrane acts as a coarse screen which exerts small control over the motion of substances such as antibiotics into the cell ( Black, 2008 ) . Furthermore, the presence of peptidoglycan or murein beds on the cell wall of Gram-negative bacteriums besides prevents the entry of bioactive compounds into the cells ( Tortora et al. , 2001 ) . Therefore, the Gram-negative bacteriums were less susceptible to the petroleum infusion of Haliclona sp. compared to the Gram-positive bacteriums which were more susceptible exhibited stronger antimicrobic activity in this survey.

In the research for new antimicrobic drugs, an of import factor that affects the findings is the inoculum size. The fluctuations in the size of inoculant are significantly act uponing the diameter of suppression zones ( Cook et al. , 1990 ) . Hence, inoculum standardisation is required in order to obtain accurate consequences and besides duplicability of the screening trial. Besides, the molten agar poured into the Petri dish on a degree organizing a unvarying deepness of 4 millimeter is of import. This is due to the deepness deeper than 4 millimeters may give false opposition consequences and overly little suppression zones whereas deepness less than 4 millimeters may give false susceptibleness consequences associated with overly big zones of suppression ( Jorgensen and Turnidge, 2007 ) . Therefore, standardisation is required during the readying of media used for testing trial in order to obtain accurate consequences.

5.3 Determination of minimum repressive concentration ( MIC )

By definition, minimum inhibitory concentration ( MIC ) is the lowest concentration of an antimicrobic drug that will suppress the seeable growing of a micro-organism after nightlong incubation ( Cook et al. , 1990 ) . MIC is employed as the most basic research lab measuring of the activity of an antimicrobic agent against a micro-organism. Therefore, MIC is the cardinal measuring that forms the footing for most of the susceptibleness proving methods ( Jorgensen and Turnidge, 2007 ) .

In this survey, the MIC values of Haliclona sp. methanolic petroleum infusion on Methicillin-resistant S. aureus ( MRSA ) , B. spizizenii, B. subtilis, B. Cereus, A. anitratus and Erwinia sp. were determined utilizing the broth dilution method. The ground for using the broth dilution method in this research was due to its standardisation and dependability which is utile for research intent to prove the susceptibleness of antibacterial drugs ( Jorgensen and Turnidge, 2007 ) . Furthermore, it is a method which is technically easy to execute and comparatively cheap. Broth dilution method for repressive finding is besides strongly recommended by Clinical and Laboratory Standards Institute ( CLSI ) to measure the microbic growing or its suppression ( NCCLS, 2003 ) . In this survey, the phonograph record diffusion method was non used as a old work performed by Rios et Al. ( 1988 ) suggesting that the phonograph record diffusion method merely allows the diffusion of polar compounds with little or average molecular size. In add-on, the diameter of suppression zone is affected by the rate of diffusion of infusion through the agar medium every bit good as the size and hydrophilicity of the infusion ( Berger et al. , 1993 ) . Likewise, a survey conducted by Klancnik et Al. ( 2010 ) besides suggested that the phonograph record diffusion method is non a dependable method for testing the antimicrobic activity of works infusions on the footing of the high MIC values obtained. This is due to the phonograph record diffusion check is non suited to the natural antimicrobic compounds that are hydrophobic in nature which prevents the unvarying diffusion through the agar media ( Mann and Markham, 1998 ) .

In this survey, the broth dilution method was chosen for finding of MIC values. However, the MIC measurings utilizing the broth dilution method are influenced by several factors including the medium composing, inoculant size, the continuance of incubation and the presence of immune subpopulations of being ( Jorgensen and Turnidge, 2007 ) . Therefore, all the parametric quantities must be standardized in order to obtain accurate measurings. In this research, the alimentary stock was employed because it is a general intent growing medium that supplies the necessary ingredients for the growing of micro-organisms and it is normally used to cultivate the non-fastidious micro-organism ( Black, 2008 ) .

However, the antibiotic resistant strains may take to mistakenly high reading of MIC values for the broth dilution method. The research done by Gaudreau ( 1997 ) exhibited that the MIC values for the immune strains of Campylobacter jejuni and Campylobacter coli are 8 to 256 times higher than the susceptible strains. This mistakenly high reading of MIC was due to the subjectiveness of reading terminal points in the broth dilution method. Harmonizing to Sauboulle and Hoeprich ( 1978 ) , a little growing ensuing from the subpopulations which were partly inhibited and a longer period required for the look of antimicrobic activity by the infusion are the part factors to this mistakenly high reading of MIC.

In this survey, the MIC values for all the trial organisms including MRSA, B. spizizenii, B. subtilis, B. Cereus, A. anitratus and Erwinia sp. were different. Therefore, this suggested that the susceptibleness of the Haliclona sp. methanolic petroleum infusion was vary for different trial being. Finally, the MIC values obtained utilizing the broth dilution method were used as a criterion to find the growing profile as it is the best method to set up the existent authority of a pure compound ( Rios et al. , 1988 ) .

5.4 Determination of minimum disinfectant concentration ( MBC )

By definition, the minimum bactericidal concentration ( MBC ) is the lowest concentration of drug that kills at least 99.9 % of cells in the original inoculant ( Levison, 2000 ) . In this survey, the MBC values recorded for B. subtilis, B. Cereus, A. anitratus and Erwinia sp. were significantly higher than MRSA and B. spizizenii. From the consequences of MBC, it suggested that the methanolic petroleum infusion of Haliclona sp. was more effectual in killing the bacterial cells of MRSA and B. spizizenii compared to B. subtilis, B. Cereus, A. anitratus and Erwinia sp. This consequence besides revealed that the trial beings possessed different susceptibleness to the infusion.

Harmonizing to Levison ( 2000 ) , if the minimum concentration of a drug that prevented turbidness has lowered the bacterial denseness from 1 x 105 cells/ml to 1 tens 102 cells/ml, the MIC that prevented the turbidness is besides considered as the MBC. In this survey, the consequences indicated that the MBC values obtained were significantly higher than the MIC values. Therefore, this consequence suggested that higher concentration of methanolic infusion was required in order to kill the bacterial cells alternatively of suppressing their growing. Generally, the MBC value for disinfectant drugs is normally the same as or non more than fourfold of the MIC value. Hence, the methanolic infusion can be said as bactericidal on all the trial organisms including MRSA, B. spizizenii, B. subtilis, B. Cereus, A. anitratus, and Erwinia sp.

5.5 The effects of add-on of infusion on the growing of bacteriums

5.5.1 Growth profiles for the Gram-positive bacteriums

For the survey of growing profile of bacteriums, the standard home base count method was employed to recite the bacterial cells present in the samples. Black ( 2000 ) described that the home base count method is by and large based on the premise in which a individual cell divide exponentially organizing a distinct settlement. In this survey, the truth of consecutive dilution and home base count method depend significantly on the homogenous dispersion of the beings in each dilution to guarantee even distribution and good growing of the being. This method was employed to find the growing profiles of MRSA, B. spizizenii, B. subtilis, and B. Cereus in this survey due to its ability to recite merely the feasible bacterial cells present in the sample as compared to the optical denseness measuring which is unable to distinguish between the dead and living bacterial cells. This is peculiarly of import as it provides the information about the effects of Haliclona sp. infusion on the bacterial cells, whether it is bacteriostatic or disinfectant ( Kueh, 2007 ) .

Based on the consequences, the add-on of Haliclona sp. methanolic petroleum infusion has significantly inhibited the growing of all the Gram-positive bacteriums in contrast to the control which is non treated with the infusion. These consequences were in understanding with the growing profile form obtained by several local research workers ( Darah et al. , 2006 ; Sasidharan, 2006 ) . Based on the growing profiles of all the 4 species of Gram-positive bacteriums including MRSA, B. spizizenii, B. subtilis, and B. Cereus, general form of growing stages with lag stage, exponential stage, stationary stage and decease stage were observed. From the consequences, the settlement organizing unit ( CFU ) obtained was observed to be diminishing at each period of intervals with the addition in the concentration of methanolic infusion. This is due to the suppression of bacterial growing by the bioactive compounds present in the infusion. The effectivity of the Haliclona sp. infusion as an antibacterial drug was increased by increasing the concentration of infusion. From the consequences, the bacterial cells were killed at the concentration of infusion that is disinfectant ( 2MIC ) .

At the concentration of half MIC, the growing profiles of MRSA, B. spizizenii, B. subtilis, and B. cereus by and large exhibited the normal growing stages. However, the growing of bacteriums by and large ceased and the stationary stage finally reached earlier and was comparatively shorter than the control. The bacterial count declined bit by bit following the short stationary stage and entered the decease stage. This consequence revealed that the infusion significantly inhibited the growing of all the 4 species of the Gram-positive bacterium. However, the suppression consequence was significantly vary for different species of bacteriums with the strongest inhibitory activity was observed on MRSA, followed by B. spizizenii, B. subtilis, and B. cereus severally with diminishing repressive activities. However, this concentration was non sufficient to exhibit important antibacterial activity of the infusion due to the high CFU count was obtained at the terminal of the incubation period. At the concentration of MIC, the growing of bacteriums did non exhibit distinguishable growing stages as shown in the control. Therefore, it suggested that the infusion exhibited important antibacterial activity on all the Gram-positive bacterium at this concentration. Harmonizing to a pharmacodynamics survey by Levison ( 2000 ) , the growing of bacteriums at the concentration of MIC must be less than tenfold addition in the bacterial denseness after 24 hours of incubation period. This statement supported that the MIC values obtained from the broth dilution method for MRSA, B. spizizenii, B. subtilis, and B. Cereus in this survey were relevant due to the low CFU count recorded after 24 hours of exposure to the infusion.

At the concentration of 2MIC, the infusion was observed to be disinfectant. In general, the growing profiles of MRSA, B. spizizenii, B. subtilis, and B. Cereus at this concentration exhibited diminishing tendency associated with low CFU count which decreased bit by bit at each exposure intervals. Therefore, the consequence indicated that higher concentration of infusion which is more than the MIC value is required to kill the bacterial cells. At this concentration, the infusion was effectual to do the lysis of the bacterial cells. The MBC values obtained by the broth dilution method were relevant because the infusion has significantly lowered the bacterial denseness to less than 1 ten 102 cells/ml ( Levison, 2000 ) .

In this survey, the coevals clip of the infusion treated civilization was a good grounds of the antibacterial activity exhibited by the methanolic petroleum infusion of Haliclona sp. From the consequences, the add-on of the infusion has significantly prolonged the coevals clip of MRSA, B. spizizenii, B. subtilis, and B. Cereus. At the MIC concentration, the coevals clip for the bacterial cells was comparatively longer than that of the control. Therefore, this consequence revealed that a longer period of clip was needed for a bacteria to bring forth two indistinguishable cells by binary fission after add-on of the infusion ( Black, 2008 ) . This status is good for the application of the infusion as an antibacterial agent.

5.5.2 Growth profiles for the Gram-negative bacteriums

Based on the consequences, the add-on of Haliclona sp. methanolic infusion has significantly inhibited the growing of the Gram-negative bacteriums in contrast to the control which is non treated with the infusion. However, the effects of suppression were significantly lower and weaker than that of the Gram-positive bacterium. Based on the growing profiles of A. anitratus and Erwinia sp. , general form of growing stages with lag stage, exponential stage, stationary stage and decease stage were observed. However, the length of each stage was changing significantly compared to the Gram-positive bacteriums. From the consequences, the settlement organizing unit ( CFU ) obtained was observed to be diminishing at each period of intervals with the addition in the concentration of methanolic petroleum infusion. Likewise, the bacterial cells were observed to be killed at the concentration of infusion that is disinfectant ( 2MIC ) .

At the concentration of half MIC, the growing profiles of A. anitratus and Erwinia sp. by and large exhibited the normal growing stages as exhibited in the Gram-positive bacterium. However, the growing of bacteriums ceased easy and the stationary stage was comparatively shorter and indistinctive than the control. The bacterial count declined in slow manner come ining the decease stage. This consequence revealed that the infusion inhibited the growing of both the Gram-negative bacteriums, but the suppression effects were much lower and weaker than that of the Gram-positive bacteriums. However, this concentration was non sufficient to exhibit important antibacterial activity of the infusion due to the high CFU count was obtained at the terminal of the incubation period. At the concentration of MIC, the growing of bacteriums did non exhibit distinguishable growing stages as shown in the control. Therefore, it suggested that the infusion exhibited important antibacterial activity on the Gram-negative bacterium at this concentration. The growing of bacteriums at the concentration of MIC is less than tenfold addition in the bacterial denseness after 24 hours of incubation period. This revealed that the MIC values obtained from the broth dilution method for A. anitratus and Erwinia sp. in this survey were relevant due to the low CFU count after 24 hours of exposure to the infusion.

At the concentration of 2MIC, the infusion was observed to be disinfectant. In general, the growing profiles of A. anitratus and Erwinia sp. at this concentration exhibited diminishing tendency associated with low CFU count which decreased bit by bit at each exposure intervals. Therefore, the consequence indicated that higher concentration of infusion which is more than the MIC value is required to kill the bacterial cells.

From the consequences, the add-on of the infusion has significantly prolonged the coevals clip of A. anitratus and Erwinia sp. At the MIC concentration, the coevals clip for the bacterial cells was comparatively longer than that of the control. Therefore, this consequence revealed that a longer period of clip was needed for a bacteria to bring forth two indistinguishable cells by binary fission after add-on of the infusion ( Black, 2008 ) .

In this survey, the chief ground for the differences in the bacterial susceptibleness to the infusion of Haliclona sp. could be due to the cell wall belongingss of the Gram-negative bacteriums. In the Gram-negative bacterium, there is an outer membrane environing their cell wall which restricts the diffusion of compounds through its lipopolysaccharide covering, as antecedently reported ( Vaara, 1992 ) . In add-on, the periplasmatic infinite contains enzymes which are capable of interrupting down foreign molecules introduced from the exterior ( Vaara, 1992 ) . Therefore, this outer membrane of the Gram-negative bacterium serves as a protective screen that prevents the bioactive compounds present in the infusion from suppressing the growing of these beings.

5.6 SEM ( Scaning Electron Microscopy ) observation of the structural devolution of the infusion treated bacterial cells

The morphology of the Gram-positive MRSA was observed under the scanning negatron microscope ( SEM ) to analyze the consequence of add-on of the methanolic petroleum infusion on the constructions of bacterial cells. SEM is a really utile tool employed to make images of the surface of micro-organisms and it besides allows the survey of external construction of the cell ( Black, 2008 ) . In this survey, the cells of MRSA exhibited a really distinguishable morphological difference after treated with 100 mg/ml of the methanolic petroleum infusion. However, the control cells which were non treated with the infusion were observed to be demoing normal coccal-shaped construction.

After 12 hours of exposure to the infusion, the morphological construction of MRSA exhibited a distinguishable difference as compared to the control cells. The bacterial cells were observed to be crumpled and shrunk. The infusion treated cells became bent together due to the secernment of gluey mucous secretion and the normal cellular maps were corrupted taking to the shrinking in the bacterial cells.

After 24 hours of exposure to the infusion, there was formation of pores or pits on the surface of the bacterial cells. This indicated that the infusion kill the cells of MRSA by organizing pores to increase the permeableness of the cell wall or by decomposition of the cell membrane which will impact the metamorphosis and physiological activities of the cells ( Sasidharan, 2006 ) . This is due to the cell wall of the Gram-positive MRSA chiefly consists a individual type of molecules that is less stiff than the cell wall of the Gram-negative bacteriums ( Tuney et al. , 2006 ) . From the SEM micrograph, it revealed that the mark of the methanolic petroleum infusion is on the cell wall due to the formation of pits. Furthermore, harmonizing to Kueh ( 2007 ) , the doomed of cellular stuffs and osmolarity control caused the surface of the bacterial cells became bent.

After 36 hours of exposure to the infusion, the construction of the bacterial cells were collapsed doing the cells lost the coccal form and the complete introversion of the cell wall was observed. From the consequence, it suggested that the cells without a normal and hardy cell wall construction explosion when exposed to low osmotic force per unit area ( Black, 2008 ) . Harmonizing to Sasidharan et Al. ( 2008 ) , the complete introversion of the cell wall indicated the doomed of cellular stuffs and organelle from the cells ‘ cytol.

Harmonizing to the research by Koprivnjak ( 2002 ) , the microbial cell wall is the mark of the infusion due to the presence of polyanionic bioactive compounds. The unity of the cell wall is chiefly depending on the Ca2+ and Mg2+ ions which ionically link the polysaccharide side ironss. The interaction between these polyanionic groups and the bivalent cations in the cell wall of bacteriums significantly disrupted the unity of the cell wall and later causes the cell lysis. Furthermore, a survey by Blondelle ( 1996 ) besides suggested that the interaction between the bioactive compounds with the sialic acid or lipid bed in the microbial cell wall will do cell lysis due to the lipid wadding in the cell wall is disrupted by the interaction.

In this survey, the morphology of the Gram-negative A. anitratus was besides observed under the scanning negatron microscope ( SEM ) to analyze the consequence of add-on of the methanolic petroleum infusion on the constructions of bacterial cells. The cells of A. anitratus exhibited a distinguishable morphological difference after treated with 100 mg/ml of the infusion. However, the control cells which were non treated with the infusion were observed to be demoing normal bacillar construction.

After 12 hours of exposure to the infusion, the morphological constructions of A. anitratus were observed to be crumpled and shrunk in contrast with the control cells which are in normal rod form. The infusion treated cells became bent together due to the secernment of gluey mucous secretion and the normal cellular maps were corrupted taking to the shrinking in the bacterial cells.

After 24 hours of exposure to the infusion, there was formation of pits on the surface of the bacterial cells which was an indicant that the infusion kill the cells of A. anitratus by organizing pores to increase the permeableness of the cell wall or by decomposition of the cell membrane which will impact the metamorphosis and physiological activities of the cells. From the negatron micrograph, it revealed that the mark of the methanolic petroleum infusion is on the cell wall due to the formation of pits.

After 36 hours of exposure to the infusion, the constructions of the bacterial cells were wholly collapsed doing the cells lost their rod form and the cell wall was wholly invaginated. From the consequence, it suggested that the cells without a normal and hardy cell wall construction explosion when exposed to low osmotic force per unit area ( Black, 2008 ) . Harmonizing to Sasidharan et Al. ( 2010 ) , the complete introversion of the cell wall indicated the doomed of cellular stuffs and organelle from the cells ‘ cytol.

5.7 Determination of in vivo toxicity utilizing the seawater runt deadliness assay

The seawater runt deadliness check was proposed by Michael et Al. ( 1956 ) . The seawater runt deadliness assay is a utile tool for the preliminary appraisal of toxicity. and it has been employed for the sensing of fungous toxins ( Harwig and Scott, 1971 ) , works infusion toxins ( McLauglin et al. , 1991 ) , heavy metals ( Mart & A ; iacute ; nez et al. , 1998 ) , cyanobacteria toxin ( Jaki et al. , 1999 ) and besides pesticides ( Barahona and S & A ; aacute ; nchez-Fort & A ; uacute ; n, 1999 ) . In this survey, the modified method of Carballo et Al. ( 2002 ) was employed to analyze the toxicity of the infusion because the seawater runt deadliness assay is more sensitive in observing the toxicity of the infusion compared to other bio-assay. Furthermore, the check was comparatively easy to execute and did non necessitate the usage of any particular equipment. However, there are several factors which critically affect the results of the toxicity trial including the H2O hardness, pH, temperature, chemical preparation, age and the developmental phase of Artemia salina ( Barahona, 2006 ) . Therefore, these parametric quantities were standardized in this survey in order to obtain accurate consequences and prevent erroneous results.

In this survey, the chief aim of the seawater runt deadliness assay is to find the LC50 ( 50 % deadly concentration ) values for both the ague and chronic toxicities. By definition, LC50 is the concentration that kills half of the sample population in a specific period of exposure. In general, the acute toxicity describes the inauspicious effects of a substance in a short period of exposure clip that is normally shorter than 10 hours. In contrary, the chronic toxicity describes the inauspicious effects of a substance over a long period of exposure clip ( Sasidharan et al. , 2010 ) . Finally, this survey is really important in finding the potency of Haliclona sp. to be developed as a safe antimicrobic drug without doing any toxic effects. In this survey, 1.5 % DMSO diluted with 3.8 % unreal sea H2O was used as the negative control. From the consequences, 1.5 % DMSO did non do any deadliness of A. Salina for both the ague and chronic toxicities. Therefore, this revealed that DMSO with the concentration less than 1.5 % was suited to be employed as the dissolver to fade out the infusion.

Based on the consequences of brine runt deadliness assay, the LC50 values for both the ague and chronic toxicities were significantly different. Most research workers came up to an understanding that there was a inclination for the LC50 to diminish with longer exposure continuance to the toxic compounds ( Barahona, 1996 ; Carballo et al. , 2002 ) . In this survey, the LC50 values recorded for the acute toxicity was 2066 µg/ml while for the chronic toxicity was 314 µg/ml. Harmonizing to Simionatto et Al. ( 2005 ) , the petroleum infusion with the LC50 value more than 1.0 mg/ml is interpreted and considered as non toxic to A. salina. In this survey, the LC50 value for the acute toxicity was higher than 1.0 mg/ml bespeaking that it is non toxic, whereas for the chronic toxicity the LC50 value recorded was lower than 1.0 mg/ml. Therefore, these consequences suggested that the petroleum infusion possessed important toxicity to A. salina.

In the seawater runt deadliness assay, the age of the runt has an influence on their sensitiveness towards the toxins, a fact that was observed by Hlywka et Al. ( 1997 ) . In this survey, for grounds of comparison and repeatability, the larvae of A. salina were employed in the seawater runt deadliness assay precisely 24 hours after hydration of the cysts. This is due to most of the eggs have already hatched within this period of incubation. Furthermore, aeration supply is another critical factor act uponing the sensitiveness of the runts towards the toxins. Therefore, it is of import for the runts to be shaken during the length of the check. Apparently, the larvae are able to stay close to the surface of solution where there is ample O supply with the solution in lasting gesture ( Hartl and Humpf, 2000 ) .

In the seawater runt deadliness assay, there may be variableness in the sensitiveness of the runts towards the poisons. Therefore, a Numberss of factors including the temperature of incubation and hatching, the minute of larvae crop, the period of clip between the crop and the oncoming of the bio-assay and the temperature, aeration supply, visible radiation, pH every bit good as the salt of the medium during the bio-assaies have to be good maintained in order to avoid the variableness in the sensitiveness of the runts ( Sorgeloos et al. , 1978 ) .

Harmonizing to the survey by Barahona ( 2006 ) , the larvae of A. salina aged 48 hours were used due to their high sensitiveness to toxic compounds. In this survey, the 48 hours old larvae of A. salina which are in their 2nd and 3rd instar larval phase were selected for the toxicity trial. This is due to the higher sensitiveness of larvae at this phase compared to the first instar phase larvae ( Soares and Calow, 1993 ) . Besides, more consistent consequences were obtained by utilizing the larvae in the 2nd and 3rd instar larval phase.

During this survey, the life and dead nauplii of A. salina were observed under the upside-down light microscope. It was observed that the dead nauplii have shrunken in their size and the liner of their variety meats seemed to be ruptured and ill-defined as compared to the life nauplii. There was besides none of any internal or external motions being observed for these dead nauplii. These observations could be due to the effects of toxication of the nauplii by the infusion of Haliclona sp. Due to this important toxicity exhibited by the petroleum infusion, therefore farther analysis and extended surveies should be carried on the infusion of Haliclona sp. in order to research the possibility of utilizing it for pharmacological intent and besides to guarantee its suitableness to be developed as a safe antimicrobic drug.

Chapter 6.0 CONCLUSION AND FUTURE RESEARCH

In this survey, the chief purpose was to analyze and pull out the antimicrobic compounds from the local Marine soft sponge, Haliclona sp. From the testing trial, the methanolic petroleum infusion of Haliclona sp. exhibited important antibacterial activity by suppressing 6 species of trial bacteriums including 4 Gram-positive and 2 Gram-negative bacteriums. Therefore, this revealed that the infusion possess a wide spectrum of activity and showed a defined potency to be developed as an effectual antibacterial drug. However, it was a comparatively inactive fungicide and antiyeasts agent due to no inhibitory activity was observed on both Fungis and barms.

On the footing of the good and important antibacterial activity exhibited by the methanolic petroleum infusion of Haliclona sp. , the in vivo toxicity of the infusion was assessed through the seawater runt deadliness assay. The LC50 values obtained for the acute toxicity was higher than 1.0 mg/ml whereas for chronic toxicity the LC50 value was lower than 1.0 mg/ml. However, these consequences suggested the important toxicity of the methanolic petroleum infusion of Haliclona sp.

In decision, the findings of this survey revealed the potency of Haliclona sp. as a beginning of antibacterial agent from the natural beginnings which is capable of controling the menaces of antibiotic opposition and outgrowth of new infective agents worldwide. Therefore, the future research should be focused chiefly on the surveies and isolation of bioactive compounds that possess important antimicrobic activity from natural beginnings to be developed as a safe, effectual and possible antimicrobic drug.