Collection and Laboratory Acclimation of Spongilla. Spp:
Protocol is design according to (Margey Tadessea, 2008) and (Prabha Devi, 2013) Spongilla. Spp shallow water sponge will manually collect from the water bodies. Sponges will carefully remove from jetty pylons with a scraper, kept wrap in plastic bags, and will immediately transport to the laboratory. Associated macro organisms (mainly algae and polychaetes) will remove from the biological material before lyophilisation. Samples of Spongilla. Spp will then identify by Polymerase chain reaction (PCR), pool, lyophilize and separately frozen at -20 o C.
Extraction of Bioactive Compound from Sponge:
Extraction protocol as describe by (Margey Tadessea, 2008), (Prabha Devi, 2013) and (G. Annie Selva Sonia, 2008) frozen sponge sample will thaw and extract exhaustively with acetone (Prabha Devi, 2013) or extract thrice with distilled methanol and the pooled organic solution made from each species will filter by suction through a Buchner funnel line with Whatman No. 1 filter paper. (G. Annie Selva Sonia, 2008). Solvent will remove by rotary evaporator. The free aqueous extract thus obtain will transferred into a separating flask and fractionate sequentially using Diethyl Ether (DE) follow by Butanol (Bu) to obtain the DE-fraction and the Bu fractions respectively (Margey Tadessea, 2008). The crude extracts will now screen for antibacterial and antifungal activity.
Pathogen collection as describe by (Prabha Devi, 2013) Fish pathogens will isolate from infected fish. Isolation will carry out using standard techniques. Briefly, one gram wet weight of the fish sample from the infected region will rinse thrice in sterile fresh water and homogenize it by using a sterile mortar and pestle in 5ml sterile freshwater.
For Bacterial Pathogens:
Serial dilutions (up to 4 dilutions) will make and spread plated on Luria agar (M. F. Mehbub, 2018) as standardize growth media due to the simplicity and accessibility of its formulation. Plates will incubate at 26°C for 2-3 days. The isolates will repeatedly sub-cultured until pure bacterial isolates will obtain and then store on Luria broth until use.
For Fungal Pathogens:
Serial dilutions (up to 4 dilutions) will make and spread plated on Sabourauds dextrose agar (SDA, Hi Media) containing 50 mgml-1 of antibiotic chloramphenicol to inhibit bacterial growth. Plates will incubate at 26°C for 2-3 days. The isolates will repeatedly sub-cultured until pure fungal isolates will obtain and then store on SDA slants until use. (Prabha Devi, 2013)
Metabolite Purification High Performance Liquid Chromatography (HPLC):
Protocol is set as per (M. F. Mehbub, 2018) and (Prabha Devi, 2013) to assess sponge metabolite profiles, and HPLC analyses will perform as per protocol defined by (M. F. Mehbub, 2018). At constant flow rate, 100 mg of freeze-dried sponge tissue was extracted three times, powdered sponge tissue was transferred to a new tube and dissolved with 1 ml methanol in an ultrasonic tank for 5 min with high energy setting, centrifuged and the pellet retained after transferring the supernatant.
The pellet was extracted twice and the combined crude extracts and finally dissolved with 1 ml methanol. This crude extract was filtered through a 13 mm 0.2 µm Syringe Filter and added to a 2ml tube with glass insert. Then, 50 µl of this filtered solution was injected into the HPLC system described above. The peaks will observe at 200 to 800nm wavelength range.
Thin Layer Chromatography (TLC):
This step is design according to protocol design by (M. F. Mehbub, 2018) and (Prabha Devi, 2013) to further elaborate the nature of the metabolites produced, we used TLC. A slurry of the Diethyl Ether (DE) fraction in silica gel was prepared by dissolving the crude extract in minimum quantity of DE and dried under nitrogen. This dry slurry was loaded onto a silica gel glass column and initially eluted with hexane followed by increasing concentration of diethyl ether in hexane. Next elution was performed using chloroform followed by increasing concentration of methanol in chloroform and finally eluted with methanol. Like fractions were combined on the basis of TLC and the combined fractions were subjected to bioassay screening against pathogenic bacteria and fungi. Separation on TLC may be detected under a UV lamp at 254 or 366 nm wavelength range.
Antimicrobial activities for the crude fractions and the pure compound against fish pathogens will determine by agar disc diffusion method. Briefly, paper discs of 6mm diameter will impregnate with 25 µg of the crude extract and 10 µg of the pure compound dissolve in diethyl ether. The zone will then measure in millimeter and scored as (– no activity; + mild activity; ++ moderate activity; +++ Significant activity; and ++++ strong activity). Positive and negative control will also use.
For bacterial pathogens discs will place on Mueller Hinton Agar (MHA) plates possessing a lawn of the different strains to be test. The cultures will incubate for 24 hours at 37°C and for fungal pathogens discs will place on Potato Dextrose Agar (PDA) plates possessing a lawn of the different strains to be test. The cultures will incubate for 48 hours at 27°C to obtain maximum growth in the culture media so as to visualize the clear zone of growth inhibition around each discs. Experiment repeat thrice to know the reproducibility of results
Minimum Inhibitory Concentration (MIC):
Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of the pure compound that inhibits visible growth of the microorganism around the disc. MIC values of crude compound against test pathogens will determine according to the Kirby and Bauer disc diffusion method. The discs load with the compound will prepare in the same way as described above. Each of the pathogen will inoculated in different plate.
For antibacterial MIC inoculum of 250µl solution will spread over each MHA agar plate surface and incubate it for 24h for 37°C and for antifungal MIC inoculum of 250µl solution of each fungi pathogen spread over the surface on potato dextrose agar and plates will incubate for 48-72 h at 28°C. An array of the discs containing different concentrations (µg/ ml) of the compound will place on the plates to determine the MIC values of the compound. Dried discs will use as negative control discs and standard drug will use as positive control. MIC will determine in triplicate.
A. TIME FRAME:
• Collection and laboratory acclimation of Spongilla. spp and its identification through PCR can take up to 3 months according to availability of reagents.
• Bacterial and fungal fish pathogens collection can be start along with Spongilla collection. As we have to collect three Bacterial and tree fungal pathogens, it can be done within 2 months after Spongilla identification.
• After identification of Spongilla, we have to wait until pathogens collection. After collection we will thaw and then extract bioactive immunomodulatory compound. Extraction of bioactive compound from sponge can be done within two week.
• Metabolite Purification by HPLC and TLC both can be done within two months to form into pure crude compound.
• Antibacterial and antifungal biological activity of crude compound done in systematic repeat manner in order to get authentic results. This step can be done in 1.5 month.
• To find out minimum inhibitory concentration of crude compound only for those against which it give antimicrobial activity, will be done in a month.
• Thesis writing can be done within 3 month after complete whole research work.