Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

Studies on the storage characteristics of Silver pomfret (Pampus argentus) transported to Bombay

An investigation on the quality of pomfrets transported to Bombay from Gujarat coast and its subsequent changes during storage at room temperature and low temperature were carried out and the results reported. The pomfrets transported in boats having insulated holds were in better condition than those having non insulated holds. In general, the transported fish can be effectively stored in ice for 2 days, while the fish is in acceptable condition up to 4 days.

Studies on the post-mortem changes in shrimp and prawn during ice storage. Pt. 2. Biochemical aspects of quality changes

Studies were conducted on biochemical changes in P. monodon and M. rosenbergii during ice storage. At the end of 10 days of ice storage, moisture and protein content of freshwater prawn slightly decreased from 78.34 to '77.35% and 18.46 to 17.10, respectively, while lipid and ash content slightly increased. The moisture, crude protein, lipid and ash content of one day ice stored tiger shrimp samples were 78.07, 18.06, 1.3 and 1.29% respectively. The protein composition of freshwater prawn immediately after killed were 36.51% sarcoplasmic, 44.63% myofibrillar, 8.12% stroma and 6.44% alkali soluble protein. At the end of 10 days of ice storage, sarcoplasmic and stroma protein slightly decreased while there was little or no changes observed in myofibrillar and alkali soluble protein. In case of one day ice stored tiger shrimp, the composition of protein were 35.32% sarcoplasmic, 46.29% myofibrillar, 7.86% stroma protein and 7.08% alkali soluble protein. At the end of 10 days in ice, sarcoplasmic protein decreased from 35.32% to 32.16% while there was slight change in other protein fractions. The TVB-N value of 1 day ice stored shrimp was 10.5 mg/100g of sample. It increased gradually with the lapse of storage period and at the end of 10 days storage in ice, the value increased up to 60 mg/100g sample. The tiger head on shrimp in ice storage were found organoleptic acceptable condition for 8 days and at that time the TVB-N values were 32.2 mg/100g which is slightly above the recommended limit for TVB-N for export.

Technological aspects of processing of edible mussels, clams and crabs. Pt. 1. Spoilage during ice storage

Rate and pattern of spoilage of some of the economically important edible species of shell fishes Mytilus edulis (Mussel), Villorita cornucopia (Clam), Neptunus pelagicus (Crab) and Scylla serrata (Crab) have been discussed in this communication. Chemical indices used for objective evaluation of quality were water extractable nitrogen (WEN), non-protein nitrogen (NPN), free α-amino nitrogen (α - NH2 -N), glycogen, lactic acid and inorganic phosphorus in addition to the subjective tests. No significant difference in the spoilage pattern of the species during ice storage was observed and these species could be preserved in ice in organoleptic acceptable condition up to 8 days, 9 days, 8 days and 11 days respectively.

Observations on changes in the major protein nitrogen fraction of prawns and sardines during ice storage

Changes in the major protein nitrogen fractions (sarcoplasmic, myofibrillar, stroma) have been studied in two species of prawns and in oil sardine held in ice storage. Myofibrillar proteins were observed to get denatured at a rapid rate as determined by salt extractability method. The sarcoplasmic proteins were not denatured to any considerable extent. With sardine however, the extraction of myofibrillar proteins was inhibited rather in the uniced condition itself presumably owing to the presence of free fatty acids.

Frozen storage characteristics of cultured Macrobrachium rosenbergii (de Man)

Cultured Macrobrachium rosenbergii (Scampi, about 30 g each) in headless shell-on form was individually quick frozen in a spiral freezer. The frozen samples were glazed and packed in polythene bags, which were further packed in master carton and stored at -18°C. Samples were drawn at regular intervals and subjected to biochemical, bacteriological and organoleptic analysis to study its storage characteristics. The data on the above parameters showed that the samples were in prime acceptable condition when stored up to 23 weeks. No appreciable change in colour and odour was noticed in the raw muscle. Afterwards, organoleptic evaluation of the cooked muscle revealed slight change in the flavour. Texture also appeared little tougher. These changes in organoleptic characters were well supported by the biochemical bacteriological changes in the muscle.

Shelf life of dried products from small indigenous fish species under various packing and storage conditions

The overall quality of five SIS products was found in good condition up to 2 months storage on the basis of organoleptic, biochemical and bacteriological characteristics and all the products was excellent in sealed packed condition up to 45 days of storage. However, quality of the products stored in open air atmospheric temperature was found excellent for first 15 days. In an average the initial moisture content was in the range of 13.5 to 15.0% with highest moisture content in puti and lowest in chapila. At the end of the 60 days the moisture content reached to the range of 18.5 to 19.0% which was more or less near the recommended limit of 16% for dried fishery products. The moisture content beyond the recommended limit as the storage period increased further and at the end of 90 days the moisture content increased to the range of 22.9 to 24% when organoleptically the product quality became very poor. The changes in the value of total volatile base nitrogen (TVB-N), peroxide value (PO), moisture and aerobic plate count (APC) of solar tunnel dried products in sealed polythene packages were investigated during 60 days of storage. There was little or no differences in TVB-N, PO and bacterial load of each species packed under various polythene density. The initial TVB-N values were in the range of 10.30 to 12.40 mg/100g of the samples. TVB-N value increased slowly up to the end of the storage period and was to in the range of 46.20 to 57.00 mg/1 00 g of sample. Initially the peroxide values (P.O.) were in the range of 6.54 to 8.40 m.eq./kg oil of the samples. During 60 days of storage, P.O. values increased slowly and at the end of the storage period these values reached to the range of 22.00 to 25.30meq./kg of sample. The initial APC was in the range 5.3xl04-7.3x104 CFU/g. The bacterial load increased slowly and at the end of the 60 days storage period reached to the range 6.6x106 - 8.6x107 CFT/g.

Freeze drying of fishery products: Part IV - Biochemical changes occurring in prawn muscle during freezing, freeze drying and prolonged storage of the freeze dried product

The changes occurring in water and salt extractable protein and non-protein fractions in prawn muscle of different species during freezing, freeze drying and subsequent prolonged storage have been studied. There is no denaturation of water extractable proteins, whereas salt extractable proteins were rendered insoluble to the extent of 21% due to freeze drying. The freeze dried products remained in good edible condition for 32 months of storage up to which storage characteristics were followed.

Iced and frozen storage of squid (Loligo sp)

The iced and frozen storage characteristics of squid (Loligo sp.) are discussed. Squid can be kept in ice in an acceptable condition for a maximum period of 2 days. Frozen squid can be stored for a maximum period of 15 weeks at -l8°C, which can be extended up to 19 weeks by suitable treatment.

Studies on frozen storage of ghol (Pseudosciaena diacanthus)

Investigations were carried out on the frozen storage of ghol (Pseudosciaena diacanthus) in the form of fillets. The results indicated that ghol fillets stored at -l8°C remained in a highly acceptable condition up to 20 weeks. However, after this stage, the acceptability steeply declined.

Packaging and storage studies of dried lizard fish

Packaging and storage studies of salted and dried lizard fish (Saurida sp.) have been conducted using different synthetic films like low density polyethylene (LDPE) of different gauges, high density polyethylene (HDPE) of 200 gauge, polyvinylidene chloride (PVDC) coated 400 MXXT cellophane, 100 gauge polypropylene (PP) and paper laminate of 100 gauge polythene. The films found most effective in the preliminary studies were subsequently used for packaging and storage of dried fish at atmospheric and lower temperature and humidity conditions for confirming their suitability under these conditions. Polyethylene films of higher gauges showed better results under both sets of conditions. PVDC coated cellophane film also performed satisfactorily under the latter conditions which under the former condition got easily attacked by insects. Lower temperature and humidity conditions in general enhanced the storage life of the dried product.

Studies on the ice storage characteristics of blood clam Anadara granosa meat

Ice-storage study of blood clam (Anadara granosa) meat in direct contact and out of contact (in 200 gauge polyethylene bag) with ice was taken up to assess the amenability of the meat to icing. Changes in moisture, total protein, non-protein nitrogen, α amino nitrogen, total volatile base nitrogen, glycogen, free fatty acid, peroxide value, total bacterial count and coliform count were followed every day. The raw and cooked meat were also subjected to organoleptic evaluation. The study showed that the clam meat can be ice-stored in very good condition out of contact with ice in polyethylene packets for 4 days and in direct contact with ice for 2 days.

Studies on frozen storage characteristics of sole fish Cynoglossus macrolepidotus

Fresh sole fish (Cynoglossus macrolepidotus) was quick frozen at -40°C and stored at -18°C. Shelf life was evaluated by following biochemical, bacteriological and organoleptic changes occurring during storage. Rapid decrease was noted in the water extractable nitrogen and salt soluble nitrogen fractions. Samples of frozen sole fish remained in acceptable condition for 20 weeks.

Preservation of Sardinella longiceps in iced and chilled seawater. Part 1 - Changes during storage with particular reference to bacterial load and nitrogenous compounds

Oil sardines in prime condition were subjected to onboard chilling. Two lots were chilled in CSW (samples C and CI), a third lot was chilled in crushed ice (sample I) and a fourth lot left not iced on deck (Sample AI). Upon landing sample AI was iced and sample CI was removed from the CSW and iced. All the four samples were kept in a chilled room for storage studies. The fish chilled and stored in CSW recorded the least, and the fish subjected to delayed icing, the highest values for all the indices of spoilage namely, free amino nitrogen, trimethylamine (TMA) and total volatile base nitrogen (TVBN). The total psychrophilic bacterial number also showed a similar trend. The organoleptic assessment of the cooked samples revealed C I, CI, AI to be the order of preference throughout the storage. This assessment was found to hold good for the rest of the parameters as well. The CSW held fishes were found to be distinctly superior to the iced ones for the first five days of storage. Such a marked prevalence in quality for five days would suffice for the fish to fetch a premium in the market over other landings of the same fish whether chilled or not chilled. Chilling on board in CSW and icing the same after landings, did not show encouraging results.

Preservation of Indian oil sardine (Sardinella longiceps) in iced and chilled seawater. Part 2 - Changes during storage with particular reference to salt penetration and lipid deterioration during CSW holding

Oil sardines in prime condition were chilled on board. Two lots were chilled in CSW (samples C & CI), one lot ice (sample I) and a fourth lot was left un-iced on deck (sample AI). Sample AI was iced after landing and sample CI was taken out of the chilled seawater and. iced. All the four samples were kept in a chilled room for storage studies. Sample C, chilled and stored in CSW, recorded a gradual gain in weight and an increase in salt content of the muscle. Presence of salt did not seem to cause any excessive protein denaturation. Salt extractability decreased at a gradual rate in all cases. Presence of salt seemed to wield no noticeable influence on lipid hydrolysis and subsequent peroxidation. Results of chemical and sensory evaluations highlight this. Holding sardines in CSW gave a product of excellent quality for the first four to five days of storage. Beyond the fifth day of storage quality deteriorated rapidly and there was no noticeable superiority for this sample (sample C) over the on board iced fish. This was evident in the sensory evaluation as well. However, a storage life of five days in a readily acceptable state is sufficient for the fish to be disposed in the market at a premium sale price over other landings of the same species.