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.

Bacterial flora of EDTA treated oil sardine (Sardinella longiceps), Indian mackerel (Rastrelliger kanagurta) and prawn (Metapenaeus dobsoni) in ice storage

The native flora of fresh oil sardine and mackerel consisted mainly of Pseudomonas spp., Moraxella spp., Acinetobacter spp. and Vibrio spp. During spoilage in ice, nearly 75% of their bacterial flora belonged to Pseudomonas spp. alone. But Na sub(2) EDTA treatment reduced the proportion of Pseudomonas spp. considerably and the major bacterial groups at the time of spoilage were Moraxella spp. and Acinetobacter spp. In the case of fresh prawn, the native flora was constituted by Pseudomonas spp., Moraxella spp., Acinetobacter spp. and Vibrio spp. At the time of spoilage of prawn in ice, Moraxella spp. and Acinetobacter spp. predominated, together constituting 74% of the total population. Na sub(2) EDTA treatment did not alter significantly the spoilage flora of prawns. Moraxella spp. and Acinetobacter spp. accounted for 86% of the spoilage flora in ice storage of Na sub(2) EDTA treated prawns.

Effectiveness of EDTA dips on the shelf-life of oil sardine (Sardinella longiceps), mackerel (Rastrelliger kanagurta) and prawn (Metapenaeus dobsoni) in iced storage

Fresh oil sardine, mackerel and prawn were dipped in 0.1% and 1% solutions of Na sub(2)EDTA, and stored in ice. Their storage-life was assessed by bacteriological, chemical and sensory methods. Even though EDTA treatment controlled the increase in bacterial counts and reduced TMA and TVBN production in oil sardine and mackerel, the consequent beneficial effect was not realised because of the deterioration of fat in these fishes, leading to rancidity. But, for prawn stored in ice, a dip in 1% solution of Na sub(2)EDTA enhanced the shelf-life by at least 8 days over the untreated control. EDTA absorbed by the muscle of fish and prawn during dip in Na sub(2)EDTA solution is not completely removed during their iced storage for 25 days.

Blackening of canned prawn and its prevention

The paper gives an account of the types of blackening associated with canned prawn in brine and their control. It was found that blackening caused by iron sulphide could be controlled by maintaining proper titratable acidity of fill brine in cans. The paper also elaborates on the factors responsible for or governing this critical titratable acidity. In regard to copper sulphide blackening, control was found to be difficult by maintaining the acidity or by additives such as EDTA when the copper content in the material went above the critical level.