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.

Quantifying global terrestrial carbon influx and storage as stimulated by an increase in atmospheric carbon dioxide concentration

EXTRACT (SEE PDF FOR FULL ABSTRACT): Measurements of spatial and temporal distributions of carbon dioxide concentration and carbon-13/carbon-12 ratio in the atmosphere suggest a strong biospheric carbon sink in terrestrial ecosystems. Quantifying the sink, however, has become an enormous challenge for Earth system scientists because of great uncertainties associated with biological variation and environmental heterogeneity in the ecosystems. This paper presents an approach that uses two driving parameters to bound terrestrial carbon sequestration associated with an increase in carbon dioxide concentration.

Faecal indicator organisms in frozen prawn products. Pt. 2. Survival of the organisms during freezing and frozen storage

The rate of survival of different types of faecal indicator organisms like Escherichia coli, enterococci etc. during freezing and frozen storage has been studied. Peeled and deveined prawns inoculated with a mixed culture of the above organisms were subjected to freezing and storage at -10̊F and examined for over four months.

Effect of aureomycin on the behaviour of certain free amino acids in oil sardine (Sardinella longiceps) held in ice storage

The course of development of a few free amino acids under the influence of aureomycin in oil sardine (Sardinella lingiceps) held in ice storage was investigated. The levels of leucines and valine regularly increased in the control and aureomycin treated fush throughout the storage period. Alanines and threonine showed similar trend in both control and fish treated with 20ppm aureomycin. These amino acids however showed a gradual fall in fish treated at 5 ppm level. The changes in tyrosine+tryptophane were found to be irregular. Most of the amino acids studied indicated a remarkable change in trend by about the 16th day of ice storage in the case of fish treated with 50ppm aureimycin.

Effects of different types of drying on the nutritive value of proteins in the fishmeals

A comparative study on the effect of different types of drying on the nutritive value of the proteins in the different fishmeals of known history was made. From the observations, it is clear that the mode of drying has got little or no effect on the nutritive value of the meal as revealed by the chemical indices of available lysine and pepsin digestibility, provided enough precautions were taken to avoid scorching during drying process. Sun dried meals are in no way inferior to the meals prepared by hot-air, steam or vacuum drying.

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.

Some aspects of freezing and frozen storage of pomfrets

It has been observed that a better frozen product can be obtained by freezing good quality pomfrets transported in insulated containers with sufficient quantity of ice. To enhance the keeping quality and to prevent dehydration and discoloration, a dip in B H A (0.005%) for 15 minutes and subsequent storage in polythene lined gunny bag at -15°c to -I8°c can be recommended. The products treated in the above manner can be stored well over six months. Periodical glazing at an interval of 3 weeks will also prevent the dehydration to a greater extent.

Preliminary observations on changes in nucleotides in oil sardine and certain Penaeid prawns during chill storage

Preliminary study has been made of the changes in common 5' nucleotides in oil sardine (Sardinella longiceps) and two Penaeid prawns of Indian waters during chill storage. The course of nucleotide degradation has been followed in the fresh fish and shell fish during ice storage. The level of inosine monophosphate (IMP) in prawns showed significant but steady decrease during ice storage and this appears to serve as useful indication of length of storage. Comparison has been made on the pattern of nucleotide changes in block frozen fish and individually quick frozen fish stored at -23°C.

Studies on technological problems associated with the processing of cooked frozen prawns. Pt. 1. Preprocess storage conditions of raw material in relation to quality

The chemical and organoleptic properties of prawn held in ice for different days prior to cooking and the changes after freezing and subsequent storage were studied with three different species of prawn viz. Metapenaeus monoceros, Metapenaeus dobsoni and Parapeneopsis stylifera. The optimum period for which the prawn can be kept under ideal conditions of icing prior to cooking has been worked out.

Studies on the storage behaviour of laminated and commercial Bombay duck

A modified method for the preparation of laminated Bombay duck is presented. Investigation was carried out to find out an effective chemical to control the discoloration of dried laminated Bombay duck. Among various chemicals tried, NDGA and BHT were found to have considerably retarded the discoloration and extended the storage life of the product. Attempt was also made to suggest the optimum humidity level for the proper storage of the commercially dried fish. It was found that a level of about 65% R. H. provided maximum storage life to commercial product.

Postmortem changes in hilsa fish. Pt. 1. Studies on rigor-mortis, typical yields and protein composition of dark and white muscle of hilsa fish (Tenualosa ilisha Ham.)

Hilsa (Hilsa ilisha) caught by gill net were immediately killed by cranial spiking. Three fish were kept in ice (0°C) and three other at room temperature (33°C) to follow development of rigor mortis and changes in muscle pH. The rest were frozen stored at -20°C. Rigor started 15 minutes after death in all fish and reached full rigor (100%) state in 2 and 4 hours respectively in fish kept at 33° and 0°C. The fish at 33°C deteriorated 16 hours after while in full rigor but those at 0°C lasted 26 hours of death without deterioration. Freshly caught hilsa had a muscle pH around 7 which decreased with time rapidly at 33°C and slowly at 0°C. The relative proportion of protein fraction in white and dark muscle of fish stored at 0°C and -20°C were also studied. The proportion of dark muscle was 30.34% of the white muscle. White muscle in fish at 0°C was found to contain 32.0% sarcoplasmic, 57.6% myofibrilla, 9.4% alkali-soluble and 1.1% stroma protein whereas these proteins in dark muscle were 29.9%, 58.4%, 9.8% and 1.9% respectively. The protein fractions of white muscle in frozen-fish were found 27.6% sarcoplasmic, 64.7% myofibrilla, 6.0% alkali-soluble and 1.7% of stroma protein whereas they were 30.6%, 58.6%, 8.9 and 1.9% for dark muscle. Some changes occurred in protein composition during frozen storage. The relative amounts of sarcoplasmic, alkali soluble and stroma protein fractions decreased while myofibrilla fraction increased in frozen condition. This may be attributed to drip loss of soluble protein during thawing.

Studies on the post-mortem changes in shrimp and prawn during ice storage. Pt. 1. Organoleptic and physical changes

The organoleptic characteristics such as appearance, textural condition, colour and odour indicated that the M. rosenbergii stored in ice for 5-6 days was acceptable for processing in the industry while P. monodon under similar ice storage condition was acceptable for 8-9 days. In both species, samples stored in headless condition in ice had longer shelf life than that of stored in head-on condition. Physical changes were evaluated by determining expressible moisture and breaking strength of sample of muscles. The expressible moisture increased continuously in both samples with the lapse of storage period. The expressible moisture increased up to around 44% in 4-5 days of ice stored M. rosenbergii muscle while it was around 40% in 8-9 days ice stored P. monodon. At the end of 9 days of ice storage, the expressible moisture content in M. rosenbergii increased up to 60%, while it was up to 47% in P. monodon after 11 days of ice storage. The breaking strength declined from 0. 78 kg/cm² to 0.53 kg/cm² in tiger shrimp after 8 days of ice storage, while in case of immediately killed prawn, the breaking strength of muscle was 0.8 kg/cm² which declined to 0.43 to 0.35 kg/cm².