Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

Mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote Indigenous communities in Australia

Background Surveillance programs and research for acute respiratory infections in remote Australian communities are complicated by difficulties in the storage and transport of frozen samples to urban laboratories for testing. This study assessed the sensitivity of a simple method for transporting nasal swabs from a remote setting for bacterial polymerase chain reaction (PCR) testing. Methods We sampled every individual who presented to a remote community clinic over a three week period in August at a time of low influenza and no respiratory syncytial virus activity. Two anterior nasal swabs were collected from each participant. The left nare specimen was mailed to the laboratory via routine postal services. The right nare specimen was transported frozen. Testing for six bacterial species was undertaken using real-time PCR. Results One hundred and forty participants were enrolled who contributed 150 study visits and paired specimens for testing. Respiratory illnesses accounted for 10% of the reasons for presentation. Bacteria were identified in 117 (78%) presentations for 110 (79.4%) individuals; Streptococcus pneumoniae and Haemophilus influenzae were the most common (each identified in 58% of episodes). The overall sensitivity for any bacterium detected in mailed specimens was 82.2% (95% CI 73.6, 88.1) compared to 94.8% (95% CI 89.4, 98.1) for frozen specimens. The sensitivity of the two methods varied by species identified. Conclusion The mailing of unfrozen nasal specimens from remote communities appears to influence the utility of the specimen for bacterial studies, with a loss in sensitivity for the detection of any species overall. Further studies are needed to confirm our finding and to investigate the possible mechanisms of effect. Clinical trial registration Australia and New Zealand Clinical Trials Registry Number: ACTRN12609001006235. Keywords: Respiratory bacteria; RT-PCR; Specimen transport; Laboratory methods

A survey of the microbiological quality of commercial frozen prawn products

The paper presents the results of a bacteriological survey carried out on 2,917 samples of frozen prawn, 55 samples of raw material, 35 samples of water, 4 samples of ice and 42 samples of various equipment used for processing. The survey covered a period of three years (1960-63) and comprised of samples collected from five of the leading processing factories in Cochin. Frozen products tested consisted of headless (marine and fresh water), peeled and deveined and cooked frozen samples. Statistical analysis of the data shows that there is no significant variation between samples and between factories with respect to product quality. The standard plate count varied between 1.0x10(4 superscript) and 1.0x10(6 superscript) per gram for headless and between 1.0x10(4 superscript) and 1.0x10(7 superscript) for peeled and deveined and cooked frozen samples. Majority of the samples had bacterial load well within the limits prescribed for such products.

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.

Effect of incubation period on plate count of raw ices and frozen fish

The total viable counts were estimated in one hundred and sixty five samples of raw, iced and frozen fish using incubation periods of 24, 48, 72 and 96h. For raw fish, 24h and for iced and frozen fish 48h incubation of the plates were found to be adequate. Variation between samples was significant at 1% level for raw iced and frozen samples.

Studies on frozen characteristics of individually quick frozen and block frozen mackerel

Studies on mackerel (Rastrelliger kanagurta) of medium (4%) and high (11%) lipid contents quick frozen individually (IQF) and as blocks (BF) and stored at -23°C showed that block frozen mackerel had higher frozen storage shelf-life than individually quick frozen samples. IQF samples of medium and high lipid contents had shelf-lives of 17 and 20 weeks whereas BF samples of both series had 23 and 24 weeks respectively based on sensory evaluation.

Caking strength of granulated clay under frozen conditions

Transport of particulate clay occurs during some extremely cold weather conditions typically in the winter in the far North area. During the transport and temporary storage time, the clay may cake inside a rail or road wagon or in a silo, and consequently be difficult to be discharged from the containers. This paper studied caking strength of a granulated clay powder with a certain water moisture content of 18% for influences of temperature, packing stress and freezing time. The temperature tested was -5 oC, -10 oC and -20 oC. Because the clay powder may be packed at different bed depth, the study was undertaken across the packing stress range at 8.3 kPa (1 m bed depth), 25.0 kPa (3 m) and 75.0 kPa (9 m). Freezing time varied between 4 hours (transport) and 18 hours (overnight). During the tests, failure of caked materials was measured using a QTS texture analyzer and the caking strength of frozen samples was calculated. Influences on freeze caking of granular clay in storage or transport are discussed briefly. Some conclusions are made at the end of the paper,including recommendations for practical methods for mitigating these problems.

Comparison between RT-PCR and the mouse inoculation test for detection of rabies virus in samples kept for long periods under different conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of plasma membrane integrity of frozen-thawed goat spermatozoa with or without seminal plasma

The aim of the present work was to evaluate plasma membrane integrity, motility, vigor and morphology of fresh and frozen goat spermatozoa with or without seminal plasma. Semen samples were diluted in Tris solution, before and after thawing, with a combination of carboxifluorescein diacetate and propidium iodide. The results showed differences (P < 0.01) for motility and minor defects in the presence or absence of seminal plasma, for both fresh and frozen samples. Periods of collection had a significant effect on motility, probably due to changes in the photoperiod. Plasma membrane integrity was significantly reduced by the freezing process, whether seminal plasma was present or absent. In conclusion, removal of seminal plasma decreased motility and vigor rates in frozen samples. The photoperiod probably decreased the testosterone level, contributing negatively to the high percentage of sperm abnormalities, mainly damaged membranes. The use of fluorescent probes allowed a better estimation of the percentage of functional cells, instead of only estimating the percentage of motile cells or morphology defects. © 2001 Elsevier Science B.V. All rights reserved.

Successful application of a simple specimen transport method for the conduct of respiratory virus surveillance in remote Indigenous communities in Australia

Objective Surveillance programs and research for acute respiratory infections in remote Aboriginal communities are complicated by difficulties in the storage and transport of frozen samples to urban laboratories for testing. This study assessed the sensitivity of a simple method for transporting respiratory samples from a remote setting for viral PCR compared with frozen specimens. Methods We sampled every individual who presented to a remote Aboriginal community clinic in a non-epidemic respiratory season. Two anterior nasal swabs were collected from each participant. The left nare specimen was mailed to the laboratory via routine postal services. The right nare specimen was transported frozen. Testing for 16 viruses was undertaken using real-time multiplex PCR. Results A total of 140 participants were enrolled who contributed 150 study visits. Respiratory illnesses accounted for 10% of the reasons for presentation. Sixty-one viruses were identified in 50 (33.3%) presentations for 40 (28.6%) individuals; bocavirus and rhinovirus were the most common viruses identified (14.0% and 12.6% of episodes respectively). The sensitivity for any virus detected in mailed specimens was 67.2% (95%CI 55.4, 78.9) compared to 65.6% (95%CI 53.7, 77.5) for frozen specimens. Conclusion The mailing of unfrozen nasal specimens from remote communities does not compromise the viability of the specimen for viral studies.

Selection of suitable diluents for bacteriological examination of fishery products

For raw, iced and frozen samples of fish and prawn, significant difference was observed in total plate counts done with various diluents, the significance level ranging from 5% to 0.1%. For raw fish, N-saline, seawater and quarter strength Ringers' solution gave maximum total plate counts. In the case of iced-fish, n-saline yielded highest total plate counts. For frozen samples, however, peptone water and n-saline gave good recoveries. Trials with suitable combinations of diluents showed that though some of them were as good as the control, namely n-saline, none were superior in any way.

Effect of pre-processing iced storage on deteriorative changes in lipids of silver pomfrets stored at -18°C

Silver pomfret (Pampus argenteus) was frozen in the fresh condition as well as after holding in ice for one, two and four days. Evaluation of changes in the quality of these samples during storage at -18°C has shown that shelf-life decreased sharply if the pre-freezing iced storage was more than one day. The shelf-life of one day iced, two day iced and four day iced frozen samples were 32, 20 and 16 weeks respectively. No correlation was observed between the peroxide value and the organoleptic detection of rancid flavour. Levels of free fatty acids were more in the samples frozen after storage in ice for one day than in all the other samples.

Incidence and low temperature survival of coagulase positive staphylococci in fishery products

Coagulase-positive staphylococci was found to be absent in all the frozen samples of lobsters, cuttle fish, cat fish, seer fish and red snapper examined. Coagulase-positive staphylococci were present in 38% of the cooked frozen shrimps and only 16% of the samples had staphylococci count more than 100/g. In the case of headless, peeled and deveined, peeled undeveined shrimps, the incidence of the organism was 6, 12 and 16% respectively. The study indicated that the incidence of coagulase-positive staphylococci is not a serious problem in frozen fishery products processed in this country. There was remarkable difference in the rate of destruction of coagulase-positive staphylococci in raw and cooked shrimps during freezing and frozen storage.

Effects of the quick and slow freezing on the quality of Nile Tilapia fillets (Oreochromis niloticus) and Red Tilapia fillets (O. niloticus and Tilapia mosambicus)

In this study, quality of fresh, slow frozen and quick frozen tilapia fillets and its changes during storage at -18C° were investigated. For preparation the samples, fresh tilapia fillets were frozen by slow and quick frozen methods. Slow frozen samples were prepared by storing the packed fillets directly in the -18 C°. The sprila freezing tunle with -30C° was also used for preparation the quick frozen sample. The quick frozen samples were then stored at -18C°for six months. Proximate composition, fatty acid profiles, TBA, PV, TVN, Total cuont, Drip loss, and sensory evaluation of the samples were determined in every month. Scanning Electron Microscopy (SEM) was used for study on the effects of the frozen condition on the microstructure of the fillets. Results indicated that two different frozen methods had significantly different effects on the quality of the fillets. Most of the proximate composition (protein, moistre and fat) reduced during the storage. Quick frozen filets had significantly (P<0.05) lower reduction than slow frozen samples. All of the chemical quality indexes (PV, TBA, and TVN) increased during the storage as compered to the fresh samples. In these paramethers, the slow freezing had higher changes than quick freezing metods (P<0.05). The microbial properties of the samples showed decrese during the storage. Lower amont of total cuont was observed at the end of the storage time in the quick frozen samples than slow frozen once (P<0.05). The large changes in the fatty acid profiles of the sample were fond in all samples. During the storage SFA and MUF of the samples increased however, the PUFA decresed. A lower change was obseved in the quick frozen samples than slow frozen samples (P<0.05). Drip loss was increased in both frozen samples during the storage period. The percentage of the drip in the slow frozen samples was significantly higer than quick frozen samples (P<0.05). SEM micrographs were also showed that the chnges in the microstructur of the samples was different in the slow and frozen samples. Slow freezing methods had higher damge in the microstructure of the sample then quick freezing mathods. Sensory evaluation of the samples indicated that a better acceptability in the quick frozen samples than slow frozen sample (P<0.05).

Identification and determination of muscle's fatty acids and amino acids in Cobia and Indian squid

The present study aims to find the effect of freezing Time on the quality of Cobia (Rastrelliger kanagurta) and Indian Squid in commercial scale during freezing and subsequent frozen storage (−18◦C). Total time for freezing was significantly different (P<0.05) between the Cobia and Indian squid samples. The difference in the freezing time could be attributed to the varied quality of the 2 samples. Upon freezing, the moisture content decreased in Indian Squide samples compared to Cobia freezer where protein content decreased in both the samples. Upon freezing and during frozen storage, lipid oxidation products (peroxide value, and free fatty acid value) and volatile bases (total volatile base nitrogen) showed an increasing trend in both the samples with values slightly higher in Indian squid samples compared to cobia frozen samples. The total plate counts showed a significantly (P<0.05) decreasing trend in both the samples. K value did not show any significant (P<0.05) difference between the samples whereas the histamine formation was significantly (P<0.05) increased in Indian squid frozen samples compared to cobia samples. The taste and overall acceptability was significantly different (P<0.05) in cobia samples compared to Indian squid frozen samples on 5th month. Both samples were in acceptable condition up to 5 month but the Cobia frozen samples quality was slightly better than the air blast frozen samples.