Microwave processing for scanning electron microscopy

The normal processing of biological samples for Scanning Electron Microscopy, includes treatment with aldehyde (1 to 2 hours), postfixation with Osmium (1 hour), followed by dehydration in a ascending grade of ethanol (30 a 100%), 10 to 15 minutes in each step, and finally drying. This procedure takes at least 8 hours. In this work, samples of mosquitoes (Aedes), protozoa (Tritrichomonas muris), bacteria (Clostridium oceanicum), murine liver, and small intestine were processed in the same manner in a domestic microwave oven for two minutes at 20% of its maximum power. The complete procedure from the initial fixation to dehydration in 100% ethanol was reduced to one hour with good preservation of the ultrastructural details of the specimens.     

A bioassay for thyroid stimulating immunoglobulins of patients with Graves' disease using porcine thyroid monolayer cells

A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2 X 10(6) cells/1.5 ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5 mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1 ml of serum-free, 0.5 mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay. Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30 min. Responses of cAMP were in a dose-related manner up to 10 mU/ml of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10 mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10 mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228 +/- 51.8%, mean +/- SD; 127-393%, range) than normal subjects (103 +/- 13.3%, mean +/- SD, n = 24; 80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI. These results indicate that this bioassay is clinically useful for detecting TSI.     

Modified enrichment-serology procedure for detection of salmonellae in soy products.

A modified enrichment-serology (MES) procedure was used to reduce the time necessary for salmonella analysis. Naturally contaminated samples of soy products were preenriched in 1% proteose peptone for 6 h at 37 degrees C followed by inoculation into tetrathionate broth for 18 h at 37 degrees C. Two drops of the tetrathionate sample were inoculated into M broth. After incubation at 37 degrees C for 6 h, 0.85 ml of the mixture was formolized, and 0.1 ml of polyvalent H antiserum was added. After incubation in water bath at 50 degrees C for 1 h, the appearance of a typical floccular flagellar precipitate was observed in tubes positive for salmonellae. Over 3,000 samples were subjected to standard biochemical and serological procedures, and the results were compared with those of the MES method with a 96.7% correlation. Eleven of the samples (0.3%) were false-negative with the MES procedure, and 3% were false-negative with the U.S. Food and Drug Administration Bacteriological Analytical Manual procedure. The 3% negative samples by this latter procedure were subsequently found to be positive by the MES procedure. The MES procedure reduced the time required for salmonella analysis from 4 days to 32 h.     

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts.

The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.     

Face Mask Sampling for the Detection of Mycobacterium tuberculosis in Expelled Aerosols

Background

Although tuberculosis is transmitted by the airborne route, direct information on the natural output of bacilli into air by source cases is very limited. We sought to address this through sampling of expelled aerosols in face masks that were subsequently analyzed for mycobacterial contamination.

Methods

In series 1, 17 smear microscopy positive patients wore standard surgical face masks once or twice for periods between 10 minutes and 5 hours; mycobacterial contamination was detected using a bacteriophage assay. In series 2, 19 patients with suspected tuberculosis were studied in Leicester UK and 10 patients with at least one positive smear were studied in The Gambia. These subjects wore one FFP30 mask modified to contain a gelatin filter for one hour; this was subsequently analyzed by the Xpert MTB/RIF system.

Results

In series 1, the bacteriophage assay detected live mycobacteria in 11/17 patients with wearing times between 10 and 120 minutes. Variation was seen in mask positivity and the level of contamination detected in multiple samples from the same patient. Two patients had non-tuberculous mycobacterial infections. In series 2, 13/20 patients with pulmonary tuberculosis produced positive masks and 0/9 patients with extrapulmonary or non-tuberculous diagnoses were mask positive. Overall, 65% of patients with confirmed pulmonary mycobacterial infection gave positive masks and this included 3/6 patients who received diagnostic bronchoalveolar lavages.

Conclusion

Mask sampling provides a simple means of assessing mycobacterial output in non-sputum expectorant. The approach shows potential for application to the study of airborne transmission and to diagnosis.     

Diagnosis of soil-transmitted helminths using the Kato-Katz technique: What is the influence of stirring,storage time and storage temperature on stool sample egg counts?

BackgroundSoil-transmitted helminths infect about one fifth of the world’s population and have a negative impact on health. The Kato-Katz technique is the recommended method to detect soil-transmitted helminth eggs in stool samples, particularly in programmatic settings. However, some questions in its procedure remain. Our study aimed to investigate the effect of storage time, storage temperature and stirring of stool samples on fecal egg counts (FECs).Methodology/Principal findingsIn the framework of a clinical trial on Pemba Island, United Republic of Tanzania, 488 stool samples were collected from schoolchildren. These samples were evaluated in three experiments. In the first experiment (n = 92), two Kato-Katz slides were prepared from the same stool sample, one was stored at room temperature, the other in a refrigerator for 50 hours, and each slide was analyzed at nine time points (20, 50, 80, 110, 140 minutes, 18, 26, 42 and 50 hours). In the second experiment (n = 340), whole stool samples were split into two, one part was stored at room temperature, and the other part was put in a refrigerator for 48 hours. From each part one Kato-Katz slide was prepared and analyzed at three time points over two days (0, 24 and 48 hours). In the third experiment (n = 56), whole stool samples where stirred for 15 seconds six times and at each time point a Kato-Katz slide was prepared and analyzed.Mean hookworm FECs of Kato-Katz slides stored at room temperature steadily decreased following slide preparation. After two hours, mean hookworm FECs decreased from 22 to 16, whereas no reduction was observed if Kato-Katz slides were stored in the refrigerator (19 vs 21). The time x storage interaction effect was statistically significant (coefficient 0.26, 95% CI: 0.17 to 0.35, p < 0.0001). After 24 hours mean hookworm FECs dropped close to zero, irrespective of the storage condition. Whole stool samples stored at room temperature for one day resulted in a mean hookworm FEC decrease of 23% (p < 0.0001), compared to a 13% reduction (p < 0.0001) if samples were stored in the refrigerator. Fecal egg counts of A. lumbricoides and T. trichiura remained stable over time regardless of storage temperature of whole stool samples. Finally, we found a significant reduction of the variation of hookworm and T. trichiura eggs with increasing rounds of stirring the sample, but not for A. lumbricoides. For hookworm we observed a simultaneous decrease in mean FECs, making it difficult to draw recommendations on stirring samples.Conclusions/SignificanceOur findings suggest that stool samples (i) should be analyzed on the day of collection and (ii) should be analyzed between 20–30 minutes after slide preparation; if that is not possible, Kato-Katz slides can be stored in a refrigerator for a maximum of 110 minutes.     

野葛试管苗茎尖玻璃化法超低温保存及植株再生

对野葛茎尖玻璃化法超低温保存技术的研究结果表明,H号野葛茎尖较佳的保存技术体系是:继代生长30 d的野葛无菌苗置于4℃冰箱炼苗5 d;在无菌条件下切取含1~2个叶原基(1.5~2.5 mm)的茎尖,转至含5% DMSO+5%蔗糖的MS培养基内,置于4℃冰箱预培养1 d;用60%、100% PVS₂(30%甘油+15%乙二醇+15% DMSO+13.7%蔗糖)分别在0℃下过渡和脱水各30 min,随即迅速投入液氮;保存24 h后,在40℃水浴中快速化冻90 s,用含41.1%蔗糖的MS培养液洗涤2次,每次停留10 min;转至再生培养基(MS+6-BA 2 mg/L+NAA 1 mg/L)暗培养7 d,然后光下培养,再生率可达60%以上。再生苗与常温苗形态指标差异不显著。     

Detection of Salmonella spp. in food by a rapid PCR-hybridization procedure.

A rapid and sensitive PCR-hybridization procedure for detection of Salmonella serovars in food samples was developed. This method is based on three subsequent steps: (1) extraction of nucleic acids from a 2 ml aliquot of the pre-enrichment medium used for the conventional culture method after 6 h of incubation at 37 degrees C; (2) amplification with primers selected from the sequences of invE and invA genes; (3) Southern blot and hybridization with a biotin labeled oligonucleotide probe. The entire procedure requires 30 h. The PCR-hybridization assay was able to detect as little as 50 fg of purified chromosomal DNA of S. typhimurium and 0.2 cfu g-1 of an artificially contaminated food sample. Of 245 food samples analyzed by culture and PCR-hybridization, 20 were positive by both methods and 16 were positive by PCR-hybridization only. None of the 209 PCR-negative samples tested positive by culture. The sensitivity, specificity, alpha and beta error values of the results of the PCR-hybridization procedure, compared with those of culture, were 100, 92.9, 0 and 7.1%, respectively. These results indicate that a short pre-enrichment and PCR-hybridization could be used as a screening test for the detection of Salmonella in food samples.     

S／D处理血浆过程中的脂包膜病毒灭活试验观察

通过有机溶剂/去污剂对血浆中指示病毒VSV灭活的观察评估有机溶剂/去污剂对脂包膜病毒死活的效果。血浆样品与VSV病毒按9:1混合,然后用1％TNBP/1％ Triton X-100在30℃处理4h,测定开始样品中的病毒总量和S/D处理后不同时间取样内的病毒总量。实验中样品内加入1％TNBP/1％ Triton X-10015min后VSV病毒已全部灭活,灭活效果≥6.0log。按所述S/D处理方法可以完全灭活血浆内所有的脂包膜病毒而没有主要血浆蛋白的损失。     

A rapid method for demonstrating skeletal muscle motor innervation in frozen sections.

Longitudinal 50-100 mum-thick frozen sections of muscle are picked up on slides coated with 3% EDTA and after drying are incubated to demonstrate acetylcholinesterase. Subsequent incubation in 0.5% K3Fe(CN)6 is followed by fixation for 30 minutes in formol-calcium or formol-saline. After washing, the slides are incubated in 20% aqueous AgNO3 containing 0.1% CuSO4 for 2-30 minutes at 37 C. Following development in a 1% solution of quinol (w/v) 5% with respect to NaSO3 (w/v), axons and subneural apparatus stain dark brown to black in contrast to the less well stained muscle fibers and nuclei. This procedure permits study of the pattern of neuromuscular innervation in skeletal muscle 3 1/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.     

Fluorescent screening of transgenic Arabidopsis seeds without germination

In this paper, we describe a reliable method for the screening and selection of Arabidopsis transgenic seeds within minutes without germination. Expression of the Aspergillus niger beta-glucosidase gene BGL1 in the plant's endoplasmic reticulum was used as a visual marker, together with 4-methylumbelliferyl-beta-D-glucopyranoside (MUGluc) as a substrate. Subsequent to incubation in a solution of MUGluc at room temperature for 2 to 15 min, transgenic seeds expressing BGL1 demonstrated a distinct fluorescent signal under UV light. Optimal screening conditions at room temperature were achieved between 75 and 450 microm MUGluc, at a pH of 2.5 to 5.0 and 2 to 5 min of incubation. No significant loss of viability was detected in transgenic seeds that were redried and stored for 45 d after incubation in MUGluc solution for 2 to 150 min. Transgenic plants expressing BGL1 displayed normal phenotypes relative to the wild type. Selection frequency was 3.1% +/- 0.34% for the fluorescence selection method, while kanamycin resistant selection resulted in only 0.56% +/- 0.13% using the same seed batch. This novel selection method is nondestructive, practical, and efficient, and eliminates the use of antibiotic genes. In addition, the procedure shortens the selection time from weeks to minutes.     

Two Quick Methods for Voges-Proskauer Test

Two previously described methods for rapidly performing a Voges-Proskauer (VP) test were modified so that results could be obtained even more quickly. On MacConkey agar, colonies of lactose-fermenting, VP-positive organisms usually contain enough acetoin to give a positive reaction when a dense suspension is prepared in creatine and tested directly with Barritt reagents. With this direct-screening test, no "false" positive results were seen, but 29 of 429 lactose-fermenting strains of Enterobacteriaceae initially gave "false" negative results. Thus, another technique was needed for quickly confirming negative results. A 0.2-ml amount of MR-VP Broth culture, inoculated with one colony or less, can be tested after 4 to 6 hr at 37 C by adding two drops of a 0.5% creatine solution followed by Barritt reagents. No "false" positive results were seen with 508 cultures, but one atypical coliform bacillus was negative with this method and VP positive after 48 hr of incubation in MR-VP Broth. With these two quick methods, the common VP positive organisms can be recognized within a few minutes or at most after a few hours.     

Reaction of the cell center to exposure to the calcium ionophore A-23187

Calcium ionophore A23187, taken at a concentration of 0.1 microgram/ml, quickly uncouples mitochondria in PE culture cells in medium 199. The cell ultrastructure undergoes reversible changes (especially that of mitochondria): maximum changes occur 2 hours after the start of the treatment; in 8 hours they become less pronounced. The adaptation of cells does not involve the ionophore inactivation in the medium. 10 micrograms/ml of A23187 induces gradual but irreversible alterations. Microtubules in PE cells are not destroyed when incubated in medium 199 containing 10 micrograms/ml of A23187 and 11 mM Ca2+. The addition of 10 micrograms/ml ionophore to the normal 199 medium (1.26 mM Ca2+) results in the formation of electron dense bodies in the cell center 30 minutes after the start of incubation. These bodies disappear in the course of a subsequent incubation. The number of cells with primary cilia decreases. The percentage of centrioles located perpendicularly to the substrate increases 30 minutes following treatment with 0.1 microgram/ml A23187 in medium 199. 2 hours after the start of treatment with 0.1 microgram/ml ionophore no such changes are detected; an electron dense halo appears around the centriolar cylinders. 8 hours after the start of treatment the structure of the cell center does not differ from the normal one.     

Effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented lesions

OBJECTIVE: To investigate the effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented melanocytic lesions. STUDY DESIGN: Twenty banal compound nevi with various grades of pigmentation were bleached by 0.5% and 1% KMnO4 for 0 to 20 minutes and by 10% H2O2 for 24 hours prior to Feulgen staining. DNA microdensitometry was performed by video image analysis to measure the integrated optical density (IOD) in nuclei from nevomelanocytes, lymphocytes and spinous keratinocytes. The DNA index of nevomelanocytes was calculated using spinous keratinocytes as the diploid controls. RESULTS: There were significant decreases in IOD (P < .05) in the nuclei of nevomelanocytes, lymphocytes and spinous keratinocytes after treatment with 1% KMnO4 for 5 and 10 minutes, but no significant changes were detected after treatment with 0.5% KMnO4 for 5 and 10 minutes. Severe tissue damage was observed in the Feulgen-stained slides treated with 1% KMnO4 for 15 and 20 minutes and with 10% H2O2 for 24 hours. There was no significant change in DNA index in any bleached sets measured. CONCLUSION: KMnO4 can affect Feulgen-DNA content if used in high concentrations or for long periods of incubation. The DNA index, which is derived from internal controls, is not affected by the bleach procedure.     

The application of viable count procedures for measuring viable cells in the various marine environments

Aims: To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony‐forming units and the contribution of capsule‐bearing bacteria to the total number of bacteria and esterase‐active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Methods and Results: Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0–30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l^?1 (at 35°C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l^?1 for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. Conclusions: The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. Significance and Impact of the Study: The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.     

Appearance of D2O in sweat after oral and oral-intravenous rehydration in men

Intravenous (IV) rehydration is common in athletics, but its thermoregulatory benefits and ergogenicity have not been elucidated. Availability of orally ingested fluid is dependent on gastric emptying and intestinal absorption rate. Deuterium oxide (D2O) has been used to demonstrate that fluid ingested during exercise appears in sweat within 10 minutes. The purpose of this study was to determine the effect of concurrent IV rehydration on D2O appearance in sweat samples after per ora rehydration with D2O labeled fluid. We hypothesized that the combination method would not be superior to the oral method. Ten fit men (age 23 ± 4, VO2max 59.49 ± 4.09 L·min(-1)) underwent 20 hours of fluid restriction resulting in 1.95 ± 0.25% body weight loss before beginning treadmill exercise and cycling. Exercise was performed in an environmental chamber (35.6 ± 0.2° C, 35.0 ± 1.8% relative humidity) for 2 hours at 55% VO2max, and the participants exhibited a mean body weight deficit of 4.50 ± 0.04%. Thermoregulatory measures were recorded while subjects were rehydrated with oral (OR) or oral combined with intravenous (IVO) fluid traced with D2O. After 30 minutes of rehydration and 30 minutes of seated recovery, the subjects began treadmill exercise at 55-60% VO2max. Forehead sweat samples were collected 0, 5, 10, 20, and 75 minutes from the start of rehydration. The samples were analyzed for D2O via isotope ratio mass spectrometry. D2O did not appear in the sweat within 20 minutes of rehydration; however, it did appear during the subsequent exercise bout. There was no significant difference between rehydration modes. Plasma volume increases and decreased volume of orally ingested fluid did not significantly alter transit time from ingestion to appearance in excreted sweat. The IVO method does not appear to be superior to the traditional OR method of rehydration.     

Evidence suggesting a role for cyclic nucleotides in acrosome reactions of hamster sperm in vitro

There have been conflicting reports concerning the involvement of cyclic nucleotides in sperm capacitation. We have examined the effects of micromolar concentrations of dibutyryl cyclic AMP (Bt2cAMP) and of the phosphodiesterase inhibitors SQ20009 and ICI63,197 on hamster sperm incubated under in vitro capacitating conditions. Washed hamster sperm were incubated in a capacitation media containing bovine serum albumin, and a protein-free "motility-factor" from bovine adrenal cortex. Incubation for 3.5 hours was followed by addition of one of the compounds (0.1-10 microM) or control buffer. At the time of addition and after 30-120 minutes further incubation, sperm were examined by phase contrast microscopy. The final motility was similar to the initial motility (50-70%) and the same in incubation of controls or experimental compounds. Bt2cAMP, SQ20009, and ICI63,197 at these concentrations stimulated acrosome reactions to a statistically significant extent (P less than 0.005) compared to controls. Activation was stimulated to a varying degree by all three experimental compounds. These results suggest a role for cyclic nucleotides in capacitation and the acrosome reaction of hamster sperm.     

Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers

The present study was performed to test the hypothesis that orally administered essential amino acids, in combination with carbohydrate, will stimulate net muscle protein synthesis in resting human muscle in vivo. Four volunteers ingested 500 mL of a solution containing 13.4 g of essential amino acids and 35 g sucrose (EAA). Blood samples were taken from femoral arterial and venous catheters over a 2-hour period following the ingestion of EAA to measure arteriovenous concentrations of amino acids across the muscle. Two muscle biopsies were taken during the study, one before administration of the drink and one approximately 2 hours after consumption of EAA. Serum insulin increased from normal physiologic levels at baseline (9.2 +/- 0.8 microU/mL) and peaked (48 +/- 7.1 microU/mL) 30 minutes after EAA ingestion. Arterial essential amino acid concentrations increased approximately 100 to 400% above basal levels between 10 and 30 minutes following drink ingestion. Net nitrogen (N) balance changed from negative (-495 +/- 128 nmol/mL) prior to consumption of EAA to a peak positive value (416 +/- 140 nmol/mL) within 10 minutes of ingestion of the drink. EAA resulted in an estimated positive net N uptake of 307.3 mg N above basal levels over the 2-hour period. Muscle amino acid concentrations were similar prior to and 2 hours following ingestion of EAA. We conclude that ingestion of a solution composed of carbohydrates to stimulate insulin release and a small amount of essential amino acids to increase amino acid availability for protein synthesis is an effective stimulator of muscle protein anabolism.     

Wellcozyme: a new kit for the detection of anti-HIV antibodies. Considerations apropos of the initial trials

We tested the Wellcozyme anti-HTLV III kit on: 600 blood donors fresh sera, simultaneously tested by Elavia (Diagnostics Pasteur) or Vironostika (Organon); 20 plasmas, known to be anti-HIV negative; 22 sera, already labelled, and a panel of 10 specimens from the "Retrovirus" study group of the French National Society of Blood Transfusion, also tested by Elavia, Vironostika, Abbott HTLV III EIA and by two confirmatory tests: Immuno-blot (Diagnostics Pasteur) and Abbott confirmatory test. The Wellcome kit is, at present, the only one to use a competitive Elisa method and not to require any sample predilution. The method is easy to perform, rapid (results in 1 h 30 time) and it tests as well serum as plasma. No interferences have been observed from high lipid concentrations or strong hemolysis on the Elisa reaction. The performances of the Wellcozyme test are satisfactory. We did not find any "false positive" result. But the study shows that this kit is not sensitive enough for the detection of anti-P25 antibodies, which appear on the beginning of seroconversion, unless the "grey range" is extended: the Wellcome Laboratories have just made this recommendation.     

Agonal phase,ischaemic times,and renal vascular abnormalities and outcome of cadaver kidney transplants.

A retrospective study of 250 cadaver kidney transplants was carried out to determine the effects of the agonal period, the warm and cold ischaemic times, and the use of kidneys with vascular anomalies on the primary success and failure and the subsequent level of function of the transplants. Kidneys with vascular anomalies or from non-ventilated donors had a primary failure rate of over 30%, whereas those with normal vasculature or from ventilated donors had a rate of 17%. An initial warm ischaemic time of more than 60 minutes was associated with a primary failure rate of 57% and a cold ischaemic time of over 550 minutes with a primary failure rate of 47%. The interrelationship between the warm and cold ischaemic times in the primary success or failure of the transplants was examined and criteria defined for selecting potentially viable cadaver kidneys for transplantation, as follows: (1) The donor should be (a) ventilated, (b) aged 6-50 years, and (c) have normal ante-mortem renal function and have secreted more than 1-5 1 of urine in the 24 hours before death (or an equivalent volume if the urinary output was recorded for less than 24 hours before death); (2) the kidney should have normal renal vasculature enabling single arterial and venous anastomoses to be performed; (3) kidneys with I.W.I.T.s of longer than 60 minutes should not be used; (4) for kidneys with I.W.I.T.s of less than 20 minutes the C.I.T. is not critical but should not exceed 12 hours; (5) for kidneys with I.W.I.T.s of 20-60 minutes the C.I.T. should not exceed 450 minutes.