Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Immature development times of the blow fly Protophormia terraenovae (Robineau-Desvoidy, 1830) were studied in the laboratory at five different constant temperatures (15, 20, 25, 30, 35 degrees C). The minimal duration of development from oviposition to adult emergence was inversely related to temperature, ranging from 9.19+/-0.3 days at 35 degrees C to 37.78+/-2.96 days at 15 degrees C. From linear regression of development rates at the five studied constant temperature regimes, it followed that the minimum development threshold (t(L)) for total immature development is 8.95 degrees C ( approximately 9 degrees C) and the overall thermal constant (K) for P. terraenovae is 240.2+/-9.3 day-degrees (DD) above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 degrees C. However, it is possible that developmental time from oviposition to adult eclosion might be different in various regions of the world, and that the thermal constant of a holarctic species like P. terraenovae is not same everywhere. Additionally, as the present paper shows, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework.     

Temperature-dependent development of Ophyra aenescens (Wiedemann, 1830) and Ophyra capensis (Wiedemann, 1818) (Diptera, Muscidae)

The influence of rearing temperature on the development rates of two "dump flies" Ophyra aenescens (Wiedemann, 1830) and Ophyra capensis (Wiedemann, 1818) is analysed. The development times of these species are determined. Flies were reared at three constant temperatures (17+/-1, 24+/-1 and 30+/-1 degrees C) with a photoperiod of 12:12 and at a relativity moisture of 75-95%. The minimum duration for each development stages, from eggs to pupae and from eggs to adult emergence of O. aenescens and O. capensis are reported. The development rate increases in both species as the rearing temperature rises. A temperature-dependent development model is calculated for each species. The larval and total development of these two species can be estimated if the environmental temperature is between 17 and 30 degrees C. Compared to O. aenescens, O. capensis has a higher threshold of development and a longer larval development time.     

Occurrences of flesh flies (Diptera: Sarcophagidae) on human cadavers in Switzerland, and their importance as forensic indicators

From 1993 to 2008, criminal investigations were conducted in the western part of Switzerland with special attention to blowfly and flesh fly species in order to estimate the post-mortem interval when requested by the police authorities. Flesh flies were found in only 33 cases out of 160. Five species of the genus Sarcophaga were identified (S. africa, S. argyrostoma, S. caerulescens, S. similis and S. sp.). The main species found on corpses (larval stage) was S. argyrostoma. The thermal constant (K) calculated for this species in Switzerland is 380.6 ± 16.3 (mean ± S.D.) degree-days. With the exception of S. caerulescens, found three times in the larval stage on corpses, the three other species are of minor forensic importance. S. argyrostoma is found during summer and indoors. This species colonises dead bodies, usually the same day as blowfly species, and it could be used to estimate the post-mortem interval. Other species are discussed in the light of current knowledge on their biology and ecology. It is recommended that voucher material be deposited in a museum, allowing further studies by relevant specialists, thereby helping investigators and avoiding misidentifications.     

Effects of refrigeration on the biometry and development of Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations

The aim of this study was to simulate the low temperatures that insects could experience between the time being sampled from cadavers and their arrival in the laboratory. This was in order to investigate the effect of low temperature on development of maggots. At different stages of development, individuals of Protophormia terraenovae (Robineau-Desvoidy) reared at 24 degrees C were submitted to a temperature of 4.0+/-0.5 degrees C for a period varying from 1 to 10 days. Independent of the stage of development at which the insects were refrigerated, the treatment induced significant changes on the duration of development. The effect of low temperature on the developmental time between the return to 24 degrees C to adult emergence depended on the larval stage that was refrigerated. When first instar larvae and prepupae were refrigerated, the time to emergence at 24 degrees C decreased with an increase of duration of the refrigeration period. Time to emergence increased under the same conditions when second instar larvae and pupae were refrigerated. These results indicate that keeping larvae of P. terraenovae at 4 degrees C does not just simply lead to a cessation of metabolism but disturbs the regular development. Ten days of cooling induced an error in estimating post-mortem interval (PMI) of more than 6h.     

Effect of Temperature on Six Different Developmental Landmarks within the Pupal Stage of the Forensically Important Blowfly Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (t_L) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework.     

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (D_z) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17–37 ⁰C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (D_z ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.     

Postmortem stability of some markers of intra-vital wounds

We have studied the diagnostic value of several markers of the intra-vital nature of wounds - cathepsin D (EC 3.4.23.5) and ions (Ca, Mg, Cu, Zn and Fe) - after the influence of putrefaction. For this purpose, we have inflicted vital wounds to six pigs, which were killed 20 min later. Ten minutes after death, wounds were excised with 5-6 cm of skin around the incision and maintained at three different temperatures (4, 18 and 28.5 +/- 13.4 degrees C). After varying periods of postmortem interval from 0 to 48 h, aliquots of each wound were taken and analyzed with atomic absorption spectrophotometry for ions and with UV-spectrophotometry for cathepsin D. Our results demonstrate that ions conserve their diagnostic ability to differentiate vital from postmortem wounds after the influence of putrefaction. Nevertheless, cathepsin D does not show this ability in these experimental conditions.     

Breath alcohol concentration determined with a new analyzer using free exhalation predicts almost precisely the arterial blood alcohol concentration

A new breath alcohol (ethanol) analyzer has been developed, which allows free exhalation, standardizes measured exhaled alcohol concentration to fully saturated water vapor at a body temperature of 37 degrees C (43.95 mg/L) and includes a built-in self-calibration system. We evaluated the performance of this instrument by comparing standardized alcohol concentration in freely expired breath (BrAC) with arterial (ABAC) and venous (VBAC) blood alcohol concentrations in fifteen healthy volunteers who drank 0.6 g of alcohol per kg body weight. The precision (coefficient of variation, CV) of the analyzer based on in vivo duplicate measurements in all phases of the alcohol metabolism was 1.7%. The ABAC/BrAC ratio was 2251+/-46 (mean+/-S.D.) in the post-absorptive phase and the mean bias between ABAC and BrAC x 2251 was 0.0035 g/L with 95% limits of agreement of 0.033 and -0.026. The ABAC and BrAC x 2251 were highly correlated (r=0.998, p<0.001) and the regression relationship was ABAC = 0.00045 + 1.0069 x (BrAC x 2251) indicating excellent agreement and no fixed or proportional bias. In the absorption phase, ABAC exceeded BrAC x 2251 by at most 0.04+/-0.03 g/L when tests were made at 10 min post-dosing (p<0.05). The VBAC/BrAC ratio never stabilized and varied continuously between 1834 and 3259. There was a proportional bias between VBAC and BrAC x 2251 (ABAC) in the post-absorptive phase (p<0.001). The pharmacokinetic analysis of the elimination rates of alcohol and times to zero BAC confirmed that BrAC x 2251 and ABAC agreed very well with each other, but not with VBAC (p<0.001). We conclude that this new breath analyzer using free exhalation has a high precision for in vivo testing. The BrAC reflects very accurately ABAC in the post-absorption phase and substantially well in the absorption phase and thereby reflects the concentration of alcohol reaching the brain. Our findings highlight the magnitude of arterio-venous differences in alcohol concentration and support the use of breath alcohol analyzers as a stand-alone test for medical and legal purposes.     

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16–34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D₀ and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D₀ = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.     

Comparison of ethanol concentrations in venous blood and end-expired breath during a controlled drinking study

Concentration-time profiles of ethanol were determined for venous whole blood and end-expired breath during a controlled drinking experiment in which healthy men (n=9) and women (n=9) drank 0.40-0.65 g ethanol per kg body weight in 20-30 min. Specimens of blood and breath were obtained for analysis of ethanol starting at 50-60 min post-dosing and then every 30-60 min for 3-6 h. This protocol furnished 130 blood-breath pairs for statistical evaluation. Blood-ethanol concentration (BAC, mg/g) was determined by headspace gas chromatography and breath-ethanol concentration (BrAC, mg/2l) was determined with a quantitative infrared analyzer (Intoxilyzer 5000S), which is the instrument currently used in Sweden for legal purposes. In 18 instances the Intoxilyzer 5000S gave readings of 0.00 mg/2l whereas the actual BAC was 0.08 mg/g on average (range 0.04-0.15 mg/g). The remaining 112 blood- and breath-alcohol measurements were highly correlated (r=0.97) and the regression relationship was BAC=0.10+0.91BrAC and the residual standard deviation (S.D.) was 0.042 mg/g (8.4%). The slope (0.91+/-0.0217) differed significantly from unity being 9% low and the intercept (0.10+/-0.0101) deviated from zero (t=10.2, P<0.001), indicating the presence of both proportional and constant bias, respectively. The mean bias (BAC - BrAC) was 0.068 mg/g and the 95% limits of agreement were -0.021 and 0.156 mg/g. The average BAC/BrAC ratio was 2448+/-540 (+/-S.D.) with a median of 2351 and 2.5th and 97.5th percentiles of 1836 and 4082. We found no significant gender-related differences in BAC/BrAC ratios, being 2553+/-576 for men and 2417+/-494 for women (t=1.34, P>0.05). The mean rate of ethanol disappearance from blood was 0.157+/-0.021 mg/(g per hour), which was very close to the elimination rate from breath of 0.161+/-0.021 mg/(2l per hour) (P>0.05). Breath-test results obtained with Intoxilyzer 5000S (mg/2l) were generally less than the coexisting concentrations of ethanol in venous blood (mg/g), which gives an advantage to the suspect who provides breath compared with blood in cases close to a threshold alcohol limit.     

Effect of storage conditions on restriction fragment length polymorphism (RFLP) analysis of deoxyribonucleic acid (DNA) bound to positively charged nylon membranes.

The ability to generate an autoradiogram from deoxyribonucleic acid (DNA) immobilized on a positively charged nylon membrane could be compromised by the storage conditions of the membrane. HaeIII-digested human DNA was size fractionated and transferred to two types of positively charged nylon membranes. The membranes were stored at -20 degrees C, 4 degrees C, and ambient temperature and humidity for times ranging from 1 day to 13 weeks, then hybridized to variable number of tandem repeat (VNTR) probes to examine the effect of the storage conditions on the membrane-bound DNAs. It was shown that such membranes could be successfully hybridized and rehybridized if they were stored at -20 or 4 degrees C, but storage under ambient conditions reduced or eliminated the likelihood of successful hybridization.     

Estimation of age from a tooth by means of racemization of an amino acid, especially aspartic acid--comparison of enamel and dentin.

In a study of age estimation from teeth by means of measuring racemization of aspartic acid (Asp), a representative amino acid, the accuracy of data from enamel and dentin in the same tooth was compared. The correlation of D/L ratio of aspartic acid with actual age gave the following parameters: r = 0.928, sigma = +/- 5.2, k = 4.47 x 10(-4) yr-1 in enamel and r = 0.995, sigma = +/- 1.4, k = 5.75 x 10(-4) yr-1. The difference in ages between one estimated by the D/L ratio and the actual one was within +/- 3 years in dentin, while in enamel an error of from 2 to 11 years was observed. Reaction rate constants of the racemization in a dry postmortem state (15 degrees C) were calculated as k = 9.70 x 10(-8) yr-1 in enamel, and k = 1.33 x 10(-7) yr-1 in dentin. Compared to rates determined from teeth recently extracted from living subjects, the rate was higher in dentin than in enamel. These data reconfirmed that dentin is superior to enamel in making exact age estimations from teeth.     

Selenium determination in mother and child's hair by electrothermal atomic absorption spectrometry

A method for the selenium determination in a mother and her child's hair using palladium as a chemical modifier was optimized. The sample was digested with nitric acid and hydrogen peroxide and diluted to 5 ml. To achieve complete mineralization the samples were ashed at 1200 degrees C in the presence of palladium as a chemical modifier. The optimum atomization temperature was 1900 degrees C. The precision and accuracy of the method were studied using the reference material CRM 397. Results of calibration using aqueous standards and the standard addition method were compared. The method was applied to the selenium determination in 30 samples of the mother's and child's hair. The levels found were 0.54 +/- 0.34 microgram/g for mother's hair and 0.77 +/- 0.25 microgram/g for child's hair.     

The nocturnal oviposition behaviour of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications

Numerous factors may cause delayed colonisation of a corpse by blowflies, leading to a discrepancy between the entomologically determined post-mortem interval (PMI) and the time of death. Blowflies, for example, are considered to be inactive at night, however, published observations are contradictory. In the present study, several field experiments and one type of indoor experiment were conducted in summer of 2004 and 2005 in order to investigate the nocturnal ovipositional behaviour of blowflies. In the field, two types of bait, dead hedgehogs and fresh beef liver, were placed at night in different urban and rural locations in Frankfurt and in Munich, Germany. For the indoor-experiments beef liver was placed in small plastic boxes containing caged Lucilia sericata females in the evening and left overnight. At night, no ovipositon was observed in the field (n=51, T=10-24 degrees C). Nocturnal oviposition in complete darkness occurred in the plastic boxes in two of six cases (T=25 degrees C). Considering the behavioural and physiological characteristics of flies we suggest that nocturnal oviposition of blowflies appears to be unlikely under natural conditions in Central Europe but may occur under certain circumstances, such as unusual high nightly temperatures and the presence of gravid flies with an appropriate arousal threshold.     

The development of the black blow fly, Phormia regina (Meigen).

The black blow fly, Phormia regina (Meigen) is a primary species commonly utilized to indicate a postmortem interval, or more appropriately a "time since colonization". Due to the importance of this species as a secondary myiasis producer in livestock operations, and more recently as a time since death indicator in the field of forensic entomology, a considerable amount of data on its growth and development has been generated. However, the developmental time as reported by these studies varies greatly, and current more detailed data is needed for use in medicocriminal entomology. Hourly developmental data is presented under constant temperatures of 10, 15, 20, 25, 30, 35 and 40 degrees C, and cyclic temperatures of 10-15, 15-25, 25-35 and 35-45 degrees C. This study is in agreement with the results reported by Kamal [Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera). I. Bionomics, Ann. Entomol. Soc. Am. 51 (1958) 261] and Melvin [Incubation period of eggs of certain musciod flies at different constant temperatures, Ann. Entomol. Soc. Am. 27 (1934) 406] only at temperatures of 25 degrees C and below. Bishopp [Flies which cause myiasis in man and animals: some aspects of the problem, J. Econ. Entomol. 8 (1915) 317] reported a shorter developmental duration for larval stages than what was produced with our laboratory rearings.     

Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram.

Developmental behavior of eggs, larva and pupa of the blowfly species Lucilia sericata (Meigen) were studied under 10 different temperature regimes. Data from these studies were used to construct the isomegalen-diagram. In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. If the temperature is roughly constant, as is the case with corpses found indoors, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable, an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. Equally, the isomorphen-diagram representing all morphological stages from oviposition to eclosion should be used, if maggots in the migratory phase or pupae or puparia are recovered from the scene. The isomegalen- and the isomorphen-diagrams could facilitate a quick and more precise estimate of the postmortem interval even for the inexperienced investigator. In addition, our results vary from those of other investigators, suggesting a different thermal behavior of the holarctic blowfly L. sericata in various zoogeographic regions.     

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.     

Experimental studies on death by fire in automobiles and exhaust gas poisoning

Studies were made on the acid-base balance, blood gases, and carbon monoxide (CO), cyanide, and sulfur dioxide concentrations in the blood of albino rabbits that died from automobile exhaust gas poisoning (group I) or fires in cars (complete combustion, group II; incomplete combustion, group III). In group I, the temperature and CO concentration increased gradually to 35 degrees C and 5.2% in 70 min. The animals died after 9 min, when the values were 20 degrees C and 5.2%, respectively. In group II the animals died after 9 min, when the values were 55 degrees C and 1.95%, respectively. In group III, the temperature was very high (870 degrees C), but the CO concentration was not (0.6-1.3%) after 4 min. The animals died after 5 min. In all experimental groups, marked acidosis and hypoxemia were seen, but the CO2 tension (PCO2) was high, in contrast to previous studies on pure CO poisoning. In group I, the level of carboxyhemoglobin (CO-Hb) was significantly higher (91.2 +/- 3.4% in arterial blood, 87.5 +/- 8.1% in venous blood; p less than 0.01) than in groups II and III. Although the O2 tensions of venous and arterial blood (PvO2, PaO2) were very low, that of arterial blood was higher, suggesting that O2 was still being utilized in the tissues at the time of death. In group II, CO-Hb was high (57.7 +/- 16.0% in arterial blood, 61.2 +/- 20.6% in venous blood) and the acid-base balance indicated marked acidosis. In group III, the CO-Hb, PCO2 and cyanide levels in the blood were very high.(ABSTRACT TRUNCATED AT 250 WORDS)     

A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine

A solid-phase microextraction-gas chromatographic-mass spectrometric (SPME-GC-MS) method has been developed and validated for measuring four club drugs in human urine. These drugs include gamma-hydroxybutyrate (GHB), ketamine (KET), methamphetamine (MAMP), and methylenedioxymethamphetamine (MDMA). These drugs are referred to as 'club drugs' because of their prevalence at parties and raves. Deuterium labeled internal standards for each of the four drugs was included in the assay to aid in quantitation. The drugs were spiked into human urine and derivatized using pyridine and hexylchloroformate to make them suitable for GC-MS analysis. The SPME conditions of extraction time/temperature and desorption time/temperature were optimized to yield the highest peak area for each of the four drugs. The final SPME parameters included a 90 degrees C extraction for 20min with a 1min desorption in the GC injector at 225 degrees C using a splitless injection. All SPME work was done using a 100microm PDMS fiber by Supelco. The ratio of pyridine to hexylchloroformate for derivatization was also optimized. The GC separation was carried out on a VF-5ht column by Varian (30m, 0.25mm i.d., 0.10microm film thickness) using a temperature program of 150-270 degrees C at 10 degrees C/min. The instrument used was a ThermoFinnigan Trace GC-Polaris Q interfaced with a LEAP CombiPal autosampler. The data was collected by using extracted ion chromatograms of marker m/z values for each drug from the total ion chromatograms (TIC) (full scan mode). Calibration curves with R(2)>0.99 were generated each day using the peak area ratios (peak area drug/peak area internal standard) versus concentration. The validated method resulted in intra-day and inter-day precision (% R.S.D.) of less than 15% and a % error of less than 15% for four concentrations in the range of 0.05-20microg/mL (MAMP) and 0.10-20microg/mL (GHB, KET, and MDMA). This method has the advantage of an easy sample preparation with acceptable accuracy and precision for the simultaneous quantification of these four drugs of abuse and shows no interference from the urine matrix.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.