Flow-batch analyser for preparation of calibration standard mixtures in simultaneous multicomponent spectrometric analysis

The application of multivariate calibration techniques to multicomponent analysis by UV-VIS molecular absorption spectrometry is a powerful tool for simultaneous determination of several chemical species. However, when this methodology is accomplished manually, it is slow and laborious, consumes high amounts of reagents and samples, is susceptible to contaminations and presents a high operational cost. To overcome these drawbacks, a flow-batch analyser is proposed in this work. This analyser was developed for automatic preparation of standard calibration and test (or validation) mixtures. It was applied to the simultaneous determination of Cu2+, Mn2+ and Zn2+ in polyvitaminic and polymineral pharmaceutical formulations, using 4-(2-piridilazo) resorcinol as reagent and a UV-VIS spectrophotometer with a photodiode array detector. The results obtained with the proposed system are in good agreement with those obtained by flame atomic absorption spectrometry, which was employed as reference method. With the proposed analyser, the preparation of calibration and test mixtures can be accomplished about four hours, while the manual procedure requires at least two days. Moreover, it consumes smaller amounts of reagents and samples than the manual procedure. After the preparation of calibration and test mixtures, 60 samples h-1 can be carried out with the proposed flow-batch analyser.

Kuumailmapolttolaitteiston numeerinen virtausmallinnus

Tämä diplomityö perustuu Lappeenrannan teknillisen yliopiston Uusiutuvien energiajärjestelmien laboratorion koelaitteistoon, jolla tutkitaan voimakkaan savukaasunkierrätyksen ja kuumailmapolton soveltuvuutta pienen kokoluokan energiantuotantoprosesseihin. Työn teoriaosassa esitellään tavanomaisesta palamisesta eroavaa kuumailmapolttoa ja tarkastellaan sen ominaisuuksia. Myös työssä käytetyn tutkimusmenetelmän, numeerisen virtauslaskennan, periaatteita ja ominaisuuksia tarkastellaan. Työssä tutkitaan numeerisella virtausmallinnuksella kuumailmapolttolaitteiston virtauskentän käyttäytymistä, kun takaisin tulipesään kierrätettävän savukaasun määrä sekä tulipesän lämpöhäviöiden suuruus vaihtelevat. Virtauskentän tarkastelu on tärkeää, sillä palamisilman ja kierrätetyn savukaasun täytyy sekoittua kuumailmapolton aikaansaamiseksi. Työn virtausmallinnus suoritettiin Finflo-virtausratkaisijalla kaksiulotteisena palamisreaktioita mallintamatta. Vaikka työssä käytetyt mallit olivat kaksiulotteisia ja niissä käytettiin yksinkertaistuksia, virtausten käyttäytymisestä tulipesässä saatiin olennaista tietoa, jota voidaan mahdollisesti hyödyntää jatkotutkimuksissa.

Potential of adjuvants to reduce drift in agricultural spraying

The reduction of pesticide spraying drift is still one of the major challenges in Brazilian agriculture. The aim of this study was to evaluate the potential of different adjuvant products, such as surfactants, drift retardants, mineral oil and vegetable oil for reducing drift in agricultural spraying. The experiment consisted of quantifying drift of sprayings of 18 adjuvants dissolved in water under controlled conditions in a wind tunnel. Tests were performed in triplicates with spraying nozzles type Teejet XR8003 VK, pressure of 200kPa and medium drops. Solutions sprayed were marked with Brilliant Blue dye at 0.6% (m v-1). The drift was collected using polyethylene strips transversally fixed along the tunnel at different distances from the nozzle and different heights from the bottom part of the tunnel. Drift deposits were evaluated by spectrophotometry in order to quantify deposits. The adjuvants from chemical groups of mineral oil and drift retardant resulted in lower values of drift in comparison with surfactants and water. The results obtained in laboratory show that the selection of appropriate class and concentration of adjuvants can significantly decrease the risk of drift in agricultural spraying. However, the best results obtained in laboratory should be validated with pesticide under field conditions in the future.

Potassium distribution in drip irrigation with fertigation for different injection distances in the main line

The purpose of this research was to evaluate the K2O distribution uniformity by surface drip irrigation at Universitat Politecnica de Valencia, Valencia, Spain (39º 29′ N, 0º 23′ W, 20 m). The irrigation was performed by drip lines with not-compensated emitters, spaced 0.3 m. The fertigation was realized using a fertilizer injector pump of electric action with injection of 0.25 h. The experimental design used completely randomized blocks with five treatments and four replications. The treatments consisted of injection in five distances, located at 10; 20; 30; 40; 50 m of the first drip line. Samples were collected in emitters located at the start, at 1/3, at 2/3 and at the end of the drip lines. The nutrient concentration was determined by flame spectrophotometry. The Christiansen's uniformity coefficients (CUC), of distribution (DUC), of statistical (SUC) and of emission (eUC) were estimated. The K2O concentration and distribution decreased linearly with the increase of the injection distance. In all treatments, the CUC, SUC and DUC were described as 'excellent'. The eUC was described as 'recommended' only at smaller injection distances.

Characterisation of Laser Beam and Paper Material Interaction

It is known already from 1970´s that laser beam is suitable for processing paper materials. In this thesis, term paper materials mean all wood-fibre based materials, like dried pulp, copy paper, newspaper, cardboard, corrugated board, tissue paper etc. Accordingly, laser processing in this thesis means all laser treatments resulting material removal, like cutting, partial cutting, marking, creasing, perforation etc. that can be used to process paper materials. Laser technology provides many advantages for processing of paper materials: non-contact method, freedom of processing geometry, reliable technology for non-stop production etc. Especially packaging industry is very promising area for laser processing applications. However, there are only few industrial laser processing applications worldwide even in beginning of 2010´s. One reason for small-scale use of lasers in paper material manufacturing is that there is a shortage of published research and scientific articles. Another problem, restraining the use of laser for processing of paper materials, is colouration of paper material i.e. the yellowish and/or greyish colour of cut edge appearing during cutting or after cutting. These are the main reasons for selecting the topic of this thesis to concern characterization of interaction of laser beam and paper materials. This study was carried out in Laboratory of Laser Processing at Lappeenranta University of Technology (Finland). Laser equipment used in this study was TRUMPF TLF 2700 carbon dioxide laser that produces a beam with wavelength of 10.6 μm with power range of 190-2500 W (laser power on work piece). Study of laser beam and paper material interaction was carried out by treating dried kraft pulp (grammage of 67 g m-2) with different laser power levels, focal plane postion settings and interaction times. Interaction between laser beam and dried kraft pulp was detected with different monitoring devices, i.e. spectrometer, pyrometer and active illumination imaging system. This way it was possible to create an input and output parameter diagram and to study the effects of input and output parameters in this thesis. When interaction phenomena are understood also process development can be carried out and even new innovations developed. Fulfilling the lack of information on interaction phenomena can assist in the way of lasers for wider use of technology in paper making and converting industry. It was concluded in this thesis that interaction of laser beam and paper material has two mechanisms that are dependent on focal plane position range. Assumed interaction mechanism B appears in range of average focal plane position of 3.4 mm and 2.4 mm and assumed interaction mechanism A in range of average focal plane position of 0.4 mm and -0.6 mm both in used experimental set up. Focal plane position 1.4 mm represents midzone of these two mechanisms. Holes during laser beam and paper material interaction are formed gradually: first small hole is formed to interaction area in the centre of laser beam cross-section and after that, as function of interaction time, hole expands, until interaction between laser beam and dried kraft pulp is ended. By the image analysis it can be seen that in beginning of laser beam and dried kraft pulp material interaction small holes off very good quality are formed. It is obvious that black colour and heat affected zone appear as function of interaction time. This reveals that there still are different interaction phases within interaction mechanisms A and B. These interaction phases appear as function of time and also as function of peak intensity of laser beam. Limit peak intensity is the value that divides interaction mechanism A and B from one-phase interaction into dual-phase interaction. So all peak intensity values under limit peak intensity belong to MAOM (interaction mechanism A one-phase mode) or to MBOM (interaction mechanism B onephase mode) and values over that belong to MADM (interaction mechanism A dual-phase mode) or to MBDM (interaction mechanism B dual-phase mode). Decomposition process of cellulose is evolution of hydrocarbons when temperature is between 380- 500°C. This means that long cellulose molecule is split into smaller volatile hydrocarbons in this temperature range. As temperature increases, decomposition process of cellulose molecule changes. In range of 700-900°C, cellulose molecule is mainly decomposed into H2 gas; this is why this range is called evolution of hydrogen. Interaction in this range starts (as in range of MAOM and MBOM), when a small good quality hole is formed. This is due to “direct evaporation” of pulp via decomposition process of evolution of hydrogen. And this can be seen can be seen in spectrometer as high intensity peak of yellow light (in range of 588-589 nm) which refers to temperature of ~1750ºC. Pyrometer does not detect this high intensity peak since it is not able to detect physical phase change from solid kraft pulp to gaseous compounds. As interaction time between laser beam and dried kraft pulp continues, hypothesis is that three auto ignition processes occurs. Auto ignition of substance is the lowest temperature in which it will spontaneously ignite in a normal atmosphere without an external source of ignition, such as a flame or spark. Three auto ignition processes appears in range of MADM and MBDM, namely: 1. temperature of auto ignition of hydrogen atom (H2) is 500ºC, 2. temperature of auto ignition of carbon monoxide molecule (CO) is 609ºC and 3. temperature of auto ignition of carbon atom (C) is 700ºC. These three auto ignition processes leads to formation of plasma plume which has strong emission of radiation in range of visible light. Formation of this plasma plume can be seen as increase of intensity in wavelength range of ~475-652 nm. Pyrometer shows maximum temperature just after this ignition. This plasma plume is assumed to scatter laser beam so that it interacts with larger area of dried kraft pulp than what is actual area of beam cross-section. This assumed scattering reduces also peak intensity. So result shows that assumably scattered light with low peak intensity is interacting with large area of hole edges and due to low peak intensity this interaction happens in low temperature. So interaction between laser beam and dried kraft pulp turns from evolution of hydrogen to evolution of hydrocarbons. This leads to black colour of hole edges.

Thermal radiation in combustion systems

A numerical procedure for solving the nongray radiative transfer equation (RTE) in two-dimensional cylindrical participating media is presented. Nongray effects are treated by using a narrow-band approach. Radiative emission from CO, CO2, H2O, CH4 and soot is considered. The solution procedure is applied to study radiative heat transfer in a premixed CH4-O2, laminar, flame. Temperature, soot and IR-active species molar fraction distributions are allowed to vary in the axial direction of the flame. From the obtained results it is possible to quantify the radiative loss in the flame, as well as the importance of soot radiation as compared to gaseous radiation. Since the solution procedure is developed for a two-dimensional cylindrical geometry, it can be applied to other combustion systems such as furnaces, internal combustion engines, liquid and solid propellant combustion.

Arc fusion of self-fluxed nickel alloys

Self-fluxed nickel alloys are usually flame fused after thermal spraying. However, due to the practical aspects of high temperatures reached during flame fusing, large structures such as the hydraulic turbines for power generation, can not be efficiently coated. An alternative is to fuse the sprayed coating with a gas tungsten electric arc. In this case, heating is much more intensive and substrate temperature during and after the fusing operation is much lower, thus reducing the possibility that any problem will occur. In this work, coatings of self-fluxed nickel alloy fused by flame and gas tungsten arc were evaluated as protection of hydraulic turbines against cavitational damage. Several tests were performed, including the ASTM ultrasonically vibration-induced cavitation, optical and scanning electronic microscopic metallography, and hardness tests. The results showed that the arc-fused coating presented better cavitation damage resistance, probably due to its finer microstructure. A field application of this new technique is also described. A self-fluxed Ni alloy was flame sprayed in critical regions of Francis-type hydraulic turbine blades and fused by a gas tungsten arc after spraying. The blades will be inspected during the next two years.

An experimental investigation of the effects of nozzle ellipticity on the flow structure of co-flow jet diffusion flames

The flow structure of cold and ignited jets issuing into a co-flowing air stream was experimentally studied using a laser Doppler velocimeter. Methane was employed as the jet fluid discharging from circular and elliptic nozzles with aspect ratios varying from 1.29 to 1.60. The diameter of the circular nozzle was 4.6 mm and the elliptic nozzles had approximately the same exit area as that of the circular nozzle. These non-circular nozzles were employed in order to increase the stability of attached jet diffusion flames. The time-averaged velocity and r.m.s. value of the velocity fluctuation in the streamwise and transverse directions were measured over the range of co-flowing stream velocities corresponding to different modes of flame blowout that are identified as either lifted or attached flames. On the basis of these measurements, attempts were made to explain the existence of an apparent optimum aspect ratio for the blowout of attached flames observed at higher values of co-flowing stream velocities. The insensitivity of the blowout limits of lifted flames to nozzle geometry observed in our previous work at low co-flowing stream velocities was also explained. Measurements of the fuel concentration at the jet centerline indicated that the mixing process was enhanced with the 1.38 aspect ratio jet compared with the 1.60 aspect ratio jet. On the basis of the obtained experimental data, it was suggested that the higher blowout limits of attached flames for an elliptic jet of 1.38 aspect ratio was due to higher entrainment rates.

Comparison of Biomass Gasification Technologies for Natural Gas Substitution in Iron Ore Pelletizing Plants

The iron ore pelletizing process consumes high amounts of energy, including nonrenewable sources, such as natural gas. Due to fossil fuels scarcity and increasing concerns regarding sustainability and global warming, at least partial substitution by renewable energy seems inevitable. Gasification projects are being successfully developed in Northern Europe, and large-scale circulating fluidized bed biomass gasifiers have been commissioned in e.g. Finland. As Brazil has abundant biomass resources, biomass gasification is a promising technology in the near future. Biomasses can be converted into product gas through gasification. This work compares different technologies, e.g. air, oxygen and steam gasification, focusing on the use of the product gas in the indurating machine. The use of biosynthetic natural gas is also evaluated. Main parameters utilized to assess the suitability of product gas were adiabatic flame temperature and volumetric flow rate. It was found that low energy content product gas could be utilized in the traveling grate, but it would require burner’s to be changed. On the other hand, bio-SGN could be utilized without any adaptions. Economical assessment showed that all gasification plants are feasible for sizes greater than 60 MW. Bio-SNG production is still more expensive than natural gas in any case.

Sähköisen taloushallinnon kehitys ja ilmiön näkyvyys alan ammattilehtikirjoittelussa

Pro gradu –tutkielman ensisijaisena tavoitteena on tutkia sähköisen ta-loushallinnon kehitystä ja miten se on näkynyt alan suomalaisessa ja kansainvälisessä ammattilehtikirjoittelussa vuosina 1997 – 2013. Tarkoi-tuksena on löytää kirjoittelun perusteella sähköisen taloushallinnon kehi-tyksen edistäviä ja hidastavia tekijöitä sekä minkälaisia tulevaisuuden näkymiä artikkelit luovat sähköiselle taloushallinnolle. Lisäksi tavoitteena on löytää yhtäläisyyksiä ja eroja kansallisen ja kansainvälisen kirjoittelun välillä. Tutkielma on laadullinen tutkimus ja tutkimusmenetelminä käytetään si-sällönanalyysia, sisällönerittelyä, teemoittelua ja vertailevaa tutkimusta. Tutkielman empiirinen aineisto koostuu Tilisanomien, Balanssi–lehden ja Accountancy–lehden sähköistä taloushallintoa koskevista artikkeleista aikavälillä 1997 – 2013. Tutkimustulosten perusteella sähköisen taloushallinnon kehityskulku näkyy myös ammattilehtikirjoittelussa. Tietojärjestelmät ja niiden käytön mahdollistava lainsäädäntö ovat sähköisen taloushallinnon perusedelly-tykset. Suurimpia kehityksen edistäjiä ovat julkinen valta, verkkolaskut ja standardointi. Kehityksen hidastajina nähdään yhtenäisten standardien puute ja asenteet. Sähköisen taloushallinnon tulevaisuuden näkymiä ovat standardoidut prosessit ja käsitteet koko taloushallinnon alueella sekä viranomaisraportointi XBRL-kielen avulla. Suurimmat erot kansalli-sessa ja kansainvälisessä kehityksessä on havaittavissa verkkolaskuissa ja XBRL-raportoinnissa. Johtopäätöksenä todetaan että kirjoittelun perusteella voidaan löytää sähköisen taloushallinnon kehitystä hidastavia ja edistäviä tekijöitä ja se luo viitteitä tulevaisuuden kehitykselle. Sähköinen taloushallinto kehittyy kunkin maan valtiovallan tahtotilan ja lainsäädännön mukaan. Jatkotut-kimuskohteena tutkielmaa voisi laajentaa kansainvälisemmäksi ottamalla mukaan tarkasteluun useampia kansainvälisiä lehtiä ja kansainvälistä lainsäädäntöä.

Koivuvanerin palonkesto-ominaisuuksien parantaminen liimaseoksen koostumusta muuttamalla

Viime vuosina puutuotteiden palonkestävyydelle asetetut vaatimukset ovat kasvaneet. Puutuotteiden käyttöä on mahdollista lisätä, mikäli niiden palonkestävyyttä saadaan parannettua. Tässä diplomityössä tutkittiin palonestoaineiden vaikutusta liimaseoksen ominaisuuksiin, liimautuvuuteen ja palonkestävyyteen. Työn tarkoituksena oli löytää liimaseos, jonka avulla saadaan koivuvanerin palonkestävyyttä parannettua. Teoriaosuudessa on käyty läpi koivuvanerin valmistusprosessi ja käytettävät liimat, ominaisuudet sekä käyttökohteet. Teoriaosuudessa käsitellään myös puutuotteiden syttymistä ja vertaillaan erilaisia palonestoaineita ja niiden vaikutusmekanismeja. Työn kokeellinen osuus koostui hartsiseoksen imeytyvyyden tutkimisesta, liimaseosten ominaisuuksien selvittämisestä, vanerilevyjen liimautuvuuden ja liimauslujuuden testaamisesta sekä palonkestävyysominaisuuksien selvittämisestä. Työssä saavutettiin pientä parannusta levyjen palonkestävyysominaisuuksiin. Tavoitteeksi asetettua palonkestävyyden kaksinkertaistumista ei saavutettu. Työ antaa kuitenkin hyvän pohjan palonestoaineiden ja vanerihartsien palonkestävyys-ominaisuuksien tutkimisen jatkamiselle. Työssä saavutettuja tuloksia tullaan hyödyntämään vanerilevyjen palo-ominaisuuksien parantamiseen tähtäävissä tutki-muksissa.

Sunflower response to repeated foliar applications of Paclobutrazol

Paclobutrazol (PBZ), a well-known growth retardant of the triazole family, is mostly used for controlling plant size and growth, resulting in more desirable compact plants for floricultural purposes; however, limited experimental data are available for use of PBZ in field sunflower. The objective of this study was to evaluate the effect of repeated foliar applications of PBZ at rates of 50 + 50 g ha-1(double application) and 50 + 50 + 50 g ha-1 (triple application) on sunflower morphology and productivity. PBZ applications corresponded to growth stages of four to five, five to six, and six to eight true leaves of sunflower plants, respectively. The double foliar application of PBZ reduced sunflower plant height at maturity by 4.4% (or by 11.7 cm). The triple foliar application of PBZ reduced sunflower plant height at maturity by 14.4% (or by 49.2 cm). However, PBZ either in the double or the triple application reduced achene yield per plant by 25.6% and 22.5% and the 100-achene weight by 11.4% and 25.0%, respectively, compared with the non-treated control. Overall, the repeated foliar applications of PBZ at the rates tested in this study, apart from providing a reduction of sunflower plant height, had major adverse effects on achene yield and weight. Thus, different application schemes of PBZ or perhaps different growth regulators should be investigated to control plant height in sunflower.

Kyberaseiden vaikutus kriittisen infrastruktuurin tietojärjestelmiin

Työn tehtävänä on selvittää lähdemateriaalin avulla hyökkäys- ja puolustusmenetelmistä valtiolliseen kyberturvallisuuteen liittyvät keskeisimmät menetelmät ja niiden vaikutukset, tarkastelemalla niitä maailmalla tapahtuneiden kyberhyökkäyksien pohjalta. Tutkimuksella pyritään vastaamaan seuraaviin alakysymyksiin: minkä tyyppisiä kyberaseita käytetään kriittisen infrastruktuurin tietojärjestelmiä vastaan, mikä on niiden vaikutus ja millaisia kyberhyökkäyksiä valtioiden kriittisen infrastruktuurin tietojärjestelmiä vastaan maailmalla on toteutettu sekä miten ne kyetään suojaamaan näiltä kyberuhilta? Tavoitteena on, että tutkimuksella kasvatetaan lukijan tietämystä kyberavaruuden vaaroista ja kriittisen infrastruktuurin tietojärjestelmistä. Keskeiset lähderyhmät ovat aiemmat tutkimukset, kirjallisuus, uutisartikkelit ja erityisesti sähköiset lähteet. Tärkeimpiä yksittäisiä lähteitä ovat muun muassa Suomen valtioneuvoston tuottama Suomen kyberturvallisuustrategia-periaatepäätös (2012) ja Mika-Jan Pullisen tekemä opinnäytetyö Kriittisten tietojärjestelmien suojaaminen kyberuhilta (2012). Tutkimusmenetelmänä käytetään kirjallisuuskatsausta. Tietojärjestelmiin hyökätessä käytetään yhä ammattimaisempia välineitä. Hyökkäystarkoituksen pohjalta suunnitellut ja valmistetut hienostuneet ohjelmistot ovat usein kyberterroristien tai jopa valtiollisten toimijoiden suunnittelemia. Kyberhyökkäyksistä vain prosentti on ollut varsinaisia valtioiden tuottamia kyberaseita. Merkittävinä kyberhyökkäyksinä voidaan pitää muun muassa Stuxnet-tietokonematoa, Flame-vakoiluohjelmaa ja Shamoon-tietokonevirusta. Kyberhyökkäyksistä Stuxnet-tietokonemadolla kyettiin tuottamaan fyysistä vahinkoa tietojärjestelmiin kohdistuvan vaikutuksen lisäksi. Kriittisen infrastruktuurin tietojärjestelmien suojaamisessa käytetään samoja menetelmiä kuin muissa yleisissä tietojärjestelmissä. Suojaus muodostuu kattavasta tietoturvasta, joka pitää sisällään hallinnollisen, henkilöstö-, fyysisen ja tietoliikenneturvallisuuden. Kriittisten tietojärjestelmien suojaus voidaan toteuttaa virustorjuntaohjelmistoilla, eristämällä kriittiset tietojärjestelmät yleisestä verkosta kokonaan, käyttämällä palomuuria, identiteetin- ja pääsynhallinnalla, vahvoilla salausmenetelmillä ja tekemällä omaa tietojärjestelmää vastaan koehyökkäyksiä, joilla löydetään tietojärjestelmän haavoittuvuudet.

Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Preparation of mitotic chromosomes of leaf-cutting ants from the genera Atta and Acromyrmex

Some modifications were made to the methodology of Imai et al. (Jpn. J. Genet. 63: 159-185, 1988) for cytogenetic analysis of the leaf-cutting ants Atta sexdens piriventris and Acromyrmex heyeri (Hymenoptera, Formicidae), shortening preparation time and improving chromosomal preparations. The brain ganglia of prepupae were dissected in a 0.0025% hypotonic solution of colchicine, placed on a glass slide on a cold plate (4 ± 1oC) for 20 min. The material was fixed directly on the cold slide (with cold fixative I), macerated with a histological needle and fixed again with fixative I, followed by fixatives II and III, all of them cold. The slide was flame-dried right after the use of fixative III, and it was allowed to air-dry at room temperature for 2 h. The resulting metaphases presented less contracted chromosomes, with separated and well defined sister chromatids at a high frequency, when the material was processed in the manner described and stained with 3% Giemsa in phosphate buffer (pH 6.8) for 15 min.