Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.     

Aceto-Orcein and Feulgen Stains for Anatomy and Cytology of Trematodes

Dicrocoelium dendriticum and Fasciola hepatica were killed in the extended condition without anesthetization by dropping them into 40% acetic acid or into aceto-orcein. By using aceto-orcein (La Cour, 1941), killing, fixing and staining were accomplished simultaneously: staining time 24 hr or more. Whole mounts were made by dehydrating, clearing and mounting in Canada balsam, or testes or the upper part of the uterus could be removed for squash preparations after as long as 2 mo in the fixing and staining fluid. For Feulgen staining, living specimens were placed in 40% acetic acid for 10—15 min and then transferred to either Gilson's fluid, for sections, or to acetic-ethanol (1:3) for squashes. Hydrolysis was either by 10% perchloric acid at 25°C for 12 hr or in 12V HCl at 60° for 10 min. The time for Feulgen staining (De Tomasi, 1936) was 1.5-4.0 hr. Squashes were made from testes and uterus in the same manner as after aceto-orcein or sections obtained after embedding in paraffin.     

Root-Tip Smear Method for Difficult Material

A technic is outlined for the preparation of difficult material for the study of chromosome number and morphology in root-tip smears. The chief objective is to obtain polar views of the metaphase plates rather than equatorial views. To achieve this end it is recommended that fresh root-tips be cut free-hand into thin cross sections. The important features are: thin freehand cross sectioning of the fresh root-tips; fixing in Belling's iron aceto-carmine solution; maceration for 2-5 minutes in 50% HCl in 95% alcohol; and mounting in “Diaphane.”     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).     

固氮植物绿豆对核桃幼苗生长、叶片气孔气体交换及水力特征的作用

采用砂培方法, 在温室内将一年生核桃(Juglans regia)嫁接苗木和绿豆(Vigna radiata)进行间作, 研究绿豆对核桃苗木生长、水分平衡和光合特性的影响。该研究设有5种处理, 即: 对照(核桃单作, 正常供应氮素); 核桃单作, 不添加氮素; 核桃绿豆间作, 不添加氮素; 核桃绿豆间作, 正常供应氮素; 绿豆单作, 不添加氮素。结果显示: 种植绿豆可以增加土壤氮含量和核桃茎内氮含量, 但对核桃叶和根系中的氮含量影响不明显。种植绿豆显著增加不施氮核桃的高生长和直径生长, 但降低了正常供氮核桃的生长。无论种植绿豆与否, 不供氮处理降低了核桃的总叶面积, 提高了根冠比。核桃叶片气孔气体交换对各处理的响应和生长有相同的趋势。缺氮显著降低了核桃叶柄在中午的导水率、提高了导水损失率; 种植绿豆显著提高不供氮核桃的导水率而且明显降低了其导水损失率。然而, 种植绿豆使正常供氮的核桃降低了导水率, 加剧了导水损失率。同时, 绿豆受到间作的竞争压力, 产量和生物量有所下降。由研究结果可知, 在贫瘠的土壤上, 固氮植物绿豆改善了间作的核桃的氮营养, 有益于核桃木质部发育、水分平衡以及光合代谢。但是在氮充足的土壤中, 种植绿豆反而降低了核桃的水分供应, 影响其气体交换和生长。     

Effects of a nitrogen fixing plant Vigna radiata on growth,leaf stomatal gas exchange and hydraulic characteristics of the intercropping Juglans regia seedlings

采用砂培方法, 在温室内将一年生核桃(Juglans regia)嫁接苗木和绿豆(Vigna radiata)进行间作, 研究绿豆对核桃苗木生长、水分平衡和光合特性的影响。该研究设有5种处理, 即: 对照(核桃单作, 正常供应氮素); 核桃单作, 不添加氮素; 核桃绿豆间作, 不添加氮素; 核桃绿豆间作, 正常供应氮素; 绿豆单作, 不添加氮素。结果显示: 种植绿豆可以增加土壤氮含量和核桃茎内氮含量, 但对核桃叶和根系中的氮含量影响不明显。种植绿豆显著增加不施氮核桃的高生长和直径生长, 但降低了正常供氮核桃的生长。无论种植绿豆与否, 不供氮处理降低了核桃的总叶面积, 提高了根冠比。核桃叶片气孔气体交换对各处理的响应和生长有相同的趋势。缺氮显著降低了核桃叶柄在中午的导水率、提高了导水损失率; 种植绿豆显著提高不供氮核桃的导水率而且明显降低了其导水损失率。然而, 种植绿豆使正常供氮的核桃降低了导水率, 加剧了导水损失率。同时, 绿豆受到间作的竞争压力, 产量和生物量有所下降。由研究结果可知, 在贫瘠的土壤上, 固氮植物绿豆改善了间作的核桃的氮营养, 有益于核桃木质部发育、水分平衡以及光合代谢。但是在氮充足的土壤中, 种植绿豆反而降低了核桃的水分供应, 影响其气体交换和生长。     

Rehydration of air-dried smears. An alternative method for cytologic analysis of exfoliative cells

OBJECTIVE: To compare the cytologic characteristics of gastric mucosal cell smears prepared by air drying and rehydration prior to alcohol fixing with cells wet fixed in alcohol. STUDY DESIGN: Gastric mucosal cells were obtained from 55 consecutive patients undergoing gastroscopy. Paired smears were made, one immediately fixed in 95% ethanol for 20 minutes (wet fixed [WF]) and the other air dried for at least 20 minutes prior to rehydration with normal saline for 30 seconds and fixation in 95% ethanol for 20 minutes (air dried/rehydrated/fixed [ARF]). Both slides were stained by the Papanicolaou method. Coded slides were examined blind and graded 1 (superior), 2 (satisfactory) or 3 (poor) with respect to staining of chromatin, nuclear membrane, nucleoli, cytoplasm/cell border and group morphology. Histology confirmed a benign disease process or normal mucosa. RESULTS: Comparing grade 1 versus grades 2 and 3, ARF slides were significantly better than WF slides for all cytologic features (P < .05). Comparing grade 1 and 2 versus grade 3, there was no significant difference between ARF and WF slides (P > .05) (chi 2 analysis). CONCLUSION: The cytologic features of ARF smears of gastric cells were equal or superior to those of WF smears. This method of preparing smears is simpler and avoids some of the problems of ethanol fixation of wet smears.     

Effect of the homokaryotic or heterokaryotic state of the uvs-2 allele in Neurospora crassa on mitomycin C-induced killing and ad-3 mutation

Mitomycin C (MC) was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 4 dikaryons of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on MC-induced killing and ad-3 mutation. These dikaryons were homokaryotic for uvs-2⁺ (H-12), homokaryotic for uvs-2 (H-59), and heterokaryotic for uvs-2/uvs-2⁺ (H-70 and H-71). MC induced killing and ad-3 mutation in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a great increase in the killing and mutagenic activities of MC. This increased sensitivity to MC-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is a different effect than that noted by others for a defect in nucleotide excision-repair in Escherichia coli and Salmonella typhimurium or in human cells. The dikaryons heterokaryotic for uvs-2/uvs-2⁺ had the same sensitivity to MC as H-12, indicating that for MC-induced killing and ad-3 mutation uvs-2 is recessive to uvs-2⁺.     

Neutrophil myeloperoxidase deficiency associated with canine hepatozoonosis

Hepatozoonosis is a very important disease in dogs in Nigeria. Hepatozoonosis was reported in Nigeria in 18 dogs. The clinical signs included fever, anorexia, loss of weight, lameness, oculonasal discharge and conjunctivitis. Hematologic findings included leukocytosis due to neutrophilia and eosinophilia. Parasitemia varied from 1 to 9% of the circulating neutrophils in the peripheral blood smears of the dogs examined. Hepatozoon canis gametocytes were identified in circulating neutrophils of dogs. Peripheral blood smears from dogs confirmed to have natural H. canis infection were cytochemically stained for myeloperoxidase. Parasitized neutrophils were myeloperoxidase deficient while non-parasitized neutrophils were myeloperoxidase positive. This is considered important, because deficiency of the enzyme may be responsible for poor response of H. canis to chemotherapeutic agents.     

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema vileae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanoi and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection offilarial infections, is discussed.     

卷荚相思根瘤菌的生物学特性

为探讨卷荚相思根瘤菌的生物学特性,从广西高峰林场卷荚相思林分采集分离到12株根瘤菌,对这些菌株进行了生理生化试验,并利用这些菌株对厚荚相思苗木进行接种结瘤试验。结果表明:(1)依据生长代时和单个菌落形成测定结果,分离获得的12个菌株可以分为9个快生菌株和3个慢生菌株。(2)大部分的供试根瘤菌菌株可以利用无机氮源作为其生长所需的氮源,但对供试用的糖源利用存在着较大的差别。(3)所有供试菌株在3一酮基乳糖反应、牛肉膏蛋白胨、淀粉水解试验中呈阴性,所有供试的12个菌株均能进行利用柠檬酸盐,在BTB试验中均产酸,在石蕊牛奶反应均产碱。(4)12个卷荚相思根瘤菌菌株接种到厚荚相思苗木后,均能使其根部结瘤,并进一步筛选出结瘤效果和固N效果比较好的供试菌株是快生型菌株(J003、J004、J005、J008)和慢生型菌株(J012)。这为从华南的酸性土壤中寻找和利用土著根瘤菌资源提供了科学依据。     

A Quick and Standardized Giemsa Stain for Wet-Fixed Cytological Material

The Giemsa stain is one of the most widely used staining techniques in cytology, especially in hematology. A standardized Romanowsky-Giemsa staining procedure using pure cationic azure B (C.I. 52010) and anionic eosin (C.I. 45380) has been described by Wittekind et al (1982). A revised standard Giemsa staining procedure was recently published (Wittekind and Kretschmer 1987). Usually the Romanowsky-Giemsa stain is applied to air dried and methanol fixed cytological material, e.g. blood smears and bone marrow films (ICSH 1984).     

Survey of Theileria parasites of sheep in eastern Turkey using polymerase chain reaction

This study provides the first molecular data on infection Theileria of sheep in Turkey. A total of 218 blood samples were collected from sheep in five distinct geographical locations of eastern Turkey. Theileria piroplasm DNAs, extracted from sheep blood, were subjected to the polymerase chain reaction (PCR) procedures, using specific primers for Theileria spp. and Theileria lestoquardi. Blood smears were examined for Theileria piroplasms by microscopic observation. In PCR analysis, the expected amplicons were obtained from 90 (41.2%) blood samples with a molecular size of 1098 bp for Theileria spp. whereas none were amplified by Theileria lestoquardi-specific primers. Piroplasms of Theileria spp. were detected in 34 (15.5%) of the samples.     

Trypanosoma avium incidence, pathogenicity and response to melarsomine in falcons from Kuwait

Epidemiological and clinical studies on Trypanosoma avium are lacking in the Middle East. The aims of this study were to determine the T. avium incidence in falcons from Kuwait, report clinical signs and find an effective therapy. Blood smears from 921 diseased and 56 healthy falcons were examined between May 2003 and April 2004. 12 birds 11.3%) were found infected by T. avium and ten of these were treated with melarsomine (Cymelarsan) at a dosage of 0.25 mg/kg intramuscularly for four days. All affected birds presented clinical signs, including incapacity of flying high, poor appetite, lethargy, loosing weight, weakness, dyspnoea and death. Signs disappeared within 1-7 days after administration of melarsomine. Trypomastigotes were not detected in blood smears made 1-7 days after the end of therapy. This study suggests that T. avium induces disease in falcons and that melarsomine can be an effective therapy eliminating both clinical signs and circulating trypomastigotes.     

Treatment of malaria in a mouse model by intranasal drug administration

The goal of this work was to investigate intranasal dihydroartemisinin (DHA) delivery as a non-invasive method for treatment of malaria. ICR female mice were infected with Plasmodium berghei ANKA, a model for severe malaria with similarities to the human disease. DHA, at a dose of 2 × 5 mg/kg/day, was administered to mice either intranasally or i.p. Two dosage regimens were tested: prophylaxis and treatment. Parasitemia was monitored every other day, from the time of infection, by thin smears prepared from tail blood. The survival rates in prophylaxis and treatment regimens were 93% and 75%, respectively, for intranasal DHA and this route was at least as effective as the i.p. route used for comparison. All mice in the untreated control and placebo groups succumbed due to the parasitemia. The results show that DHA nasal administration to mice was highly efficient in the treatment of Plasmodium infection in infected rodents. This novel mode of drug administration may be considered as an alternative to conventional treatment.     

Method for the stabilization of E-rosettes with glutaraldehyde]

A technically simple method of fixing E-rosettes is represented. Under the influence of glutaraldehyde of 0.5% the E-rosettes being themselves very fragile assume a solid form and may be firmly spread to smears. Comparative examinations made on fresh and panoptically stained preparations showed a good accordance of results. The advantages of this method consist in its easy handling, possible simultaneous or alternative application of two counting procedures and the possibility of having the test results registered in archives.     

Population status and conservation of an extremely small population species Euryodendron excelsum

猪血木(Euryodendron excelsum)是特产于中国的山茶科极危种, 但目前对导致猪血木种群结构改变的原因缺少相关的调查及研究, 保护策略不完善。该研究依据野外调查方法、编制标准生命表、存活曲线、致死力和寿命期望曲线, 利用德氏多度分析方法分析了广东省阳春市八甲镇极小种群野生植物猪血木种群的年龄结构和动态, 并与2007年的研究结果进行对比, 分析该种群近10年的变化情况及其原因。结果表明: (1)研究区域内野生猪血木种群的数量变化动态指数(V_pi和V′_pi)均小于0, 存活曲线趋于Deevey-III型, 种群结构属于衰退型, 幼树数量丰富, 但由于向小树发展过程受阻, 种群无法及时更新补充。(2)存活曲线、致死力和寿命期待曲线一致显示在幼树、小树2级、中龄树2级这3个阶段, 出现阻碍因素, 影响种群的自然更新。(3)三甲镇猪血木幼苗减少了39.3%, 八甲镇猪血木幼苗锐减了71.9%, 三甲镇幼苗转化率较八甲镇低了近1/2。人为干扰是导致猪血木野生种群衰退的主要驱动因素, 已实施的保护策略并不适合当前情况下的野生猪血木种群。     

极小种群植物猪血木的种群现状及保护对策

猪血木(Euryodendron excelsum)是特产于中国的山茶科极危种, 但目前对导致猪血木种群结构改变的原因缺少相关的调查及研究, 保护策略不完善。该研究依据野外调查方法、编制标准生命表、存活曲线、致死力和寿命期望曲线, 利用德氏多度分析方法分析了广东省阳春市八甲镇极小种群野生植物猪血木种群的年龄结构和动态, 并与2007年的研究结果进行对比, 分析该种群近10年的变化情况及其原因。结果表明: (1)研究区域内野生猪血木种群的数量变化动态指数(V_pi和V′_pi)均小于0, 存活曲线趋于Deevey-III型, 种群结构属于衰退型, 幼树数量丰富, 但由于向小树发展过程受阻, 种群无法及时更新补充。(2)存活曲线、致死力和寿命期待曲线一致显示在幼树、小树2级、中龄树2级这3个阶段, 出现阻碍因素, 影响种群的自然更新。(3)三甲镇猪血木幼苗减少了39.3%, 八甲镇猪血木幼苗锐减了71.9%, 三甲镇幼苗转化率较八甲镇低了近1/2。人为干扰是导致猪血木野生种群衰退的主要驱动因素, 已实施的保护策略并不适合当前情况下的野生猪血木种群。     

New Source for Positive Controls Used for the Identification of Spirochaetes in Tissue Sections

Lyophilized T. pallidum is reconstituted or purchased as a suspension, evenly dispersed, made into smears using alcohol-cleaned slides and a bacteriological platinum wire loop. Smears are air dried for 15 minutes and fixed in concentrated formaldehyde fumes for 30 minutes and stored at room temperature. These preparations are used as positive controls for conventional silver impregnation techniques recommended for spirochaete detection in tissue sections.     

Bactericidal Activity of Catecholamine Copper Complexes

Washed or growing E. coli cells are killed by epinephrine, norepinephrine or dopamine in the presence of non lethal concentrations of Cu(II). Killing is enhanced by anoxia and by sublethal Concentrations of H₂O₁. The rate of killing is proportional to the rate of catecholamine oxidation. The copper epinephrine complex binds to E. coli cells, induces membrane damage and depletion of the cellular ATP pool. The cells may be partially protected by SOD or catalase but not by OH radical scavengers. Addition of H²O₂ to cells which were sensitized by preincubation with the epinephrine-copper complex, causes rapid killing and DNA degradation. Sensitized cells are not protected by BSA.