尿液中细菌L型检出情况及耐药性分析

目的 了解泌尿感染患者尿液标本细菌L型的检出情况,分析尿常规结果、病原菌分布情况及耐药性特点,为临床提供诊疗依据。方法 对2014年1月至2015年12月1 532例住院和门诊泌尿感染患者的清洁中段尿标本的尿常规结果和微生物培养结果进行回顾性分析。严格按照《全国临床检验操作规程》要求采集患者尿液标本,2 h内完成尿液普通培养、高渗培养、尿常规检查及尿液离心后沉渣镜检。培养出细菌L型进行菌株鉴定和药敏试验,结果采用SPSS 13.0统计软件进行分析处理。结果 共检出细菌L型132例,检出率为8.6%。132例细菌L型阳性病例中,白细胞酯酶阳性19例,阳性率14.4%;尿沉渣镜检白细胞阳性105例,阳性率79.5%。细菌L型检出率排名前三位的分别为大肠埃希菌、粪肠球菌、葡萄球菌,分别占40.9%、22.7%、12.1%。大肠埃希菌对氨苄西林、环丙沙星、左氧氟沙星、氨苄西林/舒巴坦、头孢唑啉、复方新诺明耐药率较高;粪肠球菌对克林霉素、奎奴普丁/达福普汀、红霉素、四环素耐药率较高;葡萄球菌对青霉素G、红霉素、头孢西丁、甲氧西林、环丙沙星耐药率较高。结论 尿常规、尿沉渣镜检有助于细菌L型感染的辅助诊断,临床医生应根据尿培养结果合理、足疗程选用抗菌药物。     

City-wide screening for urinary abnormalities in schoolgirls

Screening for urinary tract infection was carried out in 23,427 schoolgirls, aged 5 to 14 years, using Uricult and, for hematuria, glycosuria and proteinuria using Hema-combistix. Cultures of 10⁵ colonies per ml. or more on two occasions were obtained in 2.3% and a positive culture was confirmed by the family physician using standard culture techniques in 82.7% of cases, giving an overall incidence of infection of 1.9%. Fifty-eight percent of these children had no previous history of any urinary tract symptoms. Of the infected group 9.5% had pyelonephritic scarring, 58.7% chronic cystitis and 58.7% urethral stenosis. Two additional cases had unilateral ureteropelvic junction obstruction with hydronephrosis. Reflux occurred in 26.6% of those investigated by voiding cystogram. In 58% of cases the urinary tract infection was not accompanied by significant proteinuria, hematuria or pyuria.Proteinuria was detected on two occasions in 1.6% of the children and confirmed by the family physician in 33% of cases, giving an overall incidence of 0.5%. In this group 9.2% had evidence of pyelonephritic scarring without a positive urine culture.Hematuria was detected on two occasions in 0.6% of the children and was confirmed by the family physician in 53%, giving an overall incidence of 0.3%. Only one case with pyelonephritic scarring was seen in this group.Of the 25 cases with pyelonephritic changes only six had been previously diagnosed radiologically.Four previously unrecognized diabetics were also detected.     

绍兴地区2型糖尿病尿路感染病原菌及其耐药性分析

摘要：目的 回顾分析本院2型糖尿病伴尿路感染患者的病原菌分布特点及其耐药性,为指导临床用药提供参考。方法 收集2013年1月至2014年1月2型糖尿病合并尿路感染患者186例,留取中段尿分离培养病原菌,用VITEK-2细菌鉴定仪鉴定,纸片扩散法（K-B）测定药物敏感性。结果 186例患者中段尿共培养出病原菌137株,其中革兰阴性菌102株（74.45%）,革兰阳性菌21株（15.33%）,真菌14株（10.22%）。革兰阴性菌以大肠埃希菌为主,检出79株占57.66%,对青霉素类,头孢菌素类,喹诺酮类抗菌药耐药率较高均＞50.00%,对头霉素类药物如头孢替坦、含酶抑制剂复合物如哌拉西林/他唑巴坦以及碳青霉烯类药物敏感率均达100.00%;革兰阳性球菌以无乳链球菌为主,检出10株占7.30%,对克林霉素及红霉素耐药率较高,耐药率＞50.00%,而对氯霉素、呋喃妥因、利奈唑烷、替加环素、万古霉素敏感率均达100.00%。除1株光滑假丝酵母菌对氟康唑和伊曲康唑中介,其余13株真菌对两性霉素B、5-氟胞嘧啶、伏立康唑、氟康唑、伊曲康唑这5种抗真菌药物均敏感。结论 2型糖尿病患者发生尿路感染的病原菌以大肠埃希菌为主,且有较高的耐药率。     

A simple spectrophotometric assay for urinary leukocyte esterase activity

We have developed a sensitive spectrophotometric method for assaying urinary leukocyte esterase activity by employing a synthetic substrate, N-toluene sulfonyl indoxyl alanine ester. This kinetic assay can be performed with a small aliquot of urine, by following the change in absorbance of the chromophore at 385 nm. It is rapid and specific for leukocyte esterase and therefore can be used in the early diagnosis of urinary tract infection.     

Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration

There is marked endogenous production of nitrate in young calves. Here we have studied the contribution of exogenous nitrate and nitrite to plasma concentrations and urinary excretion of nitrite and nitrate in milk-fed calves. In experiment 1, calves were fed 0 or 200 &mgr;mol nitrate or nitrite/kg(0.75) or 100 &mgr;mol nitrite plus 100 &mgr;mol nitrate/kg(0.75) with milk for 3 d. In experiment 2, calves were fed 400 &mgr;mol nitrate or nitrite/kg(0.75) with milk for 1 d. Plasma nitrate rapidly and comparably increased after feeding nitrite, nitrate or nitrite plus nitrate. The rise of plasma nitrate was greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. Plasma nitrate decreased slowly after the 3-d administration of 200 &mgr;mol nitrate or nitrite/kg(0.75) and reached pre-experimental concentrations 4 d later. Urinary nitrate excretions nearly identically increased if nitrate, nitrite or nitrite plus nitrate were administered and excreted amounts were greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. After nitrite ingestion plasma nitrite only transiently increased after 2 and 4 h and urinary excretion rates remained unchanged. Plasma nitrate concentration remained unchanged if milk was not supplemented with nitrite or nitrate. Nitrate concentrations were stable for 24 h after addition of nitrite to full blood in vitro, whereas nitrite concentrations decreased within 2 h. In conclusion, plasma nitrate concentrations and urinary nitrate excretions are enhanced dose-dependently by feeding low amounts of nitrate and nitrite, whereas after ingested nitrite only a transient and small rise of plasma nitrite is observed because of rapid conversion to nitrate.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative.'' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

人体泌尿系微生物感染与耐药及宿主状态分析

目的了解泌尿系感染常见病原细菌和真菌分布、耐药性及宿主相关状态,为理解相关微生物感染影响因素及临床合理用药提供资料和依据。方法对门诊及住院患者中段尿培养分离的179株病原细菌和真菌进行微生物学鉴定和K-B法药敏试验,同时记录门诊和住院的菌株相关的泌尿系感染患者情况。结果泌尿系感染微生物以大肠埃希菌居首位,占62.4%,其次为真菌和肠球菌,各占12.4%、14.0%。药敏结果显示,大肠埃希菌产ESBLs占62.2%;肠杆菌科细菌对亚胺培南、美罗培南的敏感率为100%,其次敏感性较好的为氨基糖苷类阿米卡星、头孢头霉类头孢西丁及第三代头孢类头孢哌酮/舒巴坦、头孢他啶,敏感率均大于80%。革兰阳性球菌万古霉素敏感率为100%,对呋喃妥因敏感率为70.8%。大肠埃希菌感染率与患者的身体状态和行为具有显著的相关性(P<0.01)。结论革兰阴性杆菌是泌尿系感染的主要病原菌,机体免疫状态低下、不洁等生活行为与尿路微生物感染密切相关,但与耐药性不相关。维护机体正常免疫力、注意合适的生活行为,对预防泌尿道病原细菌与真菌的感染十分重要。     

Study of urinary tract infection in children in one health district.

OBJECTIVES--To determine the number of children who had urine specimens sent for culture, who had infections or sterile pyuria, and who were investigated further. To relate the laboratory findings to the results of imaging. DESIGN--One year survey of urine specimens submitted to a laboratory; review of previous and subsequent laboratory reports; review of the findings of imaging of the urinary tract. SETTING--Portsmouth and South East Hampshire health district. SUBJECTS--An estimated population of 89,086 children aged 12 years or under. MAIN OUTCOME MEASURES--Urine bacterial count and results of imaging. RESULTS--12,551 urine specimens were submitted from 7450 children, 3138 boys and 4312 girls. 2238 children had infection or sterile pyuria at least once during the study (13.9/1000 boys, 37/1000 girls). 996 (45%) of the children with infection or sterile pyuria underwent some form of imaging. 128 children who had infection or sterile pyuria were already known to have urinary tract abnormalities and 114 children had newly identified abnormalities (1.0/1000 boys, 1.5/1000 girls). 50 (44%) of the children with newly detected abnormalities had no pyuria and 48 (42%) had bacterial counts below 10(8)/l. Eight children who had sterile pyuria on presentation were found to have abnormalities on imaging. CONCLUSIONS--Urinary tract infection is much commoner in children than is widely believed. Low bacterial counts, the absence of pyuria, or a finding of sterile pyuria should not be disregarded.     

Comparison of the quick Gram stain method to the B&M modified and favor methods

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.     

A study for the improvement of the cytological urine examination performances in upper tract infection diagnosis

Diagnosis of the location of upper and lower urinary tract infection (UTI) is necessary in defining the therapeutic conduct that has a different period and intensity according to the infection location and in prognosis. Many studies show the lack of clinical criteria peculiarity in revealing the different location of UTI. As a result, the correct location of the level in which UTI develops is the necessity of paraclinical investigations. Urinary sample examination, in which urinary sediment microscopy is essential, is a reliable technique in fast detection and localization of UTI. Finding, in pyuria context, the classic significant bacteriuria (> or = 10(5) CFU/ml) or lower value bacteriuria (< or = 10(4) CFU/ml) confirms the UTI diagnosis. The upper tract infection prognosis increases when leukocyte cylinders, characteristic for pyelonephritis, appear together with intact or degraded leukocytes, single or grouped. We settled an algorithm to examine the urine samples in order to: Concentrate and preserve the structural integrity of leukocytes and cylinders, examining the conventional urinary sediment Precisely identify and differentiate these elements by vital coloration (leukocyte peroxidase coloration and Sternheimer - Malbin coloration) to establish more accurate the UTI level. The vital coloration for leukocyte peroxidase has cytological specificity, confirming the pyuria and the cylinders that contain leukocytes (leukocytary, granular, mixed) and obviously ameliorates the reliability and reproducibility of the urinary sediment cytological exam.     

Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.     

老年女性泌尿系统感染患者的病原菌分布及耐药情况研究

目的 通过对老年女性泌尿系统感染患者病原菌类型和耐药情况进行研究,为临床合理、安全、有效使用抗菌药物提供依据。方法 收集2014年9月至2017年7月在我院进行诊治的661例老年女性泌尿系统感染患者,对其清晨清洁中段尿进行细菌培养并进行耐药试验。结果 661例样本中共分离出病原菌255株,其中革兰阴性菌165株,占64.7%,以大肠埃希菌为主,构成比为45.5%;革兰阳性菌77株,占30.2%,以屎肠球菌为主,构成比为10.2%;真菌13株,占5.1%。G-菌中检出率排在前三的依次是大肠埃希菌、肺炎克雷伯菌、奇异变形杆菌。大肠埃希菌、肺炎克雷伯菌对氨苄西林、复方新诺明耐药性较高,奇异变形杆菌对头孢唑啉耐药性较高,三者均对亚胺培南敏感性较高。G+菌中检出率排在前三的依次是屎肠球菌、表皮葡萄球菌、粪肠球菌。三种病原菌对青霉素、氨苄西林、环丙沙星耐药性较高,对万古霉素、替考拉宁敏感性较高。结论 老年女性泌尿系统感染患者的病原菌多数为革兰阴性菌,且以大肠埃希菌为主,病原菌培养和耐药试验对于临床合理有效地使用抗菌药物具有重要的指导意义。     

Mechanism of gram variability in select bacteria.

Gram stains were performed on strains of Actinomyces bovis, Actinomyces viscosus, Arthrobacter globiformis, Bacillus brevis, Butyrivibrio fibrisolvens, Clostridium tetani, Clostridium thermosaccharolyticum, Corynebacterium parvum, Mycobacterium phlei, and Propionibacterium acnes, using a modified Gram regimen that allowed the staining process to be observed by electron microscopy (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Furthermore, since a platinum salt replaced the iodine mordant of the Gram stain, energy-dispersive X-ray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. These gram-variable bacteria could be split into two groups on the basis of their staining responses. In the Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium group, few cells became gram negative until the exponential growth phase; by mid-exponential phase, 10 to 30% of the cells were gram negative. The cells that became gram negative were a select population of the culture, had initiated septum formation, and were more fragile to the stress of the Gram stain at the division site. As cultures aged to stationary phase, there was a relatively slight increase toward gram negativity (now 15 to 40%) due to the increased lysis of nondividing cells by means of lesions in the side walls; these cells maintained their rod shape but stained gram negative. Those in the Bacillus-Butyrivibrio-Clostridium group also became gram negative as cultures aged but by a separate set of events. These bacteria possessed more complex walls, since they were covered by an S layer. They stained gram positive during lag and the initial exponential growth phases, but as doubling times increased, the wall fabric underlying the S layer became noticeably thinner and diffuse, and the cells became more fragile to the Gram stain. By stationary phase, these cultures were virtually gram negative.     

Sterile pyuria in a population of wild white‐handed gibbons (Hylobates lar)

Urinalysis is an emerging method for monitoring the health and energy balance of wild primates. Here, we report the first urinalysis of wild gibbons. We used multi‐reagent test strips to monitor the health status of 52 individual white‐handed gibbons (Hylobates lar) inhabiting Khao Yai National Park, Thailand. Most urinary reference values were within normal ranges; however, regardless of age‐ and sex‐class or monthly fruit productivity, we found unexpectedly high rates of urinary leukocytes (50% and 90% of individuals in 2001–2003 and 2006, respectively). In contrast to previous studies of African apes, this finding is coupled with the near absence of urinary nitrites, demonstrating pervasive levels of sterile pyuria. This result is the first reported case of sterile pyuria in a population of wild primates. The etiology of human sterile pyuria is diverse, but in all cases it is diagnostic of systemic inflammation. We discuss the potential causes of sterile pyuria in the gibbons of Khao Yai. Am. J. Primatol. 71:880–883, 2009. © 2009 Wiley‐Liss, Inc.     

Salicylates and reversible hearing loss

Skin and soft tissue infections were studied in 21 seriously ill narcotic addicts who had been admitted to hospital. Subcutaneous abscesses were present in 14 patients; cellulitis was noted in 3, pyomyositis in 2 and necrotizing fasciitis in 2. In four patients there was septicemia. Infections in 14 patients (66.6 percent) were associated with anaerobic bacteria, which were the exclusive isolates in 6 patients. In seven patients (33.3 percent) isolates were exclusively aerobic bacteria and in eight both aerobes and anaerobes were present. The anaerobic isolates were clostridia (six), peptostreptococci (five), bacteroides (five), peptococci (three), and one of each of Veillonella, Propionibacterium, Eubacterium, Fusobacterium and Actinomyces. Staphylococcus aureus, generally thought to be the most common cause of subcutaneous infections in addicts, was found only in four (19 percent) patients. The other aerobic isolates were Klebsiella (five) and Enterobacter (four) species. When clinical features or the Gram stain of pus suggest that anaerobic bacteria may be present, antibiotic therapy should be directed against both aerobic and anaerobic bacteria until culture results are available.     

耐60℃高温乳酸菌的驯化及鉴定

目的对本实验室保存的ATCC 4356菌株进行温度梯度诱变,从而获得1株耐高温乳酸菌。方法采用温度梯度方法诱变菌株;通过革兰染色和原子力显微镜观察诱变前后菌株革兰染色特性和形态及表面结构的变化;通过生理生化特性鉴定,判断诱变前后菌株是否发生种属变化。结果在45~60℃诱变过程中,随温度升高传代次数递增,但最终均能成功驯化和稳定传代。驯化前后菌株在产酸能力、形态特征、革兰染色和生理生化特性方面无显著性变化,表明该诱导菌株未发生种属变化;原子力显微镜观察结果表明诱导前后菌株表面形态存在差异。结论温度梯度诱变技术能够成功驯化耐60℃高温乳酸菌,从而扩大其应用范围。     

Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression.

Crystal violet (hexamethyl-para-rosaniline chloride) interacts with aqueous KI-I2 during the Gram stain via a simple metathetical anion exchange to produce a chemical precipitate. There is an apparent 1:1 stoichiometry between anion (I-) and cation (hexamethyl-para-rosaniline+) during the reaction and, since the small chloride anion is replaced by the bulkier iodide, the complex formed becomes insoluble in water. It is this same precipitate which forms in the cellular substance of bacteria (both gram-positive and gram-negative types) and which initiates the Gram reaction. Potassium trichloro(eta 2-ethylene)-platinum(II), as an electronopaque marker for electron microscopy, was chemically synthesized, and it produced an anion in aqueous solution which was compatible with crystal violet for the Gram stain. It interacted with crystal violet in a similar manner as iodide to produce an insoluble complex which was chemically and physically analogous to the dye-iodide precipitate. This platinum anion therefore allows the Gram staining mechanism to be followed by electron microscopy.     

MANAGEMENT OF PATIENTS WITH URETERAL STONE

Immediate steps in the treatment of ureteral stone, beginning with the often acute onset, are relief of pain, urinalysis (including Gram stain), forcing fluids, examination of urine for the stone and urography at the earliest feasible time. If the stone causes continual pain or appears unlikely to be passed safely, it should be removed—with a cystoscope if possible; if not, by operation which may be done while the patient is still under anesthesia.To combat further stone formation a large fluid intake should be maintained, the extracted stone analyzed, an acid ash diet prescribed, serum calcium and phosphorus measured, urinary stasis corrected and urinary infection and distant foci of infection cured. Vitamin A, aluminum gels and particularly hyaluronidase appear promising as preventives to stone formation.     

A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.Gram’s staining method is considered fundamental in bacterial taxonomy. The outcome of the Gram reaction reflects major differences in the chemical composition and ultrastructure of bacterial cell walls. The Gram stain involves staining a heat-fixed smear of cells with a rosaniline dye such as crystal or methyl violet in the presence of iodine, with subsequent exposure to alcohol or acetone. Organisms that are decolorized by the alcohol or acetone are designated gram negative.Alternative Gram staining techniques have recently been proposed. Sizemore et al. (19) reported on the use of fluorescently labeled wheat germ agglutinin. This lectin binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram-positive bacteria, whereas gram-negative organisms contain an outer membrane that prevents lectin binding. Although simpler and faster than the traditional Gram stain, this method requires heat fixation of organisms.Other Gram stain techniques suitable for live bacteria in suspension have been described. Allman et al. (1) demonstrated that rhodamine 123 (a lipophilic cationic dye) rendered gram-positive bacteria fluorescent, but its uptake by gram-negative organisms was poor. This reduced uptake by gram-negative bacteria was attributed to their outer membranes. The outer membrane can be made more permeable to lipophilic cations by exposure to the chelator EDTA (4). Shapiro (18) took advantage of this fact to form the basis of another Gram stain, one which involved comparing the uptake of a carbocyanine dye before and after permeabilizing organisms with EDTA. All of these methods, however, rely on one-color fluorescence, making analysis of mixed bacterial populations difficult.An alternative to the use of stains is the potassium hydroxide (KOH) test. The method categorizes organisms on the basis of differences in KOH solubility. After exposure to KOH, gram-negative bacteria are more easily disrupted than gram-positive organisms. This technique has been used to classify both aerobic and facultatively anaerobic bacteria, including gram-variable organisms (8). In a study by Halebian et al. (9), however, this technique incorrectly classified several anaerobic strains, giving rise to the recommendation that the method should only be used in conjunction with the traditional Gram stain.In this study we demonstrate a Gram staining technique for unfixed organisms in suspension, by using clinically relevant bacterial strains and organisms notorious for their gram variability. The method uses two fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13. Sales literature (11) published by the manufacturers of HI (Molecular Probes, Inc., Eugene, Oreg.), which displays a red fluorescence, suggests that the dye selectively stains gram-positive bacteria. SYTO 13 is one of a group of cell-permeating nucleic acid stains and fluoresces green (11). These dyes have been found to stain DNA and RNA in live or dead eukaryotic cells (16). Both dyes are excited at 490 nm, permitting their use in fluorescence instruments equipped with the most commonly available light sources. We reasoned that a combination of these two dyes applied to mixed bacterial populations would result in all bacteria being labeled, with differential labeling of gram-positive bacteria (HI and SYTO 13) and gram-negative bacteria (SYTO 13 only). The different fluorescence emission wavelengths of the two dyes would ensure differentiation of gram-positive from gram-negative bacteria by either epifluorescence microscopy or flow cytometry when equipped with the appropriate excitation and emission filters. While a commercial Gram stain kit produced by Molecular Probes includes HI and an alternative SYTO dye, SYTO 9, we are unaware of any peer-reviewed publications regarding either its use or its effectiveness with traditionally gram-variable organisms.