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At least 19 recordsLinked to original sources
PubMed2006

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections.

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

Base Sequence↗
PubMed1991

Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium.

By light microscopy, Methanospirillum hungatei GP1 stains gram positive at the terminal ends of each multicellular filament and gram negative at all regions in between. This phenomenon was studied further by electron microscopy and energy-dispersive X-ray spectroscopy of Gram-stained cells, using a platinum compound to replace Gram's iodine (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Crystal violet-platinum precipitates could be found only in the terminal cells of each filament, which suggested that the multilamellar plugs at the filament ends were involved with stain penetration. When sheaths were isolated by sodium dodecyl sulfate-dithiothreitol treatment, the end plugs could be ejected and their layers could be separated from one another by 0.1 M NaOH treatment. Each plug consisted of at least three individual layers; two were particulate and possessed 14.0-nm particles hexagonally arranged on their surfaces with a spacing of a = b = 18.0 nm, whereas the other was a netting of 12.5-nm holes with spacings and symmetry identical to those of the particulate layers. Optical diffraction and computer image reconstruction were used to clarify the structures of each layer in an intact plug and to provide a high-resolution image of their interdigitated structures. The holes through this composite were three to six times larger than those through the sheath. Accordingly, we propose that the terminal plugs of M. hungatei allow the access of larger solutes than does the sheath and that this is the reason why the end cells of each filament stain gram positive whereas more internal cells are gram negative. Intuitively, since the cell spacers which partition the cells from one another along the filament contain plugs identical in structure to terminal plugs, the diffusion of large solutes for these cells would be unidirectional along the filament-cell axis.

Actin Cytoskeleton↗
PubMed1991

Comparison of acridine orange stain with culture and gram stain of needle aspirate in experimental Pseudomonas pneumonia.

The sensitivity and specificity of culture, acridine orange stain, and Gram stain were determined using needle aspiration (NA) material obtained from 82 rats with acute Pseudomonas aeruginosa pneumonia and 18 control rats. Lungs were then processed for either bacterial quantitation or histopathologic examination. NA culture proved to be the most sensitive and specific (55 and 100%, respectively). Sensitivity of acridine orange stain was 40%, whereas Gram stain was only 29%. The specificity of each stain was at least 94%. Lung bacterial concentrations influenced the sensitivities of all three techniques, with better sensitivity found in NA samples obtained from lung with bacterial concentration of at least 10(4) colony-forming units (cfu) of P. aeruginosa. Acridine orange and Gram stain results were similar except in NA samples from lung with bacterial concentration of less than 10(4) cfu in which acridine orange stain was more sensitive. The presence of stains identifying bacteria collected from animals with sterile NA culture was found in a small but significant number of samples, suggesting the presence of nonviable though stainable organisms. Use of all three techniques (culture, acridine orange stain, and Gram stain) increased sensitivity to approximately 70% with minimal decrease of specificity.

Acridine Orange↗
PubMed1977

Identification of the presence and type of biliary microflora by immediate gram stains.

Immediate gram stains were performed on gallbladder bile aspirated at the start of an operation for biliary disease in 191 consecutive patients undergoing elective biliary surgery. The results of the gram stains were telephoned to the operating theater within 20 minutes of collection. The over-all accuracy rate of the telephone gram stain reports compared with the subsequent bile cultures was 77 percent. The incidence of false-positive results was 12 percent, and false-negative results were recorded in 7 percent. The organism was identified wrongly by the gram stain in 4 percent of patients. These results have improved with experience and the over-all accuracy rate of gram stains on bile over the last 6 months have been 87 percent.

Adult↗
PubMed2002

[Reviews for Gram stain].

The Gram stain is a rapid examination method providing useful information for diagnosis and treatment of infection. However, a molecular biology inspection and rapid immunological method have been developed, and gram stains have been disregarded recently. Therefore, I explain the usefulness of gram staining for rapid diagnosis, rapid treatment, and the economy. Moreover, I will explain gram stain method "Bartholomew & Mittwer(B&M)" which replaces the Hucker method.

Bacterial Infections↗
PubMed1992

A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism.

In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.

Bacteria↗
PubMed1985

Pulmonary strongyloidiasis. Diagnosis by sputum gram stain.

Sputum Gram stain was diagnostic for pulmonary strongyloidiasis in four patients from Tennessee with chronic obstructive lung disease treated with steroids. The case reports of these patients and photomicrographs of the larval forms by Gram stain are presented. Sputum Gram stain may be a useful procedure to screen for pulmonary strongyloidiasis in steroid-treated patients with chronic lung disease who come from an endemic area.

Aged↗
PubMed1985

Comparison of the automicrobic system, acridine orange-stained smears, and gram-stained smears in detecting bacteriuria.

We compared the accuracy of the Gram-stained smear, the acridine orange-stained smear, and the AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) in screening for bacteriuria, as detected by conventional cultures. For 1,024 clinical specimens, results with the acridine orange-stained smear and the Gram-stained smear were very similar. When read for the presence of one or more microorganisms or leukocytes per 20 oil immersion fields, both smears were highly sensitive (92.1 and 93.3%, respectively) and moderately specific (70.0 and 61.7%, respectively). Sensitivity was greater for specimens yielding greater than or equal to 10(5) CFU/ml (96.1 and 98.9%, respectively) than for those with 10(3) to 10(4) CFU/ml (81.4 and 78.0%, respectively). Preliminary classification based upon the tinctorial and morphological characteristics of the Gram-stained smear was compatible with culture results in nearly all cases. The accuracy of the Gram-stained smears was not influenced by special cleaning of the microscopic slides, or the level of expertise of the microscopist. For 715 specimens, the sensitivity of the AMS in detecting bacteriuria (91.5%) was very similar to that of the stained smears (92.1 and 95.7%, respectively), but the specificity was significantly higher (83.2% versus 42.6 and 70.0%). Detection of microorganisms by the AMS took an average of 6.3 +/- 3.0 h. These data suggest that the Gram-stained smear is easily interpreted, very sensitive, acceptably specific, and still the optimal rapid method for screening for bacteriuria in most clinical microbiology laboratories.

Acridine Orange↗
PubMed1991

[Effect of heat-staining procedure on the gram staining properties of mycobacteria].

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.

Hot Temperature↗
PubMed1990

Ghost mycobacteria on Gram stain.

The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Detection of bacterial vaginosis in wet mount, Papanicolaou stained vaginal smears and in gram stained smears.

In a prospective study of 107 women, bacterial vaginosis was clinically diagnosed in 34 women. Compared with clinical diagnosis of bacterial vaginosis, detection of clue cells in Papanicolaou stained vaginal smears showed a sensitivity of 88.2%, a specificity of 98.6%, a positive predictive value of 96.8% and a negative predictive value of 94.7%. The corresponding values for detection of bacterial vaginosis in Gram stained smears compared with the clinical diagnosis were 100%, 97.3%, 94.4% and 100%, respectively. Compared with clue cells in wet smears, identification of clue cells in Papanicolaou stained vaginal smears showed a Kappa index of 0.87 and compared with Gram stain criteria a Kappa index of 0.94. The correlation between Gram stain and Papanicolaou stained vaginal smears showed a Kappa index of 0.89. In contrast to the results of earlier investigators our studies indicate that the demonstration of clue cells in Papanicolaou stained vaginal smears correlate reasonably well with the conventional clinical criteria. However, the Gram stain method may be more reliable than the Papanicolaou method.

Female↗
PubMed1975

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.

Bacteria↗
PubMed1999

A comparison of the use of Papanicolaou-stained cervical cytological smears with Gram-stained vaginal smears for the diagnosis of bacterial vaginosis in early pregnancy.

Our objective is to compare the efficacy of using Papanicolaou (PAP)-stained cervical cytology smears with a standardized method of interpreting Gram-stained vaginal smears for the diagnosis of bacterial vaginosis (BV) in pregnancy. High vaginal smears were Gram-stained and examined by a single observer to characterize 3 grades of vaginal flora and diagnose BV. Cervical smears were PAP-stained and examined for characteristic patterns of vaginal flora including evidence of BV by either a number of cytotechnicians or a single cytopathologist. The results of the 2 methods were compared. Seven hundred and forty-seven women attending an antenatal clinic in a district general hospital who consented to have a smear of vaginal secretions and cervical cytology in early pregnancy. The main outcome measure is the diagnosis of BV by different methods in a pregnant population. Compared with the Gram-stain method for the diagnosis of BV, there was good agreement between PAP-stain interpretation by a single observer but the agreement was not as good with PAP-stain interpretation by multiple cytotechnicians. When the grades were consolidated to normal (grade I) and abnormal flora (grades II and III), compared to Gram-stained smears, PAP cytology undertaken by several cytotechnicians had a sensitivity of 80.7% and a specificity of 90.7%. The sensitivity and specificity increased to 87% and 97%, respectively, when the PAP-stained smears were read by a single cytopathologist. Using kappa scores, only those readings made by a single cytopathologist were reliable. The setting in a cytopathology laboratory comprises multiple cytotechnicians, so that PAP-stain analysis of vaginal smears for the diagnosis of BV is likely to provide results which are less reliable than those obtained by Gram staining. The latter should be the first choice and every effort should be made to set up this service.

Cervix Uteri↗
PubMed2004

[Diagnostic value of Gram stain for assessment of vaginal smears during pregnancy].

The main aim of this study was to compare the diagnostic value of Giemsa stained method with Gram stained method for the evaluation of vaginal smears among pregnant women. A study population comprised 111 pregnant between 6 and 30 weeks of gestation. The vaginal smears from every subject was diagnosed according to Giemsa and Gram stained method and micro-organisms were isolated by culture. In 29.3% cases diagnosed as normal flora (2a) on the basis of Giemsa method bacterial vaginosis was detected in Gram stains according to Spiegel's criteria and pathological microflora in concentration > or = 10(5) CFU/ml was cultured among 75.9% of them. Among 31.7% women who had grade 3a (abnormal) in Giemsa stains method normal flora was diagnosed on the basis on Gram's method and from 17.1% pregnant women from this group we did not isolated any pathogens. For evaluation of vaginal smears during pregnancy the Giemsa method should be replaced by Gram stained method.

Azure Stains↗
PubMed1988

The value and limitations of the Gram stain examination in the diagnosis of intraamniotic infection.

Gram stain examination of amniotic fluid is a method used for the rapid diagnosis of intraamniotic infection in patients with preterm premature rupture of membranes and preterm labor. The management of these patients relies heavily on the Gram stain results. Therefore, it is critical that the diagnostic value and limitations, optimal technique, and outcome correlates be precisely established. Most studies have focused on culture results rather than on Gram stain results to describe neonatal and maternal outcome. However, management is based on the Gram stain because culture results are not immediately available. One hundred eighty-seven amniocenteses were performed in 131 patients with preterm premature rupture of the membranes (n = 90) and preterm labor (n = 41). Spun and unspun Gram stains were performed. Centrifugation of the sample did not improve the sensitivity of the technique significantly. The agreement between the two methods was substantial (kappa index 0.89, p less than 0.001). The sensitivity of the Gram stain was 44.8% and the specificity was 97.6%. The sensitivity of the Gram stain was directly proportional to the number of bacteria present in amniotic fluid. In the presence of greater than 10(5) colony forming units per milliliter, 80% of the Gram stains were positive. The absence of both bacteria and white blood cells in a smear was associated with a negative culture of amniotic fluid in 95% of the cases. Clinical chorioamnionitis was associated with a positive Gram stain of amniotic fluid (p less than 0.001). There was a trend toward a higher incidence of endometritis in patients with a positive Gram stain compared with those with a negative Gram stain (p = 0.07). There was no neonatal infectious morbidity in patients with a true negative Gram stain. Patients with a false negative Gram stain had a 25% incidence of neonatal infectious complications (proved and suspected sepsis).

Adult↗