[Mechanism of gram staining. II. Phenomena of gram staining of various bacterial species].
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Bacteria↗
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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↗
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↗
Pharyngeal gram stains in the treatment of sore throats.
The use of the direct pharyngeal Gram stain for diagnosis of group A beta-hemolytic streptococcal pharyngitis was evaluated. Seventy-five consecutive out-patients presented with pharyngitis (or high fever in infants) were studied with pharyngeal cultures and Gram stains. Gram stains were read as either positive, negative, or indeterminate for streptococcal pharyngitis. When these predictions were correlated with the resulting culture growth of group A beta-hemolytic streptococci, the predictive value of a throat smear Gram stain was found to be significant (p less than 0.005). There were two false positives and three false negatives. Approximately one third of the smears were in the indeterminate category. Although the Gram stain of a pharyngeal smear was found to be less sensitive than the routine throat culture, the ability to obtain inexpensive rapid microbiological information suggests that the Gram stain can be used as a quick screening procedure.
Adolescent↗
Counterimmunoelectrophoresis of blood cultures. Temporal Relationship of positive Gram stain to positive counterimmunoelectrophoresis.
Counterimmunoelectrophoresis (CIE), Gram staining, and quantitative measurements were performed on simulated blood cultures at hourly intervals after inoculation with Streptococcus pneumoniae, Klebsiella pneumoniae, or Haemophilus influenzae. The CIE became positive either at the same time as the Gram stain or within the ensuing five hours. In no case was CIE positive when the Gram stain was negative. The numbers of colony-forming units milliliter necessary for a positive CIE result varied with the types of microorganisms. The procedure of CIE appears useful as a rapid screening technic for identification of bacteria in blood cultures when microorganisms are seen in Gram-stained smears.
Antigens, Bacterial↗
Erroneous diagnosis of meningitis due to false-positive gram stains.
During a two-month period, 6% of Gram stains of predominantly CSF specimens revealed gram-negative bacilli with no growth. The source of the false-positive Gram stain results was an alcohol storage bath from which slides were taken and flamed to remove residual alcohol. All 11 patients in the outbreak had further diagnostic tests or changes in therapy. In the laboratory, false-positive slides could be created by contaminating a slide bath with 10(5) bacilli/ml, flame drying, and staining. In addition, contaminated crystal violet caused a false-positive result when applied to a warm but not to a cool slide. To prevent false-positive Gram stain results, the storage of slides in alcohol baths should be abandoned, slides should not be flamed to remove alcohol, and specimens should be Gram-stained only when the heat-fixed slide has thoroughly cooled.
1-Propanol↗
Gonorrhea: diagnosis by gram stain in the female adolescent.
The Gram-stained smear of secretions in female children and adolescents was evaluated as a diagnostic aid in acute gonorrhea. A positive smear was defined as the presence of at least eight or more pairs of Gram-negative, kidney-bean-shaped diplococci in each of at least two polymorphonuclear leukocytes. When the Gram-stained smear was compared with culture results in female patients it detected two thirds of infections and had a very high specificity. When the smear was positive, the culture was positive in 96% of the cases. The Gram-stained smear of cervical secretions is recommended as a guide to the early diagnosis of gonorrhea and its prompt therapy in the pediatric age group.
Adolescent↗
Efficacy of direct Gram stain in differentiating staphylococci from streptococci in blood cultures positive for gram-positive cocci.
A preponderance of clusters seen on direct Gram stain of blood cultures positive for gram-positive cocci was 98% sensitive and 100% specific for identification of staphylococcal species or of Peptococcus. A preponderance of chains, pairs, or both was 100% sensitive and 98% specific for identifying streptococci. Further presumptive identification of either staphylococci or streptococci based on microscopic morphology was unreliable. The direct Gram stain is highly reliable for differentiating staphylococci from streptococci and should be of considerable value to clinicians selecting initial antimicrobial therapy.
Blood↗
Selecting patients requiring antibiotics in biliary surgery by immediate gram stains of bile at operation.
The value of selecting patients for antibiotic cover during biliary surgery by the use of immediate gram stains of bile was determined in a nonrandomized prospective study which compared two groups of patients. Group A consisted of 119 consecutive patients in whom antibiotics were administered during operation according to the results of immediate gram stains on bile. Group B included 101 patients, none of whom received antibiotics. In Group A gentamicin was given for gram-negative bacteria, ampicillin for gram-positive organisms, and no antibiotics were given if no bacteria were seen on the gram stain. In Group A the incidence of wound sepsis was 7 percent, compared with 22 percent in Group B (p less than 0.005). Septicemia occured in 2 percent of Group A, compared with 8 percent in Group B. It is concluded that immediate gram stains of bile will provide a means of selecting patients requiring antibiotic cover during biliary surgery; furthermore, this procedure is a practical way of reducing postoperative sepsis while avoiding unnecessary antibiotic administration.
Adult↗
A more reliable gram staining technic for diagnosis of surgical infections.
The purpose of this study was to identify pitfalls in the Gram staining technic that limit its diagnostic value. In our clinical experience, gram-positive organisms were often decolorized too easily. Factors have been identified that alter the susceptibility of gram-positive organisms to decolorization in the Gram staining technic. The age of the bacterial culture, the preparation of the smear, the fixation technic, and the mordant have an important influence on the ease with which gram-positive organisms are decolorized. On the basis of these studies, a more reliable and reproducible Gram staining technic has been developed for the diagnosis of surgical infections.
Bacteria↗
Comparative evaluation of the Limulus assay and the direct Gram stain for detection of significant bacteriuria.
A double-blind study comparing the Limulus in-vitro endotoxin assay with the direct Gram stain of uncentrifuged urine for detection of significant bacteriuria was performed. One-thousand seventy-seven urine specimens were examined by the two methods and the results compared with results of quantitative urine cultures. Two hundred three samples produced growth of greater than 10-5 organisms per ml. urine. The Limulus assay detected 86.2% of these specimens, and 98.8% of urines that contained greater than 10-5 Gram-negative bacilli per ml. The Gram stain procedure detected only 69.5% of urines containing greater than 10-5 organisms per ml. and 74.5% of specimens with greater than 10-5 Gram-negative bacteria per ml. urine. The Limulus assay demonstrated both greater sensitivity and greater specificity than the Gram stain procedure. Moreover, the Limulus test is much less susceptible to errors of interpretation than methods involving microscopy.
Arthropods↗
A new technique for Gram staining paraffin-embedded tissue.
Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining.
Bacteriological Techniques↗
Bacterial gram staining by conventional and strip methods.
Conventional gram staining was compared with a method which uses dye impregnated paper strips for staining. The paper strip technique was judged accurate and reproducible and is recommended for use in the small laboratory and hospital ward.
Bacteriological Techniques↗
Automatic gram-staining with the microstainer II.
A comparison was made between the Microstainer II, an automatic staining machine, and the traditional, manual gram-staining method using clinical material and known organisms in a double blind study. Gram-reactions were in agreement with 98.4% of the organisms. The machine-stained microorganisms were generally found to be of the same or better quality than manually-stained organisms. Transfer of bacteria from slide to slide or smear to smear was not a significant problem. The Microstainer II would appear to be a useful addition to the large volume bacteriology laboratory.
Automation↗
Streptococcal pharyngitis: diagnosis by gram stain.
Group A beta-hemolytic streptococci were isolated from 49 (10.4%) of 472 patients with pharyngitis. Throat culture results, interpreted by five observers of varying experience, showed the mean sensitivity, specificity, and predictive value of a positive Gram-stained smear of pharyngeal secretions as 73%, 96%, and 71%. Assignment to a high-risk group by clinical algorithm gave sensitivity, specificity, and predictive value of only 45%, 83%, and 23%. The Gram-stained smear is the most accurate method of early diagnosis of streptococcal pharyngitis.
Adolescent↗
False-positive Gram-stained smears.
The rate per 1,000 smears showing nonviable Gram-negative bacilli (false-positive smears) increased from a baseline of 10.8 to 38.5 following purchase of new culture-collection devices; the rate decreased to 8.0 following replacement of contaminated culture sets. False-positive reports led to changes in therapy for five patients. In addition to being sterile, commercial culture-collection devices should be certified by the manufacturer as being free of stainable microorganisms or as unsuitable for preparation of Gram-stained smears.
Adolescent↗
Gram stain evaluation of the quality of sputum specimens for mycobacterial culture.
A group of 34 mycobacteria, consisting of 25 Mycobacterium tuberculosis and nine strains of three other species, was isolated from 400 expectorated sputum specimens submitted on 148 patients from county-wide sources. Eight strains (24% of the total) were isolated from specimens evaluated by Gram stain to be oropharyngeal fluids. The remaining 26 strains were isolated from ungradable specimens and those primarily of lower respiratory origin. It was concluded that the random examination of sputum by Gram stain to determine the specimen's quality for mycobacterial isolation is not necessary.
Humans↗
A gram stain for smears of blood cultures, body fluids and tissues.
A new Gram stain method for blood cultures was adapted from a method for tissue sections. This was accomplished by comparing various modifications of the latter method on positive smears prepared from a 24-hour negative blood culture seeded with Staphylococcus aureus and Escherichia coli. A blind study was then conducted using the new method and Hucker's method on 24-hour and 72-hour blood cultures as well as a limited number of other body fluids and tissues. Of the 35 positive blood cultures detected by the new method, the Hucker method failed to detect 14 positive specimens (40 per cent) upon initial examination. Ten additional cultures were negative using both stains, but were positive on subculture at three days. The 18 additional cultures positive after ten days were detected using only the Hucker stain.
Acinetobacter↗