Early detection of positive blood cultures by the acridine orange staining technique.

Staining 2,205 macroscopically negative blood cultures with acridine orange after 6 to 17 h of inoculation and incubation was as sensitive as an early subculture in detecting positive blood cultures. Of the 179 positive blood cultures, 30 (16.8%) were detected by acridine orange alone, 19 (10.6%) were detected by early subculture alone, 84 (46.9%) were detected by both techniques, and 46 (25.7%) were not detected by either method. The latter group includes cultures that became positive after 48 h of incubation. Acridine orange staining of smears prepared from macroscopically negative blood cultures after 6 to 17 h is a rapid, reliable method to detect positive blood cultures.     

Acridine orange staining as a replacement for subculturing of false-positive blood cultures with the BACTEC NR 660.

Despite the customization of growth index thresholds within individual laboratories, use of the BACTEC NR 660 automated blood culture system results in a number of false-positive cultures. The results of Gram staining, acridine orange staining, and subculturing to agar media were evaluated on 210 false-positive blood cultures over a 6-month period. Inclusion of acridine orange staining in the routine workup of false-positive blood cultures can eliminate the need for subculturing.     

Acridine orange stain in the early detection of bacteria in blood cultures

A total of 1,592 blood cultures without macroscopic signs of bacterial growth in the first 12–24 h of incubation were processed for both acridine orange stain and blind subculture. One hundred and twenty-one (7.6 %) blood cultures were positive by either method; of these, 105(8.68 %) were positive by both methods, 11 (9.1 %) positive by acridine orange and negative by subculture, and 5 (4.1 %) negative by acridine orange and positive by subculture. The difference between the 116 blood cultures positive by acridine orange and the 110 blood cultures positive by subculture was not statistically significant (p>0.1). Gram stain performed on all acridine orange positive cultures failed to reveal bacteria in 14 cases. Acridine orange staining is a sensitive, rapid and reliable method for detecting bacteria in blood cultures early during incubation. The method is inexpensive and easy to perform and can be substituted for blind subcultures.     

Evaluation of acridine orange staining as a replacement of subcultures for BacT/ALERT-positive, gram stain-negative blood cultures

Among 18,424 blood culture sets processed during a study period of 18 months, 85 bottles that were positive by the BacT/ALERT system were Gram stain negative. Both acridine orange staining and subcultures detected microorganisms in a total of 12 bottles. Acridine orange staining can replace subcultures of false-positive blood cultures.     

Comparison of acridine orange, methylene blue, and Gram stains for blood cultures.

Direct microscopic screening of blood cultures by Gram stain or methylene blue stain is time consuming and frequently insensitive. Therefore, we evaluated a fluorescent-staining procedure that uses acridine orange (AO) at pH 3.5 and compared it with the methylene blue and Gram stain procedures. All smears were prepared within 24 h of receiving the culture, fixed with methanol, and examined without the results of the companion smears being known. AO-stained smears were examined with incident-light fluorescence at 600 x magnification and confirmed at 1,500x magnification. All bottles macroscopically positive within 24 h were excluded from the study. Of 2,946 cultures entered into the study, 204 (6.9%) were positive within 3 days. The sensitivity and specificity of AO based on these culture results were 52 and 98%, respectively, compared with 38% sensitivity and 99% specificity by methylene blue and Gram stains. The AO staining procedure is a simple, sensitive, screening technique for the early detection of positive blood cultures.     

Blood culture gram stain,acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma

Purpose: The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic–organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. Materials and Methods: A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. Results: No ‘very major’ discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in β lactam – β lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Conclusions: Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.     

Pre-lytic release of foot-and-mouth disease virus in cytoplasmic blebs.

The pre-lytic release mechanism of foot-and-mouth disease virus was investigated by immunofluorescence, acridine orange staining, and electron microscopy in infected bovine and porcine kidney coverslip cultures. Cells with cytoplasmic fluorescence and which were positive for single stranded RNA with acridine orange staining were observed at 2 h after infection. Scanning electron microscopy showed cytoplasmic blebs in all cultures examined 2 h after infection. Rounded cells with virus inclusions began to appear 3 h after infection. Rounded cells and cytoplasmic blebs were shown to have single stranded RNA by acridine orange staining. Immunofluorescence and transmission electron microscopy with immunoferritin tagging demonstrated foot-and-mouth disease virus in cytoplasmic blebs. This study presents evidence for a pre-lytic release of foot-and-mouth disease virus through virus-containing cytoplasmic blebs emerging from infected cells.     

Differential Fluorescent Staining Method for Detection of Bacteria in Blood Cultures,Cerebrospinal Fluid and Other Clinical Specimens

The aim of this study was to evaluate a differential staining method to distinguish gram-positive from gram-negative bacteria in fluorescence. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein. With this method, gram-positive bacteria appear yellow and gram-negative bacteria appear green. In view of the importance of a rapid aetiological diagnosis in cases of septicaemia, the differential staining method in fluorescence was compared with Gram stain for the detection of bacteria in blood. Of 5,820 blood cultures entered into the study and identified by the Bactec 9120 fluorescent series instrument (Becton Dickinson Europe, France), 774 were positive. Of the 774 positive cultures, 689 yielded only a single organism. The differential staining method in fluorescence detected 626 of the 689 cultures, while Gram stain detected 468. On the basis of these results, the sensitivity of the differential staining method in fluorescence was 90.9%, while that of Gram stain was 67.9%. The difference between the two methods was statistically significant (P<0.001). The differential fluorescent staining method was more sensitive than Gram stain in the detection of bacteria in blood cultures during the incubation period. This technique provides a rapid, simple and highly sensitive staining method that can be used in conjunction with subculture methods. Whereas subculture requires an incubation period of 18–24 h, the fluorescent staining technique can detect bacteria on the same day that smears are prepared and examined. The differential fluorescent staining method was also evaluated for its ability to detect microorganisms in cerebrospinal fluid and other clinical specimens. The microorganisms were easily detected, even when bacterial counts in the specimens were low. Electronic Publication     

Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens.

Acridine orange, a fluorochrome strain, is potentially superior to the Gram stain in the direct microscopic examination of clinical specimens because it gives striking differential staining between bacteria and background cells and debris. Its value in clinical laboratories was evaluated by testing 209 cerebrospinal fluids and 288 other body fluids, tissues, and exudates by both techniques. Smears were made in duplicate, fixed with methanol, stained, and examined without knowledge of the result of the companion smear or culture. Overall, acridine orange was slightly more sensitive than the Gram stain (acridine orange, 59.9%; Gram stain, 55.8%) and equally specific in detecting microorganisms. One smear was falsely positive by the Gram stain; none was falsely positive by the acridine orange stain. We conclude that acridine orange staining is a sensitive method for screening clinical specimens and reviewing selected specimens that are purulent, but negative by the Gram stain. Bloody fluids, thick exudates, and other normally difficult-to-read specimens were easily and quickly examined. We recommend, however, that positive smears be reexamined with the Gram stain to confirm the result and determine the Gram reaction of the microorganisms.     

Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of broth blood cultures.

Bartonella quintana was isolated from 34 BACTEC nonradiometric aerobic resin blood cultures for 10 adults. Nine patients were initially diagnosed by routine acridine orange staining of routine cultures that had been incubated for 8 days. All subcultures grew on chocolate agar within 3 to 12 days (median, 6 days). The PLUS 26 high-volume aerobic resin medium, combined with acridine orange stain and subculture, is an effective system for detection and isolation of B. quintana from blood.     

Evaluation of acridine orange stain for detection of microorganisms in blood cultures.

A pH 4.0 buffered solution of the fluorochrome acridine orange was used to stain samples of 2,704 blood cultures that failed to yield visible evidence of growth after 1 day of incubation. Results obtained by the staining method were compared with those obtained by aerobic and anaerobic subcultures simultaneously performed upon the same cultures. Of the 109 culture-positive blood specimens initially detected by the acridine orange and the subculture methods, 85 (78%) were detected by both acridine orange and subcultures techniques, 14 (12.8%) were detected by subculture alone, and 10 (9.2%) were detected by acridine orange alone. The differences between the subculture and acridine orange methods were not found to be statistically significant (P less than 0.1). The acridine orange method represents a rapid and inexpensive alternative to conventional subculture techniques for the detection of bacteria in blood cultures that fail to yield visible evidence of growth after 1 day of incubation.     

Viability of organisms held in the isolator blood culture system for 15 h and their rapid detection by acridine orange staining

Sets of three Isolator blood culture tubes were seeded with low numbers of 96 strains of 26 bacterial species (fresh and stock clinical isolates). One tube was processed immediately, and the other two were held at 22 and 34 degrees C for 15 h before processing. Organism recovery was 99, 99, and 98%, respectively. Organism numbers increased at both 22 degrees C (60% of strains) and 34 degrees C (79% of strains). Especially notable was that the increases were seen with most strains of Staphylococcus aureus, streptococcal species, Pseudomonas, and all of the Enterobacteriaceae tested. Seven strains, including Streptococcus pneumoniae and Haemophilus influenzae, although viable, were recovered with a decreased number of organisms at each temperature. Acridine orange staining detected organisms in 53% of those Isolator tubes being held and 71% of those demonstrating a numerical increase, after incubation at 34 degrees C. In addition, it was noted that after processing, 48% of the strains that had increased in number while being held at 34 degrees C resulted in visible growth on agar media in 6 h. The results suggest that up to a 15-h delay in processing Isolator tubes may be possible and that acridine orange staining for the rapid detection of positive cultures may be useful in such a circumstance.     

Acridine orange staining of smears for demonstration ofMycobacterium tuberculosis

Acridine orange staining for the detection of mycobacteria was compared with staining by auramine 0 and with mycobacterial culture in a series of 1071 clinical specimens. A total of 78 (7 %) specimens were positive by staining. No false positive or negative findings were recorded by the acridine orange method. The two fluorochromes proved equal in their ability to detect mycobacteria in specimens from culture positive cases of tuberculosis. In the rapid bacteriological diagnosis of tuberculosis, acridine orange offers a good alternative to auramine 0 which is considered carcinogenic.     

Recovery of Histoplasma capsulatum from blood in a commercial radiometric Mycobacterium medium.

We report the recovery of Histoplasma capsulatum from blood specimens cultured for Mycobacterium sp. in BACTEC 13A radiometric medium. H. capsulatum was recovered from six of eight blood specimens submitted for mycobacterial cultures from five human immunodeficiency virus-positive individuals. Initial positive metabolic signals occurred at a mean of 11 days, but no organisms were detected with acid-fast stains. The bottles remained positive, and after an additional incubation (mean, 8 days), yeast cells morphologically compatible with H. capsulatum were detected when aliquots were stained with acridine orange. Therefore, when radiometric mycobacterial blood cultures with persistent positive metabolic signals and negative acid-fast stains are encountered, acridine orange staining and subculturing for a variety of microorganisms, including fungi, e.g., H. capsulatum, should be considered.     

Use of Directigen and acridine orange for rapid identification of human blood culture isolates

Of 7,871 blood cultures from hospital patients, 22 yielded growth of Streptococcus pneumoniae or Haemophilus influenzae type b. The identities of 19 (86%) of these 22 strains could be verified after 18 to 24 h of incubation by application of the Directigen meningitis test kit to the unheated, uncentrifuged supernatant from the cultures; thus, the turnaround time for these cultures was halved. Growth in 16 (72%) of the Directigen-positive cultures was detected by visual inspection, and that in 3 (14%) of the cultures was detected by acridine orange staining. Growth in the three remaining bottles (14%) was detected by blind subculturing after 18 to 24 h or incubation and, therefore, was delayed by 24 h. The systematic application the acridine orange stain was helpful in 40 (44%) of 91 cases for which macroscopic inspection failed to reveal growth after 24 h of incubation.     

Simple method for rapid diagnosis of catheter-associated infection by direct acridine orange staining of catheter tips.

Direct acridine orange (AO) staining was used to detect bacteria adherent to intravascular catheters (IVC). Samples from 710 IVC tips were first cultured on blood agar plates by a semiquantitative technique and then independently colored with AO and screened dry at a magnification of x100 for 3 min. In the absence of fluorescence, they were considered negative. When fluorescence was present, they were further examined for the presence of microorganisms at x1,000 with immersion oil. Of 710 IVC tips, 37 (5.2%) were positive upon culture (greater than or equal to 15 colonies) and 673 were negative (640 were sterile and 33 [4.6%] had 1 to 14 colonies). The AO sensitivity was 84%, and the AO specificity was 99%. When restricted to the 212 long IVC, AO sensitivity rose to 94%. AO staining was positive in all cases of catheter-associated bacteremia. The negative predictive value of the preliminary screening at x100 was 99.5%. The direct examination of IVC tips stained by AO appears to be a simple and rapid method for diagnosing IVC-associated infections. In addition, AO staining is easier to perform than Gram staining.     

Acridine orange staining and radiometric detection of microorganisms in blood cultures.

To determine whether acridine orange (AO) staining of blood cultures could be used as a substitute for blind subculture when used in conjunction with the BACTEC system (Johnston Laboratories, Inc., Towson, Md.), the two methods were compared on all BACTEC-negative specimens. Since blind subcultures were routinely performed in our laboratory on days 2 and 6 of incubation, AO staining was also performed on these days. Cultures which were BACTEC positive on day 1 of incubation were not included in the study. Of the 2,395 bottles tested after 2 days of incubation, 106 were subculture positive. Of these, 96 (90.6%) were also AO positive and BACTEC positive, 3 (2.8%) were AO positive and BACTEC negative, and 7 (6.6%) were AO negative and BACTEC positive. Of the 3,487 bottles tested on day 6 of incubation, 14 were subculture positive; 7 (50%) of these were AO positive and BACTEC positive, and seven were AO positive and BACTEC negative. Of the total of 10 culture-positive bottles missed by BACTEC, all were positive, and all 10 companion aerobic bottles were BACTEC positive. In both phases of the experiment, there was a total of only four false-positive AO stains. As a result of this investigation, we have substituted AO staining for blind subculturing of BACTEC-negative bottles.     

Value of direct catheter staining in the diagnosis of intravascular-catheter-related infection.

Ninety-nine intravascular catheters were evaluated by a semiquantitative culture and Gram and acridine orange direct stains. A diagnosis of catheter-related infection was determined by a retrospective review of clinical records. Compared with the culture method, direct examination of catheters lacked sensitivity. Surprisingly, for some patients, a positive stain for yeasts not recovered by culture was considered significant. The culture correlated with bacteremia but failed to predict infection in 42% of patients.     

Comparison of rapid urease tests, staining techniques, and growth on different solid media for detection of Campylobacter pylori.

Thirty-nine single antral biopsies (phase 1) and 99 sets of six antral biopsies (phase 2) were collected from 132 patients, and 87 (63%) yielded positive cultures for Campylobacter pylori. Of several primary media tested in phase 1, tryptic soy agar and Skirrow agar, each supplemented with 10% whole sheep blood, supported relatively good growth of C. pylori. In phase 2, four of the six biopsies in each set were tested with different urease systems. Selective urea agar for rapid identification was the most sensitive (39 of 63 [62%] at 1 h) and specific (100%); however, the difference between this system and the CLOtest was not statistically significant. The remaining two biopsies, one transported in saline and the other transported in a supplemented tryptic soy broth, were ground separately and inoculated onto tryptic soy agar and Skirrow agar, each supplemented with 10% whole sheep blood. In selected instances, 10% horse serum or 10% horse serum and 5 mM urea or 1% cholesterol were also added to the media. Smears stained with a modified Gram stain or acridine orange detected 68% of 63 culture-positive biopsies; no false-positive results were reported. Skirrow agar supported better growth of C. pylori than did tryptic soy agar; the nonselective medium was also overgrown with contaminants in 25 to 30% of the positive cultures. Based on colony size, Skirrow agar supplemented with 10% whole sheep blood, 10% horse serum, and 1% cholesterol supported optimal growth of C. pylori. Fresh media supported better growth than did prepoured commercial media (P less than or equal to 0.004). Saline was a convenient and satisfactory transport medium for antral biopsies.