Effect of incubation conditions on anaerobic susceptibility testing results.

We determined the effect of performing antimicrobial susceptibility tests in five different anaerobic incubation systems: GasPak jar, large GasPak jar, evacuated-gassed anaerobic jar, anaerobic chamber, and Bio-Bag. Growth of the anaerobes was equivalent in all five incubation systems. The results of testing 38 anaerobes against 11 antimicrobial agents were comparable for the anaerobic jars and anaerobic chamber. However, discordant results were observed for metronidazole and cefamandole tests when incubated in the Bio-Bag.     

Comparison of atmospheres of incubation for primary isolation of Campylobacter fetus subsp. jejuni from animal specimens: 5% oxygen versus candle jar.

An atmosphere with reduced oxygen tension is required for the primary isolation of Campylobacter fetus subsp. jejuni. Therefore, we compared use of the conventional atmosphere of 5% oxygen and 8% carbon dioxide with use of a candle jar (17% oxygen and 3% carbon dioxide) for primary isolation of C. fetus subsp. jejuni from 263 positive canine, cattle, and turkey fecal or cecal specimens. At an incubation temperature of 42 degrees C, the atmosphere with 5% oxygen resulted in more Campylobacter colonies per plate (P less than 0.005) and consistently larger Campylobacter colonies (P less than 0.005) than did the candle jar, whereas the growth of interfering flora was similar. Overall, 96% of the 263 specimens were positive for C. fetus subsp. jejuni with 5% oxygen, and 90% were positive with the candle jar (P less than 0.02). More striking differences in isolation rates were seen when both the temperature and the atmosphere were varied: 5% oxygen at 42 degrees C enabled recovery of 93% of the isolates from 70 positive specimens, versus 46% recovery with the candle jar at 37 degrees C. Results with 5% oxygen at 37 degrees C were intermediate. The addition of FBP supplement (0.25% each of ferrous sulfate, sodium metabisulfite, and sodium pyruvate) to Campy-BAP selective medium made no improvement over unsupplemented medium at 42 degrees C (whether in 5% oxygen or in the candle jar), but there was significant improvement over unsupplemented medium when both media were incubated at 37 degrees in the candle jar.     

Comparative evaluation of three different commercial blood culture media for recovery of anaerobic organisms.

A comparison of three different commercial media was made to assess their recovery of anaerobic organisms from the blood stream. The three media used were the 50-ml brain heart infusion broth with added CO2 (Pfizer), the 50-ml Thiol broth with added CO2 (Difco), and the 50-ml prereduced, supplemented peptone broth in a Vacutainer tube with added CO2 (Becton-Dickinson). During a period of 17 consecutive months, 12,216 specimens of blood were processed with each broth. Aerobic or anaerobic bacteria were recovered from 913 specimens (7%). Seventy-four specimens (8%) of the total positive cultures contained anaerobic organisms. When potential contaminants were removed from the totals, 7% of the positive cultures contained anaerobic organisms and 7% of the patients with positive cultures had bacteremia with anaerobic bacteria. Of the three commercial blood culture media studied, the prereduced, supplemented peptone broth recovered more anaerobic organisms than did either the brain heart infusion or Thiol broths.     

Atmospheric analysis and redox potentials of culture media in the GasPak System

Oxygen and carbon dioxide concentrations, internal atmospheric pressure, catalyst temperature, and time of appearance of water condensate were monitored for various time intervals at ambient (20 to 25 degrees C) temperature in a GasPak 100 Anaerobic System (BBL Microbiology Systems, Cockeysville, Md). Simultaneously, the redox potential (Eh) of various plated culture media in the system was also measured. The oxygen concentration was reduced to less than 0.4% in 100 min. The Eh of the media, corrected for hydrogen ion, reached -100 mV within 60 to 100 min, and the carbon dioxide concentration increased to between 4 and 7% in 60 min, depending on the number of plates of media present. Condensate appeared generally between 10 and 15 min, and the temperature of the lid reached a maximum between 20 and 40 min. Condensate time and lid temperature increase are important early indicators of a correctly functioning GasPak System. A characteristic pressure-vacuum-pressure profile is produced as a result of controlled release of hydrogen and carbon dioxide gases and the reaction of hydrogen and oxygen to produce water. Anaerobic conditions were achieved well before the methylene blue anaerobic indicator became decolorized, which required more than 6 h at 20 to 25 degrees C. At this time the Eh of media in the jar was well below -200 mV. Since the indicator is reduced within 5 h at 35 degrees C, the Eh of media in the jar would also be expected to decrease more rapidly at the higher temperature.     

The isolation of anaerobic bacteria from wound swabs

The isolation of anaerobic bacteria from routine wound swabs by three procedures was evaluated.Recovery of anaerobic organisms was doubled by immediate incubation of seeded plates, and the recovery could be further dramatically improved by the use of prereduced media, in conjunction with an anaerobic chamber.Recommendations for the treatment of swabs and cultures for anaerobic investigation are made.     

Best Practices for the Pre-Analytic Phase of Anaerobic Bacteriology

Anaerobic bacteria that cause infections contribute to patient morbidity and mortality, especially among patients with underlying conditions or who are immunocompromised. While once largely grouped together without specific differentiation, anaerobic bacteria, defined as organisms that utilize terminal electron acceptors other than oxygen, are of increasing importance in clinical microbiology. In this review, we discuss pre-analytical-phase best practices for anaerobic recovery in the clinical microbiology laboratory. The suspected infection, and in turn the type of specimen to be cultured, helps determine which sample container, specimen collection method, transport conditions, and culture media are needed for optimal recovery of anaerobic bacteria.     

Anaerobic bag culture method.

In a new method of anaerobic culture, a transparent, gas-impermeable bag is used and the anaerobic environment is established with copper sulfate-saturated steel wool. An Alka-Seltzer tablet generates carbon dioxide. The agar plate surface can be inspected through the bag at any time without interrupting the anaerobic atmosphere or disturbing other specimens. Methylene blue indicator strips are completely reduced by 4 h after the bag is set up and have remained reduced for as long as 3 weeks. Growth of 16 different stock culture anaerobes was generally equivalent by the bag and GasPak jar methods. Yield and growth of anaerobic isolates also were equivalent with 7 of 10 clinical specimens; from the other 3 specimens, 13 isolates were recovered, 5 by both the bag and jar methods and the rest by one method or the other. No consistent differences were found between the anaerobic bag and GasPak jar methods in the yield of anaerobes from clinical specimens. Early growth (24 h of incubation) of anaerobes from one specimen was detected with the bag method.     

A comparison of microaerobic systems for the culture ofCampylobacter jejuni andCampylobacter coli

The relative ability of two commercial gas generating envelopes, the evacuation-replacement technique, and a candle jar, to produce a satisfactory microaerobic atmosphere for the culture of 45Campylobacter strains on non-selective medium and five selective media (Skirrow's, modified Butzler's, Blaser's, Campy-BAP and Preston medium) was investigated quantitatively. A candle jar, and modified Butzler's medium proved to be of limited use. The ability of four commercial gas generating envelopes to produce a satisfactory microaerobic atmosphere in four anaerobic jars of different volume was investigated using five referenceCampylobacter strains. Not all of the combinations worked. The oxygen and carbon dioxide concentrations produced in the microaerobic systems studied were measured with gas analysers. The evacuation-replacement technique produced far less variable concentrations of oxygen and carbon dioxide than did the envelopes.     

Anaerobic specimen transport device.

A device is described and evaluated for the anaerobic transport of clinical specimens. The device limits the amount of oxygen entering with the sample to a maximum of 2%, which is rapidly removed by reacting with hydrogen in the presence of a palladium catalyst. The viability on swabs of 12 species of anaerobes, four strains of facultative anaerobes and a strain of Pseudomonas aeruginosa, was maintained during the length of the tests (24 or 48 h). The results demonstrated that this device protected even the more oxygen-sensitive clinical anaerobes from death due to oxygen exposure. This device can be used for swabs as well as for anaerobic collection and liquid and solid specimens.     

Comparison of lysis-centrifugation with a biphasic blood culture medium for the recovery of aerobic and facultatively anaerobic bacteria

The Du Pont Isolator tube and Roche Septi-Chek blood culture bottle employ solid media which facilitate the removal of bacteria from static or cidal substances in blood to increase recovery and decrease detection time. In a comparison of 11,567 blood culture sets, the Isolator tube and vented Roche Septi-Chek bottle were positive for 533 (80%) and 494 (74%) of the aerobic and facultatively anaerobic organisms recovered, respectively. This difference was not significant. A significant difference was found in the overall detection time. The Isolator tube recovered the bacteria ca. 1 day earlier. The earlier detection time was most notable with Staphylococcus aureus, viridans streptococci, and Pseudomonas aeruginosa. Among the 355 bacteremic episodes analyzed by a computer program, the Isolator tube was responsible more often for the first report of bacteremia in a given patient. Both systems performed well for the recovery of aerobic and facultatively anaerobic bacteria, but it is recommended that either be used in combination with an unvented broth-containing bottle.     

Evaluation of AnaeroGen system for growth of anaerobic bacteria.

The Oxoid AnaeroGen system was compared with the BBL GasPak for the production of an anaerobic atmosphere and was evaluated for its ability to support the growth of 135 clinically significant anaerobic bacteria. An anaerobe chamber was used as the "gold standard" for supporting the growth of anaerobes. The AnaeroGen requires no catalyst, produces no hydrogen, requires no water, and reduces preparation time to a minimum. The water-activated BBL GasPak generates hydrogen. For 132 of the 135 strains tested, better initial growth at 48 h was noted for the jar methods than for the anaerobe chamber. At 72 h, 113 of the 135 strains showed equal growth, and at 7 days, only marginal differences in growth patterns were noted. The AnaeroGen never failed to reduce the anaerobic indicator, while the BBL GasPak occasionally failed to do so. The AnaeroGen performed at least as well as, and sometimes better than, the established methods. The AnaeroGen is a good alternative for use in anaerobic jars.     

Evaluation of the AnaeroPack system for growth of anaerobic bacteria.

Growth of anaerobic bacteria in the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) anaerobic atmosphere generation systems, both the AnaeroPack jar and pouch and the AnaeroPack in a GasPak jar were considered equivalent to or better than growth obtained in the corresponding GasPak jar or pouch system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) for 89 (86%) of the 103 anaerobes tested. There were a total of 26 discrepancies after 48 h of incubation, with 16 discrepancies unresolved after 96 h of incubation. The AnaeroPack jar and pouch never failed to reduce the anaerobic indicator. The AnaeroPack systems are easy to use and performed at least as well as or better than the BBL GasPak systems for growth of anaerobic bacteria.     

Use of tri-gas incubator for routine culture of Campylobacter species from fecal specimens.

We evaluated a tri-gas incubator for Campylobacter isolation to be used instead of an anaerobic jar. Fecal specimens were cultured in duplicate onto charcoal selective medium and incubated at 43 degrees C for 48 h in two different environments: a tri-gas incubator (Forma Scientific) adjusted to provide an atmosphere of 10% CO2, 10% O2, and the balance N2; and evacuated anaerobic jars with a replacement gas mixture of 10% CO2, 5% O2, and 85% N2. A total of 106 Campylobacter jejuni and 8 Campylobacter coli isolates were obtained from 2,348 stool specimens. Of the positive specimens, 113 isolates came from the incubator and 111 isolates came from the anaerobic jars. An additional 32 previously positive specimens were replated onto charcoal selective medium and retested by both methods. We recovered 27 C. jejuni isolates, 26 isolates by each method. The isolates from the incubator typically produced discrete colonies, while the isolates from the anaerobic jar showed some degree of swarming in colony formation. The tri-gas incubator provided a cost-effective method for culturing Campylobacter spp.     

Comparison of methods for recovery of Clostridium difficile from an environmental surface

Survival of Clostridium difficile in an aerobic environment is possible because of spore formation. When sodium taurocholate is substituted for the egg yolk of a selective medium, cycloserine-cefoxitin-fructose-agar (CCFA), enhanced recovery of C. difficile spores is shown. This selective medium (TCCFA) does not improve recovery of vegetative forms. In this study, dry and saline-moistened swabs, adhesive paddles, and Rodac plates containing CCFA and TCCFA were compared in their ability to recover C. difficile spores from an inoculated surface. Rodac plates grew 20 to 25 times as many spores on TCCFA as on CCFA. Saline-moistened swabs recovered fewer organisms than Rodac plates. Dry swabs and adhesive paddles rarely recovered spores. Prereduction of agar in an anaerobic chamber was not necessary for optimal spore recovery. Optimal growth of vegetative C. difficile required prereduced media. Agar prereduced for 2 h supported the growth of 12 C. difficile isolates as well as agar prereduced for 18 h. Vegetative cells of C. difficile survived for only 15 min in room air. Use of Rodac plates containing TCCFA is preferred for detection of C. difficile spores in the hospital environment.     

CO2 requirement for growth of a Pasteurella strain isolated from rabbits.

A Pasteurella strain with unusual growth requirements was isolated from a rabbit abscess. Copious growth of this organism was observed only on media supplemented with serum and incubated in the candle jar. Under anaerobic conditions, growth was moderate on enriched media. The organism did not grow on unsupplemented media under these conditions or in an aerobic environment on enriched media.     

A regulated environmental perfusion system for the study of anoxic or hypoxic cultured neurons using microfluorescence imaging and electrophysiology

 We describe the design and performance of a newly developed regulated environmental perfusion system (REPS). This system allows study of the effects of anoxia or hypoxia in cultured cells at physiological temperature, without the use of oxygen-scavenging compounds or metabolic inhibitors. The REPS incorporates a ”canoe-shaped” flow-through chamber with access from above to allow positioning of pipettes for patch-clamp, microinjection, rapid-application perfusion, or microprobes for monitoring physical parameters. The combination of laminar flow and complete washout of perfusate within the chamber, and the use of a gas-tight perfusate delivery system and pressurized reservoirs containing media with pre-stabilized oxygen tensions (pO₂ values) allow rapid production of accurate perfusate pO₂ within the chamber. Perfusate pO₂ in the chamber declined monoexponentially with time constants of ≤ 20 s to stable, pre-determined levels of 0 or 2 kPa (15 Torr). Shielding the gas/liquid interface of the chamber with an argon curtain only minimally decreased time constants at flow rates ≥ 2 ml/min. The perfusion chamber of the REPS is easily mounted on the stage of an inverted microscope, for use with fluorescence imaging or electrophysiological studies of cultured cells. In tests with cultured rat cortical neurons, intracellular calcium concentration increased exponentially to values exceeding 1 μM during 10 min of anoxic insult, and returned to baseline values within 1 min after restoring normoxia. Received: 16 September 1997 / Received after revision: 18 November 1997 / Accepted: 26 December 1997     

Methods of measuring zones of inhibition with the Bauer-Kirby disk susceptibility test.

Standard Bauer-Kirby disk tests were performed with 85 selected isolates, each tested in triplicate by four different investigators. Each disk test was observed, and zone diameters were measured, under two lighting conditions (transmitted light and reflected light). The two lighting systems produced similar zone measurements (+/-2 mm) with 96% of the tests. When there were greater differences, zones appeared to be larger when observed with reflected light. Interlaboratory reproducibility was much greater when using reflected light rather than transmitted light. We concluded that zone diameters should be measured from the back of the plate while it is resting on, or held 2 to 3 inches [ca. 5.1 to 7.6 cm] above, a black, nonreflecting, flat surface, illuminated by a reflected light source.     

Continuous monitoring of oxygen concentrations in several systems for cultivation of anaerobic bacteria.

Anaerobic chambers and jars are the two conventional methods used in clinical microbiology laboratories to produce an anaerobic atmosphere. The evacuation-replacement method, the Oxoid AnaeroGen, the Merck Anaerocult A, the BBL GasPak, the BBL GasPakPlus, the Adams Scientific GasGendicator, the Difco Anaerobic, and the bioMérieux Generbox anaer systems were compared for the timed decrease in the oxygen concentration in an anaerobic jar. The experiment was repeated 10 times for each system. The oxygen concentration was measured with an oxygen analyzer series 3600 instrument (Orbisphere Laboratories, Neuchâtel-Geneva Switzerland). The BBL GasPak, the BBL GasPakPlus, the bioMérieux Generbox, the Adams Scientific GasGendicator, and the Difco Anaerobic systems contain sodium borohydride, which liberates hydrogen. The Anaerocult A system contains iron powder which binds the oxygen. The active ingredient of the AnaeroGen system is ascorbic acid. The times to reach an O2 concentration of 0.5% were 8 to 15 min for the evacuation-replacement method, 26 to 41 min for the AnaeroGen system, 60 to 93 min for the Anaerocult A system, and 22 to 419 min for the sodium borohydride systems. The AnaeroGen system, the Anaerocult A system, and the evacuation-replacement method never failed to produce an anaerobic atmosphere. The sodium borohydride systems failed in 10 of 70 runs. These results suggest that the evacuation-replacement method or the Oxoid AnaeroGen system should be used to produce an anaerobic atmosphere. The Anaerocult A system showed a good reproducibility, but the length of time required to reach an appropriately low level of oxygen was too long. Because of the high failure rate, the borohydride systems cannot be recommended.     

Ionization chamber volume averaging effects in dynamic intensity modulated radiation therapy beams

The commercial cylindrical ionization chamber ionization integration accuracy of dynamically moving fields was evaluated. The ionization chambers were exposed to long (14 cm), narrow (0.6, 1.0, 2.0, and 4.0 cm) 6 MV and 18 MV fields. Rather than rely on the linear accelerator to reproducibly scan across the chamber, the chambers were scanned beneath fixed portals. A water-equivalent phantom was constructed with cavities that matched the chambers and placed on a computer-controlled one-dimensional table. Computer-controlled electrometers were utilized in continuous charge integrate mode, with 10 samples of the charge, along with time stamps, acquired for each chamber location. A reference chamber was placed just beneath the linear accelerator jaws to adjust for variations in linear accelerator dose rate. The scan spatial resolution was selected to adequately sample regions of steep dose gradient and second spatial derivative (curvature). A fixed measurement in a 10 x 10 cm2 field was used to normalize the profiles to absolute chamber response. Three ionization chambers were tested, a microchamber (0.009 cm3), a Farmer chamber (0.6 cm3) and a waterproof scanning chamber (0.125 cm3). The larger chambers exhibited severe under-response at the small field's centers, but all of the chambers, independent of orientation, accurately integrated the ionization across the scanned portal. This indicates that the tested ionization chambers provide accurate integrated charges in regions of homogeneous dose regions. Partial integration (less than the field width plus the chamber length plus 2 cm), yielded integration errors of greater than 1% and 2% for 6 MV and 18 MV, respectively, with errors for the Farmer chamber of greater than 10% even for the 4 cm wide field.     

Rapid oxidase method for testing oxidase-variable Aeromonas hydrophila strains.

A rapid, same-day oxidase test procedure which obviates the problem of false-negative oxidase reactions of Aeromonas hydrophila removed from the surface of differential media such as MacConkey agar is described. This method allows oxidase testing to be performed within 3 h, rather than delaying the oxidase test for an additional 18 to 24 h. This procedure is applicable to any rapidly growing gram-negative rod.