Techniques for Controlling Variability in Gram Staining of Obligate Anaerobes

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1995, p Vol. 33, No /95/$ Copyright 1995, American Society for Microbiology Techniques for Controlling Variability in Gram Staining of Obligate Anaerobes MARA J. JOHNSON, 1,2 * EILEEN THATCHER, 1 AND MIKE E. COX 2 Department of Biology, Sonoma State University, Rohnert Park, California 94928, 1 and Anaerobe Systems, San Jose, California Received 25 April 1994/Returned for modification 2 June 1994/Accepted 1 November 1994 Identification of anaerobes recovered from clinical samples is complicated by the fact that certain grampositive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with quick fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes. Gram staining remains the universally accepted first step in identification of bacteria, including anaerobic bacteria (5, 9). It categorizes bacteria into two broad groups, gram positive and gram negative, a distinction based on cell wall composition. Presumptive identification of anaerobic bacteria in the clinical setting depends upon accurate Gram-staining results (13), because empirical data are often used for diagnosis of infection, and for the initial choice of antimicrobial agents for treatment (10). Certain species of gram-positive anaerobes either inconsistently, rarely, or never stain gram positive. Peptostreptococcus asaccharolyticus is described as staining consistently gram positive only at 24 h in PYG (peptone, yeast extract, glucose) broth (15). Eubacterium plautii, formerly known as Fusobacterium plautii, a gram-negative rod, was reclassified in 1982 on the basis of cell wall structural analysis and motility; however, definitive gram-positive results were unattainable (11, 16). Clostridium ramosum is described as gram positive or gram negative and is known to easily decolorize (6, 8). Clostridium symbiosum (Fig. 1) consistently stains gram negative (6) and was previously known as Fusobacterium symbiosum (12). In 1976, Bacteroides clostridiiformis was reclassified by Cato and Salmon (7), along with Kaneuchi and coworkers (12), to the genus Clostridium on the basis of observations of characteristic biochemical reactions, spore formation, motility, and DNA homology. However, Clostridium clostridiiforme continues to be misidentified as a Bacteroides sp. or Fusobacterium sp. because it stains gram negative (9, 18). Erroneous Gram-staining results necessitate further testing for spore formation (6), the use of special-potency antibiotic susceptibility disk tests (1), or the KOH test to confirm the true Gram reaction (9, 18), procedures which add substantial time and cost to the identification process. This study tested the hypothesis that culturing, fixing, and Gram staining these anaerobic bacteria under strict anaerobic conditions would yield accurate Gram stain results. Description of Gram stain results by controlled fixing and staining techniques. Test organisms P. asaccharolyticus ATCC 29743, E. plautii ATCC 29863, C. ramosum ATCC 25554, C. * Corresponding author. symbiosum ATCC 14940, and C. clostridiiforme ATCC were obtained from the American Type Culture Collection, Rockville, Md., via Anaerobe Systems, San Jose, Calif. Gram stain controls Staphylococcus aureus ATCC and Fusobacterium nucleatum ATCC were chosen as morphologically distinct from most test organisms. Pure cultures of these species were streaked on prereduced anaerobically sterilized brucella blood agar plates (Anaerobe Systems) and incubated at 37 C for 48 h within a gloveless anaerobic chamber (Sheldon Manufacturing, Inc., Cornelius, Oreg.). The anaerobic atmosphere consisted of 90% N 2,5%H 2, and 5% CO 2 (American Welding Supply, San Jose, Calif.), was kept oxygen free by a palladium-based catalyst that was reactivated daily, and was monitored by using BBL GasPak disposable anaerobic indicator strips (Becton Dickinson Microbiology Systems, Cockeysville, Md.). In the course of the study, the following parameters were examined for each test organism: heat fixing within the chamber, fixing with 95% ethyl alcohol; fixing with methanol or formalin; increasing the fixing times within the chamber; and preparing smears within the chamber, followed by staining outside the chamber. Exposure to oxygen during any stage of the fixing or staining process did not achieve acceptable results nor did increasing the time of fixation. The only acceptable methods included quick fixing techniques with methanol or formalin anaerobically, followed by anaerobic staining within the chamber. Within the working space of the chamber, suspensions of the test organisms were mixed with gram-positive and gram-negative controls in sterile deionized water on clean microscope slides and allowed to dry. The smears were then fixed by the methanol or formalin technique within the chamber. During fixing and staining, the chamber was flushed with the gas mixture every 20 min for 3 min to dilute the concentration of vapors potentially toxic to the bacteria (methanol, formalin, and ethyl alcohol). In the methanol technique, a set of slides was flooded for 30 s and rinsed briefly with deionized water, a method adapted from Mangels, Cox, and Lindberg (14). In the formalin technique, a set of slides was placed in a glass staining dish with a rack and cover and formalin was dispensed to cover the bottom of the dish. The slides were vapor fixed for 3 min and rinsed 755

2 756 NOTES J. CLIN. MICROBIOL. FIG. 1. C. symbiosum cells stained by conventional techniques. briefly with deionized water, a method adapted from Rodina (17). Gram stain reagents were then applied in a series to the fixed smears, either within the anaerobic environment of the chamber or aerobically out of the chamber. Crystal violet was applied as the primary stain; after rinsing, iodine was applied as a mordant. Decolorization was completed with 95% ethyl alcohol, and after rinsing, safranin or dilute (5%) carbol fuchsin was applied to counterstain gram-negative controls. This study used the Hucker modification of the Gram stain (1), supplemented with the procedure outlined by Engelkirk et al. (9), and used limited wash times (2) with deionized water. In addition, decolorization was limited to the minimum time that properly decolorized the gram-negative controls. For each of the test organisms, a minimum of 10 slides were prepared for each set of parameters, including the fixation method and either anaerobic or aerobic Gram staining. Slides were rejected if the controls did not stain properly. All slides were viewed under oil immersion with a 100 objective on a Nikon Optiphot microscope, and photographs were taken with a Nikon HFX-DX photomicrography system with Kodak Ektar ISO 100, 35-mm color print film. Note in Table 1 that, under anaerobic conditions, all species were described as gram positive or variable by the methanol technique and that all but E. plautii were clearly identified by the formalin technique. All species except C. ramosum were gram negative or variable under aerobic conditions. C. symbiosum, fixed with formalin, is shown in Fig. 2 as gram positive in a mixed smear with F. nucleatum, the gram-negative control organism. Compared with methanol, formalin-fixed C. clostridioforme and C. symbiosum cells appeared larger and stained more deeply purple, and the smear lacked debris from the reagents. E. plautii and C. ramosum failed to stain deeply purple in formalin vapor compared with cells fixed by methanol. Although formalin-fixed C. clostridiiforme and C. symbiosum cells stained deeply purple, formalin is a noxious chemical; therefore, the clinical application of its use as a fixative may be limited. We conclude that the methanol fixing technique is better, because it achieved acceptable results overall. For optimal results, the fixing techniques were adapted in two ways: first, by a brief rinsing with deionized water before staining rather than leaving methanol or formalin on the slide to air dry; second, by a decrease in the fixing time from 1 min to 30 s for methanol and from 10 min to 3 min for formalin vapor. Hence, the term quick fix. The rinse step was em- TABLE 1. Gram-staining results observed with anaerobic or aerobic staining conditions and anaerobic fixing techniques, with reference to published species descriptions a Test organism Staining condition Reference description Anaerobic fixation with: Methanol Formalin vapor P. asaccharolyticus Anaerobic Aerobic v v v E. plautii Anaerobic v Aerobic C. ramosum Anaerobic Aerobic v v C. symbiosum Anaerobic v Aerobic C. clostridiiforme Anaerobic v Aerobic a See text for citations., gram positive; v, variable;, gram negative. Note that control strains of S. aureus and F. nucleatum stained gram positive and gram negative, respectively. Proper staining of control cells in a smear was the basis for judging a slide acceptable.

3 VOL. 33, 1995 NOTES 757 FIG. 2. Formalin-fixed cells of C. symbiosum, shown with properly stained gram-negative cells of F. nucleatum. ployed because Basu and Biswas (3) report that alcohol may limit the initial uptake of crystal violet by the cell. The Gram stain reagents alone disrupt cells. Ethanol, an ingredient in crystal violet and the agent of decolorizing, causes lipids and proteins to leach from gram-positive and gram-negative organisms (4). Because cell wall integrity is necessary for accurate Gram staining (4), the combination of the toxic effects of oxygen by-products, along with damage from the fixatives and reagents, may cause the cells of gram-positive anaerobic bacteria to appear gram negative. Under limited conditions, if an anaerobic glove box is not available, disposable anaerobic glove bags would likely provide similar results. If no anaerobic working environment is available, laboratory workers may consider strictly limiting the time between removing the cultures from the incubation environment and Gram staining the organism. While many concepts from general bacteriology apply to anaerobes, not all techniques will readily transfer. One example is Gram staining. Anaerobic bacteria are known to lack enzymatic components necessary to neutralize oxygen free radicals. It is commonly assumed that inconsistent staining patterns in bacteria are due to excessive decolorization in the third step of Gram staining; variation in the amount of decolorization may be related to growth conditions, age of the culture, or other influences. The tendency of these anaerobes to excessively decolorize may result from the fact that anaerobes are removed from the anaerobic incubation atmosphere and are exposed to oxygen during the staining process. Some species of anaerobic bacteria may be particularly sensitive to toxic effects of oxygen, which may disrupt the integrity of the cell wall enough to cause the excessive decolorization of certain grampositive anaerobes. Fixing and Gram staining anaerobic bacteria in the absence of oxygen provide improved staining results and more accurate presumptive identification of obligate anaerobes. Additional studies will be necessary to elucidate the mechanisms underlying our observations. Cirous Samia and Lois Lindberg are graciously acknowledged for freely sharing their time and expertise. REFERENCES 1. Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.) Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 2. Bartholomew, J. W., and H. Finkelstein Relationship of cell wall staining to Gram differentiation. J. Bacteriol. 75: Basu, P. S., and B. B. Biswas Extraction of crystal violet-iodine complex from Gram positive bacteria by different solvents and its implication on Gram differentiation. Histochemie 14: Beveridge, T. J., and J. A. Davies Cellular responses of Bacillis subtilis and Escherichia coli to the Gram stain. J. Bacteriol. 156: Bottone, E. J The Gram stain: the century-old quintessential rapid diagnostic test. Lab. Med. 19: Cato, E. P., W. L. George, and S. M. Finegold Genus Clostridium Prazmowske 1880, 23 AL, p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey s manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore. 7. Cato, E. P., and C. W. Salmon Transfer of Bacteroides clostridiiformis subsp. clostridiiformis (Burri and Ankersmit) Holdeman and Moore and Bacteroides clostridiiformis subsp. girans (Prévot) Holdeman and Moore to the genus Clostridium as Clostridium clostridiiforme (Burri and Ankersmit) comb. nov.: emendation of description and designation of neotype strain. Int. J. Syst. Bacteriol. 26: Citron, D. M Specimen collection and transport, anaerobic culture techniques, and identification of anaerobes. Rev. Infect. Dis. 6(Suppl. 1): S51 S Engelkirk, P. G., J. Duben-Engelkirk, and V. R. Dowell Principles and practice of clinical anaerobic bacteriology. Star Publishing Co., Belmont, Calif. 10. Feleke, G., and S. Forlenza Anaerobic infections: the basics for primary care physicians. Postgrad. Med. 89: Hofstad, T., and P. Aasjord Eubacterium plautii (Séguin 1928) comb. nov. Int. J. Syst. Bacteriol. 32: Kaneuchi, C., K. Watanabe, A. Terada, Y. Benno, and T. Mitsuoka Taxonomic study of Bacteroides clostridiiformis subsp. clostridiiformis (Burri and Ankersmit) Holdeman and Moore and of related organisms: proposal of Clostridium clostridiiformis (Burri and Ankersmit) comb. nov. and Clostridium symbiosum (Stevens) comb. nov. Int. J. Syst. Bacteriol. 26: Magee, C. M., G. Rodeheaver, M. T. Edgerton, and R. F. Edlich A more reliable Gram staining technic for diagnosis of surgical infections. Am. J. Surg. 130:

4 758 NOTES J. CLIN. MICROBIOL. 14. Mangels, J. I., M. E. Cox, and L. H. Lindberg Methanol fixation: an alternative to heat fixation of smears before staining. Diagn. Microbiol. Infect. Dis. 2: Moore, L. V. H., J. L. Johnson, and W. E. C. Moore Genus Peptostreptococcus Kluyver and van Niel 1936, 401 AL, p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey s manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore. 16. Moore, W. E. C., and L. V. H. Moore Genus Eubacterium Prévot 1938, 294 AL, p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey s manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore. 17. Rodina, A. G Methods in aquatic microbiology, University Park Press, Baltimore. 18. Summanen, P., E. J. Baron, D. M. Citron, C. Strong, H. M. Wexler, and S. M. Finegold Wadsworth anaerobic bacteriology manual, 5th ed. Star Publishing Co., Belmont, Calif.

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