Methods for Confirming the Gram Reaction of Gram-variable Bacillus Species Isolated from Tobacco

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Abstract

Bacillus is a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity of Bacillus isolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide), the LANA (L-alanine-4-nitroanilide), and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA). Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of various Bacillus species isolated from tobacco. B. laevolacticus excepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg).

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  • 1. Gregersen T.: Rapid method for distinction of Gram-negative from Gram-positive bacteria; Eur. J. Appl. Microbiol. Biotechnol. 5 (1978) 123-127.

  • 2. Ryu E.: A simple method of differentiation between Gram-positive and Gram-negative organisms without staining; Kitasako Arch. Exp. Med. 17 (1940) 58-63.

  • 3. Cerny G.: Method for the distinction of Gram-negative from Grampositive bacteria; Eur. J. Appl. Microbiol. 3 (1976) 223-225.

  • 4. Graevenitz A. von and C. Bucher: Accuracy of the KOH and vancomycin tests in determining the Gram reaction of non-enterobacterial rods; J. Clin. Microbiol. 18 (1983) 983-985.

  • 5. Bauer A.W. W.M.M. Kirby J.C. Sherris and M. Turek: Antibiotic susceptibility testing by a standard-ized single disk method; Am. J. Clin. Pathol. 45 (1966) 493-496.

  • 6. Carlone G.M. M.J. Valadez and M.J. Pickett: Methods for distinguishing Gram-positive from Gram-negative bacteria; J. Clin. Microbiol. 16 (1983) 1157-1159.

  • 7. Fluharty D.M. and W.L. Packard: Differentiation of Gram-positive and Gram-negative bacteria without staining; Am. J. Vet. Clin. Path. 1 (1967) 31-35.

  • 8. Halebian S. B. Harris S.M. Finegold and R.D. Wolfe: Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria; J. Clin. Microbiol. 13 (1981) 444-448.

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