Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria

M anafi , M. & K neifel , W. 1990. Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. Journal of Applied Bacteriology 69 , 822–827.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy-β-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido-4-methylcoumarin (AAMC); 8-anilino-l-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 μg/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 μg/ml, and led to inhibition of all bacteria at 200 μ/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocoiitica, Bacillus cereus , and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.