Applications of fluorophore-containing microbial growth media

Ramsey, W. Scott; Nowlan, E D; Simpson, L. B.; Messing, Ralph A.; Takeguchi, Milton M.

doi:10.1128/aem.39.2.372-375.1980

Cited by 7 publications

(5 citation statements)

References 7 publications

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“…A medium containing the fluorogenic compound ANS for simultaneously enumerating gram-negative and gram-positive bacteria and for differentiating between various species was reported by Ramsey et al (185) as well as by Kwee et al (131). The mechanism of staining is thought to be that of adsorption of ANS to bacterial cell wall proteins in growing cultures (167).…”

Section: Protein-specific Dyesmentioning

confidence: 96%

“…(iii) The third group of reactions is the change in intensity of fluorescence as a result of adsorbance of fluorescent dye onto some component of the bacterial cell, e.g., acridine orange (AO) binding to DNA (174) and 8-anilino-1-naphthalene sulfonic acid (ANS) binding to proteins (185). Important extensions of this approach are the use of epifluorescence microscopy (133), direct epifluorescent filter technique (DEFT) (173), and flow cytometry (FCM) (152) for counting and characterization of cell populations.…”

Section: Introductionmentioning

confidence: 99%

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Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Manafi

Kneifel

Bascomb

1991

Microbiological Reviews

173

104

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ular emphasis on methods for specific detection of Escherichia coli. DETECTION OF ENZYMATIC ACTIVITIES General Considerations Three groups of fluorogenic and/or chromogenic reactions have been used. (i) The first is hydrolysis of synthetic substrates by bacterial enzymes, causing considerable increase in the fluorescence and/or absorption of the bacterium-substrate mixture (16). Fluorogenic synthetic enzyme substrates containing coumarin derivatives of 4-methylumbelliferone (4-MU) or 7-amino-4-methylcoumarin (7-AMC) are the most commonly used substrates. This popularity could be ascribed to availability of a wide range of substrates with different metabolic moieties, noncarcinogenicity, ease of visual detection of the 335

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Section: Protein-specific Dyesmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Manafi

Kneifel

Bascomb

1991

Microbiological Reviews

173

104

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“…As shown in this study, Gram differentiation was possible with ANS, but this effect was obtained at the expense of partial growth inhibition of the Gram-positive bacteria. As demonstrated by Ramsey et al (1980) and also by Kwee et al (1988), the optimum concentration of ANS for distinguishing the bacteria is normally rather high ( 2 500 pg/ml). For a solid medium containing this substrate these experiences were consistent with the findings reported in this study.…”

Section: Discussionmentioning

confidence: 82%

“…Compared with chromogenic substrates, it is commonly reported that fluorogenic substrates are of enhanced sensitivity. To exploit this advantage, ANS has been used as an alternative means for differentiation of Gram-negative bacteria (Ramsey et al 1980;Kwee et al 1988), based on higher fluorescence intensity than Gram-positive. According to the basic principle of adsorption of ANS to the bacterial cells during growth phase, this substrate can be used only in a culture medium, and is therefore not suited for application in rapid tests (e.g.…”

Section: Discussionmentioning

confidence: 99%

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

Manafi

Kneifel

1990

Journal of Applied Bacteriology

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Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/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 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, 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.

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Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes

Doolittle

Cooney

Caldwell

1996

Journal of Industrial Microbiology

120

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Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of Escherichia coli and Pseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity. Fluorescent antibodies were used to detect non-labeled progeny phage. Phage T4 infected both surface-attached and surface-associated E. coli while phage E79 adsorbed to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.

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Applications of fluorophore-containing microbial growth media

Abstract: Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light

Cited by 7 publications

References 7 publications

Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Fluorogenic and chromogenic substrates used in bacterial diagnostics.

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

Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes

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