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

Manafi, M.; Kneifel, Wolfgang

doi:10.1111/j.1365-2672.1990.tb01579.x

Cited by 41 publications

(20 citation statements)

References 9 publications

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“…Gram staining was performed according to Hucker & Conn (1923) and was confirmed by the L-alanine aminopeptidase assay (Manafi & Kneifel, 1990;Hernandez Molina et al, 1991). Capsule staining was performed following the methods of Cowan & Steel (1993).…”

Section: Methodsmentioning

confidence: 99%

Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov.

Bozal

Montes

Tudela

et al. 2003

International Journal of Systematic and Evolutionary Microbiology

132

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Section: Methodsmentioning

confidence: 99%

Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov.

Bozal

Montes

Tudela

et al. 2003

International Journal of Systematic and Evolutionary Microbiology

132

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“…Oxidase and catalase production and sensitivity to the vibriostatic agent O/129 (Oxoid) were performed as previously described (Gerhardt et al 1994). Gram-stain reactions were tested by using the method described by Manafi & Kneifel (1990). Colony pigmentation was determined on Marine 2216 and thiosulfate citrate bile salts sucrose (TCBS) agars (Difco).…”

Section: Conical Tube Assaymentioning

confidence: 99%

Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae

Estes¹,

Friedman²,

Elston³

et al. 2004

Dis. Aquat. Org.

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Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD 50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD 50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 × 10 4 colony forming units (CFU) ml -1 after 24 h and between 3.2 × 10 2 and 1.9 × 10 3 CFU ml -1 after 48 h.KEY WORDS: Pacific oyster larvae · Vibrio · Bacteria · Pathogenicity testing · Shellfish hatchery Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [223][224][225][226][227][228][229][230] 2004 genicity of bacteria isolated from bivalve hatcheries during routine monitoring and disease outbreaks. MATERIALS AND METHODSIsolates and reference strains. Bacterial isolates collected from Pacific oyster Crassostrea gigas larvae (n = 16) and juveniles (n = 12), European flat oyster Ostrea edulis juveniles (n = 1), and hatchery environments (n = 4) from the Pacific Northwest of the United States (Table 1) were stored at -80°C in Marine 2216 broth (Difco Laboratories) with 10% (v/v) glycerol. Subsequently, they were shipped on dry ice from AquaTechnics (Sequim) to the University of Washington (Seattle, Washington). The following control strains were included in the pathogenicity assays: RE 14 (nonpathogenic or negative control), RE 82 (pathogenic or positive control), and previously described larval bivalve pathogens Vibrio alginolyticus American Type Culture Collection (ATCC) 19108 and V. tubiashii ATCC 19106 (Tubiash et al. 1965, 1970). All cultures were maintained at -80°C for long-term storage.Isolates were grown for 24 h on Marine 2216 agar and incubated at 20 and 26°C in the first and second set of experiments, respectively. Experiments were conducted using sand-filtered Puget Sound seawater collected at the Seattle Aquarium (Seattle, Washington). Suspensions of the bacterial strains in filter-sterilized (0.22 µm) seawater were adjusted to an optical density (600 nm) of 0.2 to attain a uniform concentration of bacteria for the experiments. Bacterial density was confirmed by standard plate-count assays using Marine 2216 agar.Pathogenicity testing. Experi...

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“…Gram staining was performed according to Hucker & Conn (1923). Two alternative methods, the KOH test and the L-alanine aminopeptidase assay (Manafi & Kneifel, 1990), were also used. Oxidase, catalase and urease activities, methyl red reaction, VogesProskauer, nitrate reduction, indole production, citrate utilization, and hydrolyses of casein, lecithin, gelatin, DNA, tyrosine, starch and Tween 80 were determined following Cowan & Steel (1993).…”

Section: Strain 20cmmentioning

confidence: 99%

Paenibacillus antarcticus sp. nov., a novel psychrotolerant organism from the Antarctic environment

Montes

Mercadé

Bozal

et al. 2004

International Journal of Systematic and Evolutionary Microbiology

105

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

Cited by 41 publications

References 9 publications

Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov.

Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov.

Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae

Paenibacillus antarcticus sp. nov., a novel psychrotolerant organism from the Antarctic environment

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