Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria

Ninety bacteria isolated from raw composting materials were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. The bacteria producing the highest cellulolytic activity levels were identified by 16S rRNA sequencing as Bacillus licheniformis strain 1, Bacillus subtilis subsp. subtilis strain B7B, Bacillus subtilis subsp. spizizenii strain 6, and Bacillus amyloliquefaciens strain B31C. Cellulase activity production by the most productive strain B. amyloliquefaciens B31C was optimized in liquid culture varying the carbon source. Comparison of growth curves of B. amyloliquefaciens B31C at temperatures from 28 to 47 °C indicated its thermotolerant nature. Moreover, analysis of time courses of cellulase activity production in this thermal range showed that increase of temperature from 28 to 37 °C causes an increase of cellulase activity levels. Investigating the enzymes responsible for cellulase activity produced by B. amyloliquefaciens B31C by proteomic analyses, an endoglucanase was identified. It was shown that the purified enzyme catalyzes carboxymethylcellulose's hydrolysis following Michaelis-Menten kinetics with a K(M) of 9.95 mg ml(-1) and a v(max) of 284 μM min(-1) . It shows a retention of 90% of its activity for at least 144 h of incubation at 40 °C and exhibits a range of optimum temperatures from 50 to 70 °C.

show abstract

“…Gram-positive and gram-negative microorganisms were distinguished by means of KOH test as described by Halebian et al (1981).…”

Section: Phenotypic Characterization Of Microbial Isolatesmentioning

confidence: 99%

Industrial waste based compost as a source of novel cellulolytic strains and enzymes

Amore

Pepe

Ventorino

et al. 2012

FEMS Microbiol Lett

View full text Add to dashboard Cite

show abstract

“…Each isolate was subjected to a catalase test (Winn et al 2006). The KOH test to determine Gram positives and -negatives was performed as previously described (Buck 1982;Halebian et al 1981). Endospore staining was performed following the description of Sumbali and Mehrotra (2009).…”

Section: Milk Samplingmentioning

confidence: 99%

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

Mugadza

Buys

2017

Int J of Dairy Tech

View full text Add to dashboard Cite

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log10 cfu/mL and 4 log10 cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.

show abstract

“…The KOH test was carried out according to method of Halebian et al (1981). Gram-negative bacteria were identified, based on increased viscosity of the mixture of bacterial colonies with 3% KOH.…”

Section: K O H Test a N D Gram S T A I Nmentioning

confidence: 99%

“…As an alternative and simplified method for distinguishing Gram-positive from Gramnegative bacteria, the so-called 'potassium hydroxide test' was used (Gregersen 1978;Halebian et al 1981;Bougault & Lamothe 1988). This test is based on the cell-wall disruption of Gram-negative bacteria when exposed to dilute alkaline solutions.…”

mentioning

confidence: 99%

“…This test is based on the cell-wall disruption of Gram-negative bacteria when exposed to dilute alkaline solutions. In this case, the suspension of these bacterial cells in 3% KOH normally becomes viscous, due to the release of relatively unfragmented threads of DNA (Halebian et al, 1981). However, Blachman et al (1980) and Bourgault & Lamothe (1988) demonstrated that the KOH test does not correlate precisely with the Gram stain reaction.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Manafi

Kneifel

1990

Journal of Applied Bacteriology

View full text Add to dashboard Cite

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.

show abstract

Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria

Cited by 181 publications

References 6 publications

Industrial waste based compost as a source of novel cellulolytic strains and enzymes

Industrial waste based compost as a source of novel cellulolytic strains and enzymes

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

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

Contact Info

Product

Resources

About