A Method for Isolation of Protoplasts from Dermatophytes

Srikantha, Thyagarajan; Rao, G. Ramananda

doi:10.1099/00221287-130-6-1503

Cited by 4 publications

(1 citation statement)

References 5 publications

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“…In this treatment, cells of the IOC 3790 strain were more resistant to the enzymes than cells of the 3698R strain. Strikantha & Rao [ 17] reported that isolates belonging to the same species differed mar~edly in susceptibility to enzymatic treatment with novozym. Localized wall digestion was observed in strain IOC 3790, suggesting greater susceptibility of certain sites of the cell wall of the yeast-like phase of P. brasiliensis.…”

Section: Discussionmentioning

confidence: 99%

Paracoccidioides brasiliensis protoplast production by enzymatic treatment

Mn²,

Silva³

et al. 1994

Mycoses

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Summary The action of the enzymes novozym 234, chitinase and zymolyase 20T on the yeast‐like cells of Paracoccidioides brasiliensis was studied in an attempt to obtain protoplast release. Three enzyme systems were used: the first consisted of novozym 234 and chitinase plus 0.2 m phosphate buffer, 0.9 m sorbitol and 0.5 m sodium thioglycolate; the second consisted of novozym 234, chitinase, zymolyase 20T, buffer and osmotic stabilizer, with no sodium thioglycolate; the third consisted of the same enzymes as used in the second system but at twice the concentration, plus buffer and osmotic stabilizer. Protoplasts were only released from 72‐hold cells cultured on solid peptone‐yeast extract‐glucose medium (PYG) treated with the third enzyme system. Sodium thioglycolate used as pretreatment favoured protoplast release but had no such action when added to the enzyme solution, possibly by altering the activity of the enzymes, novozym 234 in particular. The osmotic stabilizer used, 0.9 m sorbitol, was probably one of the factors, in addition to the enzymes, responsible for the cytoplasmic changes observed by transmission electron microscopy in yeast phase cells and in their protoplasts.

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Section: Discussionmentioning

confidence: 99%

Paracoccidioides brasiliensis protoplast production by enzymatic treatment

Mn²,

Silva³

et al. 1994

Mycoses

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Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum

et al. 1989

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Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2-4 days old mycelium with Novozyme 234 using CaCl2 (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl2 were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by 3H-lysine uptake, matched the viability profile determined by fluorescence microscopy.

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