Development of a chitin assay for the quantification of fungus.
Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.
Department of Ophthalmology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2540, USA.
C A
CA
Lamps
T N
TN
Oeltmann
M J
MJ
Collins
R D
RD
Robinson
R A
RA
Logan
W S
WS
Head
D M
DM
O'Day
D002176:Candida albicans / Q000737:chemistry* / Q000254:growth & development / Q000302:isolation & purification; D002177:Candidiasis / Q000175:diagnosis / Q000382:microbiology; D002686:Chitin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002854:Chromatography, Paper; D015169:Colony Count, Microbial; D003320:Corneal Ulcer / Q000175:diagnosis / Q000382:microbiology; D003641:Deamination; D004926:Escherichia coli / Q000737:chemistry; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology; D006801:Humans; D006868:Hydrolysis; D008353:Mannitol / Q000031:analogs & derivatives / Q000032:analysis / Q000302:isolation & purification; D008828:Microbiological Techniques; D010084:Oxidation-Reduction; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity; D013210:Staphylococcus / Q000737:chemistry
D002686:Chitin; D008353:Mannitol; C031765:2,5-anhydromannitol