Barry, David

Chan, Cecilia

Williams, Gwilym A.

English

Arrow@TUDublin

Technological University Dublin ARROW@TU Dublin Articles School of Biological Sciences 2007 Nitrocellulose as a General Tool for Fungal Slide Mounts David Barry Technological University Dublin, david.barry@tudublin.ie Cecilia Chan Gwilym A. Williams Technological University Dublin, gwilym.williams@tudublin.ie Follow this and additional works at: https://arrow.tudublin.ie/scschbioart  Part of the Biology Commons Recommended Citation Barry, D., Chan, C. & Williams, G. (2007). Nitrocellulose as a general tool for fungal slide mounts. Journal of Clinical Microbiology, vol. 45, no. 3, pg. 1074-5. doi:10.1128/JCM.01609-06. This Article is brought to you for free and open access by the School of Biological Sciences at ARROW@TU Dublin. It has been accepted for inclusion in Articles by an authorized administrator of ARROW@TU Dublin. For more information, please contact arrow.admin@tudublin.ie, aisling.coyne@tudublin.ie. This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2007, p. 1074–1075 Vol. 45, No. 30095-1137/07/$08.000 doi:10.1128/JCM.01609-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.Nitrocellulose as a General Tool for Fungal Slide MountsThe microscopic examination of fungi is conventionally car-ried out by wet mount staining using lactophenol cotton blue,in combination with different modes of sample preparation:the use of mounting needles, the “adhesive tape lift,” andvarious agar block techniques are well documented in theliterature (2, 3). Such techniques require considerable experi-ence, often incur the disruption of the fragile fungal architec-ture during sampling, and do not allow observation of theearly-stage differentiation of the fungus.Nitrocellulose is a commonly used membrane for the immo-bilization and probing of nucleic acids and proteins. Used inconjunction with a filtration device, such membranes are alsoemployed for the detection of coliforms in water. An addi-tional though underused property of nitrocellulose of specificrelevance to microscopic imaging is that it may be renderedvirtually transparent by exposure to oils.Combining these observations, in this communication wereport a new method for in situ microscopic examination offungal morphology under high-power magnification, usingequipment and materials available in most diagnostic labora-tories. The method exploits the passive immobilization ofspores on a nitrocellulose membrane, which is then placed onan agar surface to facilitate organism growth. After a suitableperiod of time, the membrane is stained, rendered transparentby treatment with microscope immersion oil, and then micro-scopically examined.Aspergillus oryzae (ATTC 12891) was used as a model or-ganism. Standardized conidial suspensions were prepared frommalt agar slant cultures (after incubation for 7 days at 25°C) bythe addition of 5 ml saline (0.85% [wt/vol]) containing Tween80 (0.1% [vol/vol]). The cultures were briefly vortexed andthe conidial suspensions recovered by aspiration with a Pas-teur pipette. Conidium concentration and the absence ofhyphal elements were assessed using a Neubauer chamber,and the conidia were subsequently pelleted by centrifuga-tion at 3,000 rpm for 30 min at 4°C. The pellet was thenresuspended in saline-Tween 80 and glycerol (20% [vol/vol])to yield 1  106 conidia ml1 and stored at 20°C. Allconidium dilutions used in the experiments described wereperformed in sterile phosphate-buffered saline (PBS [pH7.2]; Oxoid Dulbecco A).Nitrocellulose squares (approximately 4 by 4 mm) wereaseptically cut from a proprietary membrane (MilliporeHAWG 047 SO; 0.45 m) by use of a sterile scissors. In thestandardization protocol, each nitrocellulose square wasoverlaid evenly onto the surface of a malt agar plate andthen inoculated either with 5 l of a standardized conidialsuspension (1  106 conidia ml1) or with a PBS control.The plates were incubated at 25°C and test squares removedafter various time intervals for drying, staining, and micro-scopic visualization. In the case of examining conidia from apreexisting fungal colony on an agar plate, the nitrocellulosemembrane was aseptically wetted with PBS-Tween 80 (0.1%[vol/vol]) and lightly touched to the growth with a sterileforceps. The membrane was then placed onto the surface ofa malt agar plate, incubated at 25°C for germination, andthen removed for drying and processing for microscopicexamination.Membranes were dried in sterile petri dishes (3 h, 25°C airincubator) and stained with lactophenol cotton blue (BBL Di-agnostic Systems). The membranes were rinsed in PBS-Tween80, redried at 25°C (3 h), and placed on microscope slides; aminimum volume of immersion oil was added (Olympus AX9602), and images were captured at various magnifications(Canon Powershot S50 camera attached to a Leica DMLS2light microscope).Standardization experiments using a lactophenol cottonblue-stained preparation established the basic imaging pa-rameters; a low level of background interference or artifactdue to the structure of the nitrocellulose was encountered.No conidium germination occurred on the nitrocellulose inthe absence of a nutrient medium, while image quality didnot noticeably vary according to the orientation of the mem-brane on the microscope slide. Using a standardized conid-ium suspension, the kinetics of early fungal developmentcould be measured easily and reproducibly. Following conidiumswelling, germ tube emergence occurred at 9 hours andhyphal branching at 16 h; there was no difference in theperformance of the method when a diluent-moistened ni-trocellulose membrane was used to sample fungal growthfrom an agar plate. The utilization of immersion oil did notadversely affect the imaging of the specimen at low-powermagnification (40, 100, and 400). Conversely, it waspossible to use the oil immersion lens directly on the sample,thereby affording the imaging of cellular details; an exampleshowing septation is provided in Fig. 1.Additional advantages of this system include the ability tomodify the nutrient environment by either simple mem-brane transfer or immersion of the membrane in a liquidmedium (thereby countering possible mass transfer limita-tions). It is well suited to investigating diffusion-mediatedprocesses, such as antimicrobial susceptibility testing, whereearly-stage differentiators (rate of germination and hyphalextension) may be of utility as indicators of interspeciesvariability (1).FIG. 1. Septation in A. oryzae visualized on nitrocellulose. Magni-fication, 1,000. Bar  5 m.1074 on September 11, 2020 by guesthttp://jcm.asm.org/Downloaded from REFERENCES1. Araujo, R., and A. Gonçalves Rodrigues. 2004. Variability of germinativepotential among pathogenic species of Aspergillus. J. Clin. Microbiol. 42:4335–4337.2. Harris, J. L. 2000. Safe, low-distortion tape touch method for fungal slidemounts. J. Clin. Microbiol. 38:4683–4684.3. Rodriguez-Tudela, J. L., and P. Aviles. 1991. Improved adhesive method formicroscopic examination of fungi in culture. J. Clin. Microbiol. 29:2604–2605.David J. BarrySchool of Biological SciencesDublin Institute of TechnologyKevin StreetDublin 8, IrelandCecilia ChanSchool of Electrical Engineering and Control SystemsDublin Institute of TechnologyKevin StreetDublin 8, IrelandGwilym A. Williams*School of Biological SciencesDublin Institute of TechnologyKevin StreetDublin 8, Ireland*Phone: (353) (0)1 402-2889Fax: (353) (0)1 402-4995E-mail: gwilym.williams@dit.ie Published ahead of print on 24 January 2007.VOL. 45, 2007 LETTERS TO THE EDITOR 1075 on September 11, 2020 by guesthttp://jcm.asm.org/Downloaded from 

Nitrocellulose as a General Tool for Fungal Slide Mounts

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Nitrocellulose as a General Tool for Fungal Slide Mounts

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