Observation Of Fungi

Observation Of Fungi

	Observation of fungi In the diagnosis of fungal infections direct methods are of more value than the indirect methods, for example, the direct observation of causative agent and its tentative identification is more helpful than demonstrating the humoral or cellular responses of the host. One of the simplest methods is to make wet mount preparation of fungi. It is easy to make, but extreme care is necessary for the collection of specimen to avoid contamination. There are all the chances of contaminant overgrowing the fungi pathogen. In addition, pathogenic fungi lose their identity features during the subculturing.
Specimens like skin scrappings, hair and thin nail pairings are observed by wet mount technique as under. Place the material on a slide in a drop of 20% sodium hydroxide solution. Warm to macerate and clear the epithilium. Apply cover slip and warm again to clear the material. Allow the preparation to stand for 15-20 minutes before examination. For more rapid method, solution of potassium hydroxide in di-methyl sulphoxide (S6) is used. The examination of the preparation should be carried out immediately, since after 15-20 minutes the disintegration of cell starts. Another alternative to observe fungi in living state is to culture it on the suitable media and examine the culture under the microscope. The use of broth culture media is not advisable to culture fungi from sample because of the possibility of the contaminant to overgrow any pathogen in the sample.
The inoculation should be made on solid media for such purpose and the specimen should be pressed into the sur-face of the medium. Malt tellurite agar or Saboraud's agar or malt agar containing antibacterial agent is used routinely for the satisfactory results. After the desired growth, fungi can be observed microscopically by wet mount or agar block culture preparation. For rapid and routine examination of almost all types of fungi, wet mount or needle mount of fungi is used. It can be prepared by following the procedure described as under.
Place a drop of 95% ethyl alcohol on a clean microscopic slide and put a small fragment of a culture from the isolated colony of the fungi to be examined. The fragment of fungal material is subsequently teased out in alcohol, using a pair of mounted steel needles, not nichrome inoculating needles, one of which having a small knife-edge in addition to a finely pointed tip. Best teasing should result into flat and even preparation. Very often it is carried out under a dissecting microscope. Let alcohol to evaporate and add a drop of Amman's lactophenol blue staining solution (S7) over the specimen and carefully place a cover slip over the preparation avoiding air bubbles and press gently. Air bubbles if trapped can be expelled out by gentle heating. Remove excess of any staining solution around the edges of cover slip with the help of blotting paper. Let the stain to penetrate, it may take few minutes, but differentiation go on improving upto 24 hours. To make a permanent record, seal the edges of the cover slip with the cellulose liquor like nail varnish. The main disadvantage of lactophenol, however, is that it has refractive index of about 1.45, very close to that of fungal hyphae. Due to this fungal haphae rendered more difficult to be observed and measured with accuracy.
	The addition of stain to lactophenol is hardly useful, since all the dyes used in this mounting medium are having tendency to stain cytoplasm rather than cell wall. Details of other stains which can be used in conjunction with lactophenol are given by Maneval (4). Butler and Mann (5) recommended the use of transparent -self adhesive tape to transfer fungal elements to slide for the examination, either in stained or unstained state, as described above. Bretz and Berry (6) gave more refined method in which the adhesive is dissolved from tape in xylene or other suitable solvent. A drop of this solution is spreaded on a cover glass and the solvent is allowed to evaporate. The treated cover glass is then pressed directly on the fungus growth and gently removed. The preparation is placed over a drop of lactophenol on a slide and examined directly. Shennon (7), recently reported the usefulness of adhesive on tape for the permanent microscopic preparations of fungi.

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