SLIDE CULTURE IN MYCOLOGY

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SLIDE CULTURE in MYCOLOGY:

SLIDE CULTURE in MYCOLOGY Dr.T.V.Rao MD

Importance of Fungal Infections:

Importance of Fungal Infections Fungi have a great importance in the field of microbiology, for they affect all living beings. There is a myriad of infections caused by these agents, ranging from superficial mycoses to diseases with high morbidity and mortality rates .

Morphology helps in Basic Identification:

Morphology helps in Basic Identification Many pathogenic fungi may be recognized grossly by their appearance in culture. The more definitive method of identification, however, is through microscopic examination of the structures on which classification depends: the spores and the spore bearing apparatus .

Precise methods of Identification of Moulds:

Precise methods of Identification of Moulds Accurate identification of filamentous fungi is based on the microscopic examination of sporulation parts of a colony since each species has a characteristic morphology in the arrangement of its spores and fruiting bodies.

Riddel’s Initiates Slide Culture:

Riddel’s Initiates Slide Culture In order to accurately identify many fungi it is essential to observe the precise arrangement of the conidiophores and the way in which spores are produced (conidial ontogeny). Riddel's simple method of slide culturing (Mycologia 42:265, 1950) permits fungi to be studied virtually in situ with as little disturbance as possible.

Slide 6:

Basic principles of Slide culturing in Mycology

Slide Culture Preparations. :

Slide Culture Preparations . One plate of nutrient agar; potato dextrose is recommended, however, some fastidious fungi may require harsher media to induce sporulation like cornmeal agar or Czapek dox agar.

Slide Culture Preparations.:

Slide Culture Preparations. 1. Using a sterile blade cut out an agar block (7 x 7 mm) small enough to fit under a coverslip. 2. Flip the block up onto the surface of the agar plate. 3. Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus to be grown. 4. Place a flamed coverslip centrally upon the agar block. 5. Incubate the plate at 26C until growth and sporulation have occurred.

Slide Culture Preparations.:

Slide Culture Preparations. 6. Remove the cover slip from the agar block. 7. Apply a drop of 95% alcohol as a wetting agent. 8. Gently lower the coverslip onto a small drop of Lacto phenol cotton blue on a clean glass slide. 9. The slide can be left overnight to dry and later sealed with fingernail polish. 10. When sealing with nail polish use a coat of clear polish followed by one coat of red Coloured polish.

Need for Slide Culture:

Most moulds will yield good results , but a few present extra difficulties. The most problematic are those that tend to disintegrate as soon as they are mounted. Species of Cladosporium , Monilia , and Alternaria have spores connected in very fragile chains that can fall apart at the slightest movement of air. Mounts of these fungi invariably reveal only loose spores and a network of hyphae. To overcome this problem it is useful to set up slide cultures Need for Slide Culture

Slide Cultures done with Simple Techniques:

Slide Cultures done with Simple Techniques Slide cultures are made by setting up a small Petri dish moist chamber containing a V-shaped piece of glass tubing resting on several layers of moistened filter-paper. A sterile block of agar medium about 1 cm square is placed on a flame-sterilized microscope slide and the slide is then set in the moist chamber on the tubing .

Slide culture of fungi :

Slide culture of fungi This procedure, which allows fungal growth to be examined undisturbed. The technique is dependent on the fungus attaching to a glass coverslip.

Cut a square of agar growth medium that is small enough to fit easily on the glass slide. :

Cut a square of agar growth medium that is small enough to fit easily on the glass slide.

Cut a square of agar growth medium that is small enough to fit easily on the glass slide. :

Cut a square of agar growth medium that is small enough to fit easily on the glass slide .

Cutting the Agar:

Cutting the Agar

Carefully transfer this aseptically to the center of the slide. :

Carefully transfer this aseptically to the center of the slide.

Carefully transfer this aseptically to the center of the slide. :

Carefully transfer this aseptically to the center of the slide.

Flame the inoculating wire and allow it to cool before inserting it into the sample. :

Flame the inoculating wire and allow it to cool before inserting it into the sample .

Sampling the fungal culture Only a small amount of material is required. :

Sampling the fungal culture Only a small amount of material is required .

Inoculate one edge of the agar square with the fungus to be examined Inoculating the agar block.:

Inoculate one edge of the agar square with the fungus to be examined Inoculating the agar block.

Agar block with all four sides inoculated:

Agar block with all four sides inoculated

Sterilize the coverslips by immersing them in alcohol. Picking up a cover-slip with forceps:

Sterilize the coverslips by immersing them in alcohol . Picking up a cover-slip with forceps

Using sterile forceps, remove and flame a coverslip to burn off the alcohol and place it on top of the agar block. In slide 4 The coverslip being placed on a slide culture:

Using sterile forceps, remove and flame a coverslip to burn off the alcohol and place it on top of the agar block . In slide 4 The coverslip being placed on a slide culture

Add a little sterile water to the base of the Petri dish to ensure that the agar does not dry out while the culture is incubating:

Add a little sterile water to the base of the Petri dish to ensure that the agar does not dry out while the culture is incubating

The duration and temperature of the incubation depend upon the species of fungus being examined Removing the cover-slip and its growth from a slide culture :

The duration and temperature of the incubation depend upon the species of fungus being examined Removing the cover-slip and its growth from a slide culture

When sufficient growth has occurred.. lose-up of cover-slip on a clean slide, fungal growth uppermost.:

When sufficient growth has occurred.. lose-up of cover-slip on a clean slide, fungal growth uppermost.

Place the coverslip, with the fungal growth uppermost, onto a clean slide. A drop of alcohol being applied to the fungal growth on the cover-slip :

Place the coverslip, with the fungal growth uppermost, onto a clean slide. A drop of alcohol being applied to the fungal growth on the cover-slip

Add a drop of 70% alcohol to the center of the coverslip.. Drop of lacto phenol cotton blue on the coverslip. :

Add a drop of 70% alcohol to the center of the coverslip.. Drop of lacto phenol cotton blue on the coverslip.

Placing a stained slide culture onto a clean slide Turn the coverslip over and place it onto a clean slide. :

Placing a stained slide culture onto a clean slide Turn the coverslip over and place it onto a clean slide .

Morphology of Chlamydospores:

Morphology of Chlamydospores

How the Chlamydospores look:

How the Chlamydospores look

Microscopic appearance of the slide culture at room temperature (Czapek-Dox solution agar) after 2 weeks. Conidia in a chain with transverse and oblique septa were observed. :

Microscopic appearance of the slide culture at room temperature (Czapek-Dox solution agar) after 2 weeks (original magnification × 200). Conidia in a chain with transverse and oblique septa were observed. Microscopic appearance of the slide culture at room temperature (Czapek-Dox solution agar) after 2 weeks. Conidia in a chain with transverse and oblique septa were observed.

Chlamydospores of Candida albicans :

Chlamydospores of Candida albicans

Candida albicans in its Hyphal Form :

Candida albicans in its Hyphal Form

Ready-to-Use, Diagnostic Slide Culture System for Identifying Fungi :

Ready-to-Use, Diagnostic Slide Culture System for Identifying Fungi The MycoVue™ System provides the laboratorian with a standardized, comprehensive method that eliminates time-consuming preparations and technical difficulties encountered with the classical slide culture technique. It simplifies the slide culture method by providing all the necessary components for this procedure in one ready-to-use disposable unit.

MycoVue™ System:

MycoVue™ System The system comes complete with a protective lid and a built-in humidifying chamber. The device is designed to fit easily onto a microscope stage, thereby allowing direct viewing of the developing fungus through the device, thus eliminating the disruption of the fungal colony. If desired, the cover slip can be removed and stained for further evaluation or preservation.

References:

Microbiology Teaching Support Faculty of Biological Support University of Leeds References

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Created by Dr.T.V.Rao MD for ‘e’ learning resources for Medical Microbiologists and Mycologists in the Developing world email doctortvrao@gmail.com