Isolation and Culture of Slime Molds

Zoospores are motile, asexual spores having one or more flagella. They may be haploid or diploid and are formed inside a sporangium, discharged from the sporangium to become a free swimming stage, and after the motile stage , the zoospore encysts in a suitable substrate or host. ( Lange & Olson, 1983). Baiting technique is a method that allows further growth of fungal mycelia on prepared baits. Major groups of fungi isolated using baiting techniques are those from Kingdom Chromista, particularly in the Class Oomycetes. The Oomycetes or “egg fungi”, also called water molds were previously grouped with fungi based on their filamentous growth and the presence of coenocytic hyphae, one of the morphological traits and characteristic of fungi. However, their life cycle, unlike that of many fungi, includes a diploid, asexually or sexually reproducing phase. (Madigan, et al., 2009). Oomycetes differ from fungi in other fundamental ways as well. The cell walls of oomycetes are typically made of cellulose, not chitin, as for fungi, and they have flagellated cells, which are lacking in all but a few fungi. (Madigan, et al, 2009) Zoospores are produced by almost all Oomycetes.

They are biflagellate bodies, having one whiplash flagellum directed backward and another tinsel type flagellum directed forward. The flagella are attached anteriorly or laterally. (Sharma, 2011). Interestingly, zoosporic fungi belonging to Phylum Chytridiomycota of Kingdom Eumycota are also commonly isolated using the baiting technique. Chytridiomycetes, or chytrids, are the earliest diverging lineage of fungi with about 100 genera consisiting of approximately 1000 species. Their name refers to the structure of fruiting body (“little pot”) which contains zoospores. Cell wall contains chitin like other true fungi. However they differ from other fungi, in producing motile, flagellated spores called zoospores, a possible remnant of their adaptation to aquatic environment, mostly freshwater and moist soils, where they are commonly found. (Madigan, et al., 2009).

According to JW Deacon & G Saxena (1997), Chytridiomycetes are very common as saprotrophs, facultative parasites and obligate parasites in moist soil and freshwater habitats. They depend on their zoospores for dispersal and site-selection. In fact, this is how the chytridiomycota can be detected – by placing baits such as plant seedlings, pollen grains, insect exoskeletons etc. in water or wet soil, because the zoospores encyst on these materials and then give rise to microscopic colonies. This study was designed with two to three objectives. The first was to assess the possible sources of fungi growing on the prepared baits, second is to discuss how these zoosporic fungi reach their substrates and thirdly, to characterize the fungal isolates based on their baits. The ultimate goal of this study is to isolate and culture zoosproic fungi from different baits like dead insects, dungs, and pollen grains. Morphological and reproductive characterization will also be conducted on the isolated fungi.

Methodology

A. Isolation and culture of zoosporic fungi from pollen and snake skin using baiting technique

Baits such as pollen grains (Pinus or any other flower-bearing plants) and snake or lizard skin (2 pieces, ½ inches squares) were obtained and brought to the laboratory. Pond waters with accompanying soil or plant debris were collected and placed inside a disposable petri plate for each baits. Baits (one bait for one plate) were placed on the pond water-filled disposable petri dishes. The cultures were incubated at room temperature under cool conditions. All of the baits were examined under the microscope after 2 days and at daily intervals thereafter. Pollen grains were examined 12 hours after culture and thereafter. To observe the cultures, the group obtained a small portion of their baits, placed them each on a glass slide and was directly observed under the microscope. The hyphae, reproductive structures and zoospores were to observe. Observations were recorded by taking pictures seen under the microscope.

B. Isolation of Zoosporic fungi from dead insects and animal dung using Baiting technique

Dead insects (2-3 pieces of cockroaches) and dried shrimp exoskeleton (2-3 decalcified bits, ½ inch square) were obtained and brought inside the laboratory. Fresh soil samples were collected and placed inside a disposable petri plates. Baits (one bait for one plate) were placed on the soil-filled disposable petri dishes. The cultures were incubated at room temperature under cool conditions for 24 hours. After 24 hours, the cultures were observed and the soil was moistened using a wash bottle. Moist environment will help for the growth of the fungi. All of the baits were examined under the microscope after 2 days and at daily intervals thereafter. To observe the cultures, the group obtained a small portion of their baits, placed them each on a glass slide and was directly observed under the microscope. The hyphae, reproductive structures and zoospores were to observe. Observations were recorded by taking pictures seen under the microscope.

C. Preparation of RDA and PDA for pure isolates of the cultures

Correct amount of potato dextrose agar was measured using the triple beam balance. The media was autoclaved before pouring into glass petri plates. The number of PDA plates that were prepared was according to the number of fungal zoospores observed on the cultures of shrimp exoskeleton and cockroach. The said zoospores were inoculated aseptically on the prepapred PDA plates. RDA media was also prepared by boiling one rabbit dung per 100 ml of distilled water. Two rabbit dungs were boiled in a beaker containing 200 ml distilled water for ten minutes and filtered using a filter paper. Correct amount of Bacto agar was measured, with the given 15g/L. This was added to the filtered dung water to serve as solidifying agent of the media. The prepared media was subjected to autoclave before pouring on plates. The spores from the rabbit dung moist culture were inoculated on the RDA plates. All of these will be stored for 1-2 days under cool conditions. The growing pure cultures were observed macroscopically and under the compound or dissecting microscope.

Results

As the result of the experiment described above, the cultures were successfully incubated at room temperature and were observed under compound or dissecting microscope. Isolates of the chitinolytic fungi (cultures of shrimp exoskeleton and cockroach) and coprophilous fungi were produced using Potato Dextrose Agar and Rabbit Dung Agar, respectively. The cultures were inoculated two to three times in PDA plates until purified, and then inoculated on test tubes with the same media. However, the results observed under the microscope shows only the resting spores, hyphae, vegetative hyphae and zoospores. The pictures taken under HPO below shows the pure isolates of the cultures.

Fig. 1 Snakeskin culture under HPO Fig. 2 Pollen grain culture under HPO

Fig. 3 Cockroach culture: Pure isolate inside a test tube and the culture under HPO

Fig. 4 Shrimp Exoskeleton culture under HPO

Fig. 5 Rabbit Dung culture under HPO

Discussion

Baiting technique was used to find chytrids on soil or water samples. Chytrids are zoosporic fungi classified in the class Chytridiomycetes. Chytridiomycetes are true fungi that require water to disperse throughout their environment. Chytrid zoospores are readily dispersed in the presence of free water but the need for water does not restrict them solely to aquatic habitats. Besides being commonly found in lakes, streams, ponds, roadside ditches and coastal marine environments, chytrids also are present in soil. [1] Accompanying organic material such as soil can also be used to moisten the set-up to be used as a source of fungal growth.

The type of zoosporic fungi that will grow on the bait will depend on the substrate used and what kind of chytrid you are seeking (keratinolytic fungi, chitinolytic fungi, cellulitic fungi). As members of terrestrial and aquatic microbial communities, chytrids play an important ecological role in seemingly ubiquitous biodegraders of recalcitrant materials like keratin, chitin, and cellulose. [1] Hence, they play a role in nutrient cycling. Chytrids live saprobically or as parasites in, or on, a number of different organisms and substrates such as in our case, pollen grains, amphibian skin and exoskeleton of shrimp and cockroaches.

The fungal isolates from the pollen grain are saprotrophic fungi that should degrade polymers. The fungal isolates from the snakeskin are keratinolytic fungi that should degrade keratin. However, observations of cultures shown under the microscope were not successful on growing fungi, may be because of the following reasons: 1) too much water flooded the whole snakeskin and pollen grain set-up; 2) the temperature is not ideal for the zoosporic fungi to grow. However, the fungal isolates from shrimp exoskeleton, cockroaches and rabbit dung were successful. Fungal isolates from dungs are coprophilous fungi that degrade the waste and other nutrients in the animal dung. On the other hand, fungal isolates from exoskeleton are chitinolytic fungi. Coprophilous and chitinolytic fungi were successfully isolated. Their cottony growth were observed on the said substrates.

Acknowledgements
The authors are grateful to the staffs of Botanical Garden and Microbiology laboratory of the University of Santo Tomas for help in obtaining the materials. The authors would also like to thank their professors, Sir Thomas Edison dela Cruz and Sir Mike Valdez, for their unending guidance and patience.

References

[1] Longcore, J.E. Morphology and Zoospore Ultrastructure of Entophlyctis luteolus sp. nov. (Chytridiales): Implications for chytrid taxonomy. Mycologia. 87: 25-33. 1995.

JW Deacon & G Saxena (1997).Orientated zoospore attachment and cyst germination in Catenaria anguillulae, a facultative parasite of nematodes. Mycological Research 101, 513-522.

Madigan, M., Martinko, J., Dunlap, P. & Clark, D. (2009). Brock Biology of Microorganisms.Twelfth Ed. Jurong,Singapore: Pearson Education South Asia Pte Ltd. 531-539. Sharma, OP (2011). Fungi and Allied Microbes. West Patel Nagar ( New Delhi): Tata Mcgraw Hill Education Private Limited.

Isolation and Culture of Plasmodial Slime Molds from Barks of Acasia tree using Moist Chamber Method

Rillera DP , Talibsao K , Talucod AC and Tan P
Department of Biological Sciences, College of Science, University of Santo Tomas, España Street, Manila 108