1. Title: COCONUT WATER-MUNG BEAN (CWMB): A MEDIUM FOR MICROBIAL GROWTH
a. Total No. of Pages: 201
b. Text No. of Pages: 148
2. Author: TUCAY, CIPRIANO JR., NEAD
3. Type of Document: Thesis
4. Type of Publication: Unpublished
5. Accrediting Institution: University of the Cordilleras
Gov. Pack Road, Baguio City
6. Key Words: Culture medium, bacteria , fungi, Escherichia coli , Staphylococcus aureus, Penicillium spp., Rhizopus stolonifer , isolation, inoculation, incubation, colonial morphology, cellular morphology, coconut water, mung bean extract, Nutrient Agar Medium, Sabouraud Dextrose Agar Medium, streak plate method, spread plate method, agar 7. Abstracts:
Culture medium for the cultivation of microorganisms is an essential tool in Microbiology. Nowadays various types of commercial media are being utilized for this purpose. Bacterial growth requirements are essential for the growth and identification of organisms in the laboratory. Coconut water and mung bean extract are cheap sources and their components are essential for microbial cultivation. The combination of coconut water and mung bean extract can support the growth of bacteria and fungi. 7.2 Summary
Four microorganisms, two of which are bacteria E. coli and S. aureus, and 2 fungi, Penicillium spp., and R. stolonifer were used as representative organisms to determine the efficacy of coconut water-mung bean with commercial agar as solidifying agent as a medium for microbial growth. The medium can be classified as solid, complex, and non-specific. As a complex medium, its chemical components vary greatly.
These organisms were cultivated in five different media preparations with different concentration of coconut water and mung bean in order to determine which among the five treatments will support the best growth of these sample organisms.
The rate of growth was measured by using the streak plate to evaluate or assess the earliest possible growth of the representative bacterial samples as determined by the presence of visible CFU’s within 24 hours incubation at 37 oC. The growth of E. coli and S. aureus was evident after 24 hours incubation period.
The growth of the microorganism in the particular medium is an indicator that will cover and satisfy the basic nutritional requirements of a successful culture medium. The incubation period was extended to 48 hours in order to obtain any change in the culture media. The fungal cultures were evaluated on day 4 and day 6 incubation periods. The rate of growth of Penicillium spp., and R. stolonifer was demonstrated differently among the treatments. 7.3 Findings
The following are the major findings of the study:
a. The rate of growth of E. coli and S. aureus in T4 (25% CW + 75% MB) is comparable to control, T0 (NA) after 24 hours incubation at 370C. b. For E. coli, the biggest colony size, which is 2.1 mm is seen in both T0 (NA) and T2 (100% MB). For S. aureus the biggest colony size which is 2.2 mm is seen in T0 (NA) followed by T2 (100% MB), T3 (50% CW + 50% MB) and T4 (25% CW + 75 % MB) which is 1.8 mm. c. The colonial morphology of E. coli in all treatments is the same, that is, the colonies are circular, convex and smooth. For S. aureus, all the colonies show clumping of cells where isolated cells are circular, pinhead, convex with entire margins in all the treatments. d. The color of E. coli and S. aureus colonies in T0 (NA) and T2 (100% MB) is white, while in T1 (100% CW), T3 (50% CW + 50% MB), T4 (25% CW + 75 % MB) and T5 (75% CW + 25% MB) the colonies are cream in color. e. The colony surface appearance of E. coli is glossy and translucent in all the treatments, while for S. aureus, the colonies are all translucent in all the treatments. f. Change in the agar resulting from bacterial growth as yellowish discoloration is seen in T2 (100% MB) initially for E. coli after 24 hours incubation. This change on the medium became widespread after 48 hours incubation. This change is not seen in S. aureus after 24 hours incubation. It is only evident after 48 hours incubation. There is no pitting of the agar plate on all the agar surfaces in all treatments.
a. Colonial Morphology
The initial growth of Penicillin spp., can be seen clearly in T0 (SDA) and T4 (25% CW + 75% MB).The growing colonies are seen as white and then blue green in color and the surface is velvety to powdery with flat, filamentous or cottony in texture. The plate reverse is seen as pale to yellowish which is characteristic of this fungus. In all the replicates of Penicillium spp., the best growth is seen in T4 (25% CW + 75 % MB) after 4 to 6 days incubation at 300C. There is growth of Penicillium spp., in less than 5 days incubation where the colonies are distinct and differentiated. Consequently, the growth of Penicillium spp., is poor in T1 (100% CW), T2 (100% MB), T3 (50% CW + 50% MB) and T5 (75% CW + 25% MB). The initial growth of R. stolonifer can be seen clearly in T0 (SDA). The colonies are fluffy, cotton-candy like growth surrounded by a white ring which became black due to maturation of the sporangiospores. The growth of R. stolonifer after 4 to 6 days incubation at 300C in all the replicates within the sample group is comparable in treatments T3 (50% CW + 50% MB), T4 (25% CW + 75% MB) and T5 (75% CW + 25% MB). The growth of R. stolonifer is moderate in T1 (100% CW) and T2 (100% MB).
b. Cellular Morphology
i. Hyphal morphology
In all the treatments, Penicillium spp., have septate hyaline hyphae with simple or branched conidiophores, metullae, phialides and conidia. The phialides are seen singly or in groups giving a brush-like appearance which is the chracteristic feature of this fungus. On the other hand in all the treatments, R. stolonifer have rhizoids that are root-like extending near the base.
ii. Spore morphology
The spores of Penicillium spp., show the same morphology in all the treatments. The spores have conidiophores that form a branching structure with each branch terminating in secondary branches from which chains of conidia are borne. In addition, in T4 (25% CW + 75% MB) there is complex method of sporulation in which conidia are borne on phialides produced on secondary branches metullae. The conidia are round, unicellular, and visualized as unbranching chains at the tips of the phialides. In all treatments, R. stolonifer spores are seen produced within the sporangium.
In light of the findings of the study, the following
are the conclusions:
1. The different concentrations of CW and MB used to cultivate E. coli and S. aureus are comparable to NA in terms of colonial morphology, color and surface appearance but not in terms of rate of growth, color size and change in agar media.
2. Different concentrations of CW and MB have different effects on the growth of Penicillium spp., and R. stolonifer in terms of colonial and cellular morphology. 7.5 Recommendations
In relation with the findings and conclusions of this
research, the following are recommended:
1. Formulation of culture media using CW and MB should be polished to make these more viable to support growth of bacteria in the Microbiology laboratory by determining the most specific ratio that will yield best results.
2. Production of culture media using CW and MB should be refined to make these more feasible in growing fungi in the Microbiology laboratory by determining the most specific ratio that will yield the best results.
a. A better method of extraction of mung bean so as not to affect the color and consistency of the medium should be experimented on. b. Further research on the production of powdered form of the materials used in this study for longer shelf life should be conducted. c. These culture media should be tested on some fastidious bacteria and other forms of fungi since coconut water and mung bean extract has the necessary vitamins and amino acids that are specifically needed to support the growth of these organisms.