Ó Springer 2005

World Journal of Microbiology & Biotechnology (2005) 21: 329–336 DOI 10.1007/s11274-004-3907-4

Use of cereals as basal medium for the formulation of alternative culture media for fungi A.O. Adesemoye1,* and C.O. Adedire2 1 Department of Microbiology, Adekunle Ajasin University, P.M.B. 001, Akungba-Akoko, Ondo State, Nigeria 2 Department of Biology, Federal University of Technology, P.M.B. 704, Akure, Ondo State, Nigeria * Author for correspondence: Tel.:+234-803-718-6133, E-mail: [email protected] Received 23 January 2004; accepted 17 September 2004

Keywords: Cereals, culture media, fungi, growth, potato dextrose agar

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliforme had an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.

Introduction Microbiological studies depend on the ability to grow and maintain microorganisms under laboratory conditions by providing suitable culture media that offer favourable environmental conditions (Prescott et al. 2002). The conditions needed for growth include good carbon source, nitrogen source such as protein, availability of enzymes, vitamins, mineral elements such as phosphorus and sulphur, suitable pH, suitable temperature, relative humidity, inorganic salt and water. The knowledge of the conditions is useful in the control of the growth of microbes that cause diseases and food spoilage but also in the effort to encourage the growth of helpful microbes and those to be studied (Tortora et al. 1997; Cappuccino & Sherman 1998). Efforts to provide suitable conditions for microorganisms, started with van Leeuwenhoek in 1675 using a fluid obtained by soaking peppercorn in water. Solid medium was developed thereafter by Koch in 1881 when he reported the use of boiled potatoes, sliced with a flame-sterilized knife in culturing bacteria (Khaled et al. 1996; Olutiola et al. 2000; Prescott et al. 2002). One of the most important agar media in modem days is potato

dextrose agar (PDA), a general purpose agar. Ali et al. (2002) reported that it comprised 200 g diced potato/l, 20 g dextrose (glucose)/l and 15 g agar/1. Glucose is the source of carbon and energy. However, information on recent studies in media formulation using locally available materials are scanty. Such studies will therefore continue for some time to come, especially in developing countries such as Nigeria, where research in microbiology is hindered by the high cost and scarcity of culture media (Poopathi et al. 2002). This study therefore investigates the possibility of using locally available cereals as substitutes for potato in PDA. Characteristics of cereals such as suitability for the growth of fungi, richness in protein, resistance to drought etc informed the choice in this study (Blum & Sullivan 1974; Brimble et al. 1982; Roni et al. 1996).

Materials and methods Proximate analysis of cereal samples The three cereal samples, namely maize (corn), millet and sorghum used were purchased from Erekesan

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Market in Akure, Ondo State, Nigeria. Whole grains were pulverised into fine powder in a large and dry mortar. Proximate analysis of each of the cereal samples were carried out according to AOAC (1980) for ash, crude fibre, moisture, fats and protein. Carbohydrate content was calculated by subtraction method. Media formulation Three different media were formulated, namely, cornmeal dextrose agar (CDA), millet-meal dextrose agar (MDA) and sorghum-meal dextrose agar (SDA). The method first recommended by Martins et al. (1955) but modified by Olutiola et al. (2000) to formulate corn– meal dextrose agar was used for all the three samples. Approximately 6 g of the pulverized yellow maize was measured into a flat bottom flask, 160 ml of clean water was added and heated in a water bath for 1 h. It was filtered through a muslin cloth. Then 6 g of dextrose and 4 g of agar–agar were added to the filtrate. The volume of the mixture was made to 200 ml with distilled water and heated on a hot plate with steady stirring until the solution boiled. The stirring was meant to achieve homogeneity. The resultant suspension was sterilized in the autoclave for about 15 min at 121 °C. The content of the flask was thereafter poured aseptically into eight pre-sterilized Petri dishes. The media were allowed to cool and solidify before inoculation. The procedure was repeated in formulating millet-meal dextrose agar and sorghummeal dextrose agar by substituting corn with millet and sorghum, respectively.

Test organisms The test organisms were chosen to cover fungi of agricultural and medical importance. Except where otherwise stated, eight fungal genera used were isolated from the following sources using malt extract agar. Aspergillus niger (onion), Fusarium moniliforme (carrot), Penicillium sp. (orange), Cercospora sp. (yam), Curvularia palescens (maize), Botryodiplopodia sp. (cassava), Rhizopus sp. (moist bread) and Rhodotorula rubra (isolated from man but the isolate was obtained from the laboratory of the State Specialist Hospital, Akure, Ondo State). Inocula were drawn from the pure cultures of the isolates just before 96th h of growth and aseptically inoculated onto all the formulated media and the control (PDA). The inocula were taken at that time to ensure that growth was still high at logarithmic phase when cells would have uniform physiological characteristics (Salmon et al. 1989; Nwachukwu & Akpata 2003). Inoculation of media

In preparing the control medium, 6.8 g of potato dextrose agar was mixed with 200 ml of water, heated and stirred until boiling before autoclaving. The physico-chemical properties of the PDA was compared with those of the formulated media and presented in Table 1. The pH test readings showed that all the formulated media were more alkaline than PDA and all contained particles. Corn-meal dextrose agar contained the least particles because it was easier to grind more finely due to its low fibre content relative to the two other cereals.

The suitability of the formulated media was estimated by culturing the isolated fungal species on them through the following procedure. The working table was maintained under aseptic condition by disinfecting with 70% alcohol (i.e 7:3 by vol. of alcohol/vol. of water). Cork borer (No. 1) and platinum wire inoculating loop were also dipped into the alcohol and flamed. The cork borer, with size approximately 0.37 cm in diameter was used to bore holes on the pure cultures of organisms. It was resterilized again before being used to bore holes in successive pure cultures. The mycelia agar plugs were then removed with the sterilized inoculating loop and transferred top down onto the centre of the formulated media. Each of the eight organisms from the pure cultures was inoculated on the plates of formulated CDA, MDA, SDA and PDA in like manner. The plates were then incubated at 31 °C inside the incubator for 96 h. The growth of the organisms were evaluated by Colony Size Method (Fawole & Oso 1995). The radius of growth was measured at 24 hourly intervals using a Venier caliper. The whole inoculation process was repeated thrice for each sample with each of the test organisms. The mean value of readings and the standard deviation were then estimated.

Table 1. Comparison of the physico-chemical properties of the formulated media and PDA

Results

Characteristics of PDA compared with formulated media

Medium

pH Value

Colour

Particles

Clarity

PDA CDA MDA SDA

5.90 7.20 7.30 7.35

Yellow Light yellow Cream Pale yellow

) + ++ ++

CC CC C C

Key: – )absent; + – present; ++ – more present; C – turbid; CC – clear; PDA – potato dextrose agar; CDA – corn dextrose agar; MDA – millet dextrose agar; SDA – sorghum dextrose agar.

The physico-chemical characteristics of the formulated media and potato dextrose agar (PDA) showed that sorghum dextrose agar (SDA) had the highest pH value (7.35) while PDA had the lowest pH value (5.90). Particles were most in SDA followed by millet dextrose agar (MDA) and least in corn dextrose agar (CDA) but absent in PDA. The media showed the following properties in terms of colour and clarity: PDA (yellow

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Alternative culture media for fungi and clear), CDA (light yellow and clear), MDA (cream turbid) and SDA (pale yellow and turbid).

(2.00 ± 0.45) (3.60 ± 0.45)

Proximate analysis of corn, millet and sorghum used in the formulation

Growth of the selected fungi on the formulated media and the control

The results of the proximate analyses of the cereals are presented in Figure 1. Corn showed the highest value in moisture content (10.36 ± 0.29) but least in ash content (1.36 ± 0.00); millet was highest in carbohydrate (76.85 ± 0.70), fat (4.50 ± 0.05) and ash contents (2.00 ± 0.45) but least in moisture content (4.83 ± 0.00) and sorghum showed the highest value for protein (13.61 ± 0.50) and fibre content

Figures 2–9 are graphical representations of the data obtained from culturing eight test fungi on the three formulated alternative culture media and PDA (radius of growth taken every 24 through 96 h). The figures are in the following order: Figure 2 (Aspergillus niger), Figure 3 (F. moniliforme) Figure 4 (Penicillium sp.), Figure 5 (Cercospora sp), Figure 6 (C. palescens), Figure 7 (Botryodiplopodia sp.), Figure 8 (Rhizopus sp.)

Figure 1. Proximate analysis of corn, millet and sorghum used in media formulation.

Figure 2. The growth of A. niger on the formulated media and PDA through 96 h.

but

least

values

in

fat

content

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Figure 3. The growth of F. moniliforme on the formulated media and PDA through 96 h.

Figure 4. The growth of Penicillum sp. on the formulated media and PDA through 96 h.

and Figure 9 (R. rubra). The growth of the test organisms on the media are compared below. Comparison of growth on corn-meal dextrose agar with potato dextrose agar Observation at the end of the first 24 h shows that all the organisms have adjusted to the culture environment and have started growing on all the formulated media and PDA. The only exception was R. rubra, which showed growth within 24 h only on CDA (Figure 9). This

period could be said to be within the lag phase of R. rubra on all the other media except for CDA where it has passed that stage. All the other organisms showed comparable growth on PDA and CDA. There was a higher rate of growth for Cercospora sp. on PDA (45.0 ± 0.29) by the 72nd h while on CDA, it was 34.0 ± 1.16 mm (Figure 5). A. niger, though grew faster on PDA than CDA within the first 24 h (9.0 ± 0.58 and 5.0 ± 0.0 respectively), the rate of growth within 48th and 72nd h was higher on CDA (Figure 2).

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Figure 5. The growth of Cercospora sp. on the formulated media and PDA through 96 h.

Figure 6. The growth of C. Palescens sp. on the formulated media and PDA through 96 h.

Comparison of growth on millet-meal dextrose agar with PDA F. moniliforme showed high rate of growth within the first 24 h on PDA relative to MDA (Figure 3). However, from the 48th h to the old culture at 96 h, the growth of the organism was better on MDA than for PDA (20.0 ± 1.16 and 12.0 ± 0 respectively). The growth rate of Curvularia sp. was higher on the MDA relative to that on PDA throughout the growth phases (Figure 6). Observations on Penicillium sp. was not significantly different on MDA and PDA (Figure 4), at

one time it was slightly higher on MDA (24 h), at another time it was slightly higher for PDA (48 h).

Comparison of growth on sorghum-meal dextrose agar with PDA Botryodiplopodia sp. grew faster on SDA between 24 and 48 h than on PDA (Figure 7), in like manner, F. moniliforme grew better up to the 96th h on SDA (21.0 ± 0.58) relative to (12.0 ± 0) on PDA (Figure 3). However, the growth of A. niger, Penicillum sp.,

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Figure 7. The growth of Botryodiplopodia sp. on the formulated media and PDA through 96 h.

Figure 8. The growth of Rhizopus sp. on the formulated media and PDA through 96 h.

C. palescens, and R. rubra were better on PDA than on SDA at the end of 96 h.

Performance comparison of each test organism on the four media The growth of the eight test organisms on all the formulated media and PDA were presented in Figure 2–9 to enhance easy comparison. A. niger for instance grew averagely well on all the media and when a graph of growth radius was plotted against incubation time, the graph reflected features similar to the logarithmic phase

(Nwachukwu & Akpata 2003) except for PDA. The growth between 48 and 72 h was insignificant on the plate but the reason for that could not be deduced. The result of the first set of experiments for PDA arouse curiosity but after the other two replicates, the suspicion of error was proved wrong as the standard deviation of (± 1.16) shows. Other organisms performed well on all the media with most showing a minimum average growth of 40 mm radius in 72 h and 45 mm radius in 96 h. F. moniliforme showed its characteristic attractive red colour on the agar but only showed a maximum growth of 21.0 ± 0.58 in 96 h recorded on SDA. R. rubra responded least on all the media among all the organism.

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Figure 9. The growth of R. rubra on the formulated media and PDA through 96 h.

Discussion Development of media using locally available materials has become imperative (Poopathi et al. 2002), since conventional media are either in short supply or very expensive in developing countries, particularly Nigeria. The high price can be traced to the poor economy, weak purchasing power of the local currency, hence forcing microbiologists to abandon much ongoing research. It was not surprising that the test microorganisms in this study performed well on the formulated media considering that cereals easily support the growth of a wide variety of flora without any additive. For instance maize naturally carries up to 43 pathogens and rice 50, the dominant organism being fungi (Kommedhl & Lang 1971; Brimble et al. 1982; Campbell 1985; Larran et al. 2002). Fungi comprise almost half of the known 157,000 species of microorganisms and show substantial variation in nutritional and biochemical attributes (Hawksworth 1994; DalBello et al. 2002). Avoidance of errors in inoculation or enumeration (Adams & Moss 1999; Boulter et al. 2002) and the impact of hydrogen ion concentration (Fawole & Oso 1995; Alcano 1997) are very important to the success of this study. There is need to emphasize that the growth of Rhizopus sp. and Rhodotorula rubra in this study may not be good bases for estimating the suitability of the formulated media because they were at the extremes relative to other test organisms. Owing to the easy growth of Rhizopus sp. on almost every medium, some microbiologists have suggested that it is not a very good test organism. Species in the genus Rhodotorula are commensals in their natural environment and in humans (Po-Ren et al. 2003), this could have accounted for the poor growth on the media. Corn-meal dextrose agar was observed to be the best among all the formulated media.

On the average it supported the growth of all the test organisms better than the other formulated media. Corn-meal dextrose agar is viewed with high prospects, more so that corn (maize) is readily available across the globe, presenting it as a base for teaching and further research with the possibility of large scale production at economic benefits. Acknowledgements The authors are grateful to Dr E.O. Igbosuah, Department of Botany & Microbiology, University of Lagos, Nigeria, for her useful criticisms of the revised manuscript and Prof. Femi Mimiko, Adekunle Ajasin University, Akungba-Akoko, Nigeria for his assistance towards the initial manuscript.

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