medium PDA

8/10/2019 medium PDA

http://slidepdf.com/reader/full/medium-pda 1/6



4528 Afr. J. Microbiol. Res.

materials to formulate media for the cultivation of micro-organisms particularly fungi, which can break down thestarch to soluble sugars which can serve as source ofcarbon and energy.

Potato tubers are often used to prepare the mediumpotato dextrose agar for culturing a wide range of fungi.

In tropical countries other starch containing tubers, sweetpotato (Ipomoea batatas), cocoyam ( Xanthosomassagittifolium) and white yam (Dioscorea rotundata), canpossibly replace the potato in a general purpose culturemedium. The need to develop alternative media tovarious culture media has become imperative as theconventional media used are either not readily availableor relatively expensive in most developing countries likeNigeria.

The experiment here reported is aimed at exploiting thesuitability of locally available sweet potato varieties,cocoyam varieties and yam tubers as substitutes forpotato in PDA.

MATERIALS AND METHODS

Sample collection

Starch containing tubers, sweet potato varieties (white andreddish), cocoyam varieties (edible and non-edible) Irish potato andyam were purchased from Ogige main market in Nsukka, Enugustate, Nigeria.

Test organisms

The test organisms used were Aspergillus niger and Aspergilluscarbonarius. Stock cultures were obtained from the department of

Microbiology University of Nigeria. The organisms were maintainedon potato dextrose agar (PDA) slants and subcultured to fresh PDAplates and incubated at 30°C for 5 days.

Experimental design

Potato dextrose Agar (PDA) can be compounded by weighing outthe media components 300 g Irish potato 20 g sugar and 20 g agarin one litre distilled water. This standard set was compared withother starch containing tubers by varying the concentration of thecomponents, substituting the Irish potato with other starchcontaining tubers and the dextrose sugar with the glucose sugarand varying their concentrations. A concentration range of 100 200,300 and 400 g for starch containing tubers and 10g 20 and 30 g of

sugar concentrations per litre were studied, However agarremained constant at 20 g per litre.

Media formulation

Ten different media were formulated, namely purple sweet potatodextrose agar (PPDA), purple sweet potato glucose agar (PPGA),whitish sweet potato dextrose agar (WPDA), whitish sweet potatoglucose agar (WPGA), edible cocoyam dextrose agar (ECDA),edible cocoyam glucose agar (ECGA), non-edible cocoyamdextrose agar (NCDA), non-edible cocoyam glucose agar (NCGA),Yam dextrose agar (YDA) and yam glucose agar (YGA). Thestandard method for preparing potato infusion (potato dextrose agar

PDA) was used by boiling 300 grams of sliced (washed buunpeeled) potatoes in water for 30 min and then, straining the broththrough cheesecloth. Distilled water was added such that the totavolume of the suspension was 1 litre. 20 g dextrose and 20 g agarpowder was then added and the medium was sterilized byautoclaving at 15 pounds per square inch for 15 minutes. In thesame manner the different concentrations (100, 200 and 400 g) othe other starch containing tubers were prepared (but yam andcocoyam tubers were peeled) and the various concentrations osugars (10, 20 and 30 g) added accordingly, only agar remainedconstant at 20 g per litre.

Inoculation of media

The suitability of the formulated media was estimated by culturingthe test fungi on them. Cork borer of an approximate size of 0.37cm in diameter was used to bore holes in the pure cultures of theorganisms. The mycelia agar plugs were then removed usingsterilized wire loop and transferred top down onto the centre of theformulated media. (cork borer and wire loop were sterilized bydipping into alcohol and flamed) . Each of the two organisms Aniger and A. carbonarius were inoculated on the plates of the

formulated media (PPDA), (PPGA), (WPDA), (WPGA), (ECDA)(ECGA), (NCDA), (NCGA), (YDA), (YGA) and PDA in like mannerPlates were incubated at 30°C for 5 days.

Measurement of mycelia sporulation

Five days after inoculation, the spores were washed off intodeionised water, and serial dilution made to a suitableconcentration and counted by using haemocytometer.

RESULTS

In this experiment we varied the concentrations of the

components of the media to determine which would givethe best conditions as well as an alternative for culturingof the fungi by substituting the other starch containingtubers with the potato in PDA. Figures 1 to 8 are thegraphical representation of the data obtained fromculturing the two test fungi ( A. niger and A. carbonariuson the various concentrations of the media component(formulated alternative culture medium and PDA). Sporecounts were taken after five days of growth. Figure 1shows the spore count of A. niger in 100 g of starchcontaining tubers (in the formulated media) usingdifferent sugar concentrations. Alternating betweendextrose and glucose had a negligible effect on funga

growth generally. One 100 grams of starch containingtuber purple sweet potato dextrose agar (PPDA) gave theoptimal spore count of 15.7 × 10

7spores/ml in 30 g

dextrose and yam dextrose agar (YDA) had the leascount of 2.7 × 10

7spores/ml in 10 g dextrose. Figure 2

shows the spore count of A. niger in 200 g of starchcontaining tuber (in the formulated media) in varyingsugar concentrations, the purple sweet potato dextroseagar (PPDA) and the purple sweet potato glucose agar(PPGA) gave the best mycelia count of 26.5 ×10

7spores/ml 30 g dextrose concentration and 25.5 ×10

7

spores/ml in 30 g glucose respectively while the edible

http://en.wikipedia.org/wiki/Cheesecloth

http://en.wikipedia.org/wiki/Distilled_water

http://en.wikipedia.org/wiki/Distilled_water

http://en.wikipedia.org/wiki/Cheesecloth



Amadi and Moneke 4529

Figure 1. Number of spores (107spores/ml) of Aspergillus niger after five day of growth in 100 g of formulated mediaand PDA in different sugar concentrations (A) dextrose (B) glucose (P ˂ 0.05 ).

Figure 2. Number of spores (107spores/ml) of Aspergillus niger after five day of growth in 200 g of formulatedmedia and PDA at different sugar concentrations (A) dextrose (B) glucose (P ˂ 0.05 ).

Figure 3. Number of spores (107spores/ml) of Aspergillus niger after five day of growth in 300 g of formulated mediaand PDA at different sugar concentrations (A) dextrose (B) glucose.

0

2

4

6

8

10

12

14

16

18

S p o r e c o n c ( s p o r e s / m l )

Formulated media

10g Dextrose

20g Dextrose

30g Dextrose

10 g Dextrose20 g Dextrose30 g Dextrose

A

0

5

10

15

20

25

30

S p o r e c o n c

( s p o r e s / m l )

Formulated media

10g Glucose

20g Glucose

30g Glucose

A

10 g Glucose20 g Glucose30 g Glucose

B

10 g Dextrose20 g Dextrose30 g Dextrose

A

0

5

10

15

20

25

30


Formulated media

10g Glucose

20g Glucose

30g Glucose

B

10 g Glucose20 g Glucose30 g Glucose

0

2

4

6

8

10

12

S p o r e s c o n c

( s p o r e s / m l )

Formulated media

10g Dextrose

20g Dextrose

30g Dextrose

A10 g Dextrose20 g Dextrose30 g Dextrose

0123456

789

10

PGA PPGAWPGA YGA ECGA NCGA

S p o r e c o n c ( s p

o r e s / m l )

Formulated media

10g Glucose

20g Glucose

30g Glucose

B10 g Glucose20 g Glucose30 g Glucose




Figure 4. Number of spores (107spores/ml) of Aspergillus niger after five day of growth in 400 g of formulated mediaand PDA at different sugar concentrations (A) dextrose (B) glucose (P ˂ 0.05 ).

Figure 5. Number of spores (107spores/ml) of Aspergillus carbonarius after five day of growth in 100 gof formulated media and PDA at different sugar concentrations (A) dextrose (B) glucose. (P ˂ 0.05 ).

Figure 6. Number of spores (107spores/ml) of Aspergillus carbonarius after five day of growth in 200 g offormulated media and PDA at different sugar concentrations (A) dextrose (B) glucose. (P ˂ 0.05 ).

0

2

4

6

8

10

12

S p o r e c o n c

( s p o r e s / m l )

Formulated media

10g Dextrose

20g Dextrose

30g Dextrose


0

1

2

3

45

6

7

8

9


Formulated media

10g Glucose

20g Glucose

30g Glucose

B10 g Glucose

20 g Glucose

30 g Glucose

0

5

10

15

20

25

30

35


Formulated media

10g Dextrose

20g Dextrose

30g Dextrose


0

5

10

15

20

25

30

35

40

45

S p o r e s c o n c ( s p o r e s / m l )

Formulated media

10g Glucose

20g Glucose

30g Glucose

B10 g Glucose20 g Glucose30 g Glucose

0

5

10

1520

25

30

35

40


Formulated media

10g Dextrose

20g Dextrose

30g Dextrose


0

5

10

1520

25

30

35

40

S p o r e c o n c (

s p o r e s / m l )

Formulated media

10g Glucose

20g Glucose

30g Glucose

B 10 g Glucose20 g Glucose30 g Glucose



Amadi and Moneke 4531



cocoyam dextrose agar (ECDA) of 2.5 × 107spores/ml 20

dextrose gave the least. Figure 3 shows the spore countof A. niger in 300 g of starch containing tubers (in theformulated media) in varying sugar concentrations, Irishpotato dextrose agar (PDA) and Irish potato glucose agar

(PGA) of 9.2 × 10

7

spores/ml at 20 g dextroseconcentration and 9 × 107

spores/ml in 30 g glucoseconcentration gave the highest spore count respectivelyfollowed by the purple sweet potato dextrose agar(PPDA) of 9.1 × 107 in 30 g dextrose concentration andthe purple sweet potato glucose agar (PPGA) of 8.7 ×107spores/ml and 20g glucose, yam dextrose agar (YDA)of 2 × 10

7spores/ml, 30 g dextrose and yam glucose agar

(YGA) of 1.7 × 107

spores/ml 20 g glucose gave thelowest spore count. Figure 4 shows the spore count of A.niger in 400 g of starch containing tubers (in theformulated media) in varying sugar concentrations, here

all the formulated media had a remarkable increase intheir spore count however Irish potato dextrose aga(PDA) of 8.6 x10

7spores/ml and 30 g and Irish potato

glucose agar (PGA) 8.1 × 107

spores/ml 20 g glucosegave the optimal spore count and yam glucose agar

(YGA) of 3 × 10

7

spores/ml giving the least spore countFigure 5 shows the spore count of A. carbonarius in 100g of starch containing tubers (in the formulated media) invarying sugar concentrations. Potato glucose agar (PGA)of 40 × 10

7spores/ml and 30 g glucose gave the highes

spore count while non-edible cocoyam glucose aga(NCGA) gave the least spore count of 4.1 × 10

7spores/m

and 10 g glucose concentration. Figure 6 shows thespore count of A. carbonarius in 200 g of starchcontaining tubers (in the formulated media) in varyingsugar concentrations. Potato glucose agar (PGA) had theoptimal spore count of 35 × 10

7spores/ml and 30 g

0

50

100

150

200

250

300

350

S p o r e c o n

c ( s p o r e s / m l )

Formulated media

10g Dextrose

20g Dextrose

30g Dextrose

A 10 g Dextrose20 g Dextrose30 g Dextrose

0

100

200

300

400

500

600


Formulated media

10g Glucose

20g Glucose

30g Glucose


0

50

100

150

200

250


Formulated media

10g Dextrose

20g Dextrose

30g Dextrose

A 10 g Dextrose20 g Dextrose30 g Dextrose

0

50

100

150

200

250


Formulated media

10g Glucose

20g Glucose

30g Glucose





glucose concentration while non-edible cocoyamdextrose (NCDA) agar of 3.2 × 10

7spores/ml had the

least spore count in 20 g dextrose concentration. Figure 7shows the spore count of A. carbonarius in 300 g ofstarch containing tubers (in the formulated media) invarying sugar concentrations. Potato glucose agar had

the best spore count of 480 × 10

7

spores/ml, 30 g glucoseconcentration and the lowest with yam dextrose agar(YDA) of 15 × 10

7spores/ml10g dextrose concentration.

Figure 8 shows the spore count of A. carbonarius in 400g of starch containing tubers (in the formulated media) invarying sugar concentrations. The results here are quitedifferent with purple sweet potato dextrose agar (PPDA)giving an optimal count of 195 × 10

7spores/ml 20 g

dextrose while edible cocoyam dextrose agar (ECDA)gave the lowest of 35 × 10

7 spores/ml 20 g dextrose

concentration.

DISCUSSION

Under the experimental conditions remarkable mycelialgrowth and sporulation was observed on all theformulated media however in varying degrees. Variationin the concentration of media components also played arole in the overall outcome of the formulated media.Cassava dextrose agar, corn meal dextrose agar andPalmyrah tuber medium have been reported to be bettermedia compared to PDA for the cultivation of some fungi(Weststeijn and Okafor, 1971; Adesemoye and Adedire,2005; Tharmila et al., 2011). In the present study onewould rather say that the purple sweet potato dextroseagar and purple sweet potato glucose agar (PPDA and

PPGA) can compete favourably with the Irish potatodextrose agar (PDA) as medium for the cultivation offungi, as optimal performance (spore count) of theformulated media depended on the concentration of themedia components. Microbiological studies depend onthe ability to grow and maintain microorganisms underlaboratory conditions by providing suitable culture mediathat offer favourable conditions (Prescott et al., 2002).The knowledge of the conditions is useful in theknowledge of the growth of microbes. The resultsobtained in this study on the cocoyam media (ediblecocoyam dextrose agar [ECDA], edible cocoyam glucoseagar [ECGA] and the non-edible cocoyam dextrose

[NCDA], non-edible cocoyam glucose agar [NCGA]showed reduced spore count which may be as a result ofthe nutritional constituents of the cocoyam. Beever andBollard (1970) suggested that elaborate growth of fungion PDA may be as a result of combined effect ofnutrients in potato in their right proportion. Modification ofthe media with dextrose/glucose had no marked effect onthe formulated cocoyam media. Similar growth was

observed though glucose thrived better than dextrose insome cases. Also worthy of note is that the ornamentacocoyam media (NCDA and NCGA) showed a higherspore count than the edible cocoyam media (ECDA andECGA). The yam dextrose agar (YDA) and yam glucoseagar (YGA) also showed less mycelial growth as

determined by the spore count. Weststeijn and Okafor(1971) reported lower mycelial growth on yam dextroseagar in an in vitro experiment conducted to comparecassava, yam and potato dextrose agar.

Conclusion

In tropical countries other starch containing tubers canreplace the potato in a general purpose culture media. Inthe present study sweet potato varieties can be used asan alternative to Irish potato to formulate media for fungacultivation. An important advantage of this medium fortropical countries is that it can be grown locally andreproduced vegetatively. However further studiesregarding the appropriate concentrations and conditionshave to be determined. The type of monosaccharidesugar, whether dextrose or glucose should depend on theavailable one.

REFERENCES

Adesemoye AO, Adedire CO (2005). Use of cereals as basal mediumfor the formulation of alternative culture medium for fungi. World JMicrobiol. Biotechnol., 21: 329-336.

Beever S, Bollard CV (1970).The nature of the stimulation of fungagrowth by potato extract. J. Gen. Microbiol., 60 273-279.

Olutiola PO, Famurewao, Sonntag HG (2000). An introduction toGeneral Microbiology: A Practical Approach. Reprinted ednHeidelberg, Germany: Hygiene-institute Der Universitat Heidelbergpp. 50, 196, 223.

Prescott LM,HarleyJP, Klein DA (2002). Microbiology, 5th edition pp105-106. London: McGraw Hill Publishers.

Tharmila TS, Jeyaseelan EC, Thavaranjit AC (2011). Preliminaryscreening of alternative culture media for the growth of someselected fungi. Arch. Appl. Sci. Res., 3(3): 389-393.

Weststeijn G, Okafor N (1971). Comparison of cassava, yam and potatodextrose agar as fungal culture media. Neth. J. PI. Path., 77: 134139.

Willey JM, Sherwood LM, Woolverton CJ (2008). Prescott Harley andKleins Microbiology, 7th edn. New york, McGraw Hill Companies, Incpp. 105-106.

medium PDA

Documents

Transcript of medium PDA