Mycopathologia 115: 9-12, 1991. 9 1991 KluwerAcademicPublishers. Printedin the Netherlands.
An evaluation of straw-extract agar media for the growth and sporulation of Madurella mycetomatis C. Rajendran, Alice Baby, S. Kumari & T. Verghese National Institute of Communicable Diseases, 22 - Sham Nath Marg, Delhi 110054, India
Received 13 September 1990; accepted in revised form 9 December 1990
Key words: Growth, Madurella mycetomatis, new medium, sporulation, straw extract agar
Abstract
Four new media, namely Wheat straw extract agar, Bajra straw extract agar, Jowar straw extract agar and Paddy straw extract agar, were evaluated for their potential to stimulate the growth and sporulation ofMadurella mycetomatis in comparison with the conventional Sabouraud dextrose agar and Soil extract agar. Vegetative growth ofM. mycetomatis on the four types of Straw extract agars was superior to that obtained on Sabouraud dextrose agar. Isolates of M. mycetomatis sporulated better and faster on the Straw extract agars than on the Sabouraud dextrose agar and Soil extract agar. Straw extract agar is recommended as a sporulation medium for M. mycetomatis. It may prove useful especially for studies of the conidium ontogeny of the fungus for elucidating its taxonomic status.
Introduction
Madurella mycetomatis is one of the commonest causes of mycetoma in many parts of the world [ 1-4]. In India, it is the main causal agent of this disease in the northern regions. Studies indicate that it occurs mostly in agricultural workers, particularly those working in farm fields [5-7]. General belief is that the organism survives primarily in the soil or plant parts or debris in the soil and through traumatic implantation during accidental injury the fungus enters the body and causes the disease [7, 8]. While studying the natural habitat and epidemiology of the causal agents of mycetoma, we evaluated the potential of the extracts of the dry shoots of four major cultivated crops in north India, namely Wheat (Triticum sativum), Bajra (Pennesetum typhoides ), Jowar (Sorghum vulgare)
and Paddy (Oryza sativum) on the growth and sporulation ofMadurella mycetomatis. The objective was to determine if there is any affinity of this organism to such plant substrates which usually form a large part in the soil of farm fields.
Materials and methods
Preparation of the media Shoot parts of the four cereal crops collected separately after harvest from the field were dried and cut into small pieces. To 250 gm each of these, 2 litres of tap water was added and the mixture autoclaved in large conical flasks at 15 p.s.i, for two hours. The extract then was filtered using a cheesecloth followed by Whatman No. 1 filter paper. In each of these straw infusions, the following reagents were added:
10 Dextrose 2.0 gm, Yeast extract 1.0 gm, Potassium phosphate 0.5 gm and agar 20 gin. The volume of each then was made up to one litre by adding water and the pH was adjusted to 7. After boiling for five minutes, the straw extract agars (25 ml each) were dispensed in 250 ml conical flasks and autoclaved at 15 p.s.i, for 15 minutes. These four media, Wheat Straw Extract Agar (WSEA), Bajra Straw Extract Agar ~(BSEA), Jowar Straw Extract Agar (JSEA) and Paddy Straw Extract Agar (PSEA) were used for evaluation in comparison to the conventional Sabouraud Dextrose Agar (SDA) and Soil Extract Agar (SEA) media.
Isolates of Madurella mycetomatis Three clinical isolates of M. mycetomatis (M17, M191, M379) were used. M17, which was isolated from a case of Madura foot 10-years ago, was received from the All India Institute of Medical Sciences, New Delhi. M191 and M379 both were isolated from two cases ofmycetoma seen by the authors at the National Institute of Communicable Diseases, Delhi during 1984 and 1985, respectively. All three isolates produced a brown pigment in culture on Sabouraud Dextrose Agar.
an illuminated light. The data on the growth rates of colonies were tabulated and analyzed statistically by the analysis of variance method [9]. For assessing the rate of sporulation, small amounts of cultures were removed randomly from 4 sites of each culture flask and were pooled and stained with 0.5 ~o trypan blue in lactophenol [ 10]. As far as possible, an equal quantity of culture was removed each time for staining and observations. The slide mounts of such preparations were examined under high power (40x) of a compound microscope and visual scoring was done as follows: No sporulation 0, light sporulation +, medium sporulation + +, good sporulation + + + and heavy sporulation + + + +. Both phialospores and blastospores were taken into account while assessing the sporulation rate.
Results
The comparative growth rates of the three isolates ofM. mycetomatis on the four types of Straw Extract Agars, SDA and SEA are listed in Table 1. Growth of all isolates on the Straw Extract Agars was superior to that on SDA. This difference in
Inoculation, incubation and observation Using a sterile cork-borer, a small amount of growth (2 mm diameter) from fresh cultures of uniform thickness and texture of each isolate was cut and inoculated carefully at the center of each medium. For the test 36 flasks of each medium were inoculated with one isolate; 18 flasks were incubated at27 ~ and 18 at 37 ~ Likewise, the other three sets of media of 36 flasks each were inoculated with the remaining two isolates of M. mycetomatis and incubated. In order to make comparison with the established media, two sets of 36 flasks each of SDA and SEA also were inoculated with the three isolates and incubated in the same way. Observations on the rate of growth and sporulation were assessed at periodic intervals of 7 and 30 days, respectively. For assessing the growth rates, the diameter of the colony was measured by holding the reverse of the flask using a scale under
Table 1. Comparative mean growth rates (in cm) ofM. mycetomatis isolates on 4 types of Straw Extract Agars, Sabouraud Dextrose Agar and Soil Extract Agar at 2 different temperatures. Media
Isolates of M. mycetomatis M17
BSEA JSEA PSEA WSEA SDA SEA
M191
M379
27~
37~
27~
37~
27~
37~
2.6 3.0 2.2 3.0 1.1 4.1
2.9 3.5 1.5 4.0 0.7 3.0
2.1 3.3 2.1 3.5 1.5 2.7
3.1 5.4 3.2 5.5 1.7 4.8
2.5 1.8 1.8 2.8 1.6 3.7
3.7 3.6 2.0 6.3 1.6 5.6
BSEA = Bajra Straw Extract Agar; JSEA = Jowar Straw Extract Agar; PSEA = Paddy Straw Extract Agar, SDA = Sabouraud Dextrose Agar; SEA = Soil Extract Agar; W S E A = Wheat Straw Extract Agar.
11 well at 37 ~ while the latter sporulated well at 27 ~ except on PSEA where the rate of sporulation was equal at both temperatures on the 90th day. The maximum sporulation was observed in M191 on the 60th day itself on PSEA.
growth rate was found statistically significant ( p < 0.01). Among the Straw Extract Agars, growth rate was maximum on WSEA. However, the growth of all the isolates on SEA, compared to those on JSEA, BSEA and PSEA (except on JSEA at 37 ~ of M17 and at both the temperatures of M 191) was superior. Among WS EA and SEA the maximum growth was in the former, except for M17 and M379 at 27 ~ The comparative rate of sporulation of the three isolates ofM. rnycetomatis on the four types of Straw Extract Agars, SDA and SEA is listed in Table 2. Up to 90 days, both at 37 ~ and 27 ~C, M17 did not sporulate at all on any of these media; both M191 and M379 did not sporulate up to 30 days. When observed on the 60th day, sporulation of M191 and M379 ranging from + to + + + + was observed on Straw Extract Agars, while the rate of sporulation of the three isolates up to 90 days on SDA and SEA was 0 to + +. Among the SDA and SEA media sporulation was better on the latter. On SDA, M379 did not sporulate at all even up to the 90th day. Between M 191 and M379, the former sporulated
Discussion
The fact that all the isolates of M. mycetomatis could grow and sporulate well on the Straw Extract Agar media indicates that there is an affinity of this organism to these plant substrates. It is possible that such shoot-remnants, which usually form a good part of the soil in the farm fields, might act as the natural substrates of these fungi. It is often a practice in many parts of India that after the plant is cut off during harvest a sharp hard stump is left over [5]. These stumps, which usually remain there until they are made to become a part of the soil while ploughing during the next planting season, may become colonized by this fungus. The farmers often go bare-foot and the stumps which are left in the field are believed
Table2. C o m p a r a t i v e rates of s p o r u l a t i o n o f M . mycetomatis isolates on 4 types o f Straw E x t r a c t A g a r s , S a b o u r a u d D e x t r o s e A g a r a n d Soil E x t r a c t A g a r at different t e m p e r a t u r e s . Isolates ofM. mycetomatisand i n c u b a t i o n in days
Media and temperatures
M17
M191
30
60
90
30
M379 60
90
30
60
90
BSEA
27 ~ 37 ~
0 0
0 0
0 0
0 0
+ + + +
+ + + +
0 0
+ +
+ +
JSEA
27~ 37 ~
0 0
0 0
0 0
0 0
+ + +
+ + + + + +
0 0
+ + + +
+ + + +
PSEA
27~ 37~
0 0
0 0
0 0
0 0
+ ++++
+ + + ++++
0 0
+ +++
+ + + +++
WSEA
27~ 37~
0 0
0 0
0 0
0 0
+ +++
+ + ++++
0 0
+ + + +
+ + + + +
SDA
27 ~ 37 ~
0 0
0 0
0 0
0 0
+ +
+ + +
0 0
0 0
0 0
SEA
27 ~ 37 ~
0 0
0 0
0 0
0 0
+ + +
+ + + +
0 0
+ 0
+ +
B S E A = B a j r a Straw E x t r a c t A g a r ; J S E A = J o w a r Straw E x t r a c t Agar; P S E A = P a d d y Straw E x t r a c t A g a r ; S D A = S a b o u r a u d D e x t r o s e Agar; S E A = Soil E x t r a c t Agar; W S E A = W h e a t S t r a w E x t r a c t Agar.
12 to be a common cause of wound infliction in those who become the victims of mycetoma [5]. During the isolation of M. mycetomatis from clinical sources, it is often a common experience that the materials inoculated on SDA have to be incubated for several days, sometimes up to 6-8 weeks, to obtain a positive culture. Many times, overgrowth of contaminants occurs causing difficulties in the isolation of this slow growing pathogen. Since the media derived from the Straw extracts were found to be more efficient than the conventional SDA medium for the cultivation of M. mycetomatis, such media, particularly WSEA, may proove highly useful for the fast isolation of M. mycetomatis from clinical as well as from environmental sources. Another notable feature of this variety of medium is that the isolates M191 and M379 sporulated on all the Straw Extract Agar media better than on the SDA and SEA media. This shows that the Straw Extract Agar media also could be used as a sporulation medium for this organism. This point is of special significance because the genus Madurella and especially M. mycetomatis is in need of revision [8]. The genus Madurella is still classified under the Mycelia Sterilia although some isolates of M. mycetomatis are known to produce phialides with collarettes. Therefore, the Straw extract agar medium may prove useful for studies of the conidium ontogeny of the fungus to elucidate its taxonomic status. Although maximum vegetative growth was observed on WSEA medium, sporulation was the highest on PSEA where vegetative growth was the lowest. This shows that there need not be any
correlation between rate of vegetative growth and sporulation in M. mycetomatis. Another striking feature is the better vegetative growth of the organism at 37 ~ compared to the growth rates at 27 ~C on all the Straw Extract Agar media except in the case of PSEA and M17. With regard to sporulation, M 191 sporulated well at 37 ~ while M379 sporulated well at 27 ~ This suggests that the temperature requirements of M. mycetomatis for sporulation can differ from isolate to isolate.
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Addressfor correspondence: Dr. C. Rajendran, National Institute of Communicable Diseases, Medical Mycology Laboratory, 22-Sham Nath Marg, Delhi 110054, India.
