Field of the Invention:
The present invention relates to a culture medium, for good growth of entire known Malassezia species, within short time span and also fit for susceptibility testing. The culture medium is homogenous, with least chances of contamination, cheaper, can be easily maintained, having long shelf life and abundant availability of medium components.
Background of the Invention:
Malassezia species have been associated with a number of diseases of human skin, such as seborrheic dermatitis, dandruff, pityriasis versicolor, folliculitis, atopic dermatitis, psoriasis etc. The genus Malassezia includes 10 anthrophilic and obligatory lipophilic spp. (M. globosa, M. restricta, M. slooffiae, M. obtusa, M. furfur, M. sympodialis, M. japonica, M. yamatoensis, M. dermatitis, M.nana ) and 3 zoophilic spp. (M. pachydermatitis, M. caprae, M. equina).
The most commonly used culture media are: Dixon's agar medium, Leeming and Notman agar (LNA), Leeming and Notman agar modified (mLNA), Modified medium by Dutta and Dikshit, Modified Chromagar Candida, RPMI 1640 medium. All of these medium are costly as well as all the known Malassezia species do not grow well on them . There are several drawbacks in the existing culture media which are mentioned below:
DIXON AGAR MEDIUM:
a) Medium is costly, besides chances of fungal contamination is more.
b) Oxoid is rarely available In Indian market, cost of 20 g oxoid in a litre of medium is Rs. 154.20
c) Bad odor of medium
d) Medium does not support good growth of all the Malassezia species in short life span.
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LEEMING AND NOTMAN AGAR ( LNA):
a) High rate of bacterial growtli
b) Medium does not support good growth of all the Malassezia species in short life span.
LEEMING AND NOTMAN AGAR MODIFIED fmLNA):
a) High amount of olive oil (20 ml) give rise disturbance of drug resistance test.
b) Olive oil alone does not mix homogeneously with basal medium.
c) Olive oil gives rise to problem of its removal from harvested cell.
d) Medium is very sensitive to bacterial and fungal contamination.
SDA MEDIUM:
a) Individual colony formation could not be obtained in SDA plate with over lay of olive oil.
b) Medium is sensitive to other yeast rather than Malassezia ( high quantity of dextrose used)
c) When olive oil used in excess the lipophilic Malassezia species incorporate in to body
d) Classical medium is unfit for culturing of lipophilic yeast.
DUTTAAND DIKSHIT MEDIUM:
a) Although the medium is comparatively cheaper but does not support good growth of entire Malassezia species in short life span.
b) Medium is sensitive to other yeast rather than Malassezia species ( high quantity of sucrose used)
RPM11640 MEDIUM:
a) Sensitive medium requires supplemented fatty acids and MOPS.
b) Cost of medium is very high (Cost of RPMI 1640 supplemented with fatty acid is Rs. 98.37 per petriplate which is about 20 ml)
c) Short shelf life of medium.
(d) Medium is very sensitive to fungal contamination.
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A lot of work has been done earlier on isolation and culturing of Malassezia spp. A few of them which are relevant to isolation and culturing of Malassezia spp. are listed below:
Isolation of 19 strains of Malassezia dermatis from healthy human skin in Korea;Yang Won LEE, Journal of Dermatology 2008; 35: p. 772-777:
discloses isolation and culturing of Malassezia strain from healthy human skin without any skin diseases on basal medium (Learning and Notman Agar), this medium is not suitable to recover all the species of Malassezia for microdilution testing as well as culturing, apart from this other drawbacks are also associated with this medium like high cost, short shelf life and chances of contamination.
An improved method for quantitative culture of Malassezia furfur; John P. Leeming ; Journal of clinical microbiology; Oct.1987; p. 2017-2019:
discloses an improved method for culturing Malassezia furfur, using medium composition as per composition of Leeming and Notman Agar medium with slight modification. This medium is unfit for BM susceptibility testing, require long incubation period , have short shelf life and high rate of contamination by other unwanted fungi and bacteria. This medium is also not suitable for all the species of Malassezia spp.
A modified Christens Urea and CLSI Broth microdilution method for testing susceptibilities of six Malassezia species to Voriconazole, Itraconazole and Ketoconazole; S. Rincon, M. C. Cepero de Garcia; Journal of clinical microbiology, Sept. 2006, p. 3429-3431: discloses two broth media (Christen urea and RPMI) used to determine MICs of Voriconazole, Itraconazole and Ketoconazole against six reference strains of Malassezia. Clinical and Laboratory Standards Institute (CLSI) recommended 1640 medium for antifungal susceptibility of fungi. But, this medium is not fit for all the species of Malassezia spp. as most of the Malassezia spp. are fastidious, they require complex nutrient for growth. RPMI 1640 is very costly
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medium, (1 litre Rs. 2095 witii fatty acid and Rs. 4879 with MOPS), have short shelf life, have chances of contamination, pure form does not support good growth of lipid dependent Malassezia spp. Also, proper use of external lipid supplement is necessary with basal RPM11640 medium.
Production of the mycelial phase of Malassezia in vitro; M. R. Saadatzadeh; Med. Mycology^ 2001 Dec; 39(6): p.487-93: discloses utility of different components like mg^*, glycine, different tween formulation, squalene, ergosterol on Malassezia spp. for production of its mycelial phase. Malassezia is a dimorphic fungi found in both form budding as well as hyphal. The basal medium used for investigation in this article is Leeming and Notman agar medium. The supplemented medium used for investigation in this article has not supported growth of all the Malassezia spp.
Suitability of lipid materials for culture of Malassezia as evaluated from its cellular fatty acid composition; Nishikawa.H.; Mycoscience;(1997):
discloses various lipid supplements for standardization of YM medium and creaming powder added medium, these medium are found good for harvesting Malassezia /uAfur (lipid dependent) and Malassezia pachydermatis (lipid non dependent spp.) only two sp. of Malassezia grew well on this medium but other sp. not grow well.
A comparative novel method of antifungal susceptibility for Malassezia furfur and modification of culture medium by adding lipid supplement; Amit K. Tiwari; Journal of Phytology, Vol 3, No 3 (2011): discloses comparative method of antifungal testing of botanicals and synthetic against Malassezia furfur using 96 well plates with slight modification of Clinical and Laboratory Standards Institute (CLSI) method. This article also discuss standardization of Dutta and Dixit medium by adding various lipid supplement such as olive oil, coconut oil, til oil and cotton seed oil with principal solid medium ( Dutta and Dixit medium). Utility of cotton seed oil with Dutta and Dixit medium was tested only for Malassezia furfur but all the other species do not
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grow well on Dutta and Dixit medium. Malassezia furfur is most common sp. among all the species and also robust comparable to other species. All the other spp. of Malassezia does not grow well and contamination by other fungi and bacteria also observed.
Indian Patent No. 217897: The principle medium used in this patented document is Dutta and Dixit medium. The main problem associated with the medium used in this document is contamination by other fungi rather than Malassezia spp. due to growth of undesired filamentous fungi. Also, this medium does not support good growth of all known Malassezia spp.
Clinical and Laboratory Standards Institute (CLSI) have recommended broth microdilution method to determine antifungal susceptibility testing of yeast using 96 well plate in year 2003 and the method is globally accepted. CLSI has recommended RPM11640 medium which is not suitable for entire range of Malassezia species.
Therefore, a need is felt for a medium suitable for the excellent growth as well as antifungal susceptibility of yeast like fungi Malassezia spp., long shelf life, economical, easy availability of its constituents and having least chances of contamination.
Accordingly, a culture medium is prepared according to present invention which show excellent growth as well as antifungal susceptibility of yeast like fungi Malassezia and solve other problems related with the already existing culture media.
Objective of the Invention:
It is an objective of the present invention to provide a culture medium for the excellent growth of the fastidious Malassezia spp.
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It is another objective of tlie present invention to provide a culture medium which show good growth of entire known Malassezia species, within short span of time(2-3 days) and also fit for susceptibility testing.
It is still another objective of the present invention to provide a culture medium which is homogenous, having least chances of contamination, cheaper, can be easily maintained, having long shelf life and abundant availability of medium components and also useful for culturing other human pathogenic unicellular yeasts.
Summary of the Invention:
Accordingly, present invention provides a culture medium for the excellent growth of the fastidious Malassezia spp.
The culture medium, comprising a disaccharide, peptone, yeast extract, ox-bile, glycerol, fatty acid, lipid source, antibiotic, surface active agent, and optionally, additives.
More specifically, 1 litre of the culture medium according to present invention comprises 10-41 g of a disaccharide, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile, 4.5-16 ml of glycerol, 3-14 ml of fatty acid, 10-22 ml of lipid source, 2-14.5 pg of antibiotic, 1.5-12 ml of surface active agent, optionally additives and balance being distilled water.
Brief Description of the figure:
Fig 1: Carbohydrate utilization by Malassezia furfur
Fig 2:Carbohydrate utilization by Malassezia globosa
Fig 3: Carbohydrate utilization by Malassezia sympodialis
Fig 4: Carbohydrate utilization by Clinical isolate of Malassezia species obtained from dandruff patient(PDI)
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Fig 5: Carbohydrate utilization by Clinical isolate of Malassezia species obtained from dandruff patient (PD2)
Detailed Description of the Invention:
The present invention provides a culture medium, for good growth of all known Malassezia species in very short duration of 2-3 days, having least chances of contamination, easy method of preparation, easy availability of all the components of the medium and their maintenance and having long shelf life. In an embodiment present invention provides a culture medium, comprising a disaccharide, peptone, yeast extract, ox-bile, glycerol, fatty acid, lipid source, antibiotic, surface active agent, and optionally additives.
In another embodiment the culture medium is broth.
In yet another embodiment the disaccharide is selected from the group consisting of sucrose, lactose, maltose, trehalose and melibiose, preferably sucrose.
In another embodiment the fatty acid is oleic acid.
In yet another embodiment the lipid source is selected form the group consisting of cotton seed oil or olive oil.
In another embodiment the antibiotic is selected from the group consisting of streptomycin, penicillin or a combination thereof.
In another embodiment the additive is selected from the group consisting of osmoregulator, glycerol monostearate or a combination thereof..
In still another embodiment the osmoregulator is selected from the group consisting of NaCI or KCI, preferably NaCI.
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In an embodiment the surface active agent is selected from the group consisting of Polyethylene glycol sorbitan monostearate (Tween 60), Polyoxyethylene sorbitan monopalmitate (Tween 40) or Polyoxyethylene sorbitan monolaurate (Tween 20) preferably Polyethylene glycol sorbitan monostearate (Tween 60) or Polyoxyethylene sorbitan monolaurate (Tween 20).
In still another embodiment the disaccharide is in the range of 10-41 g per litre of the culture medium.
In yet another embodiment the fatty acid is in the range of 3-14 ml per litre of the culture medium.
In another embodiment the lipid source is in the range of 10-22 ml per litre of the culture medium.
In still another embodiment the antibiotic is in the range of 2-14.5 pg per litre of the culture medium.
In another embodiment the osmoregulator is in the range of 1-3 g per litre of the culture medium.
In yet another embodiment the glycerol monostearate is in the range of 0.2-1 g per litre of the culture medium.
In still another embodiment the surface active agent is in the range of 1.5-12 ml per litre of the culture medium.
In yet another embodiment present invention provides 1 litre of a culture medium, comprising 10-41 g of a disaccharide, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile, 4.5-16 ml of glycerol, 3-14 ml of fatty acid, 10-22 ml of lipid source, 2-14.5 pg of antibiotic, 1.5-12 ml of surface active agent,
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optionally additives and balance being distilled water.
In another embodiment the present invention provides 1 litre of a culture medium, comprising 38-41 g of sucrose, 10-12 g of peptone, 12-14 g of yeast extract, 8-10 g of ox-bile, 14-16 ml of glycerol, 0.5-1 gm of glycerol monostearate, 12-14 ml of oleic acid, 12-14 ml of cotton seed oil, 2-2.5 |jg of streptomycin and 8-12 ml of Polyoxyethylene sorbitan monolaurate (Tween 20) and balance being distilled water.
In another embodiment the present invention provides 1 litre of a culture medium, comprising 10-15 g of sucrose, 8-10 g of peptone, 2-5 g of yeast extract, 3-6 g of ox-bile, 10-12 ml of glycerol, 6-8 ml of oleic acid, 12-14 ml of cotton seed oil, 2-2.5 pg of streptomycin and 5-6 ml of Polyoxyethylene sorbitan monolaurate (Tween 20) and balance being distilled water.
In another embodiment the present invention provides 1 litre of a culture medium, comprising 11-14 g of sucrose, 6-9 g of peptone, 1.5-4 g of yeast extract, 1.2-3.5 g of ox-bile, 4.5-7 ml of glycerol, 0.2-0.9 g of glycerol monostearate, 3-5 ml of oleic acid, 17-22 ml of cotton seed oil, 3-7 |jg of streptomycin, 4-7.5 \ig of penicillin, 1-3 g of NaCl and 1.5-4 ml of Polyethylene glycol sorbitan monostearate (Tween 60) and balance being distilled water.
In another embodiment the present invention provides 1 litre of a culture medium, comprising 12-18 g of sucrose, 6-10 g of peptone, 8-10 g of yeast extract, 5-7 g of ox-bile, 5-8 ml of glycerol, 6-8 ml of oleic acid, 14-16 ml of olive oil, 2-2.5 pg of streptomycin and 5-6 ml of Polyethylene glycol sorbitan monostearate (Tween 60) and balance being distilled water.
In still another embodiment the culture medium is a solid medium comprising a disaccharide, peptone, yeast extract, ox-bile, glycerol, fatty acid, lipid source, antibiotic, surface active agent and optionally, additives.
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In yet another embodiment lipid source is powder milk.
In another embodiment additive is selected from the group consisting of agar-agar and osmoregulator, glycerol monostearate or a combination thereof.
In still another embodiment the culture medium comprises 10-41 g of sucrose, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile, 4.5-16 ml of glycerol, 3-14 ml of oleic acid, 10-14 g of powder milk, 3-7 |jg of streptomycin, 4-7.5 |jg of penicillin, 1-3 g of NaCI and 1.5-4 ml of Polyethylene glycol sorbitan monostearate (Tween 60), 9.5-12 g of agar-agar and distilled water to make 1 litre of mixture.
In another embodiment present invention provides a process for the preparation of culture medium comprising:
(i) Mixing 10-41 g of disaccharide, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile in a pre-sterilized flask;
(ii) Adding 3-14 ml of fatty acid, 4.5-16 ml of glycerol and 1.5-12 ml of surface active agent to the mixture obtained in step (i) making final amount 1 litre by adding distilled water to obtain a crude medium by homogeneously mixing all the components;
(ili) Sterilizing the crude medium as obtained in step (ii) in an autoclave;
(iv) Adding 2-14.5 |jg of antibiotic to the sterilized mixture obtained from step (iii);
(v) Pouring lipid source into the mixture either prior to or subsequent to the step (iii) to obtain the culture medium.
Optionally, agar-agar can be added in an amount of 9.5-12 g to the medium
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before autoclaving the mixture for obtaining a solid medium. Also, the lipid source used for solid medium is powder milk in an amount of 10-14 g rather than cotton seed oil or olive oil as powder milk help to form a homogenous mixture.
In another embodiment sterilization is carried out at a pressure ranging from 68.95 KPa - 206.84 KPa.
In yet another embodiment sterilization is carried out at a temperature ranging from 115.5 °C-135 °C.
In still another embodiment sterilization is carried for time ranging from 20-30 minutes.
The culture medium of the present invention support good growth of Malassezia spp. validity is checked for 10 Malassezia spp. In case of culture medium component ratio and their amount are most important factors for supporting good growth of fastidious species like Malassezia. Therefore, in case of present invention amount of each component is determined by testing various combinations and finally the culture medium, BPLA2R0 is obtained which support faster growth of in-vitro tested 10 Malassezia spp. namely Malassezia furfur, Malassezia globosa , M. restricta , M. sympodialis, M. nana , M. obtusa, M. slooffiae, M. japonica, M. yamotensis and M. dermatis.
The culture medium of the present invention can be used in culturing and antifungal susceptibility testing procedures for all the known Malassezia spp. and other unicellular yeast.
Medium overlay with cotton seed oil and olive oil gave better result than coconut oil and til oil.
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The culture medium, according to present invention is obtained after extensive study and experimentation. The culture medium obtained according to present invention is a synergistic composition having requisite amount of each component which interact to provide the desired results. The various factors affecting the growth of Malassezia spp. are discussed below in detail:
Carbon source: carbohydrate is the main source of carbon as such listed sources have been used which are available in the KB009 Hi carbohydrate kit and observation recorded in table 1 as +/- /v as well as in the form of photographs.
Table 1: Carbohydrate utilization by Malassezia species testing through Biochemical testing kit:
S. No. Carbohydrate Pathogens
W VMg rW I Msy I PD1 \ PD2
1. Lactose + + + - + +
~2. Xylose + + + "+ + +
T Maltose + + + - + +
4. Fructose + - + + + +
5. Dextrose + + + - v v ~6. Galactose + + + + + +

7. Raffinose + _ + '^ v v
8. Trehalose + + + - v v
9. Melibiose + _ + * v ^
10. Sucrose + + + + v v ~lT. L-Arabinose + V + V V +
12. Manose + + + v v v
• PD1 and PD2 are clinical isolates of Malassezia species.
KB009 Hi carbohydrate kit.
The above mentioned kit includes following carbohydrate fermentation test
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according to given number of eacli well respectively:
1.Lactose 2. Xylose 3.Maltose 4.Fructose 5. Dextrose 6. Galactose 7. Raffinose
S.Trehalose 9.Melibiose 10.Sucrose 11. L-Arabinose 12. Mannose
( Colour of medium changes from red-pink colour to yellow colour due to acid
production, if test is positive. If the test is negative medium remains red-pink in
colour.)
Interpretation of result was done by visual observation and score sheet was made by the following given criteria-
+ = Positive (more than 90%)
- = Negative (more than 90%) V = 11-89 positive
Osmoregulator:
NaCI is used in the culture medium of present invention, other variant of osmoregulator which can be used in culture medium is KCI.
Surface active agent:
Different grades of Tween i.e. Polyoxyethylene sorbitan monopalmitate( Tween 40) , Polyethylene glycol sorbitan monostearate (Tween 60), Polyoxyethylene sorbitan monooleate (Tween 80) and Polyoxyethylene sorbitan monolaurate (Tween 20) have been used and their impact on the growth of 10 fastidious tested Malassezia species were observed. Out of 10 tested Malassezia spp , 6 species showed good growth on Tween 40. While on Tween 60 , 9 Malassezia species grow well on the other hand on Tween 80 only 4 Malassezia species
14

grow well. Based upon this observation, Tween 60 has been selected as one of the constituents in the candidate medium. Other reason for selecting the Tween 60 are its bactericidal action, lipid source as well as surface active agent. Good growth has been observed on Tween 60 and Tween 20. Thus, Tween 60 and Tween 20 has been selected for obtaining growth medium according to present invention.
Temperature :
2 ml of the pure standard culture broth of 10 tested Malassezia species was inoculated in tube containing BPLA2R0 medium and incubated for 2-3 days at 24-26°C, 32-34 °C, 35-37 °C and 38-40 °C. Thereafter, observation recorded visually in table 2. All the spp. of Malassezia grow well at the temp 32-34 °C . Thus, this temp range has been selected for candidate medium.
Table 2: Growth of tested Malassezia species on various temperature range:
Species I 24-26 "C I 32-34 "C I 35-37 "C I 38-40 "C
M. furfur - ++++ ++ ++
M. globosa - +++++
M. restricta - +++++
M. sympodialis - ++++ ++ ++
M. nana - ++++ +
M. obtusa - ++++ +
M. slooffiae - ++++ +
M.japonica - ++++ +
M. yamatoensis - ++++ +
M. dermatis - ++++ +
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++++ Excellent growth, ++ good growth,+ weak growth,- no growth.
Broad spectrum of candidate medium:
Besides excellent growth of 10 fastidious tested Malassezia spp, unicellular yeast Candida spp., Trichophyton spp., Microsporum spp. and Epidermophyton spp. are also showing good growth thereby adding the value of the present invention. Details of growth spectrum is listed in table 3.
Table 3. Growth Spectrum of IVIicroorganisms in the candidate medium of the present invention.
S. No. Species Growth in candidate medium
1. M. furfur ++++
2. M. globosa ++++
3. M. restricta ++++
4. A//, sympodialis ++++
5. M. nana ++++
6. M. obtusa ++++
7. M. slooffiae ++++
8. M. japonica ++++
9. M. yamatoensis ++++
10. M. dermatis ++++
11. Candida sp ++
12. Trichophyton sp ++
13. Epidermophyton sp ++
14. Microsporum sp ++
Note: ++++ Excellent growth, ++ good growth
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Shelf life of the Medium increases and chances of contamination reduces by various combination studied for developing the culture medium of present invention by gradually decreasing the sucrose concentration and ox bile concentration in different BPL modification and finally BPL modified medium according to present invention developed. This provide long shelf life of medium. High amount of carbon source, increases chances of contamination. Ox bile is also bacterial growth inhibitor. Antibiotic such as Streptomycin or a combination of Streptomycin and Penicillin in media inhibit the growth of bacteria and undesired fungi, thereby increases the shelf life of media but preparation methodology is also Important. Antibiotic is added in the medium only after autoclaving the medium. Oil is added in the growth medium by Millipore filter or syringe filter because variety of lipophilic microorganism may also be associated with and caused high risk of contamination. Yeast extract is rich in nitrogen source (high protein and free amino acid content)which help to grow in vitro fastidious Microorganisms like Malassezia, one of the advantages of yeast extract is comparative low cost to other nitrogen sources. Additionally yeast extract is a rich source of vitamins (Vitamin B), trace elements, and very low content of carbohydrate. Yeast extracts also discourage bacterial growth. Glycerol monosterate is a colorless, odorless, and sweet-tasting flaky powder that is hygroscopic preservative agent; an emulsifying agent for oils. Surfactant or surface active agent, also known as wetting agent, lower the surface tension of a liquid, allowing easier spreading. Glycerol and sucrose serve good preservative for medium.
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Table 4. Component ratio of various combinations of Broth Media according to present invention ( BPL modifications) for the growth of Malassezia spp.
BPL IVIodified 1: I BPL IVIodlfied 2:
Constituents are same as BPL
Sucrose = 38-41 g modified 1 medium only difference
Peptone =10-12g is tliat tlie Millipore sterilized
Tween 20 =8-12 ml Cotton seed oil is added after
Oleic acid =12-14 ml autoclaving the medium.
Glycerol = 14-16 ml
Glycerol monosterate = 0.5-1 g
Ox bile = 8-10 g
Yeast extract = 12-14g
Streptomycin = 2-2.5Mg
Cotton seed oil = 12-14 ml
Distilled water = 1 lit.
Oil is added before autoclaving the medium.
BPL modified 3: BPL modified 4:
Sucrose =10-15 g Sucrose = 24-26 g
Peptone =08-10g Peptone = 12-14 g
Tween 20 = 5-6 ml Oleic acid = 10-12 ml
Oleic acid = 6-8ml Tween 20 =10-12 ml
Glycerol = 10-12 ml Glycerol = 10-12 ml
Ox bile = 3-6 g Yeast extract = 8-10g
Yeast extract = 2-5 g Streptomycin = 2- 2.5Mg
Streptomycin = 2- 2.5|jg Distilled water = 1 lit.
Cotton seed oil = 12-14 m
Distilled water = 1 lit.
BPL modified 5: BPL modified 6:
(BPLA2R0)
Sucrose = 11-14 g Sucrose = 12-18 g
Peptone = 6-9 g Peptone =6-10g
Sodium Chloride =1-3 g Tween 60 = 5-6 ml
Tween 60 = 1.5-4 ml Oleic acid = 6-8ml
Oleic acid = 3-5ml Glycerol = 5-8 ml
Glycerol = 4.5-7 ml Ox bile = 5-7 g
Glycerol monosterate = 0.2-0.9 g Yeast extract = 8-10 g
Ox bile = 1.2-3.5 g Streptomycin = 2-2.5|jg
Yeast extract = 1.5-4 g Olive oil = 14-16 ml
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Penicillin = 4-7.5 |jg Distilled water = 1 lit
Streptomycin = 3-7 (jg Cotton seed oil = 17-22 ml Distilled water = 1 lit.
• Agar(9.5-12 g) and Powder milk( 10-14 g) is only for solid medium
preparation.
TABLE 5. Observation was taken just after inoculation in the term of absorbance OD at 530 nm wave length.
Well I Media I Initial OP I NC
1 Leeming and Notman agar 1.908475 2.132 modified(mLNA)
2 Dutta and Dikshit Modified 0.166675 0.466
3 BPL Modified 1 0.880175 1.178
4 BPL Modified 2 0.659475 0.998
5 BPL Modified 3 0.702775 1.053
6 BPL Modified 4 1.708075 2.003
7 BPL modified 5 0.479375 | 0.775
8 RPMI 1640 0.062275 0.361
"9&10 I BPL Modified 6* I 1.350 | 1.4675
^Observation for BPL modified 6 was taken in replicate, hence initial OD and negative control (NC) was given in average .
19

TABLE 6. Reading was taken after 3 days of inoculation, absorbance OD was recorded at wave length 530 nm
1 23456 789 10 11 12
Uank Broth C Mf Mg Mr Msy Mn Mo Msl Mj My Md
MLNA A .0I»2|1^ 1.4te 1.788 2m7 1J87 1^ 1.80S 1.80S IM 1,387 1^ B>*oift t>DM e -OJMO'0.197 0.r^ 1.481 1J64 1.S54 2.007 025 1.467 1575 1.574 1114 Ml^,^
....II.1I.J.I....I.I....IIM »nwiuMi»»i» iiw iino) nijiii 'i loy » Jim i iiiii)i)»»».i« i i IIDIII ni» ■Mmiiii oiiim 1)1.111)))iiramin Tini i ) i i i in |-B-rinilirni
BPLMi c OJ0O1 •0J629 0.7S4 1567 1.733 1.816 1.854 1597 1.532 1.832 1,631 1.715 AliOfflfacO
-__. -_«*-. . _—_ . _____ 1"-"--" CaiTdteC
BPLM2 D .Oi»2:0j582 1.B30 1.S64 2m im 1j891 Om 0,834 U53 1.668 i8S3 |
—-— .fM.--.... —1. ,,ii|i|..,ii, .,.,11., I. u.ii)||.).w. ui ■)) I .1 i| .».»..,.r.»»l —.■.>—r^.. .T) )ii.,,;,y—, -» ». U I HH m
BPLM3 E O.0O2 0J683 1.1S2 1i84 2J(»1 1702 1.932 1671 1559 1j816 1«61 1700 StartR^
._.«»_. 1—.—_——..™—™««_i«,_-^ »««.„ 12:15AMI
BPI-M4 F 0OT1525 1M 1^ 2354 1,7^ 2m iM 1;J58 1^0 1462 1J98
"«■ "-— - ♦■*■* ■■■' ■<"— rtwrnanwiii, j»n.w*ii>wrri.<.fniii>ii*ji ».i!iirr...M.rt.miinw. x*in..(mMir[.... in>ii*miin.iffftMniD. iwiftinrrrt.iiiinifiiiiiiiiii !•*•» naimfLmWii ■Hmi.m.tmn A,., H. rin>*
BPLM5 0 0D01 ;0.477 1.461 1056 2.137 1^2 1945 1113 1160 17^ 1074 1276 RPM11640H 4)i»0.0.4S4 tm im 1.740 1898 1.753 1197 1.735 1J670 164S 1740
NC Broth C Mf Mg Mr Msy Mn Mob Msl Mj My Md
1 2 3 4 5 6 7 8 ^ 9 10 11 12
^ 1-380 1851&^ 1.^ 1^ 1878 1894|2570 19IQ[II17|I810 1^30
°'*'"''* M^55 t^jjlT^l^ 191311^193112Ml^|li4]l^1^
Column H* Medium (RPMIM) was observed heavy Fungus and bacterial growth because control OD drastically increases, Inclusive of either filamentous fungi or yeasts other than Malassezia
Top to Bottom
A- Leeming Notman Agar Modified
B- Dutta and Dikshit Modified
C- BPL Modified 1
D- BPL Modified 2
E- BPL Modified 3
F- BPL Modified 4
G- BPL modified 5
H- RPMI 1640 supplemented with fatty acid
Left to right-
1. Column 1- Blank (without media),
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2. Column 2- Broth Media only ,
3. Column 3- Malassezia furfur + Broth media,
4. Column 4-Malassezia globosa + broth media,
5. Column 5- M. restricta + broth media,
6. Column 6-M. sympodialis + broth media,
7. Column 7-M. nana + broth media,
8. Column 8-M. obtusa + broth media,
9. Column 9-M. slooffiae + broth media, 10.Column ^0-M. japonica + broth media,

11. Column 11- M. yamotensis + broth media,
12. Column 12- M. dermatis + broth media
Comparative Growth % in various candidate media was calculated by using following formula:
% Growth = O.DtrPatmPnt-O.D.rnntrnlX 1 00
O.D ■control
Here, O.Dtreatment = O.D. of Candidate well (medium+ inoculum) after 3 days O.D.controi = O.D. of Candidate broth medium
TABLE 7: Percentage growth of different Malassezia on various medium
n FM. FM. FM. FM. TM. FM. nil nil FM. FM.
fufur globo restrict sympodial nana obtus sloofffa japonic yamotens dermatis
sa a is a e a is
LNMA -24.9 -5.19 7A7 -5.24 -2.70 -4.29 -4.29 5^35 -1.00 095
DDM 48^8 197.98 295.17 212.0 303.5 166.59 195.17 272.26 216.70 224.74
2
21

BPLM1 I 19.8 I 149.12 I 175.51 i 188.71 I 194.7 I 153.89 I 143.56 i 191.25 I 159.30 I 172.65
5
BPLM2 76^9 168.72 249.65 212.88 224.9 16.83 8^93 218.38 186.25 185.73
1
BPLM3 68.66 138.38 192.97 149.19 182.8 144.65 128.21 165.88 143.19 148.90
6
BPLM4 -12.30 ^9!28 77.66 34.49 53.96 21.43 2^49 JM 10.33 slo
BPLM5 212.15 121.38 348.0 175.05 303.7 133.33 143.0 276.93 125.15 167.50
5
RPMIM 257.97 260.50 275.0 265.94 277.8 265.73 273.92 259.91 254.52 275.0
0
BPLM6 -8.17 0.995 22 -10.3 Z87 45.23* 4.03* sloS -2.95 -29.28
Column H* Medium (RPMIM) was observed heavy Fungus and bacterial growth because control OD drastically Increases, Inclusive of either filamentous fungi or yeasts other than Malassezia
Observation for BPLM6 medium was taken in replicate, hence % growth calculated by taking average of OD of broth and candidate well.
22

Table 8. O. D. of different candidate media, treatment media and negative control media
AB - CBS Principal Medium, CD - BPLA2R0 (BPL5) Medium, EF-BPL6 Medium, GH -BPLA2RM Medium
N€ Broth C Mf M% Mr Msy Mn IVtob M^ MJ My Md
1 2 3 4 5 8 ? 3 9 ID 11 12
coc ^ 0^ 0,B 1.06o|l.289 1.B 1.055 1.075 1^ 1,031 1JK4 1.463 0154 EndpoW
B iW 0.545 1.548 1627 im 1.2% 1.295 1,375 1.319 1317 1.4^ 1.172 ^ 530
C 0.792 0.374 T.430|l.7171.^ 1213 1^9 1533 i2?1^ 1,481 1^ AUOfiiixiOnce
(BPLM5) P 0.756 0.479 1.498 1.710 1.33111357 1^5 1.455 1,309 1J3 1.517 1273
E 1.380 1.^1 1.671 IM'IM 1678 1^ 2570 1.910 1517:1.810 1230 ^^^^ _
—__^—...,.^,^_„—,,...^.„_^ _^__ i:04fn 4/1/2001
^ 1S^ 1.8S8|1.743 1.890 1.913 1157 1,931 2M) 1.9% 1514 1.7S8 1J9 3 1.463 1.777 TSlio 1.7^' 1.781 1,7^ 1586 1.979 1.747:^i7W
BPLA2RM — ————^—-p ■ " i—— • —— '- —
H| 122111.44711.74411.618 1.494|1.4^ 1.^|l224|1.5Q3|l.4^|l.568|l.052
1 2 3456 789 10 11 12
MaiJi Broth C Mf Mg Mr hfey Mn Mo M^ ^^ "V «**
BPLM2 0 ^10(1210^ 1MI1.564 2m 1J21 1.K1 (JjgaJiO,^ 1.853 1,^ !j8^ I
BPLM3 E ornkmi1S2 1.^ 2.001 1.702 1.932 1171 ll559 1116 1.681 1.700 ^^
*'*^'^* ^ QjWoMii^ 1.102p^ 1J82 2m im\tm 1.810 i.4$a i398
^^^*^^ ^Q^uij^i-^ 1-056 2.137 U^]myi3[l^1^1^^ 1.m|l276
% Actual growth = 0-Dtreatment- 0-D- Negative control X 100
O.D. Negative control
Here,
O.D. treatment = O.D. of Candidate well (medium + inoculum) O.D. Negative control = O.D of negative treated candidate well (medium + inoculum + formaldehyde)
23

Table 9. O.D of various candidate media and % Actual Growth of Malassezia spp.
CBS Principal medium (IVILNA medium without adding external lipid supplement)
I NC I Mf I Mg I Mr I IVIsy I IVIn I Mob I H/Isi I MJ I My I Md ~ 0.634^ 1.06" 1.289 1-359 1.055" 1.075 1.325 1.031 1.0234 1.463 0.854
1.267 1.548 1.627 1.306 1.239 1.295 1.375 1.319 1.317 1.435 1.172
Average 0.9505 1.304 1.458 1.3325 1.147 1.185 1.35 1.175 1.1702 1.449 1.013
of OP I
% of Actual 37.19 53.39 40.18 20.67 24.67 40.03 23.61 23.11 52.44 6.57
Growth I I I I I I I I I I
BPLA2RO (Principal Medium Supplemented with cotton seed oil)
I 0.792 I 1.43 I 1.717 I 1.294 I 1.213 I 1.219 I 1.539 I 1.236 I 1.211 I 1.481 I 1.233
0.756 1.498 1.71 1.331 1.357 1.215 1.455 1.309 1.203 1.517 1.273
Average 0.774 1.464 1.713 1.312 1.285 1.217 1.497 1.272 1.207 1.499 1.253
of O.D. I
% of Actual 89.14 121.38 69.67 66.02 57.23 93.04 64.4 55.94 93.66 61.88
Growth I I I I I I I I I I
BPL6 medium
I 1.38 I 1.671 I 1.865 I 1.887 I 1.678 I 1.894 I 2.57 I 1.91 I 1.917 I 1.81 I 1.23~
1.555 1.743 1.89 1.913 1.657 1.931 2.84 1.958 1.914 1.798 1.399
Average 1.4675 1.707 1.8775 1.9 1.6675 1.9125 2.70* 1.934 1.915 1.804 1.3145
of OD * 5
% of Actual 16.32 27.90 29.47 13.62 30.32 84.32 31.78 30.52 22.93 -10.4
Growth I I I I I I I I I I
BPLA2RM (Principal Medium Supplemented with powder Milk)
I 1.463 I 1.777 I 1.639 I 1.888 I 1.758 I 1.781 I 1.734 I 1.986 I 1.979 I 1.747 I 1.54
1.221 1.447 1.744 1.618 1.494 1.463 1.569 1.224 1.503 1.459 1.052
Average 1.342 1.612 1.691 1.753 1.626 1.622 1.651 1.605 1.741 1.603 1.296
ofOD I
% of Actual 20.27 26.00 30.62 20.86 20.86 23.02 19.59 29.80 19.44 -3.42
Growth I I I I I I I I I I
BPLM1
I 1.178 I 0.754 I 1.567 I 1.733 I 1.816 I 1.854 I 1.597 I 1.532 I 1.831 I 1.630 I 1.715
% of Actual -35.99 33.02 47.11 54.15 57.38 35.56 30.05 55.43 38.37 45.58
Growth I I I I I I I I I I
BPLM2
I 0.998 I 1.030 I 1.564 I 2.035 I 1.821 I 1.890 I 0.680 I 0.634 I 1.853 I 1.666 I 1.653 % of Actual 3.206 56.71 100.50 82.46 89.38 -31.86 -36.47 85.67 66.93 65.63
Growth I I I I I I I
_^______^ BPLM3
I 1.053 I 1.152 I 1.594 I 2.001 I 1.702 I 1.932 I 1.670 | 1.559 | 1.816 | 1.661 | 1.700
% of Actual 9.40 51.37 90.03 61.63 83.48 58.59 48.05 72.46 57.73 61.44
Growth I I I I
BPLM4
24

I 2.003 11.162 11.202 I 2.354 [1.782 I 2.040 | 1.609 | 1.358 | 1.809 | 1.462 | 1.398 ~
% of Actual -41.98 -39.99 17.52 -11.03 185 -19.67 -0.32 -19.4 -27.00 -30.20
Growth I \ I I I I
^contamination in medium by other fungal species rather than Malassezia
Table 10: Actual growth percentage of various Malassezia species in various BPL Modified media
IVIedia IVIf Mg Mr Msy Mn Mob Msl Mj My Md
BPLM1 -35.99 33.02 47A] 54.15 57.38 35.56 30.05 55.43 38.37 45.58
BPLM2 3.206 56.71 100.5 82.46 89.38 -31.86 -36.47 85.67 66.93 65.63
BPLM3 9^4 51.37 90.03 61.63 83.48 58.59 48.05 72.46 57.73 61.44
BPLM4 -41.98 -39.99 17.52 -11.03 1.85 -19.67 ^032 -19.4 ^27 -30.2
BPLM5 89.14 121.38 69.67 66.02 57.23 93.04 64^4 55.94 93.66 61.88
(BPLA2R0)
BPLM6 16.32 27.9 29.47 13.62 30.32 84.32 31.78 30.52 22.93 -10.4
BPLA2RM 20.27 26 30.62 20.86 20.86 23.02 19.59 29!8 19.44 ^3^42
Table 11. Comparative price list of the media used for Culturing Malassezia spp. {In-vitro)
Cost per Broth medium
litre in ; , , , , , , , ,
INR. A B C D E F G H I J
424.8 265.5 204.94 97.15 247.7 1143.6 89.24 2056 2095 4879.36
6 7 8 8
Agar medium*
472.8 I 337.9 I 252.94 I 155.3 I 307.7 I 1215.6 I 137.2 I 2056 2095 4879.36
6 7 5 8 8 4
*Cost of complete agar media has been considered here, except RPMI 1640 and their supplement (H, I and J) solid form of these medium not available in the market.
25

^ Comparative Cost of Broth media used for Antifungal
t- suceptibilty testing and culturing of Malassezia spp.
S 487t.36
I /
.2 Jt 1143.68 /
.f "Z^-Se 204 94 247.78^/ X^^ /
£ABCDEFGHIJ
Media
Fig. 1 (I): A- Dixon brotli IVIedium, B- SDB, C- Dutta and Dil^shit brotli medium, D- LNB Medium, E- mINB IVIedium, F- Modified Clirombroth Candida, G-BPLA2R0 broth Medium, H-RPMI 1640 I- RPMI 1640 supplemented with fatty acid J- RPM11640 with MOPS
^ Comparative cost of Agar Media used for Culturing ^
s Malassezia spp. Aji.a
i /
.3 "f *
^ \ 3^^-^^ 252.94 iVl.mjr Ny^ /
*— '1* T|F 137.24
o 155735
£ Media
Fig.1 (I): A- Dixon Agar Medium, B- SDA, C- Dutta and Dikshit medium, D- LNA Medium, E- mLNA Medium, F- Modified Chromagar Candida, G- BPLA2R Medium, H- RPM11640 I- RPMI 1640 supplemented with fatty acid J- RPMI 1640 with MOPS
In order to fully illustrate the invention, following examples are set forth. It is to be understood that the examples are only by way of illustration and are not intended as an undue limitation on the broad scope of the invention as set forth in the appended claims.
26

Examples:
Example 1. BPL Modified 1 :
A culture medium (BPLM1) according to present invention is prepared by mixing 38.5 g of sucrose, 11 g of peptone, 13.5 g of yeast extract, 9.5 g of ox bile and 0.75 g of glycerol monosterate in a flask with a capacity holding more than 1 litre and then to this mixture were added 15 ml of glycerol, 9 ml of Tween 20, 12.5 ml of oleic acid and 13 ml of cotton seed oil. The final amount was made 1 litre by adding distilled water into the flask and resultant mixture was mixed up homogeneously to obtain a crude medium and finally flask was plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and 120 degree Celsius for 20 minutes. After proper sterilization, 2.5 |jg of streptomycin was added in the sterilized crude medium to obtain the broth culture medium ready for use.
For solid medium agar-agar should be added in an amount of 11 g to the medium before autodaving the mixture . Also, the lipid source used for solid medium is powder milk in an amount of 12 g rather than olive oil because powder milk help to form a homogenous mixture.
For a solid medium, 2.5 pg of streptomycin was added in the sterilized crude medium and resulting mixture was poured in to petriplates and allowed to solidify after which sterilized powder milk in an amount of 14 g is poured (overly) on the solidified medium to obtain a solid culture medium.
Example 2. BPL Modified 2 :
A culture medium (BPLM2) according to present invention is prepared by mixing 38.5 g of sucrose, 11 g of peptone, 13.5 g of yeast extract, 9.5 g of ox bile and 0.75 g of glycerol monosterate in a flask with a capacity holding more than 1 litre and then to this mixture were added 15 ml of glycerol, 9 ml of Tween 20 and 12.5 ml of oleic acid .and . The final amount was made 1 litre by adding
27

distilled water into the flask and resultant mixture was mixed up homogeneously to obtain a crude medium and finally flask was plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and 120 degree Celsius for 20 minutes. After proper sterilization, 2.5 pg of streptomycin and 13 ml of cotton seed oil sterilized by Millipore or syringe filter were added in the sterilized crude medium to obtain the broth culture medium ready for use.
For solid medium agar-agar should be added in an amount of 11 g to the medium before autoclaving the mixture . Also, the lipid source used for solid medium is powder milk in an amount of 12 g rather than olive oil because powder milk help to form a homogenous mixture.
For a solid medium, 2.5 pg of streptomycin was added in the sterilized crude medium and resulting mixture was poured in to petriplates and allowed to solidify after which sterilized powder milk in an amount of 14 g is poured (overly) on the solidified medium to obtain a solid culture medium.
Example 3. BPL Modified 3 :
A culture medium (BPLM3) according to present invention is prepared by mixing 14.5 g of sucrose, 9 g of peptone, 4.5 g of yeast extract and 9 g of ox bile in a flask with a capacity holding more than 1 litre and then to this mixture were added 11 ml of glycerol, 5.5 ml of Tween 20 and 7 ml of oleic acid . The final amount was made 1 litre by adding distilled water into the flask and resultant mixture was mixed up homogeneously to obtain a crude medium and finally flask was plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and 120 degree Celsius for 20 minutes. After proper sterilization, 2.5 pg of streptomycin was added in the sterilized crude medium and finally 13 ml of cotton seed oil sterilized by Millipore or syringe filter was added in the autoclaved crude medium to obtain the broth culture medium ready for use.
For solid medium agar-agar should be added in an amount of 11 g to the
medium before autoclaving the mixture . Also, the lipid source used for solid
28

medium is powder mill< in an amount of 12 g rather than olive oil because powder milk help to form a homogenous mixture.
For a solid medium, 2.5 pg of streptomycin was added in the sterilized crude medium and resulting mixture was poured in to petriplates and allowed to solidify after which sterilized powder milk in an amount of 14 g is poured (overly) on the solidified medium to obtain a solid culture medium.
Example 4. BPL Modified 5:
A culture medium (BPLM5 or BPLA2R0) according to present invention is prepared by mixing 12.5 g of sucrose, 8.5 g of peptone, 3.5 g of yeast extract, 3 g of ox bile, 1.5 g of NaCI and 0.4 g of glycerol monostearate in a flask with a capacity holding more than 1 litre and then to this mixture were added 5.5 ml of glycerol, 2.5 ml of Tween 60 and 4.5 ml of oleic acid . The final amount was made 1 litre by adding distilled water into the flask and resultant mixture was mixed up homogeneously to obtain a crude medium and finally flask was plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and 120 degree Celsius for 20 minutes. After proper sterilization, 5.5 |jg of streptomycin and 5.5 |jg of penicillin were added in the sterilized crude medium and finally 18.5 ml of cotton seed oil sterilized by Millipore or syringe filter was added (overlay) in the autoclaved crude medium to obtain a culture medium which is broth.
For solid medium agar-agar should be added in an amount of 11 g to the medium before autoclaving the mixture . Also, the lipid source used for solid medium is powder milk in an amount of 12 g rather than cotton seed oil because powder milk help to form a homogenous mixture.
For a solid medium, 5.5 |jg of streptomycin and 5.5 pg of penicillin were added in the sterilized crude medium and resulting mixture was poured in to petriplates and allowed to solidify after which sterilized powder milk in an amount of 14 g is poured (overly) on the solidified medium to obtain a solid culture medium.
29

Example 5. BPL Modified 6 :
A culture medium (BPLM6) according to present invention is prepared by mixing 16 g of sucrose, 9.5 g of peptone, 8.5 g of yeast extract and 5 g of ox bile in a flask with a capacity holding more than 1 litre and then to this mixture were added 7 ml of glycerol, 5 ml of Tween 60 and 7 ml of oleic acid . The final amount was made 1 litre by adding distilled water into the flask and resultant mixture was mixed up homogeneously to obtain a crude medium and finally flask was plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and 120 degree Celsius for 20 minutes. After proper sterilization, 2.5 |jg of streptomycin was added in the sterilized crude medium and finally 15 ml of olive oil sterilized by Millipore or syringe filter was added in the autoclaved crude medium to obtain the broth culture medium ready for use.
For solid medium agar-agar should be added in an amount of 11 g to the medium before autoclaving the mixture . Also, the lipid source used for solid medium is powder milk in an amount of 12 g rather than olive oil because powder milk help to form a homogenous mixture.
For a solid medium, 2.5 |jg of streptomycin was added in the sterilized crude medium and resulting mixture was poured into petriplates and allowed to solidify after which sterilized powder milk in an amount of 14 g is poured (overly) on the solidified medium to obtain a solid culture medium.
Example 6. BPL Modified 4 (Comparative):
A culture medium (BPLM4) according to present invention is prepared by mixing
24.5 g of sucrose, 12.5 g of peptone and 9.5 g of yeast extract in a flask with a
capacity holding more than 1 litre and then to this mixture were added 11 ml of
glycerol, 10.5 ml of Tween 20 and 11 ml of oleic acid . The final amount was
made 1 litre by adding distilled water into the flask and resultant mixture was
mixed up homogeneously to obtain a crude medium and finally flask was
plugged and kept for sterilization in an autoclave at 103.4 KPa pressure and
120 degree Celsius for 20 minutes. After proper sterilization, 2.5 pg of
30

streptomycin was added in the sterilized crude mediunn to obtain the broth culture medium ready for use.
For solid medium agar-agar should be added in an amount of 11 g to the medium before autoclaving the mixture .
For a solid medium, 2.5 |jg of streptomycin was added in the sterilized crude medium and resulting mixture was poured in to petriplates and allowed to solidify to obtain a solid culture medium.
Example 7 (Comparative):
All the BPL modifications (BPL1 to 6), Dutta and Dikshit medium, modified lemming and Notman broth and RPMI 1640 supplemented with fatty acid were prepared. Components ratio of various combination of Broth Media (BPL modifications) is as given above in Table 4 and other used media as given below:
Leeming and Notman Agar modified (IVILNA) CBS
Peptone =10 g
Glucose = lOg
Yeast extract = 2g
Ox bile =8 g
Glycerol= lOmL
Glycerol monostearate = 0.5g
Tween 60 = 5mL
Olive oil = 20 mL
Agar=15g
Chloramphenicol = 0.5 g
Cycloheximide = 0.5 g
Deionized Water = 1L
Dutta and Dikshit IVIodified Culture IVIedium
(DDM)
Sucrose = 32g Yeast extracts = 8g Peptone = 2g Glycerol = 4ml Oleic acid = 4ml Penicillin = 5pg
31

Oil (olive/Coconut) = 20ml Distilled water to 1 lit Agar=13gm Streptomycin =5^9 Distilled water to 1 litre
RPM11640 medium with supplement
RPMI1640=7.2gm
MOPS = 34.72 g
Glucose = 20 g
Ox bile (Oxoid) = 4 g
Glycerol = 1 ml
Glycerol monostearate = 0.5 g
Tween 20 = 0.4 ml
0.5 McFarland standards inoculum of fastidious 10 tested Malassezia species
were prepared in different media .Observation was taken just after inoculation in
the term of absorbance OD at 530 nm wave length. Result is given in table 5. In
column 2 of 96 well plate, 200 pi broth media was added while 100 pi broth
media was added in Column 3-12, respectively. (Different tested media pipetted
in each row A-H as indicated In Table 6). In 96 well plate, Column 1 is blank
(empty, without media). Column 2 is broth control, and
Column 3-12: inoculum 100 IJI was added respectively:
Column 3- Malassezia furfur + broth media.
Column 4-Malassezia globose + broth media,
Column 5-M. restricta + broth media.
Column 6-M. sympodialis + broth media.
Column 7-M. nana + broth media,
Column 8-/W. obtuse + broth media,
Column 9-M. slooffiae + broth media,
Column ^0-M. japonica + broth media.
Column 11- A/f. yamotensis + broth media.
Column 12- M. dermatis + broth media).
32

In this way final concentration of each well become 200 |jl, except blank which was empty. Then candidate plate kept for incubation at 32-34°C for 3 days in a moist, dark chamber. Reading was taken after 3 days of inoculation; absorbance OD was recorded at wave length 530 nm (result is given in table 6). Comparative Growth % in various candidate media was calculated by using formula (result is given in Table 7). On the basis of observation of results and growth of Malassezia in various media, BPL modified medium 1, 2, 3, 5 and 6 was selected and good growth of entire range of Malassezia species was reported. Column H* Medium (RPMIM) was observed heavy Fungus and bacterial growth because control OD drastically increases. Inclusive of either filamentous fungi or yeasts other than Malassezia. Dutta and Dikshit medium not supported good growth of entire range of Malassezia species, one another problem associated with DDM medium is contamination by undesired filamentous fungi (O.D of control 0.166 after 3 days changes into 0.197 due to growth of undesired filamentous fungi rather than Malassezia) because of high amount of carbon source used in Dutta and Dikshit Medium.
Example 8 (Comparative):
The present example relates to conformation of good growth of fastidious 10 tested Malassezia species in BPL modified media 1, 2, 3, 5 and 6. The screened BPL modified media 1,2,3 and 5 was further tested for actual growth of Malassezia species along with (MLNA) CBS medium, BPL modification 6 (lipid source olive oil), BPLA2RM (lipid source Milk) in a replicate of each well. Experiment was designed as per table 8. Proper broth control and negative control was maintained. Inoculum and broth media were added in each well and each row respectively. Thereafter, the plate is kept for incubation at 32-34 °C for 3 days in a moist, dark chamber. O. D. of 96 well plate was observed after 3 days at wavelength of 530 nm. Observation was recorded (in Table 8). Per cent actual growth of each tested Malassezia species in each well was calculated by using formula result is given in Table 9. Good growth of 10 fastidious tested Malassezia species was observed in BPL modified medium 1, 2, 3, 5 and 6 .
33

Advantages of the culture medium of present invention:
1. The growth medium of present invention support good growth of all Malassezia spp. in short time span of 2 to 3 days.
2. Medium of present invention is cheaper and economical compared to other related medium.
3. The medium of present invention significantly reduces the risk of contamination by using very accurate amount of each component of the medium and without affecting the proper growth of Malassezia spp. and this requisite amount of each component is novelty of this invention.
4. The conventional bacteriological procedures such as microbial streaking of pure culture, antifungal susceptibility testing could be carried out for the Malassezia species and also useful for other Unicellular yeast.

5. The growth medium of present invention is best for direct isolation of Malassezia species from clinical specimen and subsequent culturing of Malassezia.
6. Medium has a longer shelf life as this can be used for more than 3 months, the maximum period taken into consideration provided it is kept in 5-10°C . This condition can be easily maintained by keeping it in refrigerator.
7. Good growth of Malassezia has been reported in broth medium, medium fit for susceptibility testing.
8. In case BPLA2R0 medium Penicillin and streptomycin combination is used in order to avoid costly antibiotics like Chloramphenicol or Cycloheximide.
34

Scope of medium of present invention and its uses:
1. This broth medium can be used in research labs for research purposes as well as discovery of new anti-microbials.
2. Pathological labs of Medical colleges, Private labs can be using it for diagnostic purposes for confirming the cause of Pityriasis versicolor (Senhua), Seborrheic dermatitis, Dandruff etc.
3. Susceptibility testing can also be performed by this medium and ultimately the patients will be getting early relief.
4. The Medium will play important role in economically challenged area for direct isolation of Malassezia species from clinical specimen and subsequent culturing of Malassezia.
5. Number of human pathogenic unicellular yeast can also be grown
successfully such as Candida on modified medium.
6. The cost of medium is very cheaper comparable to other solid and broth
medium for growth of Malassezia spp. So it can be positively presume that
media will succeed in Indian market as well as entire world.
35

We Claim:
1. A culture medium, comprising a disaccliaride, peptone, yeast extract, ox-bile, glycerol, fatty acid, lipid source, antibiotic, surface active agent and optionally additives.
2. The culture medium as claimed in claim 1, wherein the culture medium is broth.
3. The culture medium as claimed in any of the preceding claims, wherein the disaccharide is selected from the group consisting of sucrose, lactose, maltose, trehalose and melibiose, preferably sucrose.
4. The culture medium as claimed in any of the preceding claims, wherein the fatty acid is oleic acid.
5. The culture medium as claimed in any of the preceding claims, wherein the lipid source is cotton seed oil or olive oil.
6. The culture medium as claimed in any of the preceding claims, wherein the antibiotic is selected from the group consisting of streptomycin, penicillin or a combination thereof.
7. The culture medium as claimed in any of the preceding claims, wherein the additive is selected from the group consisting of osmoregulator, glycerol monostearate or a combination thereof.
8. The culture medium as claimed in claim any of the preceding claims, wherein the osmoregulator is selected from the group consisting of NaCI or KCI, preferably NaCI.
9. The culture medium as claimed in claim any of the preceding claims,
36

wherein the surface active agent is selected from the group consisting of Polyethylene glycol sorbitan monostearate (Tween 60), Polyoxyethylene sorbitan monopalmitate (Tween 40) and Polyoxyethylene sorbitan monolaurate (Tween 20), preferably Polyethylene glycol sorbitan monostearate (Tween 60) or Polyoxyethylene sorbitan monolaurate (Tween 20).
10. The culture medium as claimed in any of the preceding claims, wherein the disaccharide is in the range of 10-41 g per litre of the culture medium.
11. The culture medium as claimed in any of the preceding claims, wherein the fatty acid is in the range of 3-14 ml per litre of the culture medium
12. The culture medium as claimed in any of the preceding claims, wherein the lipid source is in the range of 10-22 ml per litre of the culture medium.

13. The culture medium as claimed in any of the preceding claims, wherein the antibiotic is in range of 2-14.5 pg per litre of the culture medium.
14. The culture medium as claimed in any of the preceding claims, wherein the osmoregulator is in the range of 1-3 g per litre of the culture medium.

15. The culture medium as claimed in any of the preceding claims, wherein the glycerol monostearate is in the range of 0.2-1 g per litre of the culture medium.
16. The culture medium as claimed in any of the preceding claims, wherein the surface active agent is in the range of 1.5-12 ml per litre of the culture medium.
17. The culture medium as claimed in any of the preceding claims, wherein 1
litre of the culture medium comprises 10-41 g of a disaccharide, 6-12 g of
peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile, 4.5-16 ml of glycerol, 3-
14 ml of fatty acid, 10-22 ml of lipid source, 2-14.5 pg of antibiotic, 1.5-12 ml of
surface active agent, optionally additives and balance being distilled water.
37

18. The culture medium as claimed In any of the preceding claims, wherein 1 litre of the culture medium comprises 38-41 g of sucrose, 10-12 g of peptone, 12-14 g of yeast extract, 8-10 g of ox-bile, 14-16 ml of glycerol, 0.5-1 gm of glycerol monostearate, 12-14 ml of oleic acid, 12-14 ml of cotton seed oil, 2-2.5 |jg of streptomycin and 8-12 ml of Polyoxyethylene sorbitan monolaurate (Tween 20) and balance being distilled water.
19. The culture medium as claimed in any of the preceding claims, wherein 1 litre of the culture medium comprises 10-15 g of sucrose, 8-10 g of peptone, 2-5 g of yeast extract, 3-6 g of ox-bile, 10-12 ml of glycerol, 6-8 ml of oleic acid, 12-
14 ml of cotton seed oil, 2-2.5 |jg of streptomycin and 5-6 ml of
Polyoxyethylene sorbitan monolaurate (Tween 20) and balance being distilled
water.
20. The culture medium as claimed in any of the preceding claims, wherein 1 litre of the culture medium comprises 11-14 g of sucrose, 6-9 g of peptone, 1.5-4 g of yeast extract, 1.2-3.5 g of ox-bile, 4.5-7 ml of glycerol, 0.2-0.9 g of glycerol monostearate, 3-5 ml of oleic acid, 17-22 ml of cotton seed oil, 3-7 pg of streptomycin, 4-7.5 pg of penicillin, 1-3 g of NaCI and 1.5-4 ml of Polyethylene glycol sorbitan monostearate (Tween 60) and balance being distilled water.
21. The culture medium as claimed in any of the preceding claims, wherein 1 litre of the culture medium comprises 12-18 g of sucrose, 6-10 g of peptone, 8-10 g of yeast extract, 5-7 g of ox-bile, 5-8 ml of glycerol, 6-8 ml of oleic acid, 14-16 ml of olive oil, 2-2.5 pg of streptomycin and 5-6 ml of Polyethylene glycol sorbitan monostearate (Tween 60) and balance being distilled water.
22. The culture medium as claimed in claim 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or 16 wherein the culture medium is a solid medium comprising a
disaccharide, peptone, yeast extract, ox-bile, glycerol, fatty add, lipid source,
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antibiotic, surface active agent and optionally, additives.
23. The culture medium as claimed in claim 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16 or 22 wherein the lipid source is powder milk.
24. The culture medium as claimed in claim 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 22 or 23 wherein the additive is selected from the group consisting of agar-agar and osmoregulator, glycerol monostearate or a combination thereof.
25. The culture medium as claimed in claim 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 22 , 23 or 24 comprising 10-41 g of sucrose, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile, 4.5-16 ml of glycerol, 3-14 ml of oleic acid, 10-14 g of powder milk, 3-7 |jg of streptomycin, 4-7.5 |jg of penicillin, 1-3 g of NaCI and 1.5-4 ml of Polyethylene glycol sorbitan monostearate (Tween 60) and 9.5-12 g of agar-agar and distilled water to make 1 litre mixture.
26. A process for the preparation of culture medium as claimed in any of the
claims 1-21 comprising:
(i) Mixing 10-41 g of disaccharide, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile in a pre-sterilized flask;
(ii) Adding 3-14 ml of fatty acid, 4.5-16 ml of glycerol and 1.5-12 ml of surface active agent to the mixture obtained in step (i) making final amount 1 litre by adding distilled water to obtain a crude medium by homogeneously mixing all the components;
(ill) Sterilizing the crude medium as obtained in step (ii) in an autoclave;
(iv) Adding 2-14.5 pg of antibiotic to the sterilized mixture obtained from step (iii);
(v) Pouring lipid source into the mixture either prior to or subsequent to the step
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(iii) to obtain the culture medium.
27. A process for the preparation of culture medium as claimed in any of the
claims 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or
24 comprising:
(i) Mixing 10-41 g of disaccharide, 6-12 g of peptone, 1.5-14 g of yeast extract, 1.2-10 g of ox-bile in a pre-sterilized flask;
(ii) Adding 3-14 ml of fatty acid, 4.5-16 ml of glycerol and 1.5-12 ml of surface active agent to the mixture obtained in step (i) making final amount 1 litre by adding distilled water and homogeneously mixing 9.5-12 g of agar-agar with all the components to obtain a crude medium;
(iii) Sterilizing the crude medium as obtained in step (ii) in an autoclave;
(iv) Adding 2-14.5 pg of antibiotic to the sterilized mixture obtained from step (iii);
(v) Pouring mixture obtained in step (iv) into petriplates and finally pouring powder milk to obtain the culture medium.
28. The process as claimed in claim 25 or 26, wherein sterilization in step (iii) is carried out at a pressure ranging from 68.95 KPa - 206.84 KPa.
29. The process as claimed in claim 25 or 26, wherein sterilization in step (iii) is carried out at a temperature ranging from 115.5 °C -135 °C.
30. The process as claimed in claim 25 or 26, wherein sterilization in step (iii) is carried out for time ranging from 20-30 minutes.
31. The culture medium as claimed in any of the preceding claims as and when used in culturing and testing procedures for all the known Malassezia spp. and
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other unicellular yeast.
Dated this 27'^ day of February, 2012.
(Anuradha Salhotra)
Of LALL LAHIRI & SALHOTRA
AGENTS FOR THE APPLICANT
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