chapter 2 isolation and screening of antifungal actinomycetes

25 Pages·2017·1.99 MB·English

Isolation and screening.
CHAPTER 2
ISOLATION AND SCREENING OF
ANTIFUNGAL ACTINOMYCETES
41
Isolation and screening.
CHAPTER 2
ISOLATION AND SCREENING OF ANTIFUNGAL
ACTINOMYCETES
2.1 INTRODUCTION
2.1.1 Sites for sampling
2.2 MATERIALS AND METHODS
2.2.1 Collection of samples
2.2.2 Estimation of pH of the samples
2.2.3 Determination of the temperature on the site
2.2.4 Preparation of starch casein medium
2.2.5 Isolation and preservation of actinomycetes
2.2.6 Screening the actinomycetes for antifungal activity
2.2.6.1 Giant colony technique
2.2.6.2 Agar disc diffusion technique
2.2.6.3 Agar well diffusion technique
2.3 RESULTS
2.3.1 Isolation of actinomycetes
2.3.2 Screening of actinomycetes
2.4 DISCUSSION
2.5 REFERENCES
42
Isolation and screening.
2.1 Introduction
Microorganisms are known to produce secondary metabolites, which are being
utilized by man since early 1940s. Actinomycetes have been recognized as the potential
producers of such metabolites as antibiotics, growth promoting substances for plants
and animals, immunomodifiers, enzyme inhibitors and many other compounds of use to
man (Okami, 1986). Fungi are eukaryotic and have machinery for protein and nucleic
acid synthesis similar to that of higher animals. It is, therefore, very difficult to find out
compounds that selectively inhibit fungal metabolism and exhibiting no toxicity to
humans (Glazer and Nikaido, 1995). There is an evidence that some fungal strains are
resistant to certain antimycotic drugs with resulting therapeutic failures (Macura, 1993).
There is lack of effective and safe antifungal antibiotics (Ringel, 1990). Therefore, there
is a pressing need of nontoxic and effective antifungal antibiotics. The pioneering work
of Waksman showed that actinomycetes are capable of producing medically useful
antibiotics (Nolan and Cross, 1998). Actinomycetes are diverse group of heterotrophic
prokaryotes forming hyphae at some stage of their growth, hence referred as
filamentous prokaryotes (Gottlieb, 1973). They are a successful group of bacteria that
occur in a multiplicity of natural and man-made environments and a unique group
having different morphological, cultural, biochemical and physiological characters
(Goodfellow and Williams, 1983; Lechevalier and Lechevalier, 1967). Approaches to
the search for and discovery of new antibiotics are generally based on screening of
naturally occurring actinomycetes (Okami and Hotta, 1988). Actinomycetes are well
established as producers of antimicrobial agents and are known to produce two-thirds of
today's antibiotics. These antibiotics include agents against bacteria, fungi, algae,
viruses, antitumour agents and antimalarial agents. Production of antibiotics has been
the greatest bioactivity of actinomycetes. Today also they are being screened on large
scale for finding new antimicrobial agents. Eventhough the rate of discovery of new
antibiotics has been decreased, new approaches in screening for antimicrobials, logistic
approaches in exploration of new ecological niches and varied microbial sources are
showing good results. It is being said that only 10% of the antibiotics have been
43
Isolation and screening.
discovered till now, leaving large percent of antibiotics undiscovered (Franco and
Countinho, 1991).
Actinomycetes have been described as the potential source of antibiotics. They
have provided more than 4000 of the naturally occurring antibiotics, including many of
those important in medicine, such as amino glycosides, anthracyc lines,
chloramphenicol, macrolides, tetracyclines etc (Okami and Hotta, 1988). Approaches to
the search and discovery of new antibiotics are generally based on screening of
naturally occurring actinomycetes and on biotechnological manipulation of known
antibiotics producing strains. However, current efforts to find new antibiotics faced with
the difficulty that the probability of discovering them is declining as the number of
known antibiotics is increasing. It is therefore, important to device methodologies to
enhance the probability of discovering new antibiotics. To achieve this goal, an
appropriate understanding of the actinomycetes capable of producing antibiotics in
terms of microbiology, biochemistry and genetics will be required to form basis for
future programs (Okazaki and Okami, 1976). The study of antibiotics and other
fermentation products has shown that microorganisms produce a seemingly unlimited
number of compounds with diverse structures. The continued high rate of discovery of
new chemical entities is due to creative screening procedures. These screening
procedures incorporate features such as the emphasis on unusual microorganisms, their
special propagation and fermentation requirements, supersensitive and highly selective
assays and genetic engineering both for the biosynthesis of new compounds.
Development in early in vivo evaluation, improved isolation techniques, modem
procedures for structure determination, computer assisted identification and an efficient
multidisciplinary approach has increased the rate of discovery of new antimicrobial
compounds (Franco and Countinho, 1991).
Secondary metabolites are being successfully screened for use, in the fields of
agriculture as pesticides and herbicides, antiparasitic compounds and in noninfectious
human diseases as enzyme inhibitors, immunomodifiers and antihypertensives.
Enzymes of microbial origin are another important products, which are widely used in
food processing, detergent manufacturing, the textile and pharmaceutical industries.
44
Isolation and screening.
medical therapy, bioorganic chemistry and molecular biology. Actinomycetes are being
explored for the production of enzymes. Nowadays actinomycetes are being widely
utilized in the production of some enzymes on industrial scale, due to their high yield
and cost efficiency. Many of the enzymes of actinomycetes origin yet remain to be
harnessed (Czoch and Mordarski, 1988).
Actinomycetes play a major role in producing antibiotics and other metabolites
such as extracellular enzymes, pigments and growth promoting factors (Vanajakumar et
al, 1991). Thus the unexplored environments can be a potent source for finding both
new actinomycetes and new antibiotics or biologically active substances.
2.1.1 Sites for sampling:
Mula river:
Samples were collected from the spot near Aundh area, Pune, where the water
is stagnant and polluted due to the discharge of various domestic and industrial
effluents. The pH and temperature of the water samples were found to be 7.2 and
30.2°C respectively. The flora and fauna found in this location was indicative of the
organic enrichment. Species of algae like Chlamydomonas, Chlorogonium, Pandorina,
Euglena, Oscillatoria, Microcystis, Stigeoclonium, Schizomeris, Nitzschia,
Gomphonema, Melosira, Vaucheria and Volvocales are abundant. Water hyacinth is
abundant and common snails {Viviparus sp. and Lymnaea sp.), common shrimps
{Caradina sp. and Macrobrachium sp.) are found.
Mutha river:
Water samples were collected from Mutha river near WellesJy bridge, Pune.
The site is heavily polluted especially with domestic effluents received from the
adjoining regions. The flora and fauna are nearly similar to that of Mula river. Samples
were also collected near Sangam bridge, which marks the confluence of rivers Mula and
Mutha.The water is generally stagnant throughout the year except during rainy season.
The water is polluted with industrial and domestic wastes particularly with the hutments
located at the riverbank. As a result of extensive waste disposal the river water appears
45
Isolation and screening.
dark and turbid. Water iiyacinth is common near tiie riverbanic. The pH and temperature
of the water samples were found to be 6.8 and 29.8°C respectively
Pavana river:
This river receives most of the industrial wastes from the Pimpri-Chinchwad
industrial area, near Pune. This river was highly contaminated with the domestic and the
industrial wastes. Household activities like washing of clothes and utensils at the
riverbank are prevalent. The water samples were collected from the flowing river. The
pH and temperature of the water samples were found to be 6.9 and 30.6°C respectively.
Since contamination was caused due to the industrial areas around the river, the flora
and fauna was seen to be less.
Pune University campus:
University of Poona is located on a very big piece of land north-west part of
Pune, established in the year 1948. The main building is an Italian Gothic style built
with local grey rock and surrounded by well kept green lawns. Soil samples were
collected from the Alice garden inside the university campus. The pH of this soil
sample was 7.1. The soil of Pune University allows the growth of various plants and
mushrooms such as puffballs. Shiitake, Pleurotus, Auricularia, Marasmius, Hygrocybe,
Hexagonia and Dalbergia melanaxylon leading to prevalence of animals such as
monkeys, cows, sheeps, mongoose and snakes such as Ratsnake, Sheildtail, Trinket,
Wolfsnake, Racer, Russesl's viper, Binocellate cobra and Kukri.
Sinhagad:
An unmistakable landmark, the towering Sinhgad fort (1,290 meters from sea
level) is one of the most popular picnic spots situated at about 25 kms from Pune.
Sinhgad is surrounded by thickly forested valleys and rises tall at one end of the
Khadakwasala lake. The water from this lake enriches the soil of the fort leading to
thick plantations on and around the fort. Soil samples were collected from the eastern
part of the fort. The pH of the soil sample was 6.9.
46
Isolation and screening.
Ulsoor lake:
Ulsoor lake which is located in the Eastern side of Bangalore, spreads over an
area of 1.5 sq km, 11'-12' deep and is an ideal place for sight seeing and boating. It is
said to date back to the time of Kempe Gowda I and II in the 16'^ century. Two springs
exist at the bottom of the lake, but they are now covered with heavy silt. It was
constructed by Sir Lewin Bentham Bowring, the then commissioner of Bangalore. This
lake contains soft aquatic grass, weeds, algae, zooplanktons and mosquito larvae. It has
a variety of fish of grass carp, silver carp, catia and mrigal varieties. These manage to
control the weed growth which is major problem in the lake leading to milky green
colour, foul smell and slimy layer. The quantity of silt on the lake bed is about five lakh
metric tonnes. Water samples were collected from Ulsoor lake in the month of
September. The pH of the water sample was 7.4.
Cubbon Park :
In 1864, Lord Cubbon, the then Viceroy of India, laid out 300 acres of verdant
tranquility. Today, it remains an oasis of greenery in the heart of modem Bangalore.
This beautiful park is the haunt of the seekers of pleasure and tranquility and is highly
popular with walkers, joggers and nature lovers. Soil samples were collected from the
southern part of the park where the pH of the soil was found to be 7.0.
Lalbagh Botanical Garden:
This 240 acres expanse of greenery forms one of India's most beautiful botanical
gardens. The credit for this goes to the rulers Hyder Ali and his son Tipu Sultan, who
laid out an 18th century paradise with rare trees brought from far off Italy, Persia,
Afghanistan and France. At the heart of the gardens is situated the Glass House. Its
design was obviously inspired by the Crystal Palace, London. The garden has a very
good and rich quality soil that supports the growth of various plants especially red rose
and so the name 'Lalbagh'. The annual flower show is a major attraction of Lalbagh.
47
Isolation and screening.
The soil sample was collected from the green house, which is well known for its various
kinds of flora. The pH of the soil sample was 7.2.
Bharat river:
liharat river is the second longest river in Kerala, after Periyar river, and Hows
b\ the southern border of Malapuram district and drains into the sea of Ponnani. There
are a plcnt> of tlsh in this river and it is known to enrich a variety of tree plantations
such as teak, rosewood, choropin and mahogany and surrounded by animals such as
elephants, deers, tigers, monkeys, reptiles and birds. Samples were collected from the
river where the aquatic flora and fauna was high and the contamination caused due to
industrial and domestic waste was relatively ver>' low. The pH of the water sample was
7.2.
Nilanibur Teak Plantafion:
Nilambur is a beautiful town on the bank of the Chalixar river at a distance of
about 70 km from C'ahcut and 100 km from the hill-station Ooty (Uddagamandalam).
It is located in the Malappuram district of Kerala. The world's first teak plantation was
raised in the Conoll> Plot in Nilambur about 150 years ago. Named in honor of H.V.
Conolly. the then Collector of Malabar, the plot covers an area of 2.3 1 hectares of teak
plantation. It is maintained by the Kerala Forest Research Institute in collaboration with
the Kerala Forest Department. Soil samples were collected from the western part of this
plantation, where the pFl of the soil was 6.8.
Since actinomycetes play a major role in producing antibiotics and other
metabolites, the diverse soil and water environments can be a potent source for finding
new actinomycetes and novel antifungal antibiotics with less or no to.xicity to human
beings.
48
Isolation and screening.
2.2 Materials and methods
2.2.1 Collection of samples
Water samples were collected from different locations as seen from table (2.1)
using point-integrating sampler and b> the procedures recommended in APHA (Cleseri
et uL. 1989). Samples were collected in sterile containers and were held at 4''C until
analysis in case of any delay, which was avoided during most samplings. Automated
point integrated samplers were used to obtain samples at desired depth of shallow water
bodies. A sampling bottle and two lengths of bent tubing acting as intake and exhaust
pipes are the simplest form of this type of sampler. The bottle starts to till till the depth
of flow rises to the outlet of the air exhaust pipe.
For the collection of soil samples hand-held scoops (10 to 100 g capacity),
spoons (100 to 300 g capacity) and shovels were used for sampling near surface soils.
The top layer of the soil was removed to the desired sample depth with a clean spade
and then using a clean stainless steel scoop, plastic spoon, or trowel, a thin layer of soil
from the area which came in contact with the spade was removed and discarded. I'he
sample was transferred to an appropriate, labeled sterile sample container with a sterile
laboratory spatula, or equivalent.
2.2.2 Estimation of pH of the samples
Ihe pH of the water samples was measured using a digital pH meter (Orion Co.,
USA). The system was calibrated with the standard buffers provided with the unit. The
samples were stirred to ensure homogeneity prior to recording the pH. Soil samples
were mixed with distilled water in 1:1 ratio and mixed vigorously, this slurr\ was
allowed to stand for 3-4min and then the pi 1 of the suspension was recorded.
2.2.3 Determination of temperature on the site
The temperature of the samples was determined on site using a digital
thermometer. Ihe thermometer probe was retained in the sample till constant reading
was attained. The temperature was recorded in degree C elsius.
49
Isolation and screening.
2.2.4 Preparation of starch casein medium
Starch casein medium (SCM) of pH 7 was used for the isolation of
actinomycetes. The medium contained (g/IOOml) soluble starch 1, casein 0.1, KH2PO4
0.05, MgS04.7H20 0.05, agar 3. The medium was sterilized at 12TC for 15 minutes.
2.2.5 Isolation and preservation of actinomycete isolates
Actinomycetes from the soil and water samples were isolated on starch casein
agar by using standard plate count method (Collins et al, 1995). Isolation of
actinomycetes was done by suspending Ig of soil sample in 10ml sterile saline, which
was vigorously shaken and then allowed to settle for 5 min. The supernatant was
serially diluted and 0.1 ml of each dilution plated on starch casein agar (SCA) of pH 7.
Similarly the water samples were analyzed. The plates were incubated at 37°C for a
period of 7-10 days. Actinomycetes were recognized by their characteristic tough,
leathery colonies branched vegetative mycelia and spore formation. They were isolated
by repeated transfer on starch casein agar. All isolates were purified by using streak
plate technique and confirmed by colony morphology (Casida, 1995).
Pure strains of actinomycete isolates were maintained at 4°C on starch casein
agar. They were also preserved at -20°C in 20% glycerol in distilled water, where
glycerol acts as a cryoprotective agent.
2.2.6 Screening the actinomycetes for antifungal activity
All the actinomycete strains isolated were screened for their antifungal activity
by three methods.
2.2.6.1 Giant Colony technique
The actinomycete isolates were inoculated on starch casein agar plates as a
straight line and incubated at 37''C for 7 days. After 7 days, different test
microorganisms such as Aspergillus niger NCIM586, Aspergillus flavus NCIM1028,
Aspergillus fumigatus MTCC2544, Fusarium oxysporum NCIM1072, Candida albicans
50