Isolation and screening of antibiotic producing Actinomyces species from soil sample

 

Abhijit Ray

HOD, Department of Biotechnology, Raipur Institute of Technology, Raipur (CG)

*Corresponding Author E-mail: abhijitray_2001@yahoo.com

 

ABSTRACT:

Actinomyces species were successfully isolated from soil sample in and around Raipur Institute of technology, Raipur, CG. The strains were morphologically studies and their antibiotic potential was observed. Later plasmid curing was done to perform the location of antibiotic resistant genes.

 

KEY WORDS: Actinomyces, plasmid curing, antibiotic, isolation, soil

 


INTRODUCTION:

Most bacteria (singular, bacterium) are very small, on the order of a few micrometers (10-6 meters) in length. Bacteria come in a wide variety of shapes and sizes, called the morphology of the organism. The most common shapes are rod-like, called the bacillus form, or spherical, called thecoccus form. The rod forms vary considerable from very short rods that almost look like cocci, to very long filaments thousands of microns in length. Bacteria also form spirals and corkscrews, ovals (coccoid), commas, and elaborately branched structures. The cocci often take on multi-cell forms; as two cocci joined together (diplococcic), as chains of cocci (streptococci), or as tetrads (four cells in a cube). 

 

Bacteria can be distinguished based on the cell wall structure. Bacteria with a thin wall layer and an outer membrane stains red with gram staining and are called gram negative. Bacteria with a thicker wall layer, lacking the outer membrane, stain violet and are called Gram positive. Many organisms belong to the class of Actinobacteria, such as Mycobacterium leprae were considered as species somewhere between fungi and bacteria. The Actinomyces are commonly known as producers of bioactive compounds in medicine, agriculture, ecology and industry. In the most of the cases the ability to produce one or another bioproduct is taxonomically determined, which makes taxonomic identification of the isolates so important.

 

 

Recently, the scientists have turned to investigate some more specific biotopes for new microorganisms, perspective both for industry and for science.Actinomycetes are aerobic, gram-positive bacteria. They are one of the major groups of soil population and are very widely distributed (Kuster, 1968). Actinomycetes populations are relatively lower than other soil microbes and contain a predominance of Streptomyces that are tolerant to acid conditions (Davies and Williams, 1970).

 

Antibiotic Producing Bacteria and Fungi

The term "antibiotic" literally means "against life".  British scientist Alexander Fleming is credited with being the first to notice that another organism could inhibit bacterial growth in 1928.  He noticed that growth of the bacterium Staphylococcus aureus was inhibited by a mold (fungus) that had contaminated his plate. The mold was later identified asPenicilliumnotatum and the antibiotic, isolated a short time later, was named penicillin. The value of penicillin was immediately recognized, but it wasn't until 1940 before the first clinical trials of penicillin were tried on humans.  The reason for the long delay was because of the difficulty in producing large enough quantities of pure penicillin. While research was progressing World War Two reached Britain and the entire research project was given the highest priority and moved to the United States for safety.  This was before the time of genetic engineering; so one of the aspects of the research project was to find a mutant strain ofPenicillium that would produce massive amounts of penicillin.

 

MATERIAL AND METHOD:

Soil samples were collected from four different locations. These habitats include the rhizosphere of plants, agricultural soil, preserved areas and forest soils. The samples were taken up to a depth of 20 cm after removing 3cm of the soil surface. The samples were placed in polyethylene bags, closed tightly and stored in a refrigerator. The following screening procedure was adopted for the isolation of Streptomyces.Numerous media have been used for the isolation of actinomycetes from soil and other natural materials. Glycerolarginine medium (Porter et al., 1960), starch casein agar medium (Kuster and Williams, 1964) and colloidal chitin agar medium (Linagarppa and Luckwood, 1962) have been widely used for isolating soil actionmycetes. In our study soil was pretreated with CaCO3 (10:1 w/w) and incubated at 37 for 4 days. It was then suspended in sterile Ringer solution (1/4 strength). Test tubes containing a 10-2 dilution of the samples were placed in a water bath at 45 for 16 hours so that the spores would separate from the vegetative cells.The dilutions were incubated on the surface of the Actinomycete isolation Agar plates. The plates were incubated at 28 until the sporulation of Sreptomyces colonies occurred. Streptomyces colonies were then removed and transferred to the Yeast extract Malt extract Agar slants. Pure cultures were obtained from selected colonies for repeated sub culturing. After obtaining pure culture (working & mother culture both), bacteria’s antibiotic activity against 4 unknown bacteria’s Lawn culture to observe cell lysis.

 

RESULT AND DISCUSSION:

Actinomyces species were isolated successfully from four soil samples collected from different locations. All the cultures have screened for their potential as a source of antibiotics active against antibiotic resistant bacteria. A total of four different Streptomycetes isolates were shown to have a very potent in-vitro antimicrobial activity against the test organisms. All of the isolates were tested for their ability to produce inhibitory substances against 4 different test bacteria. Two bacterial zones showed in the Lawn culture, i.e. two bacterial strains have been identified successfully and then after plasmid curing depicted that the responsible genefor antibiotic secretion was present in chromosome.

 

We success fully isolate the various strains of Actinomycesfrom different types of sub cultures. Our aim was to identify and isolate the antibiotic producing bacteria. Antibiotic is widely used in pharmaceutical industry. Our work was to screen a few of those species which could be later used in industrial production.

 

Figure-1 mother culture

 

Figure-2 working culture

 

Figure.3Actinomycesculture (white dotes are the streptomyces bacteria).


 

Figure.4 - Actinomycesculture of various dilutions.

 


 

Figure- 5- Zone appearing on Lawn culture.

 

REFERENCES:

1.       Davies FL, Williams ST (1970). Studies on the ecology of actinomycetes in soil. The occurrence and distribution of actinomycetes in a pine forest soil. Soil Biochem. 2: 227-238.

2.       Kuster E (1968). The actinomycetes. In: Soil Biology, eds. Burges (A.) & Raw (F.), Academic Press, London. pp. 111-124.

3.       Kuster E, Williams ST (1964). Selective media for the isolation of Streptomycetes. Nature, 202: 928-929.

4.       Linagarppa Y, Lockwood JL (1962). Chitin media for selective isolation and culture of actinomycetes. Phytopathology. 52:317-323.

5.       Porter JN, Wilhelm JJ, Tresner HD (1960). A method for the preferential isolation of actinomycetes from soil. Appl. Microbial. 8: 174-178.

 

 

 

Received on 05.02.2012        Accepted on 27.03.2012        

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Research J. Engineering and Tech. 3(3): July-Sept. 2012 page 199-201