www.globaljournal.asia GJESR RESEARCH PAPER VOL. 2 [ISSUE 10] NOVEMBER, 2015
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ISOLATION AND CHARACTERIZATION OF ANTIBACTERIAL COMPOUNDS PRODUCING MICROBES FROM SOIL AND WATER SAMPLES 1. Zainab Siddiqui, Graduation student 2.Saima Azmi, PhD scholar Department of biotechnology Integral university, Lucknow 227105 Abstract Actinomycetes are one of the most attractive sources of antibiotic.In the present studies total of four Actinomycetes strains were isolated from two different places in Lucknow, U.P, India. Isolated strains were identified for their activity but only two isolated show good resultsthey were Neisseria mucosa and Streptococcus equisimilis. Isolation of bacterial strain was done by serial dilution method. Primary screening of the culture was done by sub-culturing through simple streaking and quadrant streaking. Identification of bacteriawas done with help of Bergey’s manual and antibiotic sensitivity test was performing by agar will diffusion method. The pathogens were used E.coli, S.aureus, and P.aeruginosa against intracellular and extracellular antibacterial compounds which were isolated from the microbes. Best results were obtained for Neisseria mucosa against E.coli. Further Growth Kinetics study was done to know the log phase because in log phase culture will produce maximum secondary which works like antibacterial compounds. The present study is carried out by isolation of anti-bacterial compound producing microbes from the soil sample and characterization as well as production for this culture to get maximum secondary metabolites
1. INTRODUCTION The actinomycetes are gram positive, high G+C (>55%) organisms that tend to grow slowly as branching filaments. Many actinomycetes will grow on the common bacteriological media used in the laboratory, such as nutrient agar, trypticase soy agar, blood agar, and even brain-heart infusion agar. Actinomycetes encompass a wide range of bacteria. They can be terrestrial or aquatic.Actinobacteria is one of the dominant phyla of the bacteria. Analysis of glutamine synthetase sequence has been suggested for phylogenetic analysis of Actinobacteria. Actinomycetes are best known for their ability to produce antibiotics and are gram positive bacteria which comprise a group of branching unicellular microorganisms. They produce branchingmycelium
which may be of two kind’s viz., substrate mycelium andaerial mycelium. Among actinomycetes, the streptomycetes are the dominant. The non‐streptomycetes are called rare actinomycetes, comprising approximately 100 genera .Actinobacteria are well known as secondary metabolites producers and hence of high pharmacological and commercial interest. In 1940 Selman Waksman discovered that the soil bacteria he was studying made actinomycin, a discovery for which he received a Noble Prize. Since then, hundreds of naturally occurring antibiotics have been discovered in this terrestrial microorganism, especially from the genus Streptomyces. Antibiotics are compounds produced by microorganisms that are able to inhibit the growth of other microorganisms. Antibiotics are medicinal products that have an anti-bacterial effect, they either kill bacteria in the system or keep away them from reproducing, allowing the infected body to heal by producing its own defenses and overcome the infection. Screening of antibiotics has been widely performed for about last 50 years and new antibiotics are still being found. In screening of new antibiotics, new approaches are required and following three factors must be considered i.e. detection of antibiotic producing microorganisms, selection of producing microorganisms and cultivation methods. These are closely related to each other, and their efficient combination is essential for successful screening of an antibiotic interest 2. SOURCE OF ANTIBIOTICS Soil are cheap source of antibiotics, many bacteria are present in soil. Many antibiotics produce in microorganisms. People have been using herbal antibiotics traditionally for centuries to combat microbes that cause illnesses. Recently, scientists have studied and reported results supporting the use of these antimicrobial plants. Herbal antibiotics are milder than their pharmaceutical counterparts, which
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makes using them for minor illnesses a logical option. According to the Centers for Disease Control and Prevention, the overuse of pharmaceutical antibiotics causes microbes to mutate and grow stronger. As a result, some experts say it makes sense to use herbal treatments in place of pharmaceutical antibiotics when appropriate.
4.
Reishi mushrooms medicinally for centuries, Reishi Mushroom has antibacterial properties, Tea Tree Oil can effectively treat and prevent fungal infections, Goldenseal have been traditionally used goldenseal to treat skin disorders, digestive problems and eye infections, Garlic can effectively treat and prevent the common cold., Neem is a multi-purpose Ayurvedic herb that is especially effective against skin diseases, Myrrh has been used by herbalists for centuries and is even mentioned in the Bible. It is antiseptic, antibiotic and antiviral. It can be taken internally, used as a gargle or used as a wash for wounds; Echinacea is one of the most popular herbal remedies for colds, flues and other bacterial infections. It has been used by herbalists as a blood purifier and to treat a number of infections.
5.
6.
3. DIFFERENT TYPES OF ANTIBIOTICS Of the 100 plus antibiotics substances produced naturally or synthetically,very few have been proven safe and effective. The commonly used antibiotic types are: 1.
2.
3.
Penicillin:Penicillin is a group of antibiotics derived from Penicillium fungi. It is an antibiotic which destroys the cell walls of the bacteria, while they are in the process of reproduction. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases, such as syphilis, and infections caused by staphylococci and streptococci . Cephalosporin: Cephalosporins are bactericidal and have the same mode of action as other beta-lactam antibiotics (such as penicillins) but are less susceptible to penicillinases. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The Cephalosporin is a class of β-lactam antibiotics originally derived from the fungus Acremonium, which was previously known as "Cephalosporium". Aminoglycosides: Thistype of antibiotics hinders protein formation of bacteria
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invading cells. This antibiotic encompasses gentamicin, streptomycin and neomycin. As aminoglycosides are effective in inhibiting protein production in invading bacterial cells, they are administered to treat typhus. Macrolides: Macrolides are protein synthesis inhibitors. The mechanism of action of macrolides is inhibition of bacterial protein biosynthesis, and they are thought to do this by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid (similarly to chloramphenicol) as well as inhibiting ribosomal translocation.Another potential mechanism is premature dissociation of the peptidyl-tRNA from the ribosome. Sulfonamides: The sulfa-related group of antibiotics, which are used to treat bacterial infections and some fungal infections. The sulfonamide family includes sulfadiazine, sulfamethizole, sulfamethoxazole , sulfasalazine , sulfisoxazole, and various high-strength combinations of three sulfonamides. Sulfa drugs kill bacteria and fungi by interfering with cell metabolism. Tetracyclines: Tetracycline is a broadspectrum polyketideantibiotic produced by the Streptomycesgenus of Actinobacteria, indicated for use against many bacterial infections. It is a protein synthesis inhibitor. It is used to treat various eye infections.
A simplified enrichment method for the highly selective isolation of the zoosporic actinomycetes Actinoplanes spp. From soil. The method consists of baiting the species with Pinus pollen grains, desiccating (300C, 2 h) the baits bearing sporangia in dried soil particles with the aid of silica gel and following the spore liberation upon immersion in water. Portions of the liquid enriched with zoospores are plated out on humic acid-vitamin (HV) agar supplemented with nalidixic acid at a concentration of 10 g/ml. The desiccation stage had enabled the almost complete elimination of associated bacteria from colonized baits while allowing the Actinoplanessporangia to survive and still possess the ability to release many spores. A total of four different soil samples from fields of corn, peach, vegetable and paddy rice were examined. The pollenbaiting and drying method consistently resulted in the highly selective isolation of Actinoplanes spp. which accounted for over 83% of the total number of microorganisms recovered on HV agar containing nalidixic acid [1]. An actinomycete, designed strain KS3-5, was from a soil sample collected from Kaohsiung,
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Taiwan, ROC. This organism is capable of producing a series of antibiotics that strongly inhibit the growth of gram-positive and gram-negative bacteria and yeast like fungi. The spore morphology and cell wall chemotype suggest that strain KS3-5 is a Streptomycete. Further cultural and physiological characterization and the DNA homology suggest that strain KS3-5 is identical to Stretomycestoxytricini [2]. The presence and types of antibiotic-producing bacteria, fungi and actinomycetes using nutrient agar, potato Dextrose agar and starch casein nitrate agar respectively as culture media was determined in university of lagos from the compost soil. A variety of bacteria were isolated and these included Staphylococcus aureus, B. subtilis, B. pumilis, B. lactesporus, B. megaterium, B. pulvifaciens, B. licheniformis, Streptococus spp., Corynebacterium spp. and E. coli. The fungal isolates encountered were Aspergillusniger, A. flarus, T. viridae, P. chrysogenum, P. pinofylum and Absida spp., while the following actinomycetes were identified: Norcadia spp., Micromonospora spp., Streptomyces scabies, S. reticuli and S. hygroscopicus. When these organisms were screened for antibiosis, the following species were found to be antibiotic producers: B. licheniformis, B. subtilis, Penicilliumchrysogenum, Streptomyces reticuli, S. hygroscopicus and Micromonospora spp. The fungus Penicilliumchrysogenum had the highest rate of antibiotic production with an inhibitory zone width of 17mm while Trichodermaviridae produced toxins lytic to other fungal hyphae[3]. Bacillus subtilis and Bacillus pumiluswere isolated from soil and screened for the production of antibiotics by plate assay and then cultured in shake flask fermentation at 300C for further studies. Identification of antibiotics was done by paper chromatography. Bacitracin was found to be produced by both the strains against Micrococcus luteus(ATCC#10240),whereas;Staphylococcusaureus (ATCC# 6538) proved to be resistant to Bacitracin produced by Bacillus pumilus. The maximum production of Bacitracin from B. subtilis and B. pumilus against Staphylococcus aureus and Micrococcus luteus at different pH (6-9), incubation time (0-144 hours) and glucose concentration (1-5%) was checked by agar diffusion assay as detected by the size of zones of inhibition. Maximum zones of inhibition were observed at pH 8, 5% glucose and after 24 hours of incubation at 30oC against Staphylococcus aureusand Micrococcus luteus [4]. Isolation of different microorganisms from some soils' rhizosphere in Al-Madinah AlMunawwarah, viz. corn (Zea mays), datepalm (Phoenix dactylifera), catnip (Menthapiperita), sunflower (Helianthus), balessan
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(Amyrisgileadensis), nabk-tree (ZiziphusSpinaChristi Willd)and basil (Marrubiumvulgare) was carried out. All microbial isolated were then screened for their antagonistic activity against the most resistant eight target bacteria isolated from caesarean section site infections (E.coli, Klebsiellaspp., Pseudomonas spp., Proteus spp., Citrobacterspp., Acinetobacterspp., methicillin resistant Staphylococcus aureus MRSA, and coagulase negative Staphylococcus). Among the total 86 fungal and bacterial isolates, only 15 of them (17.44%) were capable of biosynthesizing antimicrobial metabolites. One of the actinomycetes that was obtained from catnip rhizosphere, Al-Ouayna district in Al-Madinah Al-Munawwarah, found to exhibit the highest antimicrobial activity and it matched with Streptomyces ramulosusin the morphological, physiological and biochemical characters. Thus, it was given the suggested name Streptomyces ramulosus, A-MM-24[5]. 4 .MATERIAL AND METHODS Soil sample (C1, C2, C3, C4) was collected from garden soil of different places of Indira Nagar and Aliganj area of Lucknow city. Actinomyceties Agar ,Nutrient Agar medium, Nutrient Broth, Production media, different Pathogens(Pseudomonas , E.coli , S.aureus) , Antibiotics(Tetracycline), Crystal violet, Gram’s iodine, Ethanol, Saffranine , Malacnite Green were used for the purpose of study.
4.1Isolation of antibiotic actinomycetes from soil
producing
Serial dilution agar plating method or viable count method is one of the most commonly used procedures for the isolation and enrichment of the most prevalent micro-organism such as fungi, bacteria. This method is based on the principle that when sample containing the micro-organism is cultured each viable micro-organism will develop into a colony; this method use is number of reduced bacterial colonies in order to get pure cultures. The numbers of micro-organism are diluted because the soil sample which was collected had a population of micro-organism. The numbers of colonies are same as the number of organism contain in the sample. Microbes from soil were isolated by serial dilution method and mixed culture was obtained by spreading as shown in figure 1. Colony morphology of culture from sample C1, C2, C3 and C4 is also depicted in table 1.
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3.1 PRIMARY SCREENING After incubated overnight four types of colonies were present in the plates, Zone of inhibition around the colonies was observed. Colonies showing zones of inhibition were selected for streaking and secondary screening.
A. Sub culturing Pure culture is usually derived from a mixed culture (containing many species) by methods that separate the individual cells so that, when they multiply, each will form an individually distinct colony, which may then be used to establish new cultures with the assurance that only one type of organism will be present. Pure cultures may be more easily isolated if the growth medium of the original mixed culture favours the growth of one organism to the exclusion of others. 60 ml nutrient agar was prepared and poured in to four sterile petriplates. Simple or zigzag streaking of four cultures was done on petriplates with the help of inoculation loop. All Figure 1- Mixed colonies in spread plate
the plates were incubated at 370C for overnight. After
Table 2- Colony morphology of culture from sample
zigzag streaking quadrant streaking is done.
C1, C2, C3 and C4. Colony morpholog y
Culture C1
Culture C2
Cultur e C3
Cultur e C4
Shape
Circular
Circular
Circul ar
Circul ar
Margin
Entire
Entire
Entire
Entire
Opacity
Transluce nt
Transluce nt
Opaqu e
Opaqu e
Pigmentati on
White
Off White
Off White
Off White
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C4 Fig 2: Pure colonies obtained through simple streaking
C2 Quadrant streaking is done when colonies are not clearly identified. For this, 30 ml nutrient agar was prepared and poured in to two sterile petri plates. Quadrant or four ways streaking of the two cultures was done on petriplates with the help of inoculation loop. Both the plates were incubated at 370C for overnight. C3 and C4 pure colonies obtained through Quadrant Streaking. The cultures C1, C2, C3 and C4 obtain through primary screening were purified by zigzag or simple streaking was shown in fig. 2. C3
C3
and C4 cultures obtained after quadrant streaking is shown in fig.3
C3
C4 Fig 3:- Pure colonies obtained through Quadrant streaking Š Virtu and Foi Better for Humanity
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E.coli
B. STAINING AND BIO-CHEMICAL CHARACTERIZATION OF SAMPLE Gram staining was given by Hans Christian Gram. It is a method of differentiating bacterial species into two large groups i.e. Gram-positive and Gramnegative. It is based on the chemical and physical properties of their cell wall. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive bacteria. A gram positive results in a purple/blue color while Gram negative results in a pink/red color. Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell envelope), which are stained purple by crystal violet, whereas Gramnegative bacteria have a thinner layer (10% of cell envelope), which are stained pink by the counter-stain.a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. After gram’s staining slide were observed under microscope and some colonies (C1, C2, C3) get pink colour and some get purple colour (C4). The colonies showing pink colour are gram negative cocci and colony which shows purple colour are gram positive cocci bacteria.
S.aureus
3.2 SECONDARY SCREENING A. Antibiogram Analysis Activity of biological compounds agent pathogen an important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. Antibiogram of purified cultures C1, C2, C3 and C4 was performed against various pathogens (E.coli, P.aeruginosa and S.aureus). There were zones of inhibition obtained in each culture. Each cultures show positive result against pathogensas shown in fig 4 and table 2.
P.aeruginosa Fig 4: Antibiogram analysis of cultures against various pathogens. Table 3 Antibiogram of Isolated cultures against various pathogens. S.No. Pathogen C1 C2 C3 C4 Dia(mm) Dia(mm) Dia (mm) Dia (mm) 1
E.coli
10
10
0
8
2
S.aureus
10
0
9
0
3
Pseud omona s
10
10.5
0
9
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B. Endospore Test We observe pink color cocci under microscope, which confirm that these are non-endospore forming bacteria. C. Catalase Test In this test bubble formation was not observed. By this we confirmed that the bacteria are from group Streptococcus. D.6.5% NaCl Test No growth was observed in 6.5% NaCl. Therefore, further glycerol test was done to confirm the species of Streptococcus. E. Acid from Glycerol Test After 72 hrs appearance of yellow halo surrounding the streak was appeared showing that these gram positive bacteria are producing acid, which confirmed Streptococcus equisimilis species as shown in fig 5.
Blank
Fig 5:- Gram positive bacteria are producing acid. F. Of Glucose Test
Colour change
The oxidative-fermentative (OF) test was developed by Hugh and Leifson. This test determines whether an organism metabolizes a given carbohydrate (usually glucose) by oxidation or by fermentation. The power of breakdown of carbohydrates is possessed by a large number of bacteria, fungi and yeasts. After performing this test blue colour of the solution changes to yellow due to glucose fermentation and confirm Neisseria group, the of glucose test results are shown in fig 6.
Fig 6:- glucose fermentation G. Nitrate Test: This test is done to confirm the species of Neisseria. In this test the red colour of solution was observed which shows that nitrate was reduced and confirmed Neisseria mucosa species. Result is shown in figure 7.
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S.No. Pathogen
Intracellular Extracellular Intra + Extra Diameter(m Diameter(m Diameter(mm m) m) )
1
E.coli
0
32.5
27.5
2
S.aureus
14.5
32.5
29
3
Pseudom onas
14.5
29
29
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3.3ANTIBIOTIC SENSITIVITY TEST A. Antibiogram of Intracellular and Extracellular Antibiotics Extracts Antibiogram analysis of intra-extracellular from our sample C3 and C4 was performed against various pathogens. The results can be seen in table 3 and fig 8 below For Neisseria. Antibiogram for of intracellular and extracellular antibiotic extract from S.equisimilis is shown in fig 9 and table 4.
For Neisseria
Table 3- Antibiogram analysis of intra-extracellular Antibiotic extracts from N.mucosa.
Initial colour
E.coli
Final colour showing nitrate reduction Fig 7: Nitrate reduction.
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monas
P.aeruginosa S.Aureus S.aureusP.aeruginosa
S.aureus Fig 8: Antibiogram of intracellular and extracellular antibiotic extract from N.mucosa For S.equisimilis Table5.Antibiogram analysis of antibiotic extract from S.equisimilis
intra-extracellular
E.coli
S.No. Pathogen Intracellular Extracellular Intra + Extra Diameter( mm)
Diameter( mm)
Diameter(m m)
1
E.coli
27.5
0
22
2
S.aureus
18
0
12.5
3
Pseudo
19
0
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0.5
Decline
8
P.aeruginosa
Fig 9:- Antibiogram for of intracellular and extracellular antibiotic extract from S.equisimilis
Growth kinetics of S.equisimilis and N.mucosa is graphically represented for better understanding in figure.10&11 along with table 6&7 showing optical density at different intervals of the day and stages of them. Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosome, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. 1.2
3.4GROWTH KINETICS OF CULTURE Growth kinetics process is applied to determine the time period at which the culture show its optimum activity (stationary phase). Growth of any microbes occurs in different stages which are indicated by growth curve. Growth shows different four stages Lag, Log, stationary and decline phase.
1
O.D
0.8 0.6 O.D
0.4 0.2
Table 6- Growth Kinetics of S. equisimilis
0
DAYS O.D OF THE CULTURE GROWTH PHASE
0 0.77
Lag
1.03
Log
1.05
Log
10
No. of days
1
Fig 10: Graph of Growth Kinetics of S. equisimilis
2
Table 7- Growth Kinetics of N. mucosa
3
DAYS O.D OF THE CULTURE GROWTH PHASE 1.03
Stationary
4
0.24
Lag
0.57
Log
0.95
Log
0.94
Stationary
1 1
Stationary
5
2 0.89
Decline
6
3 0.84
7
5
Decline 4
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Stationary
5 0.87
Decline
0.83
Decline
S.no
Pathoge n
Tetracy cl-ine (mm)
Intracell ular (mm)
1
E.coli
41
24
2
P.aerugi nosa
26
29.5
22
3
S.aureus
35
22
14.3
6
Intraextra mixture (mm) 16.5
7 For determining growth kinetics of the bacteria we have to prepare 50 ml NB in two conical flasks then it is Autoclaved and cool to ensure the impurity autoclave is the method for steam sterilization generally used for media sterilization. After that Inoculatation of the first culture in first flask and second culture in another flask is done. Incubate the media in shaker incubator and do check the O.D day by day for 4-5 days at 600 nm. Finally Plot the graph of growth kinetics. 1.2 1
O.D
0.8 0.6 O.D
0.4
E.coli 0.2 0 0
5
10
No. of days Fig 11: Graph of Growth Kinetics of N.mucosa 3.5 ANTIBIOGRAM ANALYSIS Antibiogram analysis of intracellular and extravellular antimicrobial extract agsinst various pathogens is sown in table 8 and figure 12. By Antibiogram analysis of intra and extra cellular against various pathogens and by comparing with tetracycline, intracellular componentshows better result than tetracycline against P.aeruginosa. Intracellular give 29.5mm of zone of inhibition and tetracycline give 26mm. Table 8- Antibiogram analysis of intra and extra cellular antimicrobial extract against various pathogens. Š Virtu and Foi Better for Humanity
P.aeruginosa
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5. REFERENCES
S.aureus Fig 12: Antibiogram analysis of culture sample against various pathogens.
1. Masayuki Hayakawa, Takayuki Kajiura and Hideo Nomomura, 1991, new methods for the highly selective isolation of Streptosporangium and Dactylosporangium from soil, Journal of Fermentation and Bioengineering, 72(5):327-333. 2. Rong-Yang Wu and Ming-Ho Chen, 1995, Identification of theStreptomyces strain KS3-5. Bot Bull Acad Sin, 36:201-205. 3. Adeleye IA, Eruba S, Ezeani CJ2004, Isolation and characterization of antibiotic producing microorganisms in composted Nigerian soil. J Environ Biol.; 25(3):313-6. 4. MuhammadAwais2007, Isolation, identification and optimization of bacitracin produced by Bacillussp., Pak. J. Bot., 39(4): 1303-1312. 5. Abo-Shadi Al-RahmanMahaAbd2010, Antimicrobial Agent Producing Microbes from some Soils' Rhizosphere in Al-Madinah AlMunawwarah, KSA,Journal of American Science, 6(10):915-925.
4. CONCLUSION AND FUTURE EXPECTS Screening of antibiotics has been widely performed for about last 50 years and new antibiotics are still being found. In screening of new antibiotics, new approaches are required and following three factors must be considered i.e. detection of antibiotic producing microorganisms, selection of producing microorganisms and cultivation methods. Finally it can be concluded that the Actinomycetes are the best source for antibiotic isolation. In this project by Antibiogram analysis intracellular component shows better result than tetracycline against P.aeruginosa. Intracellular give 29.5mm of zone of inhibition and tetracycline give 26mm. As the antibiotics are secondary metabolites, they are synthesized in trace amounts. Moreover the synthesis of antibiotic is regulated by tight metabolic and genetic regulation. Therefore it is the task to the biotechnologists to modify the wild type strain and to provide cultural conditions to improve the productivity of antibiotics. Improvement of the microbial strain offers the greatest opportunity for cost reduction without significant capital investment. The problem of the bacterial resistance to antibiotics had evolved and new compounds or derived from the known antibiotics had to be found to replace existing ones. Water and air microbes can also be used for the isolation, characterizing, purification and production of antimicrobial compound.
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