... Hello Annoymous can you please provide me with a few works cited for this su
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Hello Annoymous can you please provide me with a few works cited for this summary, like a few resources about this science project ?? Thank You so much!!!!!
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Anonymous
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ANSWER:
TOPIC
ISOLATION,SCREENING OF MANGROVE ACTINOMYCETE STRAIN L3 FOR THE PRODUCTION OF ANTIBACTERIAL ACTIVITY
SLIDE 1
•INTRODUCTION
•MATERIALS AND METHODS
•ISOLATION OF ACTINOMYCETES
•ANTIBACTERIAL ACTIVITY STUDIES
•RESULTS
SLIDE 2
INTRODUCTION
ØMangrove ecosystems are increasingly being investigated as a source of microorganisms with potential to produce novel bioactive compounds and enzymes.
ØMangrove ecosystems represent a largely untapped source for isolation of new microorganisms.
ØActinomycetes are of particular interest, since they are traditionally known for their unparalleled capacity to produce biomolecules with diverse biological activities.
SLIDE 3
MATERIALS AND METHODS
STUDY AREA AND SAMPLING
ØMangrove sediment samples were collected from the two sampling sites
1. Machilipatnam
2.Nizampatnam
ØThe mangrove sediment samples were collected with a soil corer during low tides and transferred to a precleaned polycarbonates glass bottles stored at -20oC and kept frozen for further analysis.
SLIDE 4
ISOLATION OF ACTINOMYCETES
ØMangrove sediment samples were stored at 4°C until isolation.
ØActinomycetes were isolated by plating out the samples in proper dilutions.
Ø Actinomycetes colonies can often be distinguished on the plate from those of fungi and true bacteria.
They are often compact, leathery giving a conical appearance and have a dry surface
SLIDE 5
ØAbout 1g of the sample was taken into a 250ml conical flask containing 100ml of sterile water and the flasks were kept on a rotary shaker for 15 min.
ØThe suspension in each flask was serially diluted up to 10-5 level.
ØIsolation was carried on Starch Casein Agar plates supplemented with rifampicin 2.5µg/mL and cycloheximide 75µg/mL to inhibit bacterial and fungal contamination respectively.
SLIDE 6
ØThe plates were seeded with a sediment sample suspension of 1.0ml each and incubated at 28°C for 14 days.
ØAfter 14 days, actinomycetes colonies formed on the SCA plates were picked and transferred onto SCA slants and incubated at 28°C for 7 days.
ØOnly those isolates that appeared different from one another others were selected.
ØThe isolates were further maintained by periodically subculturing in SCA slants.
SLIDE 7
Constituent
Concentration (g/L)
Soluble starch
10.0
Vitamin free casein
0.3
Potassium nitrate
2.0
Sodium chloride
2.0
Dipotassium hydrogen phosphate
2.0
Magnesium sulfate
0.05
Calcium carbonate
0.02
Ferrous sulfate
0.01
Agar
20.0
pH
7.0 ± 0.2
Composition of starch casein agar (SCA) medium
SLIDE 8
The isolates were pooled together and cultures which appeared identical to naked eye in respect of color of aerial mycelium, reverse color, soluble pigment and colony texture were eliminated.
SLIDE 9
ANTIBACTERIAL ACTIVITY STUDIES
ØThe isolates obtained were screened for antibacterial activity.
ØThe test organisms used for antibacterial activity studies are given in the table
SLIDE 10
List of test organisms used in the study
S. No
Bacteria
Gram -ve / +ve
1.
Bacillus cereus
Gram +ve
2.
Bacillus subtilis
Gram +ve
3.
Staphylococcus aereus
Gram +ve
4.
Escherichia coli
Gram –ve
5.
Enterobacter aerogenes
Gram –ve
SLIDE 11
Primary screening by Cross-streak method
ØThe marine actinomycete isolates were screened for antibacterial activity by cross streak method on agar plates containing starch casein agar (SCA) and nutrient agar in equal proportions.
ØEach plate was streaked with a single isolate at the center along the diameter of the plate and incubated at 28°C for 5 days.
ØAfter 5 days, test organisms were streaked perpendicular to the growth of the actinomycete culture.
SLIDE 12
Secondary screening by well diffusion method
ØThe active isolates identified in the primary screening using cross streak method were selected for secondary screening.
SLIDE 13
Submerged fermentation
ØThe active isolates were first cultivated under submerged conditions and then their ability to produce extracellular antibacterial metabolites was tested by well diffusion method.
SLIDE 14
Composition of production medium
Constituent
Concentration (g/L)
Sucrose
20.0
Malt extract
10.0
Yeast extract
4.0
Dipotassium hydrogen phosphate
5.0
Sodium chloride
2.5
Zinc sulfate
0.04
Calcium carbonate
0.4
pH
7.0
SLIDE 15
Well diffusion method
ØThe antibacterial activity against the test bacteria was quantified by using well diffusion method on nutrient agar medium.
ØNutrient agar plates were used for well diffusion method.
ØMolten sterile nutrient agar was cooled to about 45°C, inoculated with test bacteria, mixed thoroughly, poured into sterile petriplates and allowed to settle.
ØWells were made in the solidified agar plates using a sterile cork borer.
SLIDE 16
ØThe clear supernatant from the fermentation broth was added to each well (50 µl) using a micropipette.
ØThe plates were kept in the refrigerator for about 2h for antibiotic diffusion and then incubated at 37°C.
Ø After 24 h, the inhibition zones were recorded.
ØThe procedure was repeated for all the promising active isolates obtained after primary screening.
SLIDE 17
RESULTS
Antibacterial activities of the selected actinomycete isolates bywell diffusion method
SLIDE 18
Isolate L3 has shown good antibacterial activity against
Bacillus subtilis and enterobacter aerogenes and has been selected for further studies
SLIDE 19
Isolate
Gram positive
Gram negative
Bacillus licheniformis
Bacillus subtilis
Staphylococcus aereus
Escherichia coli
Enterobacter aerogenes
C1
17
16
-
16
21
C2
22
14
16
-
-
C5
-
29
20
19
21
L1
13
-
14
-
-
L2
15
-
-
-
-
L3
17
32
33
26
36
L4
-
-
20
-
20
M1
19
20
22
12
-
M4
22
20
-
35
-
M6
36
-
29
30
19
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Summary:
Mangrove Actinomyces strain L3 was screened for the presence of antibacterial activity. Mangrove sediment samples were taken from the sampling sites, Machilipatnam and Nizampatnam. Isolation of Actinomycetes was carried on Starch Casein Agar plates supplemented with rifampicin and cycloheximide. The plates were seeded with a sediment sample and incubated at 28°C for 14 days. After 14 days, colonies were transferred onto SCA slants and incubated at 28°C for 7 days. The isolates were pooled together and cultures which appeared identical were eliminated. Primary and secondary screening of actinomycete isolates were done by cross streak method and well diffusion method respectively. The plates were kept in the refrigerator for about 2h for antibiotic diffusion and then incubated at 37°C. After 24 h, the inhibition zones were recorded and the Isolate L3 was found to have good antibacterial activity against Bacillus subtilis and enterobacter aerogenes.
Explanation of the last table:
The table summarises the values of inhibition zones obtained by different isolates of actinomycetes namely C1, C2, C5, L1, L2, L3, L4, M1, M4 and M6 against gram positive (Bacillus licheniformis, Bacillis subtilis and Staphylococcus aureus) and gram negative (Escherichia coli and Enterobacter aerogens) bacteria. Antibacterial activity against Bacillus licheniformis is maximum with M6 isolate. Isolate L3 was found to have good antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Enterobacter aerogenes. Isolate M4 had good antebacterial activity against escherichia coli.
Constituent
Concentration (g/L)
Soluble starch
10.0
Vitamin free casein
0.3
Potassium nitrate
2.0
Sodium chloride
2.0
Dipotassium hydrogen phosphate
2.0
Magnesium sulfate
0.05
Calcium carbonate
0.02
Ferrous sulfate
0.01
Agar
20.0
pH
7.0 ± 0.2
Explanation / Answer
Following publications are related to the study showed in slides. These articles discuss in detail about the isolation, identification and screening of actinomycetes strains from mangrove plants.
https://www.google.co.in/url?sa=t&source=web&rct=j&url=http://www.rsisinternational.org/IJRSI/Issue30/54-59.pdf&ved=0ahUKEwjCxcPtiebTAhWBpI8KHZsTDKUQFggoMAI&usg=AFQjCNE4Z0V0KJSJ6QBNXl708uyGAMBwag
http://www.ajol.info/index.php/ajb/article/view/137881
http://www.ajol.info/index.php/ajb/article/view/82733
http://link.springer.com/article/10.1007/s11274-009-0113-4
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