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Isolation and Screening of industrially important microbes.pdf

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Isolation and Screening of industrially important microbes.pdf

  1. 1. Isolation and Screening of industrially important Microorganisms - Prepared by:- Mrunali.B Subject:- MBT-302 Bioprocess engineering PRN:- 223251025 Class:- MSc.Biotechnology Part-2 -Guided by:- Dr. Uttara Oak 1
  2. 2. Contents • Introduction • Introduction to research article • Isolation of Actinomycetes isolates • Isolation of MDR wound infection bacterial isolates • Screening • Antimicrobial susceptibility test • Observation • Observation table • Result • Reference 2
  3. 3. Introduction • Industrially important microorganisms are the one which are typically grown on a large scale in order to produce commercially important products such as vaccines, antibiotics, enzymes, etc. • They may include bacteria, algae, fungii, viruses. • Major source for isolation of such microorganisms is soil. • Other sources may include water, body fluids, feces, etc. • Isolation and screening of microorganisms is a process of isolation, seperation and detection of these industrially important microorganisms. • Microbe isolation:- serial dilution agar plate method • Different methods for microbe screening:- Ø Primary screening:- a technique that is used to determine the microorganisms capable of producing desired products. Example:- cross streak method Ø Secondary screening:- a technique that is carried out after primary screening of microbes to determine the quantitative and qualitative information about microbes. Example:- disc diffusion method. 3
  4. 4. Introduction to Research article • Research article- developed by L.Ashokkumar, R.Balaraghunathan, P.Palanivel and D.Jegadeeshkumar from Periyar University, Tamil Nadu; which includes the study of antibacterial activity of Actinomycetes against MDR wound infection bacterial isolates. • Actinomycetes- gram positive, filamentous, anaerobic, non-motile bacteria with high G+C content. • Most common source- soil and marine habitat. • Well known source for- antibiotics production. • MDR wound infection- bacteria are multi-drug resistant bacteria which are associated with wound infections. example:- Methcillin resisitant S.aureus • Different MDR wound infection isolates used in this experiement are:- Klebsiella species, E.coli, Streptococcus mutans, Staphylococcus aureus and Pseudomonas aeruginosa. • Ampicillin- used as a standard reference for antibiotic susceptibility testing 4 Fig 1:- Actinomycetes stained with methylene blue
  5. 5. Isolation of Actinomycetes • Total 5 different isolates of Actinomycetes were recovered from mine soil samples collected from Salem, Tamil Nadu. • Serial dilution agar plating method - used for isolation and identification of actinomycetes using starch casein agar medium. • pH of medium is set at 7.2 • Cyclohexamide-added innto medium as antifungal agent. • 1 ml aliquots of serially siluted soil sample were prepared and added over solidified caesin agar medium and incubated at 28°C for atleast one week. • ISP2 medium is also used for actinomycetes isolation. • Identification of actinomycetes were done with the help of different morphological and biochemical test. • Morphological test:- carried out by methylene blue test • Different biochemical tests:- gram positive test, methyl red test, urase test, oxidase test, catalase test, sucrose test, lactose test and mannitol sugar test. • after carrying out morphological and biochemical test, the results acquired from these tests were determined and it was concluded that colony produced is of actinomycetes. 5 Fig 2:- Actinomycetes on starch casein agar Fig 3:- ISP2 Medium
  6. 6. Isolation of MDR wound infection bacterial isolates • Pus sample was collected aseptically with the aid of sterile swab sticks from different patients with wound infections from Namakkal Dist surrounding hospitals. • Different culture media were used for culturing different isolates of MDR wound bacteria such as eosin methylene blue agar, Macconkey agar, Mannitol sugar agar, cetrimide agar and nutrient agar. • Streaking method was used for isolation of bacterial isolates in which, swab sticks with different isolates were directly streaked on respective and labelled agar plates. • Incubated at 37°C for 24hrs. • Cultures were then examined for significant growth and subcultures were then prepared on nutrient agar and incubated for another 24hrs. • Identification and characterization- was made by colony appearance, pigmentation, different biochemical methods. 6
  7. 7. Screening 1. Primary screening:- • Primary screening of Actinomycetes for thier antibacterial activity against MDR wound infection is carried out by Cross-streaking method. • Helps to determine whether the isolates are capable of producing antibiotics. • Actinomycetes isolates were cultured on Multi nutrient agar(MNA) and incubated for 7 days. • After 7 days, MDR wound infection isolates were streaked in perpendicular manner on MNA cultured with actinomycetes isolates. • Incubated at 37°C for 24hrs. • The plates were observed with zone of inhibition of MDR wound infection bacterial isolates by Actinomycetes. 7 Fig 4:- Cross-streaking
  8. 8. 2. Fermentation:- a. The Actinomycetes was inoculated into 250 ml Erlenmeyer flasks containing 75 ml of liquid starch nitrate medium. b. The flasks were incubated on a rotary shaker (200 rpm) at 30°C for 6 days. c. 20lit total volume was filtered through Whatman No.1 filter paper and followed by centrifugation at 5000 rpm for 20 minutes to remove cell debris. d. The clear supernatant used as an antibacterial substance. 8
  9. 9. Screening 3. Secondary Screening:- it is carried out to determine susceptibility of MDR wound infection bacteria against Actinomycetes isolates. • Disc-diffusion method used • Isolates of MDR wound infection bacteria were inoculated and cultured on nutrient broth. • Incubated at 37°C overnight. • Bacterial cultures were then swabbed on Muller-Hinton agar plates. • 6mm diameter of 5 wells were punched on each agar plates containing 5 different isolates of MDR bacteria. • These wells in each plate were then dispensed with 100μl of 5 different Actinomycete isolates. • Incubated at 37°C for 24hrs. • Diameter of zone of inhibition around the wells is measured and recorded. 9
  10. 10. Antimicrobial susceptibility test • For standard reference, MDR wound infection bacterial isolates were screened for thier sensitivity against Ampicillin by disc diffusion method. • The nutrient broth was prepared and inoculated with the MDR wound infection isolates. • Incubated at 37°C for 24hrs. • After incubation period the broth culture was swapped onto the surface of the Muller-Hinton agar plates and antibiotic disc of Ampicillin antibiotic were placed. • Incubated at 37°C for 18 to 20 hrs. • The zone of inhibition was measured and recorded and compared with secondary screening of antibacterial activity of Actinomycetes isolates. 10
  11. 11. Observations • Antibacterial activity against wound isolates:- 11
  12. 12. Observation Table 12 Table:- Antibacterial activity of Actinomycetes against wound isolates
  13. 13. Result • Among 5 actinomycete isolates, A2 isolate has the highest antibacterial activity against S.aureus and E.coli. • Pseudomonas aeruginosa and E.coli MDR wound infection bacteria were highly suppressed by Actinomycetes. 13
  14. 14. Reference • https://www.biotechnologynotes.com/microorganisms/screening/screening-of- microorganisms-primary-and-secondary-techniques-industrial-biotechnology/13697 • https://www.researchgate.net/publication/260559107_STUDIES_ON_ANTIMICRO BIAL_ACTIVITY_OF_ACTINOMYCETES_AGAINST_MDR_WOUND_BACTE RIAL_ISOLATES 14
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