Metode Mikrobiologis -1

1. Metode Mikrobiologis-1 Penanganan Mikrobia Metode Kultur murni dan Teknik Aseptis Isolasi, Purifikasi Penumbuhan mikrobia: Culture media Sterilisasi dan desinfeksi Mikroskopi Pengecatan mikrobia Enumerasi mikrobia Karakterisasi dan Identifikasi

2. 1. Penanganan mikrobia Safe handling microbes: • Sebagian besar mikrobia bermanfaat • Sebagian kecil mikrobia merupakan agensia penyakit e.g. HIV, Neisseria meningitis • Kultur mikrobia tidak terlepas ke lingkungan • Kultur mikrobia tidak terkontaminasi oleh mikrobia lain • Teknik saftey harus dipenuhi dalam bekerja dengan mikrobia • Bahan dan alat dan tempat bekerja harus steril (teknik aseptis) • Kultur mikrobia yang tidak dipakai lagi harus dimusnahkan

3. 2.Metode kultur murni (Pure culture) • Mikrobia yang dipelajari harus dalam bentuk kultur murni • Kultur murni: kumpulan sel yang merupakan keturunan sel tunggal • Strain : kumpulan sel kultur murni yang berasal dari satu sel tunggal • Untuk memperoleh kultur murni →Teknik Aseptis • Teknik Aseptis: semua bahan, alat, dan tempat bekerja yang digunakan serta cara kerja harus bebas dari kontaminasi(steril)

4. Kultur murni mikrobia Inokulasi Koloni

5. Aseptic Technique • A pure culture is a culture consisting of only one kind of microorganism • Aseptic technique is technique which involves avoiding any contact of the pure culture, using sterile medium, and using sterile surfaces of the growth vessel with contaminating microorganisms • How to accomplish it? • Sterilization of work area • Sterilization of working instruments and medium • Work must be done quickly and efficiently

6. Aseptic Technique in Practice

7. 3. Isolasi dan Purifikasi: Teknik Enrichment dan Isolasi Selektif

8. Enrichment Technique • Method used to isolate specific groups of microorganism • Culture medium and incubation conditions are designed to support target microorganism • Microorganisms other than the target will grow poorly or unable to grow in enrichment medium

9. Isolation of Microorganism • Isolation of pure cultures involves: • Separating samples of microorganisms into individual cells • Allowing the individual cells to reproduce and form clones of single microorganism • The success of isolation depends on: • Ability to do physical separation of the microorganism • Ability to maintain viability and growth of pure culture of microorganism

10. Isolation Diagram Samples Enrichment Dilution Platting Streak Plate Spread Plate Pour Plate Maintenance

11. Dilution Technique

12. Streak Plate Technique

13. Spread and Pour Plate Technique

14. 4. Penumbuhan Mikrobia : Culture Media • A suitable culture media for microorganisms growth must: • Containing substances which support their needs. Substances (nutrients) divided into three types: macronutrients (C, N, P, S, K, Mg, Ca, Na, Fe), micronutrients(Cr, Co, Cu, Mn, Mo, Ni, Se, W, V, Zn), and growth factors (vitamins, amino acids, purines, pirimidines) • Having suitable environment conditions (pH, temperature, oxygen level)

15. Types of Culture Media • Defined media: media in which all components are known( eg. Azotobactermedium, BG-11 Medium) • Complex media: media in which the components are not totally known and may vary (eg. NA, PDA) • Selective media: media which only favor the growth of specific microorganisms (eg. Salmonella-Shigella Agar) • Differential media: media which contain substances that can detect microorganisms with specific metabolic activitise (eg, MacConkey Agar)

16. Maintenance and Preservation of Pure Cultures • Maintenance of microorganism is important to preserve cultures which have special features • The purposes of maintenance are to maintain viability and genetic stability of the microorganisms • Several methods of maintenance are: subculture, drying, freeze-drying, and freezing

17. 5. Sterilisasi dan disinfeksi • Steril: bebas dari segala bentuk kehidupan • Sterilisasi: upaya untuk mencapai keadaan steril • Bactericidal • Fungicidal • Viricidal (seolah virus jasad hidup !) - inactivator • Germicides • Bacteristatic • Fungistatic • Antibiotics

18. 5. Sterilisasi dan disinfeksi • Desinfection • Desinfectan • Antisepsis • Antiseptic • Sanitation

19. Metode Sterilisasi Metode Fisikawi: • Mekanis : filtrasi • Pemanasan: • Pemijaran: e.g. pemijaran ose • Udara panas kering: e.g. oven • Uap air panas: e.g. Arnold –steam sterilizer • Uap air panas bertekanan: e.g. Autoclave • Penyinaran : • Radiasi UV, gamma

20. Sterilization • Sterilization is a process to eliminate contaminant microorganisms • Chemical sterilization (alcohol, xylol, H2O2 etc) --> work surface and working instrument • Radiation sterilization (UV, gamma, etc) --> work surface and working instrument • Filtration --> heat-sensitive growth medium • Heating (autoclave, oven) --> working instrument, heat-resistant growth medium

21. Oven

22. Autoclave

23. Metode Sterilisasi Metode Kimiawi: • Disinfectant: HgCl2, betadine • Antiseptics: fenol, cresol • Antibiotics • Ozonization (ozon)

24. 6. Mikroskopi Light microscopy: • Bright-field microscopy • Dark-ground microscopy • Phase-contrast microscopy • Fluorescence microscopy Electron microscopy: • Scanning Electron Microscopy (SEM) • Transmission Electron Microscopy (TEM)

25. Microscopy Technique • Microscopy is the use of microscope to view objects too small to be visible to naked eye • Type of microscope: • Light microscope (brightfield, darkfield, ultarviolet, fluorescence, phase contrast, nomarski differential interference, confocal) • Electron microscope (Transmission Electron Microscope, Scanning Electron Microscope)

26. Light Microscope

27. Electron Microscope

28. Confocal microscopy • Confocal scanning laser microscopy (CSLM) • Memakai sinar LASER • Bayangan tiga dimensi dan lebih jelas Scaning Probe Microscopy • Scanning tunneling microscope (1980) • G. Binnig & H. Rohrer : Nobel Prize (1986) • Perbesaran 100 juta kali • Dapat melihat atom pada permukaan padat • Atomic force microscope (lebih baru) • Spesimen yang non-konduktor: interaksi protein, letak restriction site pada plasmid

29. 7. Pengecatan mikrobia • Simple staining • Gram staining • Ziehl-Neelsen staining • Granules staining • Negative staining • Spore staining

30. Staining • Staining is important to discerns object from its background, hence it is become viewable under light microscope • Staining procedure: • Simple staining (positive staining, negative staining) • Differential staining (Gram staining, acid-fast staining, endospore staining)

31. Simple Staining • Single stain is used • All cells and structures generally stain the same color • Positive staining: stain attracted to the cell • Negative staining: stain repelled by the cell, background takes on the color

33. Differential Staining • Multiple staining reactions are employed • Differentiate types of cells or cell’s structures base on their staining reactions, hence given different color • The specimen must be “fixed” by heating or chemical • Examples: • Gram stain: staining based on composition of lipids and peptidoglycan in bacterial cell wall • Acid-fast stain: staining based on composition of mycolic acid, fatty acids, waxes, and complex lipids in bacterial cell wall • Endospore stain: staining to visualize bacterial endospore

34. Gram staining Acid-fast staining

35. Acid-fast Staining Gram Staining Endospore Staining