Environmental Microbiology -Laboratory Manual-

1. Environmental Microbiology-Laboratory Manual- I Isolation and Purification Use of Microscope Simple Stain Environmental Management technology Faculty of civil and environmental engineering Itb, 2010 anindrya.pahlawan@ymail.com

2. Isolation and Purification Exp. 1 - Aseptic Culture Transfer Exp. 2 – Pure Culture Isolation Technique Exp. 3 – Isolation from Environment

3. Why Isolating ? • Microorganism is everywhere • Isolation : microorganism population  individual species for study purpose  Pure Culture

4. Autoclave Culture Tube Petri Dish Laboratory Apparatus (1)

5. Inoculation Needle Bunsen Burner Incubator Laboratory Apparatus (2)

6. Broth Semi-Solid Solid Medium

7. Deep Tube Agar Slant Agar Plate Agar Solid Medium

8. Making Media

9. Aseptic Culture Transfer

10. Exp. 1 - Aseptic Culture Transfer • Transferring m.o. culture from a medium to another  standard procedure in microbiology lab • M.o. are everywhere  external contamination  ASEPTIC TRANSFER : • Disinfection • Bunsen Burner

11. Sterilizing Loop Using Bunsen burner ) Position the handle end of the wire in the light blue cone of the flameii) Draw the rest of the wire upwards slowly into the hottest region of the flame – immediately above the blue cone.iii) Hold there until it is red hot.iv) Ensure the full length of the wire receives adequate heating.v) Allow to cool for a few seconds in the air, then use immediately.vi) Do not put the loop down, or wave it around.vii) Re-sterilize the loop immediately after use

12. Inoculation

13. Sterilizing Tube Mouth Using Bunsen Burner

14. Transfer from Liquid Medium to Broth Tube In tube to tube transfers by loop or straight wire, both the tube containing the inoculums source and the tube to be inoculated are usually in the hand at the same time.

15. Transfer from Slant Agar to Another

16. Transfer from Agar Media to Petri Dish

17. Pure Culture Isolation

18. There is no individual species living by its own • Laboratory  separated using pure culture to study biochemical, morphology, etc • Developed by Robert Koch • Colony  m.o. mass growth seen on solid medium, each colony represents multiplication of singe organism  transferred to agar medium • Streak plate, pour plate, spread plate

19. Streak Plate • the dilution of bacteria by systematically streaking them over the agar surface in a petri dish to obtain isolated cells • During incubation, the isolated microbes multiply • Excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media. If this water drops down onto the agar surface, spreading and merging of colonies can occur one should INVERT the plates after streaking them and when they are incubated.

20. Streaking Method

21. After Incubation

22. Pour plate • also called the loop dilution method • involves the successive transfer (serial dilution) of bacteria from the original culture to a series of tubes of liquefied agar • the concentration of bacteria in the first tube is lower than the concentration in the original culture • the process is repeated for a third tube of liquefied agar • the contents of each tube is poured into a separate Petri plate • a major concern in preparing pour plates is the temperature of the agar

23. Spread Plate

24. Isolation from Environment Isolation from Air Isolation from Soil

25. Colony Counter

26. Using Microscopes

27. Terms & Definition • A microscope  (from the Greek: mikrós, "small" and skopeîn, "to look" or "see") = is an instrument to see objects too small for the naked eye • Microscopy the science of investigating small objects using such an instrument • Microscopic  invisible to the eye unless aided by a microscope. • Optical, electron, etc.

28. Optical Microscope • Using visible wavelengths of light • the simplest and most used • have refractive glass to focus light into the eye or another light detector • Typical magnification of a light microscope is up to 1500x with a theoretical resolution limit of around 0.2 micrometers or 200 nanometers • Caution  • use of immersion oil in 100x magnification • Coarse focus and fine focus

29. Components of A Microscope A microscope consists of non optic components & optic components

30. Simple Stain

31. Staining • Bacteria  extremely small m.o., transparant • Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image, highlighting structures in biological tissues for viewing • Basic principle  ion bonding of cellular component and active component of dye • Simple Stain & Differential Stain • Acid and base stain • Acid : dye (-), chinese ink, Nigrosin • Base : dye (+), fuchsin carbol, methylene blue

32. Preparing Smear Heat fixation aims to attach m.o. to the slide, and to preserve the shape of the cells or tissue involved as much as possible After placing several drops of water on the slide, smear the bacteria from the inoculating loop from the top to the bottom of the slide

33. Stain

34. Morphological Shapes of Bacteria

35. SAFETY FIRST !` Work steril is the success of microbiology laboratory practice.