Culturing microorganisms

Transcription

1 Culturing microorganisms I. Historical development II. Problems and Solutions III. Studying microorganisms without a microscope -- culturing techniques A. How do you do it? B. Inoculation and isolation C. Media

2 I. I. Two main microbiological questions led to the historical development of new technologies (like culturing bacteria). Does spontaneous generation occur? What is the nature of contagious?

3 A. Spontaneous generation Does arise from non-living matter (abiogenesis( abiogenesis) or only from living matter (biogenesis( biogenesis)? See Text, page 12 ( Spontaneous Generation and the Battle of the Hypothesis ).. Note especially increasing refinement of experiments to disprove abiogenesis.

4 B. The Germ Theory of Disease and Koch s Postulates

5 What are the potential methodological difficulties with this step? onal safety iculties of detection

6 B. Koch s postulates (cont.) However: mere of the bacterium with the disease did not prove that it the disease. What else could be happening? How to prove cause and effect?

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8 hy is this step necessary? Non-pure culture would who s causing what? hat are the potential methodological problems with this step? Not all microorganisms can be.

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11 B. Koch s postulates (cont.) Why is this 4th step necessary? Koch s (and Pasteur s ) approaches were so successful that the causative agents for at least 20 other diseases were discovered between 1875 and Led to development of culturing methods (why? why?)

12 II. Problems inherent in studying microorganisms They are and widely. Usually need to grow under controlled situations, in lab. Strains must be.

13 III. Studying microorganisms without a microscope to microscopy -- how does one study physiology, genetics, etc.? Most major discoveries in microbiology not based on anything one can see. Many (perhaps most) microbiologists never look in a microscope!

14 A. How do you do it? Make billions of cells to study rather than one. BUT (big but [as opposed to big butt]): all the cells. Big breakthrough: Needed: culturing techniques, aseptic technique Led to development of broth and, especially, solid culture and techniques, sterilization, etc.

15 B. Inoculation and isolation Inoculation: : introduction of small sample into a medium for. Culture: : the observable growth in the medium Medium: : a solution - -,, or semi- solid -- used to grow microorganisms

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17 Mixed culture

18 Aseptic technique

19 aseptic technique (cont.)

20 How do we get a pure culture? Isolate cells

21 Method 1: The Streak Plate Method

22 Isolating cells Method 2: Loop dilution or pour Method 2: Loop plate dilution method or pour plate method (see text, pp )

23 The goal: isolated colonies for further purification

24 C. Media Liquid Semisolid Solid Development of agar as solidifying agent (Fanny Hesse/Walther Hesse) Petri plate

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26 Enterococcus faecalis broth

27 Sulfur indole motility (SIM) agar. Left,, negative; middle,, motility; right,, motility + H 2 S ga

28 Chemical content of media 2 Basic types: 1) (or Synthetic) media 2) or Complex (or Non- synthetic) media

29 Examples efined culture medium (for E. li): K 2 HPO 4 7 g KH 2 PO 4 2 g (NH 4 ) 2 SO 1 g MgSO g CaCl g Glucose 4 g Trace elements (Fe, Co, Mn, Zn, Cu, Ni, Mo) ) 2-10 µg each Distilled water, ph ml Complex medium (5% PTYG): peptone 0.25 g trypticase 0.25 g yeast extract 0.25 g glucose 0.5 g MgSo g CaCl g Distilled water, ph ml

30 Selective and differential media medium: : One in which compounds have been added to selectively inhibit the growth of some kinds of organisms but not others. Example: Mannitol salts agar for Staphylococcus medium: : One to which some sort of indicator, usually a dye, has been added to allow one to differentiate between various chemical reactions during growth. Some media are both selective and differential. Example: : Eosin methylene blue (EMB) agar for E. coli and other lactose fermenters

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32 Media that are both selective and differential Mannitol salts agar Selective for halotolerant bacteria, like Staphylococcus Differentiates species of Staph that ferment mannitol MacKonkey agar Selective for Gramnegative bacteria, like E. coli differentiates bacteria that ferment lactose.

33 Final caveat: culturing or microscopy are still not the only ways to study microorganisms