1. Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Chapter 3 (p.55-65) Chapter 7(p. 168-180) The Methods of Culturing Microbial Nutrition

2. Learning Objectives: Describe the five steps followed to characterize microorganisms in the laboratory. Define colony and pure culture. Describe streak method of isolating pure cultures. Identify the purpose and the advantage of using agar in a culture medium. Distinguish between chemically defined and complex media, selective and differential media.

3. Learning objectives: Identify the use of the following: general purpose medium, enriched medium, reducing medium. Define essential nutrients; distinguish between macronutrients and micronutrients. Provide a use for each of the four macroelements Describe microbial nutritional strategies by carbon and energy source Define simple diffusion, facilitated diffusion, osmosis, active transport, and group translocation.

4. Problems to overcome: How to separate mixtures of microbes? Growth under artificial conditions: does it reflect the reality? How to make sure that the bacteria you grow are from the sample you study

5. Culturing Microorganisms: The Five I’s We generally use five specific steps to characterize microorganisms in the laboratory: Inoculation Incubation Isolation Inspection Identification

6. Specimen Collection Sources: body fluids or tissues, food, water, soil. Sampling devices: swabs, syringes, transport systems. Has to be done aseptically!

7. Inoculation is adding small sample (the inoculum) into a container of medium Incubation promotes growth by maintaining proper conditions ( temperature, humidity, O 2 pressure) Inoculation and Incubation

8. Isolation Isolation: The separation of different microorganisms from each other As the microbes grow, they will form visible masses of cells (colonies). If formed from a single cell, each cell in the colony will be genetically identical (pure culture).

9. Isolated Colonies Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mixture of cells in sample Microscopic view Macroscopic view Incubation Separation of cells by spreading or dilution on agar medium Growth increases the number of cells. Microbes become visible as isolated colonies containing millions of cells. Parent cells

10. Streak Plate Method A sample spread along a large surface using inoculating loop Isolated colonies are eventually obtained 12345 (a) Steps In a Streak Plate Note: This method only works if the spreading tool (usually an inoculating loop) is resterilized after each of steps 1–4. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

11. Pour Plate Method 213 213 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cultures are diluted beforehand in liquefied agar Poured into plates Colonies form in and on the agar

12. Inspection Colonies observed: Macroscopically (size, color, texture), and Microscopically (morphology, staining)

13. Identification Cultures can be maintained using stock cultures Once cultures are no longer being used, they must be sterilized and destroyed properly. Identification: Using appearance as well as metabolism (biochemical tests) and sometimes genetic analysis or immunologic testing to identify the organisms in a culture.

14. Conclusions: The five I’s: inoculation, incubation, isolation, inspection and identification are procedures used to culture microorganisms. A colony is a visible mass of microbial cells that theoretically arose from one cell. Pure cultures are usually obtained by the streak plate method

15. Types of Media There are hundreds of different types of media Contained in test tubes, flasks, or Petri dishes Media can be classified in three ways: Physical state Chemical composition Functional type

16. Physical States Liquid Solid (agar) Semi-solid Reversible (a) © Fundamental Photographs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © Kathy Park Talaro (b)

17. What Is Agar? Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C

18. Classification by Chemical Content Defined Media: the chemical composition is precisely known. (synthetic) e.g. 0.1 M sodium phosphate, pH 7.0, 0.1 M glucose, and 0.01 M ammonium sulfate Complex Media: one or more components is not chemically definable. (nonsynthetic) e.g. 5% yeast extract

19. Classification by Function General purpose media - to grow as broad a spectrum of microbes as possible Usually non-synthetic Contain a mixture of nutrients to support a variety of microbes Examples: nutrient agar and broth, brain- heart infusion, trypticase soy agar (TSA).

20. Enriched Media Enriched media- contain complex organic substances (growth factors) to support the growth of fastidious bacteria. Examples: blood agar, Thayer-Martin medium (chocolate agar)

21. Selective media lets some grow but not others. Differential media allow many microorganisms to grow but display visible differences (usually colors) Selective and Differential Media

22. Other Types of Media Reducing medium: to grow anaerobes and to determine oxygen requirement. Carbohydrate fermentation media: contain sugars and pH indicator. Assay media: to test effectiveness of antimicrobial drugs

23. Bacterial Cultures Pure culture: contains only colonies of the same identified species(axenic). Mixed culture: contains two or more identified species. Contaminated culture: contains colonies of unwanted microbes of uncertain identity

24. Conclusions A culture medium is any material prepared for the growth of bacteria in a laboratory. Microbes that grow and multiply in or on a culture medium are known as a culture. Agar is a common solidifying agent for a culture medium. A chemically defined medium is one in which the exact chemical composition is known. A complex medium is one in which the chemical composition varies slightly from batch to batch. By inhibiting unwanted organisms, selective media allow growth of only the desired microbes. Differential media are used to distinguish among different organisms.

25. Microbial Nutrition Microbes must get essential nutrients from the environment Macronutrients: Major elements C, O, H, N, P, S Ions Mg 2+, Fe 2+, K + Micronutrients: Trace elements necessary for enzyme function

26. Microbial Nutritional Strategies Carbon source: Autotroph: self-feeders use carbon dioxide Heterotroph: other-feeders use organic carbon Energy source: Chemotroph: use organic molecules Phototroph: use light Lithotroph: use inorganic molecules like H 2 S Every combination is possible and does exist…

27. Nutrient Transport Some substances move across cell membrane by diffusion, determined by concentration gradient and permeability of the substance. Most nutrients are polar, and not cross the membrane alone Requires a carrier Need to concentrate essential nutrients Requires energy

28. Osmosis The movement of water across a selectively permeable membrane from an area of high water concentration to an area of lower water concentration. Osmotic pressure: The pressure needed to stop the movement of water across the membrane.

29. Cell Response to Osmotic Content Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Early Direction of net water movement. Late Isotonic SolutionHypotonic SolutionHypertonic Solution Cell membrane Cell wall Cell membrane Water concentration is equal inside and outside the cell, thus rates of diffusion are equal in both directions. Water diffuses out of the cell and shrinks the cell membrane away from the cell wall; process is known as plasmolysis. Net diffusion of water is into the cell; this swells the protoplast and pushes it tightly against the wall. Cells with Cell Wall Cell membrane Cells Lacking Cell Wall Late (osmolysis) Rates of diffusion are equal in both directions. Diffusion of water into the cell causes it to swell, and may burst it if no mechanism exists to remove the water. Water diffusing out of the cell causes it to shrink and become distorted.

30. Facilitated Diffusion Used to transport hydrophilic molecules Protein carrier No energy required Does not accumulate against a gradient Extracellular High Intracellular Concentration Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

31. Active Transport Protein carrier and energy required Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (a) (b) Membrane Protein Extracellular IntracellularExtracellular Protein IntracellularExtracellularIntracellular Protein IntracellularExtracellularIntracellularExtracellular Membrane Group translocation modifies substrate

32. Active Transport Large substrates engulfed by eukaryotic cells Endocytosis and pinocytosis 1 2 3 4 PinocytosisPhagocytosis Liquid enclosed by microvilli Oildroplet Pseudopods Vacuoles Vesicle with liquid Microvilli (c) 1 2 3 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

33. Conclusions All organisms require a carbon source; chemoheterotrophs use an organic molecule, and autotrophs typically use carbon dioxide. Nitrogen is needed for proteins and nucleic acid synthesis. It is obtained from decomposition of proteins or from NH 4 + or NO 3 -, a few bacteria are capable of nitrogen fixation. Movement across the membrane may be by passive processes, in which materials move from area of higher to lower concentration and no energy is expended by the cell. In simple diffusion, molecules and ions move until equillibrium is reached

34. Conclusions Osmosis is the movement of water from areas of high to low concentration across selectively permeable membrane until equillibrium is reached In facillitated diffusion, substances are transported by transporter proteins across membranes from areas of high to low concentration. In active transport, materials move from areas of low to high concentration by transporter proteins and cell must expend energy. In group translocation, energy is expended to modify chemicals and to transport them across the membrane