Supplemental Lecture (98/04/06 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Culturing Microbes
    1. A list of vocabulary words is found toward the end of this document
  2. Culture
    1. A culture is the microorganisms that grow in a culture medium.
    2. To culture means to grow microorganisms in a culture medium.
  3. Culture medium [pl. media]
    1. Culture media are solutions containing all of the nutrients and necessary physical growth parameters necessary for microbial growth.
    2. Note that not all microorganisms can grow in any given culture medium and, in fact, many can't grow in any known culture medium.
    3. In addition to chemical and physical characteristics, media can be distinguished qualitatively as:
      1. solid vs. broth
      2. non-synthetic vs. chemically defined
      3. reducing
      4. selective
      5. differential
  4. Solid medium [agar]
    1. Solid medium is media containing agar or some other, mostly inert solidifying agent.
    2. Solid medium has physical structure (broth lacks structure) and this allows bacteria to grow in physically informative or useful ways (e.g., as colonies or in streaks).
    3. Solid medium is usually used as:
      1. slants
      2. stabs
      3. petri dishes
    4. See illustration below.
  5. Illustration, solid medium
  6. Colony
    1. A colony is a pile or mass of a sufficiently large number of cells, growing on or in solid medium, that they are visible to the naked eye.
  7. Broth medium
    1. Broth medium is media lacking a solidifying matrix (see solid medium).
  8. Non-synthetic [chemically undefined] medium
    1. Chemically undefined ingredient:
      1. Non-synthetic medium contains at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch.
      2. Often these are partially digested proteins from various organismal sources.
      3. Nutrient broth, for example, is derived from cultures of yeasts.
    2. May be broth or solid.
  9. Simple non-synthetic medium
    1. A simple non-synthetic medium, operationally defined, is capable of meeting the nutrient requirements of organisms requiring relatively few growth factors.
  10. Complex non-synthetic medium
    1. Undefined with more growth factors:
      1. A more complex non-synthetic medium, on the other hand, supplies more (additional types of) growth factors.
      2. Complex non-synthetic medium are consequently able to support the growth of more fastidious microorganisms than can simple non-synthetic medium.
  11. Chemically defined [synthetic] medium
    1. A chemically defined medium is one prepared from purified ingredients and therefore whose exact composition is known.
    2. Not trivial for fastideous:
      1. To use a chemically defined medium you must know exactly what your microorganism's growth requirements are.
      2. If this is not known it must be discovered through trial and error and therefore is not a trivial process for fastideous microorganisms.
  12. Preprepared medium
    1. Media of many types can be obtained premixed, in an often dehydrated and powdered state. Such media are called preprepared media.
    2. Ready to use:
      1. It is even possible to obtain sterilized media that is ready to use.
      2. Such media can be especially desirable if one lacks an autoclave, filtration devices, or the desire to prepare even preprepared media from a can.
  13. Enriched medium
    1. Added growth factors:
      1. Enriched media is preprepared (or equivalent) media to which additional growth factors have been added.
      2. These may be added individually or in complex mixtures.
      3. Enriched medium therefore may be chemically defined or chemically undefined, simple or complex.
    2. An example of enriched medium employing the latter would be blood agar which is made from complex medium to which whole blood has been added.
  14. Reducing medium
    1. Oxygen scavenging media:
      1. Reducing medium is employed for growing obligate anaerobes.
      2. Reducing medium particularly contains chemicals (reducing agents) that deplete molecular oxygen.
  15. Selective medium
    1. Differential growth suppression:
      1. Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others (i.e., they select for certain microbes).
      2. Solid medium is employed with selective medium so that individual colonies may be isolated.
    2. Examples of selective media include:
      1. mannitol salts agar (selects against non-skin flora)
      2. MacConkey agar (selects against gram-positives)
      3. eosin-methylene blue agar (selects against gram-positives)
      4. phenylehyl alcohol agar (selects against gram-negatives)
  16. Differential medium
    1. Differential appearance:
      1. Differential media allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies.
      2. Note that almost any medium containing a specific substrate and well tailored indicator can be used as a differential medium.
    2. Examples of differential media include:
      1. mannitol salts agar (mannitol fermentation = yellow)
      2. blood agar (various kinds of hemolysis)
      3. MacConkey agar (lactose fermentation = yellow)
      4. eosin-methylene blue agar (various kinds of differentiation)
  17. Enrichment culture
    1. Techically enrichment culture differs from selective medium in not out and out suppressing the growth of non-enriched microorganisms (e.g., no selective poisons are employed).
    2. Enrichment culture is used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium.
    3. Unlike selective media, enrichment culture is typically used as broth medium.
  18. Pure culture technique
    1. Clonal populations:
      1. Pure culture technique is a method of culturing microorganisms in which all of the individuals in a culture have descended from a single individual.
      2. This is done so as to:
        1. inhibit evolutionary change within cultures
        2. allow the characterization of types microorganisms without the confounding presence of other, different types of microorganisms
    2. Colony isolation:
      1. The basis of pure culture technique is the isolation, in colonies, of individual cells, and their descendants, from other colonies of individuals.
      2. This is usually done by culturing methods employing petri dishes such as:
        1. streaking
        2. pouring
        3. spreading
    3. Isolation from the wild:
      1. When isolating microorganisms from complex mixtures it is always a good idea to repeat the isolation procedure at least once (e.g., restreak an isolated colony) to make sure that an isolated colony is truly derived from only a single cell (i.e., closely overlapping colonies can be indistinguishable from colonies founded from single cells).
      2. Following their isolation from the wild, microorganisms may be characterized by inoculation into differential medium to determine what type of nutrients they require or can use, and what types of by-products they produce. This aids in identification.
  19. Pouring a plate
    1. A pour plate is a method of melted agar inoculation followed by petri dish incubation.
    2. Steps include:
      1. cultures are inoculated into melted agar that has been cooled to 45C
      2. the liquid medium is well mixed then poured into a petri dish (or vice versa)
      3. colonies form within the agar matrix rather than on top as they do when streaking a plate
    3. Pour plates are useful for quantifying microorganisms that grow in solid medium.
    4. Because the "pour plate" embeds colonies in agar it can supply a sufficiently oxygen deficient environment that it can allow the growth and quantification of microaerophiles.
  20. Spreading a plate
    1. Quantification technique:
      1. Spreading a plate is an additional method of quantifying microorganisms on solid medium.
      2. Instead of embedding microorganisms into agar, as is done with the pour plate method, liquid cultures are spread on the agar surface using a devise that looks more or less like a hockey stick.
    2. An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45C+ melted agar temperatures.
  21. Preserving cultures
    1. Preserving cultures is important for:
      1. scientific reasons
      2. identification
      3. vaccine production
      4. industrial use
      5. etc.
    2. Methods of preserving cultures include:
      1. refrigeration
      2. stabs
      3. slants
      4. lyophilization
      5. freezing
  22. Refrigeration (a.k.a., 4C)
    1. Effective short term preservation:
      1. Refrigeration can be effective for short periods.
      2. All of the following may be refrigerated:
        1. broth cultures
        2. stabs
        3. slants
        4. streaks
  23. Stabbing
    1. Cultures are stabbed deeply into agar using a inoculating needle.
    2. The stabs are incubated until visible cultures form, then sealed and stored at room or lower temperature.
    3. See illustration.
  24. Slant method
    1. Cultures may be streaked onto the surface of the solid medium in a slant tube.
    2. The slants are incubated until visible culture formation then sealed and stored at room or lower temperature.
    3. See illustration.
  25. Lyophilization
    1. Freeze drying:
      1. Lyophilization is the freeze-drying of cultures.
      2. Cultures are first frozen and then dried under high vacuum.
      3. To revive cultures they are rehydrated by broth.
    2. Lyophilization can be an effective long term method of storage.
  26. Freezing
    1. Freezing with protection:
      1. Broth cultures are mixed with various ingredients (e.g., glycerol, DMSO) to limit damage upon freezing and then frozen to temperatures ranging from -50C to -95C.
      2. To revive cultures they are thawed, pelletted, and resuspended into broth.
    2. Freezing can be an effective long term method of storage.
  27. Colony morphology
    1. Differentiating colonies:
      1. Colony morphology gives important clues as to the identity of their constituent microorganisms.
      2. Important classes of characteristics include:
        1. size
        2. type of margin
        3. colony elevation
        4. colony texture
        5. light transmission
        6. colony pigmentation
  28. Colony size
    1. Colony size is dependent not just on the type of organism but also on the growth medium and the number of colonies present on a plate (that is, colonies tend to be smaller when greater than ascertain amount are present) and on culture medium characteristics.
    2. Usually stabilizes after few days:
      1. Colony size usually stabilizes after a day or two of incubation.
      2. Exceptions include:
        1. slow growing microorganisms
        2. during growth under conditions that promote slow growth
      3. With slow growth colonies may continue to experience growth past this time, especially if an effort is made to prevent solid medium from drying out.
  29. Type of margin
    1. Colonies can vary in the shape of their margins.
    2. See illustration below.
  30. Illustration, variation in colony margins
  31. Colony elevation
    1. Colonies can vary in their elevations both between microorganisms and growth conditions, and within individual colonies themselves.
    2. See illustration below.
  32. Illustration, variations in colony elevation
  33. Colony texture
    1. Surface appearance:
      1. Colonies can vary in their texture.
      2. Possible textures include:
        1. shiny to dull
        2. smooth to wrinkled
        3. rough
        4. granular
        5. mucoid
    2. A shiny, smooth, and/or mucoid appearance tends to be associated with the presence of capsular material.
  34. Colony light transmission
    1. The light transmission through colonies can range from:
      1. complete (transparent)
      2. through intermediate (translucent)
      3. through completely lacking (opaque)
  35. Colony pigmentation
    1. Colonies can come in a rainbow of colors.
  36. Petri dish
    1. Petri dishes are circular, vertical sided plates used to contain agar and with tops for aseptic purposes.
  37. Loop
    1. A platinum wire formed into a loop is heated to an orange glow to sterilize it then is used to transfer a culture from one physical location to another.
    2. See illustration below.
  38. Streaking
    1. Streaking is a method of applying cultures to solid medium:
      1. a sterile loop is cooled and brought into contact with a culture
      2. the loop is then brought into contact with the surface of solid medium whereupon it is streaked (i.e., dragged) along the surface of the solid medium
      3. colonies grow along the points of the streak
    2. See illustration below.
  39. Streaking a plate
    1. Colony isolation:
      1. A petri dish is streaked in manner such that individual colonies may be isolated.
      2. See illustration below.
  40. Illustration streaking a plate
  41. Vocabulary
    1. Broth medium
    2. Chemically defined medium
    3. Colony
    4. Culture
    5. Culture medium
    6. Differential medium
    7. Enriched medium
    8. Isolation from the wild
    9. Non-synthetic medium
    10. Pouring a plate
    11. Preserving cultures
    12. Pure culture technique
    13. Reducing medium
    14. Selective medium
    15. Solid medium
    16. Solid medium, illustration
    17. Spreading a plate
    18. Streaking a plate
    19. Streaking a plate, illustration
    20. Variation in colony elevation, illustration
    21. Variation in colony margins, illustration
  42. Practice questions
    1. To purify a microorganism, which would you very likely utilize (circle only one correct answer)? [PEEK]
      1. solid medium
      2. broth medium
      3. non-synthetic medium
      4. chemically defined medium
      5. all of the above
      6. none of the above
    2. You have a microorganism you know is both fastidious and which normally lives in human lungs. Which medium might this organism have the highest probability of growing on? (choose best answer) [PEEK]
      1. simple non-synthetic media
      2. complex non-synthetic media enriched with fresh, whole blood
      3. chemically defined, minimal media
      4. complex non-synthetic media
      5. all of the above
      6. none of the above
    3. Design an enrichment medium and a procedure for growing an endospore-forming bacterium that fixes nitrogen and uses cellulose for its carbon source (from Tortora et al., 1995, p. 166). [PEEK]
    4. A medium which lacks physical structure would be called _____________? [PEEK]
    5. You figure that if you could come up with a bacteria that decomposed PCBs you'd make zillions (PCBs---a.k.a., polychlorinated biphenyls---are a heterogeneous kind of toxic waste that are at least potential carbon and energy sources). In setting forth on this task you note that PCB spills often occur into environments both lacking in sufficient oxygen supplies and having relatively low temperature (e.g., the muck at the bottom of bodies of water), though it would be handy if the organism was also capable of growth in the presence of atmospheric oxygen and at 25C. You figure that for handling reasons you would also prefer that this organism be capable of producing endospores. How would you isolate bacteria that could decompose environmental PCBs? [PEEK]
    6. List two methods of efficient long term bacterial storage. [PEEK]
    7. If you were interested in isolating a wild microorganism in pure culture, one which you suspect makes up approximately half of the cells in your crude mixture, name a technique (other than the use of selective media or enrichment culture) that you would have to employ in order to accomplish this isolation, and tell me a key aspect of how you would have to use it in order to assure the preparation of a truly pure culture. [PEEK]
    8. Fill in the blanks with (a) reducing medium, (b) differential medium, (c) selective medium, (d) enriched medium, or (e) enrichment culture. [PEEK]
      1. Agar containing 8.5% CaCl2 to increase osmolarity of medium: ___________.
      2. Broth containing molecular oxygen (O2)-scavenging chemical: __________.
      3. Agar containing factors which inhibit gram-positive bacteria growth, such as bile salts and crystal violet: __________.
      4. Agar which turns blue when galactose is fermented: __________.
    9. Which method is least able to maintain cultures for the long term (years): [PEEK]
      1. stabbing
      2. slant culture in refrigerator
      3. lyophilization
      4. storage at -85C
      5. storage at -70C
      6. stab culture in refrigerator
    10. What does the noun "isolate" mean? [PEEK]
    11. A __________ medium is one that aids the growth of obligate anaerobes. [PEEK]
    12. A __________ medium is designed to suppress the growth of some microorganisms while allowing the growth of others. [PEEK]
      1. reducing
      2. selective
      3. differential
      4. enriched
      5. complex
      6. chemically defined
  43. Practice question answers
    1. i, solid medium
    2. ii, media enriched with fresh, whole blood. The fact that it's fastidious and lives in/on you suggests that it might require growth factors normally present in the human body. A good sampling of those growth factors often can be found in whole blood.
    3. No nitrogen except from the atmosphere and no carbon source except from cellulose. Boil the mixture at some point, preferably before inoculating, to kill all vegetative cells. Then you would isolate the surviving endospores perhaps on selective media (i.e., no nitrogen with cellulose as its sole carbon source).
    4. broth medium
    5. Start with the muck at the bottom of a lake (or a soil sample). Boil sample to kill vegetative cells. Use selective media containing only PCBs as both the carbon and energy source. Incubate at low temperatures and under anaerobic conditions. Plate differentially on PCB-containing plates (sole carbon and energy source) at 25C, exposed to the air.
    6. Freezing, lyophilization, and to a lesser extent stab culture.
    7. Colony isolation by streak or pour plate and repeated isolation of individual colonies in order to assure purity.
    8. selective medium, reducing medium, selective medium, differential medium.
    9. ii, slant culture in refrigerator.
    10. An isolate is a (preferentially pure) culture of microorganisms which was derived from a heterogeneous wild population.
    11. Reducing
    12. ii, selective
  44. References
    1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 144-148.
    2. Cappuccino, J.G., Sherman, N. (1996). Microbiology. A Laboratory Manual. Fourth Edition. The Benjamin/Cummings Publishing Co., Inc. Menlo Park, California. pp. 13-16, 21-23, 89-90.
    3. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 147-154, 158-166.