Supplemental Lecture (98/04/14 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Identification of Bacteria
    1. A list of vocabulary words is found toward the end of this document
  2. Bacterial species
    1. Similar individuals:
      1. A bacterial species is "a population of cells with similar characteristics." (p. 257, Tortora et al., 1995)
      2. Note that this definition is very different from how plant and animal species are often defined, which usually involves something to do with sex.
    2. Resemble fossil species:
      1. Bacterial species resemble the way fossil species are distinguished (i.e., phylogenetic species concept).
      2. Normally plant and especially animal species are defined as populations of interbreeding or potentially interbreeding individuals that don't breed with individuals of other, like-defined populations.
      3. For fossils, on the other hand, it cannot be determined who interbred, or could have, with whom. Consequently, fossil species are defined only in terms of character resemblance, just as are bacteria.
  3. Bergey's manual
    1. Bergey's manual is a guide to distinguishing bacterial species based on phenotypic differences between isolates.
  4. Strain
    1. A strain is a subset of a bacterial species differing from other bacteria of the same species by some minor but identifiable difference.
    2. A strain is "a population of organisms that descends from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways." (p. 392, Prescott et al., 1996)
    3. Strains are often created in the laboratory by mutagenizing existing strains or wild-type examples of bacterial species.
    4. The term strain is also applicable to eucaryotic microorganisms , as well as to viruses .
  5. Type strain
    1. "One strain of a species is designated as the type strain. It is usually one of the first strains studied and is often more fully characterized than other strains; however, it does not have to be the most representative member. Only those strains very similar to the type strain are included in a species." (p. 392, Prescott et al., 1996)
  6. Serovar [serotype]
    1. A serovar is a strain differentiated by serological means.
    2. Individual strains of Salmonella spp. are often distinguished and distinguishable by serological means.

  7. Biovar [biotype]
    1. Biovars are strains that are differentiated by biochemical or other non-serological means.

  8. Morphovar [morphotype]
    1. A morphovar is a strain which is differentiated on the basis of morphological distinctions.

  9. Isolate ('i-so-lit)
    1. An isolate is a pure culture derived from a heterogeneous, wild population of microorganisms .
    2. The term isolate is also applicable to eucaryotic microorganisms as well as to viruses .

  10. Classification
    1. Placement of an organism within a scheme relating different types of organisms, such as that presented in Woese's universal tree , is know as classification.
    2. Organisms are classified for scientific purposes.

  11. Identification
    1. Identification is the determination of whether an organism (or isolate in the case of microorganisms ) should be placed within a group of organisms known to fit within some classification scheme.
    2. Organisms are identified for practical purposes, such as diagnosis of disease .
    3. Many identification techniques:
      1. Many different criteria may be employed for identification, though it is often desirable to employ the easiest techniques possible.
      2. What techniques and tests may be necessary, however, depend on what organism is being identified and how much detail into the organism's classification you are interested.
      3. Techniques include:
        1. morphological identification
        2. differential staining
        3. use of differential media
        4. serological methods
        5. flow cytometry
        6. phage typing
        7. protein analysis
        8. comparisons of nucleotide sequences

  12. Morphological identification
    1. A number of morphological characteristics are useful in bacterial identification. These include the presence or absence of:
      1. endospores
      2. flagella
      3. glycocalyx
      4. etc.

    2. Additional considerations include:
      1. colony morphology
      2. cell shape
      3. cell size
      4. etc.

  13. Serological methods [agglutination test, ELISA, Western blot]
    1. Antibodies:
      1. Serological methods employ antibodies and include:
        1. agglutination tests
        2. ELISAs
        3. Western blots

      2. It is antibody binding that all serological tests ultimately detect.

    2. The basic premise behind all of these tests is that antibodies are highly selective in terms of the proteins (or other cell structures) to which they bind, to the point that they are able to distinguish the proteins coming from one bacterial species among many species, or even one strain among many strain.

  14. Flow cytometry
    1. Flow cytometry is a technique that can employ serological methods (but doesn't necessarily) that analyzes cells suspended in a liquid medium by light, electrical conductivity, or fluorescence as the cells individually pass through a small orifice.

  15. Phage typing
    1. Bacteriophage (or phage ) are viruses that infect bacteria .
    2. Phage can be very specific in what bacteria they infect and the pattern of infection by many phage may be employed in phage typing to distinguish bacterial species and strains.

  16. Protein analysis [gel electrophoresis, SDS-PAGE, establishment of clonality]
    1. The size and other differences between proteins among different organisms may be determined very easily employing methods of protein separation using methods collectively known as gel electrophoresis.
    2. SDS-PAGE:
      1. One popular technique goes by the name SDS-PAGE which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
      2. Note that another name for SDS is sodium lauryl sulfate, a detergent you will find in many shampoos.

    3. Such methods are very good at detecting small differences between isolates and are especially good at establishing clonality.

  17. Comparison of nucleotide sequences [Southern blot, nucleic acid hybridization, RFLP, DNA fingerprinting]
    1. The actual sequence of bases (nucleotides) in the genome of organisms may be inferred or actually determined (nucleotide sequencing) by a variety of methods.
    2. Various methods of inference include:
      1. Southern blotting
      2. nucleic acid hybridization
      3. RFLP comparison (restriction fragment length polymorphism or DNA fingerprinting)

    3. Another technique that is worth knowing about is PCR which stands for polymerase chain reaction, a method of amplifying specific regions of DNA found in an organisms genome by selectively catalyzing the replication of those regions.
  18. Vocabulary
    1. Agglutination test
    2. Bacterial species
    3. Classification
    4. Bergey's Manual
    5. Biotype
    6. Biovar
    7. Nucleotide sequences
    8. DNA fingerprinting
    9. ELISA
    10. Flow cytometry
    11. Gel electrophoresis
    12. Establishment of clonality
    13. Identification
    14. Isolate
    15. Morphological identification
    16. Morphovar
    17. Morphotype
    18. Nucleic acid hybridization
    19. Nucleotide sequencing
    20. Phage typing
    21. Protein analysis
    22. RFLP
    23. SDS-PAGE
    24. Serological methods
    25. Serotype
    26. Serovar
    27. Southern blot
    28. Strain
    29. Type strain
    30. Western blot

  19. Practice questions
    1. You would consult Bergey's Manual if you were (circle only one correct answer) [PEEK]
      1. repairing your dune bergey
      2. classify a bacteria isolate
      3. identifying a bacteria isolate
      4. determining whether bacteria species A is more closely related to species B or species C
      5. all of the above
      6. none of the above

    2. Two pure cultures of bacteria differ only in terms of the stream (i.e., running water) in which they resided prior to their domestication. The bacteria in those cultures are considered to be separate __________. (chose best answer) [PEEK]
      1. strains
      2. isolates
      3. species
      4. serovars
      5. biovars
      6. all of the above
      7. none of the above

    3. Two pure cultures of bacteria are created by the splitting of a single broth culture in two and then growing the resulting broth cultures for 24 hours. The bacteria in those cultures are considered to be different __________. (chose best answer) [PEEK]
      1. strains
      2. isolates
      3. species
      4. serovars
      5. biovars
      6. all of the above
      7. none of the above

    4. Two bacteria differ by only a single genetic change, one which has no noticeable effect on the bacteria. Nevertheless, one is considered to be a mutant of the other. These two bacteria are considered to be different __________. (chose best answer) [PEEK]
      1. strains
      2. isolates
      3. species
      4. serovars
      5. biovars
      6. all of the above
      7. none of the above

    5. Name a circumstance in which consultation of Bergey's Manual might be helpful. [PEEK]
    6. Given that all of the following comparisons are between two stocks of bacteria of the same species, fill in blanks with the appropriate letter (i.e., best answer): [PEEK]
      1. strains
      2. isolates
      3. serovars
      4. biovars
      5. none of the above
        1. Different antibody reactivity: __________
        2. Different cell shape: __________
        3. Differentiable, but not as living cells (no phenotypic differences): __________
        4. Obtained from different locations, otherwise identical: __________
        5. Differentiable, but both descended from single isolate: __________
        6. Not differentiable, both descended from single isolate: __________

    7. A microorganism purified from a wild, heterogeneous mixture of microorganisms is identified as being a member of a particular species, but is morphologically distinct from other members of that species. How might you describe such an organism? As ________. (CIRCLE ALL APPLICABLE ANSWERS) [PEEK]
      1. an isolate.
      2. a serovar.
      3. a morphovar.
      4. a biovar.
      5. a strain.
      6. a new species.
      7. a type strain.

    8. Describe a reason you might resort to protein analysis to distinguish two similar strains. [PEEK]
    9. Agglutination tests, ELISAs, and Western blotting all detect ___________ binding to samples, and thus are considered serological techniques. [PEEK]
    10. Usually one of the first strains of a species studied, the __________ is often more fully characterized than other strains. [PEEK]
    11. Two pure cultures of virus are obtained from two different patients. By all possible criteria, biological, morphological, serological, and genetic, the two viruses are deemed identical. Consequently, we classify the two viruses as being two examples of the same strain of virus. Given this information, we infer that the virus infecting each of the two patients had a common source, either one patient infected the other, or both were infected by a common third party. Since the two pure cultures of virus were obtained from different patients, despite the otherwise identity of the two viruses (extremely high degree of organismal similarity), we would still classify the two viruses as distinct and separate __________. [PEEK]

  20. Practice question answers
    1. ii, identifying a bacteria isolate
    2. i, isolates since they must be the same species and even same strain (since they differ minimally) but they do differ specifically in where or when they were isolated.
    3. vii, none of the above. Ideally, at least, there should be no difference between the bacteria making up the two cultures.
    4. i, strains. The lack of phenotypic differences is consistent with they not being serovars or biovars, both kinds of strains.
    5. You are attempting to identify an unknown bacteria; you are interested in knowing the characteristics of an identified microorganism.
    6. (1) iii, serovars; (2) iv, biovars; (3) i, strains; (4) ii, isolates; (5) i, strains or iv, biovar; (6) v, none of the above.
    7. i, iii, v, and also, even, iv: an isolate, morphovar, biovar (since morphovars are also biovars), and strain.
    8. (i) establishment of clonality, (ii) epidemiological reasons, (iii) determination of whether an epidemic is occurring due to a change in an organism (increase in virulence---establishment of clonality), or some sort of organism-independent procedural change, (iv) linking of contamination to potential source, etc.
    9. antibody
    10. type strain
    11. isolates

  21. References
    1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 227-228, 235-240, 249-250.
    2. Postlethwait, J.H. and Hopson, J.L. (1995). The Nature of Life. Third Edition. McGraw-Hill, Inc. New York. pp. 433-441.
    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. 255-272.