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The Identification of Bacteria

Spencer Reames


In this activity, students are given a two week period to put to use the knowledge they have gained while studying bacteria. As a culminating activity, the students each receive three unknown bacteria to identify. The students are given a list of over thirty potential unknowns that they might have. They are responsible for designing the strategy that they will use to approach the identification of the unknowns (discussions as to how one might approach such a task take place throughout the course of the semester).

This activity does require the use of techniques and knowledge which had been gained prior to being given the unknowns. The students will have successfully demonstrated sterile technique and pure culture methods as well as staining methods by the time they receive their unknowns. In addition,they will have conducted a number of biochemical tests including: phenol red carbohydrate, catalase production, oxidase production, tests, methyl red test, Voges-Proskauer test nitrate reduction,starch hydrolysis, tryptophan hydrolysis, hydrogen sulfide production, citrate utilization, and litmus milk reactions. In addition they will have had experience in the characterization of colony characteristics, growth on slants, and the characterization of growth in liquid media. They will have determined motility of bacteria and demonstrated proficiency in Gram's stain and simple stains.

The procedures for running biochemical tests, staining bacteria, and the composition of the necessary reagents and bacteriological media are given in the references listed below. The necessary procedures and techniques are given in a wide variety of microbiology lab manuals.



  • hands on lab
  • authentic assessment

  • to demonstrate proficiency in handling bacteria
  • to demonstrate proficiency in use of various tests in order to identify bacteria
  • to develop problem solving skills
  • to help students develop ability to monitor their own learning

  • Bacterial cultures
    a few examples of bacteria that might be used include: Alcaligenes aecalis, Bacillus brevis, E. coli, Serratia marcescens, Micrococcus luteus, Enterobacter aerogenes, Staphylococcus epidermidis, Rhodospirillum rubrum

  • Bacteriological media
    Phenol red dextrose broth, phenol red lactose broth, MR-VP medium, nutrient broth, nutrient agar, nutrient broth, tryptic soy agar, nitrate broth, starch agar, SIM medium, triple sugar iron agar, Simmon's citrate agar, litmus milk, EMB agar (the students make all the media they need during the time that they are working on their unknowns)

  • Methyl red
  • Barritt's reagents
  • 3% hydrogen peroxide
  • sulfanilic acid
  • dimethyl-alpha-naphthylamine
  • zinc powder, Kovac's reagent

  • Crystal violet stain (Hucker modification)
  • 95% alcohol
  • Gram's iodine
  • safranin staining solution (for Gram staining)
  • 5% malachite green for spore staining

  • culture tubes and closures (16x 150 mm 0r 18x150 mm)
  • 100 x 15 mm sterile petri dishes
  • bunsen burners
  • inoculating loops
  • hot plate or stirring hot plate mixer
  • pressure cooker or autoclave
  • deionized water
  • microscope slides
  • serological pipets
  • Pasteur pipets
  • microscope with 100x oil immersion objective
  • balance
  • refrigerator (not necessary but it is helpful to have one)
  • incubator (this is not necessary for most of the bacteria that might be used)

    It is absolutely necessary that the students understand and be able to carry out the practices used in a microbiology lab before they be given an unknown bacteria. It is also important that known pathogenic strains be used in this activity. All cultures should be sterilized after use and before cleaning of the glassware.


    The students are given copies of charts which give characteristics of a number of bacteria such as 'Differentiation of Enterobacteriaceae by biochemical tests' from some of the references given below. In addition, they have access to all of the publications listed below for their use.

    Anonymous. 1984. Difco Manual: Dehydrated Culture Media and reagents for Microbiology. Difco Laboratories, Detroit.

    Baron, Ellen Jo, and Sydney M. Finegold. 1990.Bailey and Scott's Diagnostic Microbiology. The C.V. Mosby Co., St. Louis.

    Bartholomew, James. 1977. Laboratory Textbook and Experiments in Microbiology. Kendall /Hunt Publishing Co., Dubuque.

    Benson, Harold J. 1990. Microbiological Applications: A Laboratory Manual in General Microbiology. Wm. C. Brown Publishers, Dubuque.

    Blazevic, Donna J., and Grace Mary Ederer. 1975. Principles of Biochemical Tests in Diagnostic Microbiology. John Wiley and Sons, New York.

    Boyd, Robert. 1988. General Microbiology. Times Mirror/Mosby College Publishing, St. Louis.

    Boyd, Robert F. and J. Joseph Marr. 1980. Medical Microbiology. Little, Brown and Co., Boston.

    Buchanan, R.E., and N.E. Gibbons(editors). 1974. Bergey's Manual of Determinative Bacteriology, Eighth Edition. The Williams and Wilkins Co., Baltimore.

    Gordon, Ruth E., William C. Haynes, and C. Hor-Nay Pang. 1973.The Genus Bacillus: Agriculture Handbook No. 427. ARS-USDA, Washington, D.C.

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