Koch's postulates are regularly included in the lecture portion of microbiology courses, but rarely are they demonstrated in a microbiology teaching lab. This is understandable given the logistical challenges of undergraduates working with pathogenic bacteria, ethical concerns using animals, and limited time constraints of a weekly lab period. Here we present a cost-effective, time-friendly lab activity that demonstrates the principles of microbial isolation and infection assays that are part of fulfilling Koch's postulates. The disease is “peep pox” caused by a gelatinase-positive bacterial species hydrolyzing marshmallow peeps that proxy as infected animals.

Introduction

Koch's postulates are four steps that, if satisfied, will identify which microbe is the etiological agent of a disease:

  1. The suspected microbe is present in all instances of individuals with the disease.

  2. The microbe must be isolated in pure culture.

  3. A test animal inoculated with the purified microbe will show disease symptoms resembling those of the original host.

  4. The same microbe must be re isolated from the test animal showing similar symptoms.

Others have used plants to demonstrate Koch's postulates in a teaching laboratory (Fulton, 1981; Hogue, 1971; Lennox, 1985; Mitchell et al., 1997; Ringel, 1968). However, we wanted to simulate the original generation of these postulates as formulated by Robert Koch while isolating Bacillus anthracis from sheep to induce anthrax in rabbits (Brock, 1999).

While recently teaching a general microbiology lab course, our students isolated several environmental microbes that demonstrated strong abilities to hydrolyze gelatin in a traditional gelatinase test (Leboff & Pierce, 2010). One of these environmental isolates was later determined to be Bacillus amyloliquefaciens by sequencing the 16S rRNA gene (Sanders & Miller, 2010). Because of its very strong ability to hydrolyze gelatin, this bacterial species can simulate disease symptoms in an animal host comprised of gelatin. This surrogate creature is the marshmallow peep (see Figure 1) that is readily available and can be stored indefinitely on the shelf. Using marshmallow peeps as hosts, gummy bears as surrogate test animals, and B. amyloliquefaciens as a virulent microbe, we propose a simple lab exercise demonstrating the principal of Koch's postulates. A flow chart of the procedure of this lab is shown in Figure 2.

Figure 1.

Time lapse photography of a peep showing symptoms of peep pox. Incubation times from left to right are 0, 2, 4, and 6 days.

Figure 1.

Time lapse photography of a peep showing symptoms of peep pox. Incubation times from left to right are 0, 2, 4, and 6 days.

Figure 2.

Diagram of steps needed to fulfill Koch's postulates for “peep box.”

Figure 2.

Diagram of steps needed to fulfill Koch's postulates for “peep box.”

Supplemental Material online includes a user-ready presentation of the lab exercise and representative results.

Procedure

Lab Period 1

  1. Working in pairs, students are presented with a group of peeps that have been potentially exposed to peep pox. One subset of students receives marshmallow peeps (in empty petri plates) that have been briefly submerged in nutrient broth (NB) containing Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923D-5). Both these strains are designated as BSL-1 microbes. A second subset of students is given peeps exposed to these two bacteria and also Bacillus amyloliquefaciens (ATCC BAA-390), which is also a BSL-1 microbe. Students record the condition of their peeps, as many will be showing early symptoms of “necrotizing fasciitis” that accompanies the disease.

  2. Students touch the surface of their moistened peep with an inoculating loop and streak for isolated colonies on the following agar plates that are incubated at 37°C degrees:

    • NA (nutrient agar)

    • PEA (phenylethyl alcohol agar)

    • MAC (MaConkey agar)

    • EMB (Eosin Methylene Blue agar)

    • MSA (Mannitol salt agar)

  3. Place inoculated peeps into plastic Ziploc plastic bags and incubate for 1 week at 30°C. Include one Ziploc bag with a peep incubating only with NB as a control. (An alternative approach is incubate peeps in containers of NB with bacteria. Peeps left incubating with B. amyloliquefaciens will eventually hydrolyze in NB at room temperature.)

Lab Period 2

  1. Record growth patterns on agar plates (see Figure 1).

  2. Expected results (colony sizes and appearance) are shown in Table 1.

  3. Pick an isolated colony for each bacterium to restreak on separate NA plates and incubate at 37°C for 1–2 days. (satisfies postulate 2)

  4. Each student records the morphology of their peep inoculating in container with NB. (Have students compare appearances of their peeps and note if some are beginning to dissolve more than others.)

Table 1.
Expected colonly size and appearance.
bacteriumNAPEAMACEMBMSA
E. coli medium small dark pink metallic green no growth 
S. aureus small medium no growth no growth small, yellow hallo 
B. amylo. large large, mucoid no growth no growth large, no yellow halo 
bacteriumNAPEAMACEMBMSA
E. coli medium small dark pink metallic green no growth 
S. aureus small medium no growth no growth small, yellow hallo 
B. amylo. large large, mucoid no growth no growth large, no yellow halo 

Note: Images of expected results are available in the online supplementary material.

Lab Period 3

  1. Perform gram stains and spore stains on isolated bacteria from lab period 2.

  2. Use purified bacteria (step 3 from Lab 2) to “inoculate test animals.” This means using sterile swabs to inoculate tubes containing 3–5 ml NB and a gummy bear submerged in the NB. Include a tube with NB and a gummy bear but without bacteria as a control. Incubate at 30°C until lab period 3. (satisfies postulate 3)

  3. Record which of the peeps in the original Ziploc bags (step 4 from Lab 2) show signs of “peep pox” (have visible signs of hydrolysis compared to the control).

  4. As a class share observations on peeps:

    • Physical appearances of their peeps.

    • Whether or not their peep contained bacteria that produce large, mucoid colonies on PEA and large colonies without yellow halos on MSA. (satisfies postulate 1)

Lab Period 4

  1. Place gummy bear inoculations at 4°C for 15–30 min before determining if gummy bear shows signs of “peep pox” (hydrolysis). (satisfies postulate 3)

  2. Use liquid from gummy bear tube to streak for isolated colonies on PEA or MSA plates. (satisfies postulate 4)

  3. B. amyloliquefaciens should be identified as the etiological agent of “peep pox.” Reveal to the students the identities of the two other test bacteria used in the exercise, and have them generate a dichotomous key for identification of all three bacterial species based upon the results of their differential staining and growth on selective and differential agar plates.

  4. Encourage students to generate a confirmatory test to show that B. amyloliquefaciens is the microbe that causes “peep pox.” Three suggestions for testable hypotheses are:

    • Since the spore stain showed B. amyloliquefaciens forms endospores, but E. coli and S. aureus do not, it is expected that B. amyloliquefaciens should survive pasteurization of peeps.

    • Purified B. amyloliquefaciens is expected to hydrolyze gelatin using the gelatinase test.

    • B. amyloliquefaciens is expected to grow on a “peep plate” (analogous to the creation of a blood agar plate using red blood cells).

Advantages of this approach to teach Koch's postulates in a microbiology teaching lab

  1. Safety. All three bacterial species used are BSL-1 level organisms.

  2. Speed. The three bacteria grow quickly, and Koch's postulates can be taught in a 2–3 week period—longer if lab classes meet once per week.

  3. The three bacterial species are readily differentiated by light microscopy, colony morphologies, and differential growth on selective media.

  4. There is a clear “disease phenotype” of peep pox: it is hydrolysis of gelatin-based candies such as marshmallow peeps and gummy bears.

  5. This multilab protocol combines multiple techniques and can be used to introduce or to strengthen the following core microbiology skills: sterile technique, light microscopy, pasteurization, gelatinase test, and differential and selective media.

References

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