Laboratory Exploration of Survival of Probiotic Cultures Inside the Human Digestive Tract Using In Vitro Models

Abstract

Scientists often model complex biological phenomena in vitro, mimicking conditions found in living organisms. Understanding the power and limitations of biological models is an important topic in undergraduate science. In this activity, students develop their own in vitro model for testing the survival of bacteria from commercial probiotic supplements. Students work in groups to decide which factors are important for survival of bacteria in a chosen portion of the human digestive tract. Groups of students create their own in vitro models of organs such as stomach and/or intestines. Students expose a probiotic supplement to conditions mimicking the chosen portion of the human digestive tract, and measure the effect of those conditions on the survival of bacteria found in the supplement. Students choose to focus on conditions such as low pH found in stomach or pancreatic enzymes found in the upper intestine. Through this activity, students gain experience with serial dilutions and calculations of colony forming units (CFUs). This project also provides the students with the valuable experience of designing experiments in small groups. Students present their findings in a poster session, which provides a venue for discussing the validity and limitation of various models.

INTRODUCTION

This resource is meant to be a guided, yet open-ended set of laboratory research activities for undergraduate students. The goal is for students to think about what it takes to design and carry out a meaningful in vitro experiment in order to model and investigate a complex biological phenomenon, in this case survival of bacterial in the environment found inside the digestive tract. Building and evaluating models is integral to modern research practices in all biological sciences, and yet very few students get first-hand exposure to such experiences in undergraduate classroom or laboratory. Furthermore, the exercise ends with a poster session, which not only motivates the students to think about results in a clear and connected way, but gives them practice at presenting their findings. They may have to create and present posters at scientific conferences, at later dates, and this gives them an initial try.

Intended audience

This activity was developed for use by microbiology/biology majors. With modifications, the activity can also be used with allied health majors and non-majors.

Learning time

This is a three-week, lab-based project. A minimum of three three-hour laboratory sessions are needed for completion of the whole module. Alternatively, some of the activities of week 1 and week 3 may be conducted during non-lab class time. Most of the first week is spent on experimental design, and most of the third week is spent on counting colonies, data analysis and poster presentation. During the second week, it is helpful if students have access to lab outside of regularly scheduled lab hours to evaluate and count their plates. At institutions where this is not possible, the instructor may have to perform these tasks on student’s behalf: take the plates out of the incubator and place them in the refrigerator (approximately two days after plating).

Background

Students should have previous experience with sterile technique, serial dilutions, pH measurements and basic laboratory safety procedures. Students should also be familiar with the concept of selective media. It is helpful if biomolecules and bacterial cellular structures, including cell wall and cell membrane, are covered in class prior to this exercise, so that the students have an understanding of the effect of pH on proteins and living cells. Prior to this exercise, the microbial ecology of the human digestive tract should be briefly introduced, either in class or in lab session.

If this lab is the first time students encounter the process of serial dilutions and corresponding calculations, the instructor may use the handout “Serial Dilutions and CFU Calculations” (Supplementary Material) to teach these concepts. Students with no prior experience in dilutions or spreading technique will need a lab period to learn the technique.

Learning objectives

At completion of this activity students will:

1. Develop a specific research question to investigate survival of probiotics inside the human digestive tract.

2. Correctly perform serial dilutions and calculate the number of colony forming units (CFUs) in the probiotic supplement.

3. Explain major factors that influence survival of probiotic cultures in the human digestive tract.

4. Develop their own model for testing survival of probiotic cultures inside the human digestive tract.

5. Calculate and analyze the survival of bacterial cultures in their model system.

6. Present their data orally to their peers in a poster session.

7. Critically evaluate benefits and limitations of their own and their peers’ models.

PROCEDURE

Materials and methods

1. Probiotic supplements such as BIO-K Plus, Probiotic Complex 365, Power-dophilus and Solaray Acidophilus may be purchased in health food sections of many grocery stores, such as Whole Foods. Products may be in liquid, pill or capsule form. Probiotic supplements may also be ordered on-line from distributors such as http://www.drugstore.com or http://www.gnc.com.

2. At least 10 Lactobacillus Selection (LBS) or deMan, Rogosa and Sharpe (MRS) agar plates per group (for selection and growth of Lactobacillus, most common culture in probiotic supplements). LBS agar solution is prepared by suspending 84 g of LBS agar into 1 L of water, followed by autoclaving. MRS agar solution is prepared by suspending 70 g of MRS agar in 1 L of water, followed by autoclaving. Both types of ready-made media mixes can ne ordered from BD (http://www.bd.com/). The manufacturer’s website also contains complete recipes for making the media from scratch.

3. 0.2 mL of pepsin solution at 2 mg/mL at pH 2.0 per group (made by dissolving powdered pepsin in 10 mM HCl, followed by filter sterilization) (Sigma Aldrich).

4. 10 mLs of autoclaved 100 mM HCl per group (Sigma Aldrich).

5. 0.2 mLs of 5 mg/mL Trypsin solution per group; pH 6.5, made by dissolving powdered trypsin in 10 mM sodium phosphate buffer, followed by filter-sterilization (Sigma Aldrich).

6. 0.2 mLs of 5 mg/mL Chymotrypsin solution per group, at pH 6.5 made in 10 mM sodium phosphate buffer, filter sterilized (Sigma Aldrich).

7. 10 mLs of autoclaved 10 mM Na Phosphate Buffer pH 6.5 per group (Sigma Aldrich). It is helpful to first make a 1000 mM buffer stock (100x stock) by dissolving 9.64 g of monosodium phosphate and 8.08g of disodium phosphate in 1L of water; and then use the stock buffer to make the 1x NaPhosphate buffer solution by diluting with distilled water.

8. 5 mLs of 1 M NaOH solution per group (Sigma Aldrich).

9. 100 mL of autoclaved distilled water per group.

10. pH paper and/or pH meters with a small electrode.

11. 15 mL plastic test tubes, 1.5 mL eppendorf tubes (VWR) (3–4 per group, with more available as needed by individual protocols). The tubes will be used as in vitro modeling vessels, and also for performing serial dilutions.

12. Pipetters (100–1000uL, 20–200 uL, and 1–10 uL range) and corresponding pipette tips (VWR).

13. 37°C water bath (at least one per class).

14. 0.2 mLs of 1 mg/mL filter-sterilized Lysozyme solution in water (Sigma Aldrich).

15. Mortar and pestle (one per group). The mortar and pestle do not need to be sterile. Grinding of supplements mimics chewing of food, which is not a sterile process.

16. Scales, weigh boats, spatulas (at least one weighing station per class).

17. L-shaped spreaders (preferably disposable to avoid open flame).

18. 10mL plastic syringes and 0.2 um filters for sterile-filtering of enzyme solutions (VWR).

Faculty and student instructions

Faculty and student instructions for this guided, yet open-ended set of laboratory research activities for undergraduate students are available in the supplemental materials for this manuscript.

Suggestions for determining student learning

I suggest not grading the initial project proposals, because students usually do not have previous exposure to writing experiment proposals. I only provide written and oral feedback to the proposals, and assign a participation grade for the proposal (full credit if the proposal was turned in on time). Final poster presentations may be graded using the poster presentation grading rubric (Table 1). The interactive nature of a poster presentation allows the instructor to gain an insight into the students’ understanding of the experimental process and data analysis. This grading rubric may also be used with written laboratory reports.

TABLE 1.

Grading criteria for evaluating poster presentations.

Group Members:_______________________________________________________________________________
Presenter:______________________________________Evaluator:______________________________________
CRITERIA	0 Points	1 Point	2 Points	3 Points	4 Points	5 Points
1. Clearly defined question or hypothesis	no hypothesis or research question	vague hypothesis or research question	implicit question/hy pothesis, or student unsure about the exact hypothesis/question	hypothesis/question stated but there is confusion about details or assumptions	hypothesis/question stated, but there might be some misun derstanding	clearly stated, well understood research hypothesis or question
2. Backgroud ideas are well researched	no background information	some general background information, sources lacking	some general background information from textbooks or popular websites	background information adequate	background information is clear and detailed, multiple sources used, but not chosen selectively/critically	background information is clear and detailed, multiple sources used, student shows clear understanding why some sources might be more appropriate than others
3. Model parameters (experimental details) clearly presented	no model specified	general model, but no specific parameters	general model, some parameters	model mostly makes sense, but some parameters not clear	parameters clearly explained	excellent choice of parameters for the model, complete explanation of each parameter choice
4. Data clearly presented	no data	essential data missing	some data missing	most of the data included, hard to follow what they represent	all the data presented in a way that makes sense	excellent choice of graphs, charts and tables to present the data
5. Conclusions supported by data and calculations	no conclusion	conclusion does not make sense, given the data	conclusion supported by data, some calculations wrong	conclusion makes sense, minor calculation problems	clear conclusion and calculations	excellent conclusions, fully supported by data, all calculations clearly explained
6. Analysis/discussion includes evaluation of model and suggestions for further improvement	no analysis/discussion	analysis/discussion vague, or not related to the model	some attempt to comment on the quality/implications of the model	mostly adequate analysis of the model, but misses major limitations or assumptions	meaningful analysis of the model, might have missed some minor assumptions or limitations	thorough analysis of the model, awareness of limitations, assumptions, and implications of the chosen model

To encourage collaborative learning throughout this activity, students work in groups. Because the design, execution and presentation are truly group efforts, students are also evaluated as a group. To ensure fair participation of all group members, the instructor should meet with each student to talk about his or her specific role in the group, and to find out if everybody participated in the activity.

Sample data

Sample data with three sample lab reports and six sample posters are available in the supplemental materials for this manuscript.

Safety issues

Even though this lab uses food-grade ingredients as a starting material, general microbiology safety rules should be followed. Aseptic techniques should be followed throughout the experiment. Students should be trained in BSL-1 and BSL-2 safety protocols (1). Students should wear gloves, goggles and closed-toed shoes during the laboratory portion of this module. All biological waste (plates, disposable spreaders, pipette tips, etc.) should be autoclaved prior to disposal. Students use dilute solutions of strong acids and bases in this lab, and should be familiar with chemical spill emergency protocols.

DISCUSSION

Field testing

I have completed this project with three classes of students at Agnes Scott College, twice with an upper-level microbiology class (prerequisites include one year of introductory biology and organic chemistry), and once with a non-majors biology class. A total of 33 upper-level microbiology students distributed into 14 groups of two or three students each have completed this project. Eleven non-major students also completed a more guided version of this activity (as described under “Possible Modifications”). When implementing this project with non-majors, I specified the experimental conditions for testing the probiotic supplements, rather than having the students chose the conditions. I used the open-ended project, as described here, when I used this activity with biology majors.

Evidence of student learning

Pre-project Survey

A pre-project survey (Appendix 1) was administered to all students. Familiarity with the concept of probiotics (Question 1: Are you confident that you could explain what probiotics are to a friend with no microbiology background?) differed between biology majors in my microbiology class and non-majors in my microbiology-centered survey course ( 69% majors answered yes, and only 15% non-majors answered yes). This demonstrates the need for introducing the topic and this project at different levels to these different groups of students.

There was also a difference in perceived understanding of the role of physical and chemical factors in the digestive tract on survival of ingested probiotics. (Question 2: Do you understand what challenges ingested live cultures face as they travel down our digestive tract?). Thirty-eight percent of non-majors and 80% of majors that responded stated that they understood these factors.

Responses to the question: “If you were a researcher studying probiotics, what probiotic-related question or hypothesis would you like to test in your research?” were indicative of the students’ ability to think about a question broadly, without taking specific feasibility details into account.

Questions such as: What effect do probiotics have on our regular diet? Are probiotics actually helping to make us healthier? What benefits can be seen from taking probiotics? Are probiotics harmful if you take too much in your system? Do probiotics in products do what the maker says they do? show that the students are interested in exploration of the role probiotics may play in human health. I used the survey results to motivate an in-class discussion during week 1. When I asked the students if they could carry out studies to answer their questions safely and ethically, they soon realized that their proposed questions were difficult to study.

Our inability to study these questions in a safe and controlled experiment in undergraduate classroom motivates the development of a safe, controlled in vitro experiment.

I only introduced the last question (Do you understand how you could tell if a supplement could be beneficial to human health?) the second time I taught this lab to biology majors. Twenty-one students answered the question. The majority of the students chose answers that indicated that they did not fully understand the topic. Specifically, 11 students answered “I think I have some idea about how I could assess if a supplement could be beneficial” and five students answered “I am not sure how I could tell if a supplement is beneficial”. Interestingly, five students thought that you could learn about the safety and efficacy of supplements by reading the “FDA-approved product info”. This shows that a large fraction of microbiology students are not aware of the fact that supplements are not regulated the same way drugs are, and that manufacturer’s claims are often not supported by extensive studies –and provides a wonderful opportunity to discuss this topic.

Instructors may choose to administer the same survey after the activity is completed. I did not administer the same survey post-activity. Instead, I used the poster to assess the student understanding of the concepts covered in this activity.

Project proposal

Fourteen project proposals (one from each student team of two or three students) were analyzed for the following criteria:

1. Does the proposal contain a clear research question?

2. Does the proposal contain sufficient experimental detail?

3. Are there major experimental flaws with the proposal?

   1. Of the 14 proposals, only three did not have a clearly defined research question. After meeting with the instructor, the groups successfully redefined their questions.
   2. Twelve out of 14 proposals lacked some experimental detail. This shows the importance of evaluating the proposals prior to letting the students start with their experiments.
      The data typically lacking were:

      • ♦ Volumes of solutions added were not specified
      • ♦ Volumes of sample plated was not specified
      • ♦ Times of exposure to certain conditions were not specified
      • ♦ Concentrations of solutions were not specified
   3. There were four out of 14 proposals that had some flaws or problems in the experimental protocol. These problems are summarized below

      • ♦ One group did not realize that plating a strongly acidic solution on MRS plates might affect the actual growth of bacteria by affecting the pH of the plate itself. This was easily resolved by suggesting neutralization of the solutions prior to plating.
      • ♦ Two groups were not aware that their data would be of quantitative nature rather than just simply of the “yes/no” type. Claims such as “If there is no growth with these two enzymes, then the supplements are not capable of living in the digestive tract” should be discussed with the group to help them realize that they might have some survival, and that they can quantify their data.
      • ♦ Two groups proposed experiments that would take too long. I suggested simplifying their protocol and shortening their incubation times.

Poster presentations

Each group of two to three students presented their poster orally. A total of 14 poster presentations were evaluated using the six criteria shown below, and further explained under “Suggestions for determining student learning” found in the supplemental materials for this manuscript. The presentation and poster were evaluated together (i.e., there were no separate rubrics for visual assessment of the poster itself versus the quality of the oral presentation). I used the 0–5 grading scale for each criterion as specified in Table 1.

Poster-evaluation data show encouraging trends in the students’ ability to develop their own experimental models, collect and analyze the data, and interpret the data in the context of benefits and limitations of models they developed. Table 2 shows the results of poster analysis using the criteria explained above. Although the data are limited by the sample size (33 students in 14 groups), some positive trends can be observed. In all categories, the percentage of students who earned at least 3 points is 67% (Figure 1). The percentage of groups of students who earned at least 3 points is even higher in categories directly related to development of research skills. All of the 14 groups earned at least 3 points in category 1 (experimental question and/or hypothesis). This demonstrates that all of the students were able to develop and clearly pose their own research question as a result of this activity. Also, 13 out of 14 groups were able to choose and implement meaningful experimental model parameters, as demonstrated by a score of 3 or higher in the third grading category. Finally, perhaps the most challenging aspect of this project was critical evaluation of results and of the model itself. In this category (category 6), 13 out of 14 groups also earned a score of 3 or higher.

TABLE 2.

Evaluation of poster presentations. Number of groups/posters who earned a certain score in each category is shown below the respective grading category.

Score	1. Research Question/Hypothesis	2. Background Research	3. Model Parameters	4. Data Presentation	5. Conclusions	6. Analysis/Discussion
0	0	0	0	0	0	0
1	0	1	0	1	0	0
2	0	2	1	3	1	1
3	5	1	5	3	3	4
4	5	6	6	5	7	5
5	4	4	2	2	2	4

FIGURE 1.

Overall success of poster presentation in each grading category (as explained in Table 1)

SUPPLEMENTAL MATERIALS

Lab Student Version

Lab Instructor Version

Student Data

Student Data: Proposal #1

Student Data: Proposal #2

Student Data: Proposal #3

Student Data: Posters

Possible Modifications

Appendix 1: Pre-Lab Assessment Survey

Appendix 2: Serial Dilutions and CFU Calculations Handout

Appendix 3: Recipes Supplemental Materials References