Using Flow Cytometry to Measure Phagocytic Uptake in Earthworms

Abstract

This laboratory module familiarizes students with flow cytometry while acquiring quantitative reasoning skills during data analysis. Leukocytes, also known as coelomocytes (including hyaline and granular amoebocytes, and chloragocytes), from Eisenia hortensis (earthworms) are isolated from the coelomic cavity and used for phagocytosis of fluorescent Escherichia coli. Students learn how to set up in vitro cellular assays and become familiar with theoretical principles of flow cytometry. Histograms based on fluorescence and scatter properties combined with gating options permit students to restrict their analyses to particular subsets of coelomocytes when measuring phagocytosis, a fundamentally important innate immune mechanism used in earthworms. Statistical analysis of data is included in laboratory reports which serve as the primary assessment instrument.

INTRODUCTION

First discovered by Elie Metchnikoff, phagocytes provide a critical first line of defense for the innate immune system by engulfing and killing microbes. Phagocytosis commences with binding of the phagocyte to its target (or “prey”) using microbe recognition receptors (e.g., mannose, scavenger and opsonin receptors) on the host cell surface, followed by engulfment into a phagosome. Engulfment requires the activation of signaling pathways that facilitate the rearrangement of the cytoskeleton. The internalized phagosome fuses with a lysosome to form a phagolysosome where microbial killing of the prey occurs. Killing involves the activation of a respiratory/oxidative burst (a dramatic increase in nonmitochondrial oxygen consumption) and the production of reactive oxygen species (ROS) which are toxic to ingested pathogens.

This curriculum module focuses on the use of E. hortensis as an invertebrate model to study phagocytosis. E. hortensis possesses a coelomic cavity which runs the length of the earthworm and contains coelomic fluid and coelomocytes which, together, employ highly effective cellular and humoral innate defense mechanisms to combat microbial infections. The coelomocytes share many of the same functions as mammalian leukocytes including the ability to phagocytize, induce inflammation and graft rejection, and stimulate agglutination and cytotoxicity reactions. The cellular component is comprised of leukocytes known as coelomocytes. The coelomic cavity of E. hortensis consists of three major subpopulations of coelomocytes: hyaline amoebocytes (large coelomocytes consisting of neutrophils and basophils), granular amoebocytes (small coelomocytes comprised of granulocyes, type I and II acidophils, and transitional cells), and chloragocytes (also referred to as eleocytes which consist of type I and II chloragogen cells). Although the granular amoebocytes also phagocytize, it is the hyaline amoebocyte subpopulation that exhibits the most significant phagocytic activity. This laboratory exercise will focus primarily on the hyaline amoebocyte subpopulation and its role in phagocytosis using a flow cytometer to track the ingestion process.

Intended audience

Microbiology/Biology majors, Biotechnology majors

Learning time

Three lab sessions: Three three-hour labs (Lab 1: Mini lecture on flow cytometry and explanation of how to set up in vitro phagocytosis assay; Lab 2: Extrusion of coelomocytes, phagocytosis assay and data acquisition on the flow cytometer; Lab 3: Analysis of data using flow cytometry software).

Prerequisite student knowledge

The laboratory curriculum module described here was used in an advanced microbiology course (BIO 308) at Cabrini College for undergraduates majoring in biology with concentrations in biological sciences, biotechnology, and pre-medicine, and also for those students enrolled in pre-nursing. Students were familiar with innate immune responses through material covered in lecture. Students were already familiar with micropipetting, fluorescence microscopy, hemacytometry, and aseptic technique. The basic concepts and theory of flow cytometry were introduced during a mini-lab lecture preceding this exercise. Prior to attending the mini-lecture, students were encouraged to read Chapters 1–5 of “Introduction to Flow Cytometry: A Learning Guide” from BD Biosciences (available online at http://www.scribd.com/doc/11553056/introduction-to-flow-cytometry-a-learning-guide), a thirty-seven page, easy-to-read summary of the basics of flow cytometry. Information about light scatter, fluorescence, fluidics, optics, electronics and data analysis was introduced in this brief review. Students were encouraged to answer the questions embedded within the reading assignment to facilitate their understanding of flow cytometry.

Learning objectives

(Refer to Appendix 4B for an explanation of how each of the following eight student learning outcomes is assessed within the formal laboratory report.)

1. Students research the biological process of phagocytic engulfment.

2. Students isolate and enumerate coelomocytes from E. hortensis.

3. Students devise a testable hypothesis and then collect and analyze the data to test the hypothesis.

4. Students set up in vitro phagocytic assays including appropriate controls and run samples on the flow cytometer.

5. Students use flow cytometry software to analyze their data by assigning regions, using gating options and overlaying histogram profiles.

6. Students use quantitative reasoning skills to calculate the percent specific phagocytosis of bacteria, and determine statistical significance between experimental and control groups using the student t-test.

7. Students write a formal scientific paper incorporating methodology, results and conclusions of their experiment.

8. Students generate and answer questions that reflect their mastery of the cellular mechanism involved in phagocytosis and flow cytometry technology.

PROCEDURE

Materials

Materials, Instrumentation, Recipes and Flow Cytometry Settings (Appendix 1) describes the laboratory reagents, equipment and media composition for this exercise. It also contains important information for setting up the flow cytometer for data acquisition. This information should be shared with the flow cytometer operator, providing extra untreated earthworm coelomocytes (without E. coli/GFP) and E. coli/GFP for instrument setup.

Student instructions

Students are given copies of: 1) Materials and Methods for Phagocytosis and Flow Cytometry (Appendix 2) for the first laboratory meeting; and 2) WinList Protocol (Appendix 3) for the third laboratory meeting.

Faculty instructions

Undergraduate microbiology courses for biology majors generally reserve the discussion of host defense mechanisms for combating microbial infections for the lecture component of the course, and do not usually implement laboratory procedures to explore the cellular physiology of adaptive and innate immune responses. Some labs incorporate a serological procedure (e.g., ELISA), which provides an excellent opportunity to link lecture material on adaptive immune responses with a hands-on laboratory activity. This curriculum module describes a laboratory exercise appropriate for an advanced microbiology course that investigates a universal innate immune response that is fundamental to the survival of a wide range of eukaryotes. Specifically the process of phagocytosis (4, 9, 10) will be introduced to students using a combination of flow cytometry and short-term in vitro cell culture methods using leukocytes derived from the invertebrate Eisenia hortensis, also known as the European nightcrawler (earthworm). Intellectual discovery guides the students to a more comprehensive understanding of the importance of innate immunity in invertebrates through an experimental approach.

This curriculum module was introduced to two junior/senior-level microbiology classes of 20 and 17 students in fall 2008 and spring 2010. The fall 2008 class had two labs of 10 students each, while the spring 2010 class had a single lab with 17 students. The module focused on the use of E. hortensis as an invertebrate model to study phagocytosis. E. hortensis possesses a coelomic cavity which runs the length of the earthworm and contains coelomic fluid and coelomocytes which together employ highly effective cellular and humoral innate defense mechanisms to combat microbial infections (2). The coelomocytes share many of the same functions as mammalian leukocytes including the ability to phagocytize, induce inflammation and graft rejection, and stimulate agglutination and cytotoxicity reactions. The cellular component is comprised of leukocytes known as coelomocytes. The coelomic cavity of E. hortensis consists of three major subpopulations of coelomocytes, hyaline amoebocytes (large coelomocytes consisting of neutrophils and basophils), granular amoebocytes (small coelomocytes comprised of granulocyes, type I and II acidophils, and transitional cells), and chloragocytes (also referred to as eleocytes which consist of type I and II chloragogen cells) (7). Although the granular amoebocytes also phagocytize, it is the hyaline amoebocyte subpopulation that exhibits the most significant phagocytic activity (3, 5). Figure 1 shows the three major coelomocyte types found in E. hortensis, which are distinguished easily by flow cytometry based on their relative size and granularity. The laboratory exercises described in this paper will focus primarily on the hyaline amoebocyte subpopulation and its role in phagocytosis using a flow cytometer to track the ingestion process.

FIGURE 1.

Shown are coelomocytes from E. hortensis after extrusion from the coelomic cavity. The large cells resembling fried eggs are the hyaline amoebocytes (large coelomocytes). The smallest cells with the white halo around their perimeter are the granular amoebocytes (small coelomocytes). The cells containing multiple small vesicular inclusions are the chlorogocytes (eleocytes). (Phase contrast microscopy, 400×)

The flow cytometer is an instrument that permits the operator to generate multiparameter data from particles (or cells) in suspension based on light scattering and light excitation/emission properties using fluorochrome molecules. Flow cytometry enables rapid acquisition of experimental data (up to ∼ 5000 particles per second), permitting the collection of numerous student samples in a short period of time, with the cost per sample being relatively low. The light source is a laser. An argon laser is commonly used in flow cytometry; it has an excitation wavelength of 488 nm, appropriate for the excitation of GFP described in this exercise. The laser is aligned to intercept a laminar sheath of particles that are focused hydrodynamically in a saline stream. [See (6) for an introductory textbook on the principles of flow cytometry.] As particles enter the laser interrogation point, they scatter light in both a forward and side direction, referred to as forward scatter (FSC) and side scatter (SSC), respectively.

Forward scatter is an indicator of particle size (cell volume), while side scatter is an indicator of particle granularity (internal complexity). Both measurements are obtained even in the absence of a fluorochrome molecule. If a fluorochrome molecule [such as green fluorescent protein (GFP)] is bound to the particle, it is excited by the laser and absorbs energy (higher energy state) followed by emission (or release) of that energy as a photon of light at a longer wavelength (lower energy state), a transition referred to as Stokes shift. The emission spectral properties obtained are unique to different fluorochromes, allowing a single laser to be used to excite multiple fluorochromes providing their emission spectra have different Stokes shifts for detection by different photomultiplier tubes. The emitted light, or fluorescence, is detected in the flow cytometer using a variety of filters and mirrors that direct the emitted light to photomultiplier tubes (e.g., FL-1, FL-2, FL-3) that amplify the signal. Finally, the optical signals are converted to electronic signals which, in turn, are assigned digital values that are proportional to light intensity. Listmode data files are collected which list all of the events corresponding to all of the parameters collected (forward scatter, side scatter and fluorescence) for each particle. Data analysis is carried out using WinList 5.0.

Because of the ability to gate specifically on desired cell populations using flow cytometry software, it was not necessary to fractionate the subpopulations to investigate the phagocytic properties of hyaline amoebocytes in the laboratory exercise described in this paper. This feature was afforded by the different forward scatter (size) and side scatter (granularity) properties of the three principal cell types in earthworm coelomic fluid. Therefore, the changes that occurred in the hyaline amoebocyte population during phagocytosis were studied in isolation from the other two cell types using listmode analysis software employing gating options. To gate a desired population of cells, the process is very straightforward. For this exercise, the total coelomocyte population is first run on a flow cytometer and analyzed for forward scatter (FSC - abscissa) versus side scatter (SSC -ordinate). The flow cytometer operator will need to adjust the voltage and amplifier gain settings so that the three sub-populations are visible. Figure 2 shows what this will look like and explains the process of gating in more detail.

FIGURE 2.

A forward (FSC) versus side (SSC) scatter profile of coelomocytes from E. hortensis. Note the rings [called regions (R)] drawn around the three major subpopulations: R1 = hyaline amoebocytes; R2 = granular amoebocytes; and R3 = chloragocytes. A region is a boundary drawn around a subpopulation for analysis. Once the regions have been established, analysis (e.g., FSC versus fluorescence detected by the FL-1 photomultiplier tube) can be carried out specifying which particular regions to include, a process known as gating. A gate may include one region or more than one region.

This curriculum module describes how the ingestion of fluorescent bacteria by hyaline amoebocytes was determined using a flow cytometer. E. coli-expressing GFP was used as the “prey” and ingestion was confirmed by measuring the increase in relative fluorescence intensity of the hyaline amoebocytes following phagocytosis; ingested prey caused a corresponding increase in the geometric mean of fluorescence measured by the FL-1 detector.

It is acknowledged that the instrumentation needed to carry out these exercises requires a significant capital outlay, however, with the acquisition of these instruments, justification of embarking upon or expanding undergraduate research programs is warranted – a direction gaining momentum in higher education and recognized as an important strategy for recruiting talented students interested in challenging themselves beyond the traditional curriculum. If the instructor does not have access to a flow cytometer, the samples can be chemically fixed with 1% paraformaldehyde at the end of the procedure, and stored at 4°C for a period of about 1 week until analysis can be carried out at an off-campus flow cytometry facility. Many larger colleges and universities have shared-use flow cytometry facilities which are available to neighboring colleges who do not have these facilities at their disposal. It is worthwhile investigating whether these types of collaborations are feasible for smaller colleges interested in teaching flow cytometry techniques.

Additional notes for instructor

If hemacytometers are not available, or if there are not enough of them to facilitate timely enumeration of coelomocytes, assume that the coelomocyte number is ∼ 1 × 10⁶ per earthworm if extrusion occurs (look for cloudy extrusion buffer and a noticeable pellet upon centrifugation).

If access to a flow cytometer is not an option, this protocol could be modified for fluorescent microscopy. Be aware, however, that the chloragocytes (eleocytes) are highly autofluorescent, and they should be excluded from analysis when viewing, to avoid obtaining false positives. Their characteristic morphology (multivesicular) under phase contrast, and even distribution of autofluorescence, facilitates their identification.

Instead of using E. coli/GFP obtained through the BioRad kit, the instructor could label heat-killed bacteria with fluorescein isothiocyanate. This would also eliminate the need to use paraformaldehyde for chemical fixation. Briefly the procedure for making heat-killed FITC-labeled bacteria is: centrifuge 10⁹ bacteria at ∼ 3200 × g, discard supernatant and resuspend pellet in 1 ml FITC/NaHCO3 [0.1 mg ml⁻¹ FITC (fluorescein isothiocyanate) isomer 1 (Sigma) in 0.1 M NaHCO3, pH 9.0]. Incubate 60 min at 25°C. Wash bacteria by four repeats of centrifugation, removal of supernatant, and resuspension in phosphate-buffered saline (PBS) containing 5% fetal calf serum and 5 mM glucose. After enumeration, bacteria are ready to use in the phagocytosis assay. The disadvantage to this methodology is the need to prepare the bacteria fresh for each laboratory meeting. In contrast, once amplified and chemically fixed with paraformaldehyde, E. coli/GFP is stable for 6–12 months at 4°C in the dark, enabling the same batch to be used with reproducibility over extended periods of time.

The Super Dulbecco’s Modified Eagle Medium (SDMEM) used is highly recommended if the students wish to adopt an inquiry-based, hypothesis-driven approach (see below) to carrying out this procedure, which involves culturing the earthworm coelomocytes for extended periods of time (2–5 days). It is possible, however, to modify the SDMEM by omitting the tetracycline, chloramphenicol, ampicillin and kanamycin, without altering the results. This medium can also be supplemented with newborn calf serum instead of fetal calf serum, to reduce costs.

If 96-well V-bottom plates are not available, students could simply use the flow cytometry tubes to carry out their phagocytosis reactions. The “buzz-spin” can be carried out directly in the flow cytometry tubes. A “buzz spin” involves bringing the centrifuge speed up to 150 × g for only a few seconds before braking.

Instead of using WinList 5.0, there are a number of other software programs that can be used for analysis of listmode data generated by the students on the flow cytometer. For example WinList 6.0, which is an updated version of WinList 5.0 that uses the same basic steps for histogram generation and analysis outlined in Appendix 3 (Verity Software House, Topsham, ME; ), is available. Additional software programs include CellQuest, WinMDI, EXPO, Paint-A-Gate, FloJo and MFI. Some of these software programs are available as free downloads, helping with budgetary constraints.

Instructors can also use Eisenia fetida, also known as redworms (Carolina Biological Supply Company, Burlington, NC), a closely related earthworm. The forward and side scatter properties are very similar between the two species. The coelomocyte yield is a lower compared to E. hortensis, but should be sufficient for this exercise.

Appendix 1 includes a comprehensive description of all materials needed and advance preparation required by the instructor. Note that the background information in the “Instructor Version” above has been incorporated into Appendix 2, which should be distributed to students in the form of a handout.

Suggestions for determining student learning

Assessment of student learning is based on the completeness and quality of a laboratory write-up (see Formal Laboratory Report and Laboratory Rubric - Appendix 4A & B) and their performance on a twenty-question quiz. This quiz is given as an unannounced pre-test early in the semester prior to the introduction of the mini-lecture, and again as an announced post-test a few days after the introduction of the mini-lecture (see Flow Cytometry Pre-Test and Post-Test for General Microbiology Laboratory Exercise – Appendix 5). A student evaluation questionnaire is also administered as part of assessment (Appendix 8).

Student data

Student data for the spring 2010 class is shown in Fig. 3 and Tables 1–3. The results of the pre- and post-test (Appendix 5) are shown in Fig. 3. Out of 17 students enrolled in the General Microbiology (BIO 308) spring 2010 course, five were also co-enrolled in my Theory & Practice in Biotechnology (BIO 312) course. Those five students had already learned the basics of flow cytometry, used the instrument and software, and written lab reports based on several exercises employing flow cytometry. Because their prior learning would skew the interpretation of student learning when comparing the pre-test and post-test averages, Fig. 3 shows statistical data that includes all students (n = 17), as well as data that includes only those students who were enrolled in General Microbiology but not in Theory & Practice in Biotechnology (n = 12). In both datasets, class averages were statistically significant (p < 0.01) when comparing pre-test with post-test averages. Tables 1–2 are based on the grading rubric for the formal laboratory report (refer to Appendix 4B). Table 1 indicates the performance in each category of the formal laboratory report for every student, while Table 2 shows the statistical analysis of student performance based on the criteria: exemplary, satisfactory or needs improvement. Table 3 shows the results of the Student Evaluation (Appendix 8), a ten-question questionnaire distributed to students after completion of data collection and analysis.

FIGURE 3.

Results of pre-test and post-test are illustrated. Left to right: pre-test for all students in class; post-test for all students in class; pre-test for students in class excluding students enrolled in BIO 312 (Theory & Practice in Biotechnology); post-test for students in class excluding students enrolled in BIO 312.

TABLE 1.

Student Performance on Formal Laboratory Report – Spring 2010 Class. Individual scores and averages awarded for each category on the grading rubric (see Appendix 4B) and the overall grade earned are indicated for each student (n=17). Maximum possible points for each category are shown in parentheses.

Student	Abstract (10)	Format (5)	Intro (20)	Obj (5)	M&M (15)	Results (20)	Disc (10)	Refs (5)	Q&A (10)	%/Grade
#1	8	3	19	1	15	16	5	5	9	81/B−
#2	8	3	20	3	12	18	9	4	5	82/B−
#3	5	3	19	1	12	15	5	2	8	70/C−
#4	8	5	18	4	14	18	5	3	10	85/B
#5	8	1	13	3	12	16	5	3	9	70/C−
#6	5	2	0	2	11	10	5	3	8	46/F
#7	5	3	11	1	9	10	1	4	7	51/F
#8	7	1	19	4	13	17	8	1	10	80/B−
#9	9	1	20	4	13	19	7	4	10	87/B+
#10	9	3	19	1	13	18	8	3	10	84/B
#11	5	0	6	3	12	16	7	0	6	55/F
#12	9	1	19	4	10	16	10	3	8	80/B−
#13	9	3	18	4	14	19	10	3	10	90/A−
#14	8	4	19	3	15	16	7	5	10	87/B+
#15	8	3	19	5	14	16	7	5	10	87/B+
#16	9	3	19	2	15	20	10	5	10	93/A
#17	8	3	19	4	14	15	7	4	10	84/B
Average	7.5	2.5	16.3	2.9	12.8	15.4	6.8	3.4	8.8	77.2/C+

Intro = Introduction; Obj = Objectives; M&M = Materials & Methods; Disc = Discussion; Refs = References; Q&A = Student-generated Questions & Answers. Grading scale (%): 93–100 = A; 90–92 = A−; 87–89 = B+; 83–86 = B; 80–82 = B−; 77–79 = C+; 73–76 = C; 70–72 = C−; 67–69 = D+; 60–66 = D; < 60 = F.

TABLE 3.

Student Evaluation of Flow Cytometry Laboratory Exercise (n = 17)

Student Evaluation of Flow Cytometry Laboratory Exercise
(5) Strongly Agree (4) Agree (3) Neutral (2) Disagree (1) Strongly Disagree (NA) Not Applicable
	(5)	(4)	(3)	(2)	(1)	(NA)	No Response
1. This laboratory exercise enhanced my general knowledge of flow cytometry.	47%	53%
2. This laboratory exercise enhanced my ability to understand microbiology.	29%	47%	24%
3. The written material for this laboratory exercise was useful.	41%	47%	6%				6%
4. The instructor was well prepared and knowledgeable.	100%
5. The facilities and instrumentation for this laboratory exercise were adequate to support the class.	71%	29%
6. I would like to see flow cytometry used in other biology classes.	17.5%	59%	17.5%	6%
7. My experience with flow cytometry has enhanced my ability to understand innate immune responses.	23.5%	70.5%	6%
8. My experience with flow cytometry has enhanced my ability to analyze scientific data quantitatively.	47%	29%	24%
9. My experience with flow cytometry has enhanced my appreciation for computer-aided data analysis.	35%	59%	6%
10. As a result of this laboratory exercise, I am more interested in becoming involved in undergraduate research.	29%	47%	18%		6%

TABLE 2.

Statistical Analysis of Student Performance Based on Criteria Established for Exemplary, Satisfactory, or Needs Improvement. Where each student learning outcome was achieved is indicated. The average score for each criterion is shown with % students achieving each criterion in parenthesis.

Category (maximum possible points)	Student Learning Outcomes*	Exemplary	Satisfactory	Needs Improvement
Abstract (10)	7	9 (29%)	7.9 (47%)	5 (24%)
Format (5)	7	4.5 (12%)	2.9 (59%)	0.8 (29%)
Introduction/Background Sources (20)	1, 7	19 (76%)	12 (12%)	3 (12%)
Objectives/Purpose (5)	3, 7	4.1 (41%)	2.7 (35%)	1 (24%)
Material & Methods/Procedures (15)	2, 4, 5, 7	14 (59%)	11.5 (35%)	9 (6%)
Results/Detail/Evidence (20)	2, 3, 5, 6, 7	18.4 (41%)	15.8 (47%)	10 (12%)
Discussion/Summary (10)	3, 7	9.8 (24%)	7.3 (41%)	4.3 (35%)
References (5)	7	4.5 (47%)	2.9 (41%)	0.5 (12%)
Student-Generated Questions (10)	1, 7, 8	9.8 (71%)	7.4 (29%)	(0%)

^*Student Learning Outcomes:

¹Students research the biological process of phagocytic engulfment

²Students isolate and enumerate coelomocytes from E. hortensis

³Students devise a testable hypothesis and then collect and analyze the data to test the hypothesis

⁴Students set up in vitro phagocytic assays including appropriate controls and run samples on the flow cytometer

⁵Students use flow cytometry software to analyze their data by assigning regions, using gating options and overlaying histogram profiles

⁶Students use quantitative reasoning skills to calculate the percent specific phagocytosis of bacteria and determine statistical significance between experimental and control groups using the student t test

⁷Students write a formal scientific paper incorporating methodology, results and conclusions of their experiment

⁸Students generate and answer questions that reflect their mastery of the cellular mechanism involved in phagocytosis and flow cytometry technology

Appendix 6 (Student Data and Expected Results) includes representative samples of flow cytometry data obtained by students (fall 2008) for this laboratory exercise. I have provided explanations of how to interpret these results to provide instructors with a better sense of expected outcomes.

Appendix 7 (Excerpts from Student Lab Reports) includes samples of student-generated questions and answers (Part I), abstracts (Part II), objectives sections (Part III), discussion sections (Part IV), and sample data (Part V) from formal laboratory reports (from fall 2008 and spring 2010 courses).

Microorganisms

Escherichia coli – HB101 (BSL-1) transformed with pGLO plasmid and expressing green fluorescent protein (GFP) (BioRad) (E. coli/GFP). Note: any fluorescent bacterium or fluorescent beads can also be used.

Safety issues

Gloves: Students should wear gloves at all stages of this exercise.

E. coli – HB101: Aseptic technique and BSL-1 safety precautions should be practiced when handling E. coli/GFP.

Paraformaldehyde: The instructor must exercise extreme care and appropriate handling technique in a chemical fume hood when working with paraformaldehyde which may be fatal if swallowed, inhaled or absorbed through the skin. It causes irritation to skin, eyes and the respiratory tract. Paraformaldehyde emits formaldehyde which may cause cancer. If samples need to be stored in paraformaldehyde, use Parafilm to seal the top of each flow cytometry tube.

Cytochalasin B: Extreme care should be taken when making up and handling cytochalasin B, a cell permeable mycotoxin used to inhbit phagocytosis (1,8). The instructor should make up the cytochalasin B solution (in DMSO) and make up the aliquots for students to use in advance. Cytochalasin B may be fatal if inhaled, absorbed through skin or swallowed. It may cause birth defects (based on animal data) and may cause damage to the liver. Disposal of hazardous waste should follow the chemical hygiene plan in place at the academic institution.

Note: Because of the safety issues connected to cytochalasin B, the instructor could omit the inclusion of this drug without major alterations to data acquired. I have been performing phagocytosis assays with E. hortensis for over two years and consistently see only a marginally higher relative fluorescence intensity value (∼ 3%–5%) for cytochalasin B-treated samples compared to the negative control (earthworm coelomocytes incubated in the absence of E. coli/GFP). The instructor should determine whether it is worth the safety risks to include this control. An alternative control would be to add 1% sodium azide, but that also has associated safety risks.

Disposal of samples: After running samples on the flow cytometer, the flow cytometry tubes should be soaked in 10% bleach solution before discarding.

Antibiotics: Students should wear gloves at all times to avoid contact with Super Dulbecco’s Modified Eagle Medium (SDMEM – see Appendix 1) that contains a variety of antibiotics to which students might have allergies (e.g., penicillin).

Sodium azide (if used): Sodium azide is an extremely toxic and powerful poison. It is an irritant and very hazardous in case of skin or eye contact, ingestion or inhalation. When it comes in contact with solid metals, sodium azide can change into a toxic gas. Do not dispose of sodium azide down the sink as explosive deposits can accumulate. Contact your chemical hygiene officer for best practices for disposal at your institution.

DISCUSSION

Field testing and evidence of student learning

The phagocytosis laboratory exercise outlined in this paper has provided thirty-seven students enrolled in General Microbiology (BIO 308) at Cabrini College with the opportunity to work with the immune cells of live organisms and to gain an investigative-based understanding of innate immune mechanisms during the fall semester of 2008 (n = 20) and spring semester of 2010 (n = 17). The spring 2010 class engaged in all of the activities described in this exercise; many revisions were implemented between the fall 2008 and spring 2010 classes. Evidence of student learning in the 2010 class is provided in the form of data from the pre-test/post-test (see Fig. 3 and Appendix 5), an analysis of scores obtained on the formal laboratory report based on a grading rubric (see Tables 1–2 and Appendix 4A/B), and a student questionnaire evaluation (see Table 3 and Appendix 8). Importantly, my research focuses on innate immune responses in invertebrates, specifically earthworms, and I use routinely a phagocytosis assay very similar to the one that I have described in this curriculum module to study the effects of various stimulatory and inhibitory substances on immune responses in E. hortensis(5). In addition, I have used the WinList 5.0 software program with great success in my Theory and Practice in Biotechnology class (BIO 312) over the past four years for the purpose of analyzing clinically-derived flow cytometry data.

There are several advantages to using earthworms to study innate immunity in invertebrates. First, the immune cells (coelomocytes) are easily extruded from live earthworms without the need to dissect or euthanize the animal. In addition, the animals can be returned to their habitats and re-extruded four to five weeks later after a recovery period. Another advantage of using earthworms is that they are inexpensive to purchase and maintain, and are easy to handle. In addition, because they are invertebrates, experimental procedures used to study earthworms do not require the need for an Institutional Animal Care and Use Committee (IACUC), helping to simplify the introduction of this type of methodology to a microbiology laboratory course.

It was necessary to introduce the students in advance to the fundamentals of flow cytometry through reading assignments which were also reinforced during a mini-lab lecture. Students were very comfortable with using WinList 5.0 software for data analysis after an introductory tutorial. Restricting analyses to three parameters, namely forward scatter (FSC), side scatter (SSC) and fluorescence (FL-1), simplified the introduction of the software to students, and the concept of establishing regions and gating on those regions was grasped without difficulty.

It is clear from the level of complexity of the student-generated questions and answers and the quality of the formal laboratory reports that the students possessed a level of understanding of the basic theoretical principles appropriate for this introductory flow cytometry exercise. (See Appendices 6–7 for examples of student data and excerpts from formal laboratory reports.) The single parameter histograms and two-dimensional dot-plots generated by the students were instrumental in teaching students about the types of blood cell analyses that are carried out in research and clinical settings using the flow cytometer as a mainstream instrument, thus preparing them for those arenas. In addition, the flow cytometry software and Microsoft Excel exercises permitted students to acquire quantitative reasoning skills through statistical analyses. Finally, data obtained from the pre-test/post-test, the formal laboratory rubric scores and the student evaluation questionnaire clearly demonstrate that students learned the basics of flow cytometry technology and data analysis (see Student Data) and achieved the eight student learning outcomes developed for this laboratory exercise.

Possible modifications

Research-based approach

This lab is amenable to a hypothesis-driven investigative pedagogy by encouraging the students to explore how phagocytosis can be enhanced or inhibited through experimental alteration. For example, my fall 2008 class altered temperature, while my spring 2010 class examined the effect of cadmium chloride on phagocytosis. Students could also alter either the multiplicity of infection (m.o.i.), or the incubation time, and investigate the effects of adding different stimulators (e.g., phytohemagglutinin, lipopolysaccharide or cytokines), or inhibitors (e.g., corticosterone, heavy metals or polycyclic aromatic hydrocarbons such as dimethyl benzanthracine) on phagocytosis compared to standard conditions. A research project to learn more about these stimulatory or inhibitory compounds could be assigned in advance of the laboratory exercise to arm students with appropriate knowledge to formulate testable hypotheses.