A basic microbiology course for high school students.
I describe a 5-day basic microbiology enrichment course for high
school students. In this course, students learn microbiological
techniques such as preparation of agar plates, isolation of bacteria
from food, serial dilution, and plating. Additionally, they experience
the steps involved in the identification of an unknown bacterium and
learn about the modes of action of common antibiotics against different
types of bacteria. Feedback indicates that this course provided
invaluable lessons and experiences for students who had no prior
hands-on experience with microorganisms.
Key Words: Microbiology; bacteria; serial dilution; API20E kit; antibiotics.
(Study and teaching)
Sciences education (Curricula)
Penicillin resistance (Research)
Microbial mats (Identification and classification)
|Author:||Yip, Cheng Wai|
|Publication:||Name: The American Biology Teacher Publisher: National Association of Biology Teachers Audience: Academic; Professional Format: Magazine/Journal Subject: Biological sciences; Education Copyright: COPYRIGHT 2010 National Association of Biology Teachers ISSN: 0002-7685|
|Issue:||Date: Oct, 2010 Source Volume: 72 Source Issue: 8|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
At the high school level, microbiology is not often experienced hands-on in the laboratory Although high school students learn about the properties of microorganisms such as bacteria, fungi, protists, and viruses, and the differences among them, they do not apply their knowledge in identifying an unknown microbe in a medical diagnostic laboratory setting.
At the Hwa Chong Institution, a school in Singapore, students participate in 1-week sabbatical courses that are held three times a year. The sabbatical courses are enrichment courses in various disciplines, ranging from science and mathematics to leadership, sports, and the arts. The Basic Microbiology course is an example of a class that is offered to students in grades 9 and 10.
The Basic Microbiology course uses an inquiry-based approach; the students identify an unknown bacterium and discover how antibiotics work against bacteria. According to Borich et al. (2006), inquiry-based learning is achieved through questioning and actively searching for information and knowledge. Students attempt to find answers by conducting various hands-on experiments. They make observations; gather, analyze, and interpret data; and propose answers and explanations (National Research Council, 1996). For learning to take place, students must think about what they have observed and draw logical conclusions from the results of the experiments (Lord, 1998).
Outline of the Basic Microbiology Course
In this course, students learn the theory and practical aspects of the following experiments, the details of which are listed in Table 1:
* Isolation of microorganisms from food samples
* Identification of the species and genus of unknown bacteria through a series of experiments involving Gram staining, growth on selective and differential media, oxidase test, and API20E test
* Comparison of the effects of the antibiotics ampicillin and kanamycin on Gram-positive and Gram-negative bacteria
The students were provided with gloves during the experiments. All experimental work was classified as Biosafety Level 1 work. Plates containing unknown microorganisms isolated from food samples remained sealed throughout the counting process and were discarded in a biohazard bag, to be autoclaved at 15 psi (121[degrees]C) for 20 minutes before disposal. In the identification exercise, the students were provided with a mixed culture containing laboratory Biosafety Level 1 stock cultures (i.e., Escherichia coli MM294; Carolina Biological Supply) and bacteria purchased from the American Type Culture Collection (Citrobacter freundii ATCC 8090, Enterobacter cloacae ATCC 23355, and Providencia stuartii ATCC 35031).
Isolation of Microorganisms from Food
* Nutrient agar plates (prepared by students)
* Normal saline (0.85% sodium chloride solution)
* Sterile 10-mL pipettes
* Sterile disposable tubes
* Sterile L-shaped spreaders
* Sterile micropipettes
* Sterile pipette tips
* Food samples used were minced meat (10 g in 90 mL sterile saline), milk (10 mL in 90 mL sterile saline), and soft-rot vegetables (10 g in 90 mL sterile saline). They were blended in saline with a Waring blender, giving [10.sup.-1] dilution of the original concentration.
The students did serial tenfold dilutions and plating in groups of three. Using a sterile 10-mL pipette, 9 mL of saline was placed in each of four sterile tubes labeled [10.sup.-2], [10.sup.-3], [10.sup.-4], and [10.sup.-5]. Using a sterile micropipette tip, 1 mL of the [10.sup.-1] food suspension was transferred into 9 mL of saline to give a [10.sup.-2] dilution and mixed well. Serial dilution was carried out until [10.sup.-5]. Then, 0.1 mL each of the [10.sup.-4] and [10.sup.-5] dilutions were plated on nutrient agar plates in triplicates. The plates were incubated in an inverted position in the incubator at 37[degrees]C overnight.
The following day, the students counted the number of colonies. The number of colony-forming units per milliliter was calculated as follows: mean number of colonies per plate x dilution factor x 10. Plates containing unknown bacteria remained sealed during the counting process, and the bacteria were not subcultured further. They were autoclaved after counting was done.
Identification of Unknown Bacteria
* Gram stain reagents (crystal violet, Lugol's iodine, ethanol, safranin)
* MacConkey agar and mannitol salt agar
* Oxidase test disks
* API20E test strips (bioMerieux)
* Mixed cultures (E. coli, C. freundii, E. cloacae, and P. stuartii) streaked on nutrient agar plate
* Yogurt containing Lactobacillus
* Laboratory stock culture of Staphylococcus epidermidis ATCC 12228 (mannitol salt agar test)
* Laboratory stock culture of Pseudomonas putida ATCC 31800 (oxidase test)
Unknown bacteria isolated from food were not studied further, as that would constitute a Biosafety Level 2 study. Instead, mixed cultures of Biosafety Level 1 laboratory stock cultures belonging to the Enterobacteriaceae (a group of bacteria that are common inhabitants of the intestinal tracts of humans and animals) were given to the students. The mixed culture consisted of E. coli, C. freundii, E. cloacae, and P. stuartii streaked on a nutrient agar plate.
The following steps were taken to identify the unknown bacterium. The methods were modified from Benson (2002) and Colome et al. (1986).
1. A pure culture was first obtained by streaking a well-isolated colony of bacteria from the mixed-culture plate onto a fresh nutrient agar plate.
2. The first step in the identification of an unknown bacterium was to observe its morphological characteristics, for example, the color, shape, and features of the colonies on agar.
3. Gram staining was done to differentiate bacteria largely into two groups, Gram-positive and Gram-negative. The morphology of the bacterial cells was then observed under the microscope. Gram-positive bacteria used were S. epidermidis and Lactobacillus from yogurt.
4. Following the morphological studies of bacterial colonies and cells, the biochemical properties of unknown bacteria were explored through their growth and colony color on two selective and differential media, MacConkey agar and mannitol salt agar, and the results of the oxidase test. MacConkey agar contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria. This medium also contains lactose; thus, Gram-negative bacteria that can ferment lactose (red colonies) can be differentiated from the non-lactose-fermenters. Mannitol salt agar contains the selective agent 7.5% sodium chloride, which inhibits many bacteria except those in the genus Staphylococcus. The medium also contains 0.5% mannitol and the pH indicator phenol red. When S. aureus grows on this medium, it ferments mannitol, releasing acidic products that cause the pH indicator surrounding the colonies to change from red to yellow. Staphylococcus epidermidis, however, does not ferment mannitol; hence, no change in the color of the culture medium is observed. The oxidase test is used to differentiate among Gram-negative rods. Bacteria belonging to the genus Pseudomonas are oxidase positive, whereas all the Enterobacteriaceae are oxidase negative.
5. Finally, the API20E System was used in the identification of the species and genus of members of the family Enterobacteriaceae, based on biochemical properties of the bacteria. Only Gram-negative and oxidase negative isolates were identified using the API20E System (Figure 1).
Figure 2 summarizes the tests done in the identification of unknown bacteria, in the form of a concept map constructed with CmapTools (available at http://cmap.ihmc.us).
This series of experiments allows students to gain an understanding of the logical sequence in the identification of unknown bacteria. Students feel a sense of achievement when they have successfully identified an unknown species to greater than 95% homology through the seven-digit identification number using the API20E kit and software.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Antimicrobial Susceptibility Test (Disk Diffusion Test)
This method is also known as the "Kirby-Bauer method" for testing the effect of antibiotics on bacteria. The objective of this experiment was to determine the relative effectiveness of the antibiotics ampicillin and kanamycin on Gram-positive and Gram-negative bacteria.
Ampicillin (10 ug) and kanamycin (30 [micro]g) disks
Laboratory stock culture of E. coli MM294
Laboratory stock culture of Micrococcus lysodeikticus ATCC 4698
Mueller-Hinton agar plates
Sterile forceps and sterile cotton swabs
Micrococcus lysodeikticus (Gram-positive) and E. coli (Gram-negative) were grown overnight in nutrient broth at 37[degrees]C. The cell suspensions were swabbed evenly on the surface of Mueller-Hinton agar plates using sterile cotton swabs. Antibiotic disks were placed on the agar surface using sterile forceps. The plates were incubated at 37[degrees]C overnight. The diameters of zones of inhibition were compared. A negative control consisting of a blank disk was used.
Investigation of the MIC & MBC of Antibiotics
The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic that will inhibit the growth of microorganisms. A loopful of culture from each tube that shows no growth is transferred to fresh medium without antibiotic. The lowest concentration of the antibiotic that stops growth on medium without antibiotic is the minimum bactericidal concentration. This means that the bacteria have been killed by the antibiotic.
Ampicillin or kanamycin (128 [micro]g/mL in nutrient broth)
Nutrient broth culture of E. coli and M. lysodeikticus
Sterile nutrient broth
Micropipette, sterile pipette tips, sterile microfuge tubes Shaking incubator (37[degrees]C)
Serial twofold dilutions of 128 ug/mL of each antibiotic were carried out. Then 0.1 mL of bacterial broth cultures of E. coli and M. luteus were added separately to various concentrations of each antibiotic. The tubes were placed in a shaking incubator at 37[degrees]C overnight. The next day, a loopful each of broth cultures with dilutions of the antibiotics that did not show growth in the MIC test were streaked on Mueller-Hinton plates. The plates were incubated at 37[degrees]C overnight.
In the disk diffusion test, the zone of inhibition of both E. coli and M. lysodeikticus was the same for kanamycin. However, the zone of inhibition of M. lysodeikticus was significantly larger than that of E. coli for ampicillin (Figure 3).
The MIC of ampicillin was also at a lower concentration compared with kanamycin. It was also observed that ampicillin was bactericidal toward M. lysodeikticus, as the MBC value was close to the MIC value. On the other hand, bacteriostatic drugs have a much lower MIC than the MBC value. On the basis of these observations, the students were guided to deduce the mode of action of ampicillin and kanamycin on the bacteria. These were the questions raised:
1. Compare the relative effectiveness of the two antibiotics against Gram-positive and Gram-negative bacteria. (Ampicillin is more effective against Gram-positive bacteria, whereas kanamycin shows the same effectiveness against both types of bacteria.)
2. What is the main difference between the structures of Gram-positive and Gram-negative bacteria? (Students would have learned in the
Gram stain procedure that Gram-positive bacteria have a thicker peptidoglycan cell wall than Gram-negative bacteria.)
3. On the basis of the difference described above, explain why ampicillin is more effective against Gram-positive bacteria. (Ampicillin is known to inhibit bacterial cell-wall synthesis, leading to osmotic rupture; thus, it is more effective against Gram-positive bacteria.)
4. What is the mode of action of kanamycin? (Kanamycin binds to the 30S ribosomal subunit, interfering with protein synthesis in bacteria.)
5. How do the different modes of action result in the antibiotics being either bactericidal or bacteriostatic? (Antibiotics that cause osmotic lysis of bacteria are bactericidal, whereas those that inhibit growth of bacteria are bacteriostatic.)
6. Comparing the two modes of action of the antibiotics, which type is a broad-spectrum drug? (Antibiotics that interfere with protein synthesis are broader-spectrum drugs, as they affect both Gram-positive and Gram-negative bacteria to almost the same extent.)
[FIGURE 3 OMITTED]
The course provided basic understanding and hands-on skills in microbiology beyond the high school biology syllabus. The students analyzed the data obtained to draw conclusions regarding the unknown isolate and mode of action of antibiotics in an inquiry-based setting. The identification of the unknown bacterium represented an authentic task that medical microbiologists in diagnostic laboratories experience. Further discussions on the emerging antibiotic resistance in bacteria can be carried out. Feedback from course participants has been encouraging, with many of them commenting that they have learned useful hands-on techniques and that the course had enriched their understanding of microorganisms.
I thank my school for supporting this work.
Benson, H.J. (2002). Microbiological Applications: Laboratory Manual in General Microbiology, 8th Ed. New York, NY: McGraw-Hill.
Borich, G.D., Hao, Y.-W. & Aw, W.-L. (2006). Inquiry-based learning: a practical application. In A.-C. Ong & G.D. Borich (Eds.), Teaching Strategies That Promote Thinking: Models and Curriculum Approaches (pp. 29-52). Singapore: McGraw-Hill.
Colome, J.S., Kubinski, A.M., Cano, R.J. & Grady, D.V. (1986). Laboratory Exercises in Microbiology. St. Paul, MN: West Publishing.
Lord, T. (1998). Cooperative learning that really works in biology teaching: using constructivist-based activities to challenge student teams. American Biology Teacher, 60, 580-588.
National Research Council. (1996). National Science Education Standards. Washington, DC: National Academy Press.
CHENG-WAI YIP is Senior Biology and Research Consultant at the Hwa Chong Institution, 661 Bukit Timah Road, Singapore 269734; e-mail: firstname.lastname@example.org.
Table 1. Experiments carried out within the 5 days. Day Experiments 1 Preparation of culture media (agar plates) Isolation of microorganisms from food samples by serial dilution and plating 2 Enumeration of microorganisms isolated from food Pure culture techniques (streak plate method) Antimicrobial susceptibility test by disk diffusion method Minimum inhibitory concentration (MIC) of antibiotics 3 Gram staining of bacterial isolates and observation of bacterial morphology Streaking of isolates on selective and differential media Oxidase test for bacterial isolates Read results of disk diffusion and MIC tests Minimum bactericidal concentration (MBC) of antibiotics 4 Assessment of growth of isolates on selective and differential media Identification of bacterial isolates using the API20E system Read results of MBC test 5 Read results of API20E test strip
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