1. Chris Mitchell Benton High School Advanced Biology

2. Goals Introduce you to some of the new microbiology labs that I use in my classroom. Take a look at the technology available to demonstrate tough concepts to students. Review the labs and the concepts which they help to reinforce. Share examples of what works and what doesn’t work.

3. Background Labs are used in my 12 th grade Advanced Biology course. Students have a double lab period, twice a week on back to back days. The other days consist of 42 minute class periods, used to cover the theory. Much of the equipment in the lab was purchased as we began to offer Advanced Placement Biology, with required labs as part of the curriculum. Many manufacturers offer similar lab kits as well as replacement materials.

4. Lab # 1 An Introduction to Microbiology Techniques Learning Objectives: Students will use aseptic technique to culture microorganisms. Students will use the pour plate technique to prepare agar dishes. Students will distinguish between colonies of bacteria and fungi. Students will heat fix and stain bacteria for identification purposes.

5. Rationale This is a good introductory lab which can be used to teach sterile technique, omnipresence of microorganisms, and simple culturing techniques. Students get hands-on practice using the instruments needed for collecting and transferring bacteria. You can introduce bacterial staining techniques i.e.. Gram Staining, which can be used for identification purposes. Groups can prepare slides to identify the general shape of bacteria which they have collected. Students enjoy being able to choose sites for collecting and comparing which group(s) obtained the largest growth.

6. Procedure Part 1: Media Preparation: Can be done with all types of agar (I usually just use what I have left over from last year) Be sure to emphasize sterile technique (washing hands, wiping down tables, care for Petri dishes) Agar can be melted in hot water bath (heat ahead of time), or a microwave works well. Allow plates to cool before attempting to transfer any microbes.

7. Procedure Continued Part II: Collection and Culture: Allow students to choose a sampling sight of interest around the school (remember to inform staff of what is going on!) Provide students with sterile swabs to collect samples. Use streak plate technique to transfer microbes from swab to plates. (No poking holes in agar) Incubate at 30⁰C for 4-5 days or at room temp for 5 days, having students check plates daily for growth. Tip: Plates may have to be incubated upside down to prevent moisture from dropping on agar and inhibiting growth.

8. Procedure Continued Part III: Observing Colonies and Isolating Pure Cultures Compare bacterial growth (circular colonies) to any fungal growth (white or yellow fuzz). Students can now prepare slides for examination using various staining techniques. ( we Gram stain to ID Gm+ or Gm -). Have students practice flame sterilizing inoculating loops for bacterial transfer. (Stress sterile technique here!) Have students classify bacteria collected by shape (cocci, bacilli, or spirilla and by Gm+ or Gm-) Colonies can be isolated and transferred to additional agar plates for growth and further study.

9. Bacterial Culturing

10. Clean Up / Analysis/ Extension Clean up: Autoclave (if available) or place in autoclave bag and soak with bleach. I would strongly recommend using disposable plates. Also, sterile disposable inoculating loops are available. Analysis: Students can report on the type of bacteria which was cultured and the area of the school which the sample was taken from. Also, the teacher can confirm their findings by examining their stained slides. Extension: Now is also a great time to introduce the concept of antibiotics. Antibiotic soaked discs (or varying antibiotics?) can be placed on areas of growth and then incubated to see the possible effect. Sanitizers (germ-x, hand sanitizers, and other “antibacterial” products) can also be tested for effectiveness.

11. Lab #2: Colony Transformation Learning Objectives: Students will genetically modify a strain of E. coli bacteria by inserting a plasmid. Students will gain an understanding of the principles used in bacterial transformation. Students will use a selectable marker found in bacterial DNA to alter the phenotype of bacteria. Students will transfer antibiotic resistance to a strain of bacteria to use for selection purposes.

12. Rationale This lab satisfies AP Lab #6 requirements for AP Biology. Several different plasmids are available for genetic modification (pBlu, pGreen, pAmp, pVIB,…) from several manufacturers. I recommend using the pBlu or Pgreen, since both have easily observable phenotypes. The plasmid not only alters the overall appearance of the bacteria, but also makes the bacteria antibiotic resistant. This activity allows students to gain an understanding of the process used to insert plasmids into “competent” bacteria and also the effectiveness of such techniques.

13. Practice Before the Lab I have students practice the theory behind the lab before doing the actual activity. Students access the free Prentice Hall – Lab Bench website at http://www.phschool.com/science/biology_place/labb ench/lab6/design1.html http://www.phschool.com/science/biology_place/labb ench/lab6/design1.html Students work through online tutorial and complete quiz at the end to show me they “know what they are doing”. ( I highly recommend doing this before attempting to do the lab.)

14. Transformation Lab Procedure Part 1: Plate and Colony Preparation: Students need to prepare their agar and pour their plates. Label plates before pouring. They need to prepare 3 different types of plates: Plate 1 = Simple Luria Broth Agar plates (LB) Plate 2 = Luria Broth Agar + Ampicillin (LB + Amp) Plate 3 = Luria Broth Agar +Ampicillin + X-Gal What is X-gal? – a substance when metabolized by the transformed bacteria which makes them turn blue! Once plates are poured, they will need to be cooled down before use.

15. Part 1 Procedure Continued Colony Transfer: The E. coli bacteria supplied in the stock culture need to be transferred onto individual Luria Broth agar plates and grown. Plates need to be incubated at 37⁰C for a 22 hour period. Timing is critical, since bacteria need to be in the most rapid phase of growth for the transformation to work! Tip: Plates need to be incubated upside down to prevent moisture from falling onto the agar.

16. Day 2 Procedure Day 2: Colony Selection and Transformation After 22 hours of incubation, bacteria should be in the log phase (most rapid growth) Locate areas of the plate that show individual (satellite) colonies – avoid areas where lawns have formed. Using a sterile inoculating loop, transfer a loop full of bacteria to the test tube provided and follow the transformation procedure. Also – as a Control, some of the non-transformed (- plasmid) bacteria should be applied to each of the three types of plates. (If I am running low on materials, we have just used one set of plates as a “class control”) It is extremely important to follow the times listed in order to “heat – shock” the bacteria to get them to take the plasmid. If temps are too hot or too cold, or if not enough time is allowed, transformation will not occur!

17. Testing for Transformation Once bacteria have been transformed, they will need to be applied to the three types of agar: LB, LB+Amp, and the LB+Amp+X-Gal. To do so, glass beads are supplied with most kits to spread bacteria evenly over the surface of the agar. Plates need to be incubated at 37⁰C overnight or at room temp. if no incubator is available. Have students check the results on each of the plates for a three day period. ( I have students do a daily count for each plate) I also have students predict the outcomes on each of the plates before viewing their results.

18. Expected Results Control Group (- Plasmid) Experimental Group (+ Plasmid) LB agar = Lawn of Growth LB + Amp = No growth – bacteria were not transformed and were thus killed by ampicillin LB+Amp+X-Gal = No Growth –bacteria were not transformed and were killed by ampicillin and thus couldn’t metabolize X-gal LB agar = Lawn of Growth LB+Amp = many clear-white circular colonies formed, indicating resistance to ampicillin was achieved. LB+Amp+X-Gal = several blue colonies formed, indicating ampicillin resistance and blue color due to metabolizing X-gal!

19. Questions? Comments? Has anyone done either of these experiments? How did they work out for your students? What changes would you make? What other labs are offered for senior biology students? Does anyone else make use of the Prentice Hall Lab- Bench website? Any other lab simulation web-sites? Contact email: cmitchell@bentonsd.k12.pa.uscmitchell@bentonsd.k12.pa.us