Lesson 4 – Conducting the Halo Salinity Experiment

Description: Halobacterium are extremophiles thriving in high saline environments. An ecological disturbance in a single abiotic or biotic factor could affect an entire ecosystem. Natural events and human activities affect the Earth’s capacity to sustain biological diversity. Spectrophotometers are used to measure cell density.

 

 

Objectives

What Students Learn:

  • Halobacterium are extremophiles thriving in high saline environments.
  • An ecological disturbance in a single abiotic or biotic factor could affect an entire ecosystem.
  • Natural events and human activities affect the Earth’s capacity to sustain biological diversity.
  • Spectrophotometers are used to measure cell density.

What Students Do:

  • Implement a scientific investigation using appropriate technology, multiple measures, proper controls, systematic data collection and safe approaches.
  • Compare, contrast and critique divergent results from their investigations.

 

Instructions

  1. The students should have already been introduced to their Case Study. (See Lesson 4- Introduction to GSL case study).
  2. Halobacterium Salinity Lab

    Using Slide 18 (from the previous day’s GSL Case Study.ppt)(Google Slides | PowerPoint), introduce students to the concept of producing an optimized procedure. Review the definition of optimize and discuss how scientists design optimized procedures. Ask students, “Why is this of value to scientists?”

  1. Present students with the optimized Halobacterium lab procedure.

    Discuss with the students how this procedure was optimized by scientists to increase the efficiency or effectiveness of a process as much as possible. Discuss the idea with the students that in the perfect setting they would perform their procedure many times until it was optimized. In the interest of time, the class will use a procedure that has been optimized by scientists.

  2. Students receive the protocol for the Halobacterium lab (Student Halo Protocol.doc).

    Please carefully review the teacher document for further laboratory explanation (Teacher Halo Protocol.doc) (Google Doc | Word Doc).  If you will not be using an incubator, please refer to this student document: Student Halo Protocol for Stir Plates and ISB Handheld Spectrophotometers (Google Doc | Word Doc)

TEACHER PREPARATION INSTRUCTIONS FOR THE HALOBACTERIUM SALINITY LAB

PREPARATION OF THE CULTURE – Note: Please read through the entire direction set before preparing your culture. Important and needed instructions are clarified and more specifically stated below the item with bulleted points.  For a print out of these instructions click here.

  1. You must first prepare the Halobacterium culture for the students to use. This is a fairly easy process, but care must be taken in order to prepare a viable sample for the students to use.
  2. Open the kit at least 4 days prior to when the students are completing the lab.
  3. Use one of the enclosed, sterile disposable pipets to add 5 mL of 4.3 M growth medium (from the stock solution bottles in the kit) to 1 Falcon tube. Repeat this step again with a second Falcon tube. Since the graduations on the pipet only go to 1 mL, you will have to transfer 1 mL, 5 times. Be sure to avoid contamination. Using this 1 pipet and the 10 mL of 4.3 M will allow your students to have enough media to complete their experiment. However, if you have a glass pipet and bulb or a suitable micropipette, feel free to use that if you prefer. Make sure there is no soap residue in anything you use. Soap will cause the Halobacterium to lyse.
    • Do not add more than 5 mL of growth medium to the Falcon tube. The cells need to have a great deal of empty “air” space in the tube so that oxygen can be forced to cycle through this high saline sample without losing water through evaporation.
  4. Locate the agar stab enclosed in the kit. This is contained in a Falcon tube and is labeled, “Halobacterium Culture.” You should see pink colonies growing on the surface of the agar.
  5. Use the enclosed toothpick to GENTLY remove a single pink colony from the agar. Carefully remove the toothpick (containing the colony) from the Falcon tube and gently drop the entire toothpick and colony into the Falcon tube that has the 5 mL of growth medium in it. The toothpick should remain in the Falcon tube while the culture is incubating. Place the Falcon tube cap TIGHTLY on the tube (to the second stop) by firmly pressing the cap with your thumb. When completing this, pay careful attention to the bulleted points below that further describe this step.
    • If red colonies are present, do not use them. The pink colonies have intact gas vesicles and will form a more viable culture for your students to use.
    • It is best to only remove 1 colony with your toothpick. This is because it is likely these organisms are identical genetically. If only one colony is used, we will be limiting the variables in this experiment. However, it is okay if you remove more than 1 colony.
    • Try not to transfer agar with the colony. A gentle swabbing motion with the toothpick (instead of a gouging motion) should achieve this. If some agar is transferred, your culture will likely still be viable.
    • You should have 2 toothpicks in your kit. However, if toothpicks are not available, you may use a similar object (such as a micropipette tip). Be certain that the object is small enough to allow the Falcon tube to tightly close. We are not terribly worried about contamination in this step, because it is unlikely that anything besides Halobacterium will be able to grow in the 4.3 M medium.
    • There are 2 “stops” to closing a Falcon tube. Be certain that the cap is closed to the 2nd stop (as tightly as it can be). Due to the high saline solution, evaporation is likely. This cap placement prevents a great deal of evaporation while the shaking motion of the incubator encourages enough oxygen to cycle through the sample.
  6. Using a new toothpick and your second Falcon tube containing 5 mL of growth media, repeat the above steps listed in step #5.
  7. Take the two inoculated Falcon tubes and place them in a shaking incubator at 37°C and 220 rpm for 3-4 days.
  8. Remove the Falcon tubes from the incubator. You now need to test the sample to make sure you have enough cells for your experiment.
    • Set the mode of your spectrophotometer to absorbance and to a wavelength of 600 nm. This will allow you to measure the optical density (OD) of your sample.
    • Transfer a sample of your inoculum to a clean cuvette. Use the guidelines listed below.
      • Be certain to use a sample size that is appropriate for your spectrophotometer. For the “Fisher Visible Spectrophotometer,” at least 2.5-3.0 mL must be used.
      • In order to clean your cuvette (before and after testing the OD), use copious amounts of deionized water. Do not use soap on the cuvette. If there is soap residue on the cuvette, it will cause the cells to lyse.
      • It is okay to simply pour your sample into your cuvette if it holds 5 mL.
      • Use Kim wipes to remove any fingerprints, etc. from the exterior of the cuvette.
    • Use a sample of your 4.3 M growth medium to blank your spectrophotometer. Again, use an appropriate amount for your spectrophotometer.
    • Insert your sample. Record your optical density. Repeat with your second culture.
      • Ideally, the OD should read between 0.6 and 0.8 for a great sample. If your OD is lower than 0.6, place your samples back in the incubator for another 24 hours and run the OD again. The sample can be used if the OD is as high as 1.2 or 1.5, but this is not ideal.
  9. Return your samples to the Falcon tubes.
    • Leave your culture at room temperature. Be certain the cap is tightly on the Falcon tube and keep the sample out of direct sunlight. The cells will continue to grow very slowly. If your sample’s OD is extremely high, you may choose to dilute it. However, remember to use 4.3 M medium, not water.
    • Before using the culture in the lab, gently swirl the solution to thoroughly mix the cells.
    • The original stab can remain at room temperature and will remain a viable culture source for many months.

*Media Prep     See this document for directions to make your own media and dilutions.

If you do not have a shaking incubator, you can put the tubes in a non-shaking incubator at 37 degrees and then allow for more incubation time.  Or you can have students use flasks with stir bars and stir plates to run the experiment.  See this protocol for more information: Student Halo Protocol for Stir Plates and ISB Handheld Spectrophotometers (Google Doc | Word Doc)

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Assessment

How will I know they know…

  • Students conduct the laboratory investigation successfully and safely. The assessment of the laboratory write-up is in the following lesson.

Resources

Accommodations

Choose student lab groups appropriately to strengthen lab skills and promote equal participation. Use verbal and written cues while students are in the lab. Write key items on the board to remind students how to properly use equipment. Use verbal cues throughout the lab to help students stay focused on both making predictions and on the purpose of the lab. Feel free to use other appropriate materials as needed to physically help students with new techniques such as pipetting. If the lab procedure needs to be broken down further, have individual students build a checklist within the pipetting procedure to ensure each tube receives what is needed (growth media + halo versus just growth media).

Extension

Students learn in this lab that Halobacterium are extremophiles thriving in high saline environments. However, they do not learn why they thrive in this environment, yet die in others. The reason is due to their internal composition (see the Teacher Background Information in Lesson 4- GSL Case study introduction ). As an extension, the students could research and learn more about osmosis in order to thoroughly explain why Halobacterium will lyse when placed in low salt concentrations.

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