Crassulacean acid metabolism titrations




Louis Santiago

Additional Authors

Modified from Katia Silvera and Klaus Winter


CAM is the only known physiological process that causes nocturnal variation in the acidity of plant tissue. Therefore, analysis of plant tissue acid accumulation between the evening and morning hours is a sound technique for determining the presence and magnitude of CAM activity.


Crassulacean acid metabolism (CAM) is one of three metabolic pathways found in vascular plants for the assimilation of carbon dioxide. In contrast to C3 and C4 photosynthesis, CAM is characterized by CO2 uptake at night. The CAM pathway is widespread among vascular plants and is found in many succulent species in semi-arid regions, as well as in tropical epiphytes. Uncertainty exists regarding the total number of CAM species among the more than 260,000 species of vascular plants. Nocturnal acidification of plant tissue is the ‘smoking gun’ for determining CAM activity.


  • Ph Meter
  • Ph buffers 4 & 7
  • Deionized or DI Water
  • NaOH 0.1 N and NaOH 0.01N
  • Cork Borer
  • Balance Scale
  • Glass Beakers
  • Erlenmeyer flasks
  • Pipettor with tips or Burette with stand and clamps
  • Hot Plate with magnetic stirrer and stir bar
  • Kimwipe
  • Notebook

Units, terms, definitions

Fresh Weight – FW
Specific Leaf Area – SLA
Crassulacean Acid Metabolism – CAM


1. Set Up
Set up burette with stand and clamps if not utilizing pipettor. Always calibrate Ph Meter with buffers 4 & 7 before doing a set of measurements. Clean the electrode after each measurement with deionized or DI water and blot dry with Kimwipe.

2. Taking Samples and Measurements

  1. Fill Erlenmeyer flask with 20 ml of deionized or DI water (check with Ph Meter to be sure the water has a Ph of around 7).
  2. Take chlorenchyma (photosynthetic parenchyma tissue) samples using the cork borer and weigh the fresh weight of chlorenchyma with the balance scale being sure to get 1-2 g of sample in the Erlenmeyer flask and record.
  3. Place on hot plate without stirring or stir bar to boil at a low enough setting so that sample does not boil over.
  4. Boil for 30 minutes.
  5. Remove flasks from hot plate and allow cooling for 30 minutes.
  6. After cooling, pour remaining liquid into a glass beaker and add more DI water to reach 50 ml.
  7. Add stir bar and place on hot plate with stirring mechanism on at a speed where the mixture is being stirred, but not too violently.
  8. Take initial Ph with Ph Meter and record.
  9. Fill Burette or Pipettor with NaOH 0.1 N for adults and NaOH 0.01 N for seedlings. Add 5 µl of NaOH 0.1 N (for adults) and obtain a Ph measurement and record.
  10. Continue adding 5 µl of NaOH 0.1 N at a time, obtaining and recording the Ph measurement.
  11. Once the measurement gets close to 7 Ph, decrease the amount of NaOH 0.1 N to 3 µl to be careful not to miss the Ph of 7.00. Remember to fill the Burette after each measurement to obtain an accurate recording of the millimeters or microliters used to bring the sample to 6.5 and 7.0. Only the measurement taken at 7.0 will be used but the 6.5 Ph measurement must be recorded if 7.0 Ph is passed.

3. Analyze Data

Calculate µequivalents of H+:

H+ = ml titrated (pH 7.0) x (mM of solution used)

i.e. 5 if 5 mM used, 10 if 10 mM used, and divide by weight (g) to give µmolH+ g-1 FW. Alternatively, the data can be presented per unit area, I chose to calculate SLA per species and present titration data by FW. Perform a t-test using the 3 replicates for the day and 3 replicates for the night. If you find a significant difference between morning/night you found CAM! Correlate with δ13C to determine whether weak CAM or strong CAM.

Literature references

Osmond CB, Adams WWI, Smith SD (1989) Crassulacean acid metabolism. In: Pearcy RW, Ehleringer J, Mooney HA, Rundel PW (eds) Plant Physiological Ecology. Chapman & Hall, London, pp 255-280

Silvera K, Santiago LS, Winter K (2005) Distribution of crassulacean acid metabolism in orchids of Panama: evidence of selection for weak and strong modes. Functional Plant Biology 32:397-407

Ting IP (1985) Crassulacean acid metabolism. Annual Review of Plant Physiology 36:595-622