& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Guide to the study
Lab part 1
Lab part 2
Amputation of Flagella from Chlamydomonas
To study microtubule assembly in Chlamydomonas, we need a reliable method to amputate flagella without damaging the cells, allowing the cell membranes to reseal and flagella to reassemble. We have tried two methods to remove flagella from Chlamydomonas, and present the results here. Your experiment will use one or the other method, so for the materials and methods you need to know which was used.
We purchase all of our chemicals from the Sigma Chemical Co., St. Louis, Mo., spring water and cultures of Chlamydomonas reinhardii from the Carolina Biological Supply Co., Burlington, NC., and basic culture solution from Ward's, Rochester NY. We grow motile Chlamydomonas by inoculating spring water in a shallow glass container with an active culture, then placing the container in a bright window. We sometimes have difficulty obtaining cells that are mostly motile, but unless you hear otherwise you can assume that we achieved nearly 100% motility. Motility will be confirmed just before the lab starts by examination of a drop of culture in a depression slide using dark field at 100x.
Deflagellation by pH shock
When we use the pH shock method we use a magnetic stirrer to stir about 100 ml culture vigorously while monitoring the pH. We add 0.5M acetic acid dropwise to reduce the pH to <4.5, doing so within 30 sec. After another 30 sec. we add 0.5M KOH dropwise, to restore the pH to near 6.8. After distributing the suspension into six 15 ml disposable capped conical centrifuge tubes we centrifuge at high speed (500 x g) in an International Model HN tabletop centrifuge for 3 min, resuspending and combining the pellets in spring water to a concentration 2.5x the original concentration of cells. Note that all of the deflagellated cells are prepared in one batch, with volumes of the suspension separated out after deflagellation in order to make different treatment groups.
When we first tried it, success with the pH shock method varied widely from one preparation to the next. The most frequent result was persistent motility – many of the cells failed to shed flagella at all. Near 100% amputation was obtained by prolonging the time at which the cultures were kept at pH 4.5, however those preparations often failed to regenerate flagella. Success has been vastly improved by growing cells in 10% Ward's basic culture solution in spring water, with pH adjusted to 7 if necessary.
To remove flagella by shearing them off, we use a Janke & Kunkel Ultra-Turrax (Polytron type) tissue disruptor homogenizer with 20-mm shaft (Tekmar, Cincinnati, Oh). This type homogenizer was designed to rapidly disrupt suspensions of minced solid tissue. A rotor inside a hollow stainless steel shaft rotates at high speed, sucking tissue through openings in the end of the shaft. Tissue is then "whacked" by the rotor and sheared. Minced tissue is homogenized quickly this way, fragmenting the cells. Single cells in suspension typically slip through the gap between rotor and shaft and are unharmed, however flagella are quite fragile. The end of the shaft is barely submerged when touching bottom when we use one hundred ml of culture in a 250 ml beaker, maximizing efficiency of the treatment. Eight treatments at a setting of '70' for 15 sec. at at one minute intervals usually shears most of the flagella to a length of less than one µm. We then centrifuge and resuspend as described for the pH shock method.
Mechanical deflagellation typically results in amputation of flagella from 95% of cells, with partial amputations among the remainder. Eighty percent or more of the cells typically regenerate flagella, although a significant percentage consistently do not. Following a lag period of 10 to 20 min. flagella usually elongate rapidly to 90% of full length within an hour.
Even with mechanical deflagellation we encounter failures. We need very healthy cultures and very clean spring water. A failure to regenerate flagella among a majority of cells is the most common problem.