Scoring Euglenid Pellicles
From Protists
Contents |
Basic Pellicle Scoring Guidelines
- Go around and number each strip. Be sure not to skip any reduction strips or count the same strip twice.
- Using one color, highlight the first pellicle strip reduction (PSR; going from the middle of the cell)strips, because they are usually the easiest to recognize. For Trachelomonas and Strombomonas strains they generally are every other strip.
- Find a good place where a pattern of the second PSR occurs. If there is only one other set of strips that are neither in the first or second PSRs that reach to the farthest point, then color the longest strips another color. If there is a point where it gets confusing, go the other way until you get back to the confusing part. If it follows a pattern evenly, then you can score the strips as they follow the other more visible and/or less confusing strips.
- Write down the total number of strips (p value), then the number of any that are either the second PSR or the longest strips, then the number of the longest strips only.
- If anything is weird or confusing, be sure to clarify with someone with more experience scoring for advice. If any are uncertain or seem to be undergoing division, then do not score those cells or only score what is clear and put question marks on the other values and do not include those values in the statistics.
Example Pellicle Scoring Table
| Strain | Farmer 3-letter Code | Rotation | P Value | Anterior/Posterior | Ta(i) | Ta(ii) | Tp(i) | Tp(ii) | Image Source |
|---|---|---|---|---|---|---|---|---|---|
| Strombomonas ovalis NJ blah | SOB | CCW | 40 | A | 20 | 10 | n/a | n/a | EF Sp04 SOB01B |
| Strombomonas ovalis NJ blah | SOB | CCW | 40 | P | n/a | n/a | 40 | 10 | EF Sp04 SOB02B |
Pellicle Statistics
- Try to get at least 30 anterior and 30 posterior images of different cells in the same uniprotistan scanning electron microscopy (SEM) fix. This will give a Poisson distribution of the population of the strain you are analyzing to be sure we get a good idea of the variation within that population.
- Calculate the mean, mode, range and standard deviation of the mean for each of the following items (be sure to make a note of what the sample size is for each...example, n = 30):
- P value
- Ta(i)
- Ta(ii)
- Ta(etc. as needed for your strain)
- Tp(i)
- Tp(ii)
- Tp(etc. as needed for your strain)
Things to Take into Consideration When Taking Images of Cells to Score
- Take several images of a single cell at low (sometimes medium) and high magnifications to be sure that you get all the details of the pellicle that you need as well as to catalog the whole cell so you don't count it a second time.
- Work at a intermediate working distance so that you can get a good depth of field (the whole cell will look in focus even at low mag) and so that you can get a good focus quality even at high magnifications. Usually about 7-8mm should work well.
- Use the in-lens secondary electron detector, especially when you are imaging inside of the flagellar canal in order to get as much information inside the canal as possible to be sure that you are scoring pellicle strip reductions that may be occuring inside the canal. This will cause a virtual flattening of the cell and pellicle strips sometimes, especially when there is too much white level in the image (mostly due to charging artifacts).
- Take pictures for publication quality using the secondary electron detector and use manual adjustment of the contrast and brightness to have a good grayscale balance (try not to have too much of the extremes of black and white). You can always adjust the contrast in Photoshop at a later date.
- Make sure you take pictures where all the PSRs that you are trying to image are actually all in the picture.
- Leave space all around the cell in case you have to crop out the cell to make it appear vertical or horizontal in the publication-quality images. You can use a virtual rotation of the image to change the position of the cell in the picture to minimize the amount of the cropping that would be necessary later.
- If there are any confusing pellicle strip areas, make sure that you get several images using several different detectors, contrast/brightness level options, magnifications, etc. in order to get all of the clarified information that you will need to score the cell at a later date.
Possible Problems
- If a cell is dividing, then it will duplicate its strips prior to dividing and will have abnormal pellicle statistics. If this the cell is just about to divide, then it will have exactly double the normal number of strips. However, this is not normally the case.
- If you are working with loricate strains, then you have to fix and image the cells just after cell division in order to have as little mucilage and lorica obstructing the pellicle as possible.
- Flagella can obstruct pellicle information.
- If strains are in culture too long they might start to have abnormal division and therefore pellicle information. This will be the hardest thing to identify unless there are SEM pictures of cells early in culture to compare current cells to.
- Some PSRs go very far down the cell and all of them cannot be imaged because other parts of the cell obstruct them.
- Some PSRs are very close together and you can confuse a strip associated with one particular PSR and mistakenly score it with a different PSR.
- The posterior strip reductions tend to have more variation in where they occur relative to each other and may jump around a bit.
