Overview
This protocol is designed to standardise the collection and processing of foliage before chemical analysis.
Background
Much of our research relies on the chemical analysis of eucalypt leaf, either by various traditional analytical procedures such as ((Kjeldahl Nitrogen Analysis)) or an extraction followed by HPLC or, alternatively, by ((Measuring chemical composition with NIR spectrophotometry|near infrared reflectance spectroscopy)). In most cases, we freeze-dry the leaf and then grind it using a Cyclotec 1093 Mill (Tecator, Sweden) fitted with a 1 mm sieve. An exception is the analysis of terpenes for which we place a known mass of finely chopped fresh foliage directly into a solvent containing an internal standard (often tridecane or dodecane). Thus, if you are leaves collecting to analyse for several components then you may need to split samples and prepare them in different ways. The purpose of this protocol is to standardise the collection and processing of foliage before analysis. It is written both for people in our laboratory as well as for those who send us foliage.
Materials/Equipment
__NOTE: Comprehensive analysis of leaves requires 30-40 g of fresh leaves__
*Cutting pole (we do occasionally use other techniques (slingshot with attached line or a rifle) to obtain leaves.
*Sharpener for pole cutting mechanism
*Lubricant for cutting head
*Paper or plastic bags
*Portable freezer or other cooler
*Generator or large capacity battery
*Dry ice and shipping container
*Freeze-dryer or drying ovens
*Grinder e.g., Cyclotec 1093 mill
*Storage vials e.g., Sarstedt specimen vials, yellow lids, 70 mL (cat no 75.9922.745)
*Sample master sheet
Units, terms, definitions
((NIR spectrophotometer|Near infrared reflectance spectroscopy NIRS))
((HPLC))
Procedure
__How should I collect the foliage __
While we often collect foliage directly from young plantation eucalypts, most forest and woodland eucalypts are much taller and leaf collection requires the use of a cutting pole. Our cutting poles are built in three telescopic sections (in our workshop using tubular aluminium and bicycle quick release levers to lock and unlock each section) so that they can be extended to about 8 m. They operate by pulling on a rope that closes the secateurs cutter. This is an unwieldy piece of equipment and you will need to be shown how to use it so that you don’t hurt yourself or damage the pole. This pole enables us to cut foliage at about 10 m because one can lift it off the ground. In most cases, this means that we can sample leaf from roughly the same position in the canopy from all trees of interest. Note that at this stage we do not have any evidence that position in the canopy has any effect on the chemistry of the foliage. There are exceptions: resistant branches on trees that are otherwise susceptible to herbivore damage and differences between juvenile and adult foliage. Also, we might expect differences between leaves exposed to differing amounts of light. Using a pole reduces the temptation of collecting foliage from the one very low branch on the tree.
__How much leaf should I collect __
While it is appealing to collect large samples of foliage, this is extremely wasteful in terms of the time taken to pluck leaves, the freezing capacity in the field, transport volume, freeze-drier capacity and the time to grind the leaf. On several occasions, we have discarded excess sample just before freeze-drying. This involves standing in a cool room throwing out leaf that someone painstakingly collected! It is wiser to collect the amount of leaf needed for the analyses with generous allowance for the losses that occur during grinding. How much is this
Most analyses do not require much sample. For instance, ((Near infrared reflectance spectroscopy (NIRS))) is a non-destructive analysis; ie the sample is not spoiled in any way and is available for other analyses. The sample chambers we use hold about 2 g of ground leaf and so 15 g of material clearly supplies ample for unbiased triplicate analyses. Nitrogen and dry matter analyses each require 1 g of sample whereas something like our superseded extraction of formylated phloroglucinol compounds (FPCs) requires 3 g. In the unlikely event that all of these analyses need repeating, then the 15 g of sample is still plenty.
Eucalypt leaf typically contains 40-50% dry matter with the remaining 50-60% being water. Note that very young leaf may contain relatively more water. We require 15 g of dry ground leaf or about 30 g of “wet” leaf. However, some losses may occur during grinding so it is advisable to collect 50 g of “wet foliage”. We suggest that you weigh a sample of your leaves so that you can gauge 50 g.
__How should I collect the leaf __
It is important not to damage the leaves during sampling. Leaves vary in fragility. Sometimes leaves may be stripped many at a time; others need to be plucked one by one.
__In what should I store the fresh leaf __
The answer to this question should consider factors like the condition of the leaf and the methods of storing and drying. More often than not, we collect leaves into paper sandwich bags, fold the top and sometimes staple it. Samples need to be in a porous container for drying, but there is no sense in placing wet leaf into a paper bag. Likewise, collecting into unperforated plastic bags is not ideal because the samples need to be transferred to a porous container before freeze-drying. We have encountered problems with simply opening plastic bags for freeze drying, especially when large numbers of samples are packed into the drier. Transferring samples is a waste of time and requires re-labelling of bags. Thus, to answer the original question, collect fresh leaves into paper bags or into perforated plastic bags. If you anticipate long delays before drying then place leaves in high-quality freezer bags (those with a zip seal and a place for writing a label). __Note that if you do not have freeze – drying facilities then we recommend air-drying leaves. Paper bags are perfect for this. If possible, hang them up in a dry atmosphere where there is reasonable airflow. Once dry, store them in the dark.__ If samples are to be freeze-dried then they should be placed on ice immediately. Typically we freeze 2 L plastic bottles of water because they stay frozen much longer than do bags of crushed ice and do not leak. We then place batches of paper bags inside a plastic bag. This method is preferable because by packing bags tightly you can exclude most of the air and fit many more samples into an esky (portable cooler). An esky of samples packed in this way can be stored in a freezer room for days or even weeks. That said, it is advisable to process samples as soon as possible after collection.
__In what should I store the fresh leaf __
It is important not to damage the leaves during sampling. Leaves vary in fragility. Sometimes leaves may be stripped from the branch many at a time; others need to be plucked one by one.
__How should I label the bags __ There is no point writing a descriptive essay on each bag (eg ””Eucalyptus sideroxylon” ssp ”sideroxylon”, young leaf collected on 15/7/2002 at Coonabarrabran”). Instead, use a simple labelling system (eg 1, 2, 3 or A1, A2, A3) supported by a master list that describes each number. This label can then be used to follow a sample through analyses. Furthermore, one can then label the bags before collecting the samples. It is easier to read the label if it is written midway down the bag.
__How should I store samples in the field __
This is extremely hard to answer. Storage in the field is probably not important in cool places. However, it is important in warm weather and this is when it is most difficult. If working close to home then eskies (cool boxes) with ice are good. However, if working further afield or on large collecting trips then storage becomes difficult. We are now using car freezers that we run from large capacity wet-cell truck batteries in the field and plug them into mains power at night. Note that car freezers discharge normal car batteries very quickly so a generator is a much better option. On these longer trips, collecting the right amount of sample becomes important, to ensure that you have sufficient freezer space. Make sure that you take battery chargers to recharge the truck batteries. It is sensible to fill empty freezers with frozen goods to precool them. __Finally, a warning about using dry ice:__ It is appealing to use dry ice or frozen CO{SUB()}2{SUB} to rapidly freeze leaves or to keep them frozen during transportation. We did this recently on a week-long trip using a special insulated container to ship the dry ice and still had a supply at the end of the trip. This is fine as long as the dry ice is outside the cabin of the car – secured to the roof or in the back of a utility. Note that it is unsafe to carry it in the boot (trunk). Carbon dioxide is lethal at low concentrations in air (less than 5%) and vertebrates lose consciousness (and crash cars) at much lower concentrations. One mole (44 g or a small handful) of dry ice CO{SUB()}2{SUB} sublimes to 22.4L of CO{SUB()}2{SUB} so it is obvious that several kilograms represent an extreme hazard. If you do use dry ice, make sure that you remove all of it before placing a container of frozen samples into a confined space such as a walk-in freezer. Additionally you must use gloves and take other precautions required so that you do not “burn” yourself with the dry ice.
__Do you need leaf area and mass __
It is common practise to measure the areas and masses of fresh leaves. Depending on the leaves, it is best to do this soon after collecting them. One method to reduce change is to store the leaves for a short time in plastic bags with some moist paper.
__Should I freeze-dry or oven-dry samples __
This depends largely on convenience because the ideal drying method seems to depend on the particular analysis (see Julkunen-Tiito and Sorsa, 2001). Our studies suggest that it is preferable to freeze-dry samples because, even at 40∘C, small losses of sideroxylonals occur. However, freeze-drying is slow and expensive (we pay for our freeze-drying). Also, if you don’t have a freeze-drier you will need to deliver your samples for analysis by refrigerated transport. In Australia, such transport is harder to find than you might think and may involve several carriers who probably care much less about your samples than you do. Samples may arrive frozen but you have little idea of the incidents between the points of dispatch and delivery. In this situation and especially if you are interested in relative values for analytes, there is much to be said for air-drying samples. This is much easier if you inhabit a warm dry place. Alternatively, if you have access to large forced draught ovens then dry leaves at 40∘C.
__How should I grind the leaf __
The method of grinding is extremely important for NIRS, which gives much better results when the particle size is as uniform as possible. Thus, using a cyclone grinder like the Cyclotec 1093 mill, fitted with a 1 mm sieve, is essential.
__In what should I store the ground leaf __
We find the standard urine specimen vials, made by companies like Sarstedt (specimen vials, yellow lids, 70 mL; cat no 75.9922.745), perfect for storing ground leaf samples. These vials are about 44 mm in diameter and 55 mm high and comfortably hold 15 g of sample. Please remember to fill vials about 75% full. Mixing and sampling is difficult if vials contain any more than this. Perhaps the one drawback of these vials is that they are clear and ground leaf turns from green to brown rather quickly when left exposed to light. Thus, store samples in the dark.
__Is there any merit in keeping samples in order __
One might argue that samples should be analysed randomly. I have done this at least once to account for machine bias with time. If you want random order, however, then do it properly with a random number generator. If not, collect, label and analyse the samples in order. In this case, you have a much better chance of pinpointing where problems occur during many analyses. Whatever the case with analyses, it is still worth keeping the samples in order. Invariably it is necessary to repeat some analyses and there is little entertainment searching for a few samples in a box of 500 jumbled vials.
Links to resources and suppliers
[http://www.foss.dk/Solutions/ProductsDirect/SamplePreparationMills/Cyclotec1093SampleMill.aspx|Cyclotec 1093 Mill (Tecator, Sweden)]
[http://www.sarstedt.com/php/main.php|Sarstedt (specimen vials, yellow lids, 70 mL; cat no 75.9922.745)]
Literature references
Julkunen-Tiito, R., Sorsa, S., (2001). Testing the effects of drying methods on willow flavonoids, tannins and salicylates. J. Chem. Ecol. 27, 779-789.
Health, safety & hazardous waste disposal considerations
Keep in mind that lopping branches from trees, particularly eucalypts, is hazardous. Some of the hazards that you need to be aware of and manage are:
- __Falling branches:__ We typically lop branches that have a diameter of less than 30 mm, but even a branch of this size falling from 10 m is dangerous. Keep in mind that it is easy to break off eucalypt branches and get a lot more than you bargained for. This may occur at any time but particularly when hooking the pole onto a branch and then heaving on it. It is possible for the whole branch to fall rather than just the thin tip. Thus, it is compulsory to wear a hard hat.
- Another less obvious danger is __driving to the collection site__. If using a utility (pickup truck) make sure the pole is firmly tied down and that there is no risk of the rope coming free and potentially dragging the pole onto the road. Finally, be extremely careful looking for trees while driving. Park safely and walk!
- __Back, shoulder and arm injuries__ can happen if you don’t handle the pole safely, particularly when it is fully extended. A pole extended to nine metres places a large amount of stress on your arms, shoulders and back. Whenever possible, get help raising the pole from horizontal to vertical or walk the pole up by wedging its base against a solid object such as a tree. Take regular breaks whenever you are collecting leaves for long periods. Remember, fatigue leads to accidents! A sharp cutter and lubricated pulleys make the job much easier.
- __Carrying the collected samples__ and pole back to the car can cause back and shoulder injuries. Make sure that you collect only what you need (see ‘How much leaf should I collect ‘ above). Also, it may be better to carry smaller loads back to the car. Make two safe trips rather than one unsafe trip!
- __Dry ice__: ‘See How should I store samples in the field ‘ above.