Dear Mr. Safeirad,

please contact my colleagues Dr. C. Heunisch

Geological Survey of Lower Saxony, P.O. Box 510163, D-30631 Hannover, Germany

or

Dr. J. Strahl

Landesamt fuer Geowissenschaften und Rohstoffe Brandenburg,

Stahnsdorfer Damm 77, D-14532 Kleinmachnow, Germany

Both ladies are experts on palynology and I benefited from their palecological and paleontological studies in Nepal and Jordan

Best regards

Harald G. Dill

Dear Dr. Dill,

Thank you for your helpful answer.

sieve first 120 mu (about 2 minutes a sample), catch sample in bucket and put sediment and water over 7 mu sieve. Add sample residue, with water from sieve to porcelain dish. Put dish in ultrasoon bath, leave in there for several minutes. Heavy material will sink to bottom. Top of upper level with light fraction and put into HF-resistant beaker. Centrifuge of the water, and add 40% HF. Let samples shake for about an hour or two. If ready add water, centrifuge and decant. Put into glass tubes and add about 10-20 ml KOH (10%), put into heater for about 5 minutes. After heating (let it cool down) and add water and centrifuge and decant. Add water to residue and sieve again over 7mu. Repeat ultrasoon step and after sieveing (7mu), remove residue from sieve with alcohol and put it into vial to store by adding 3 or 4 droplets of glycerine. Let alcohol evaporate and mark sample appropriately. Depending on equipment and experience you could prepare up to 40 (or more) samples a day.

Another method, does not include HF but uses instead Ca-acetic and acetic acid, and separates with heavy liquid (NPW). If interested I could give you the method, it is even faster but is also depends on the equipment you use.

Nico

p.s. if further questions, contact me on [email protected]

Different palynology companies working offshore use a variety of proprietary processes to do preparation of palynology samples (and micro-pal and heavy-min and nano-palaeo) from the same set of original samples. I haven't precisely followed the sample prep numbers, but typically I'm seeing 15 to 20 sample descriptions and analyses come through in a 24 hour period, using two sample techs and two scientists on 12 hour shifts. For a total of 4 bedspaces and around 25cu.m of equipment space, there is obvious pressure to drive down those costs.

The details of the methods are proprietary, as I say : they don't take care to blindfold me when I go into the 'bugs' unit, but equally I don't go around reading the labels on their chemicals bottles. I do know that they go to fairly strenuous efforts to avoid doing HF preps because of the inherent hazards and the copious paperwork associated with the chemicals and hazardous waste. Also, when they're doing non-acid preps we can shunt another specialist into their unit, but when the HF comes out of the storage locker, the lab is emptied of all non-company personnel.

I do know that an important preliminary step in most such "acid-free preps" is to digest the sample with strong (20vol or 50vol?) hydrogen peroxide, with agitation or in a utlrasound bath. After that, with several stages of neutralising and filtering the sample is ready for mounting and counting. For critical operations I've seen samples ready and analysed in a bit under 5 hours.

Unfortunately, as I said, these techniques are proprietary. I have considered getting trained in doing these preps as a useful additional string to my bow, but I'd have to sign NDAs before being given the training. So these notes are from an interested observer. If any of your other correspondents could use them to deduce (and test) an acid-free prep in the public literature, that would be great.

One thing that your initial question didn't mention was the nature and age of your original sample. We find these acid free preps are good throughout Tertiary mudrocks and silty sandstones. (I honestly don't know if they work at all in limestones/ Chalk.) Normally they're good into the Lower Cretaceous mudrocks too, but start having problems with recovery by the time we get to Lower Cretaceous sandstones and Jurassic sandstones (obviously, we're using them to position oil wells in the stratigraphy). Doing biosteering in Jurassic clastic sequences of various grain sizes is manageable, but we have to integrate the palynology with petrophysics (gamma)  to manage crossing faults.

Going much deeper, we have to resort to "full acid" preps with increasing frequency.

Hi Reza,

I have processed a lot of samples for pollen and they do typically take 3 days to complete ~ 18 samples.  I do recommend sieving (as you have done) but this won't really cut down your time unless you are doing multiple treatments of the steps above.  In that case, sieving typically will reduce how many times you have to do each step.  Most of my samples are from lake sediments, so I actually can skip the KOH step and start with HCL.  That saves a bit of time, but skipping KOH depends on what kind of material you have. If you have humic acids (e.g., peatland material) you need to used KOH or NaOH. Depending on the samples I am processing, I can sometimes cut the processing time to 2.5 days by leaving my samples in HF overnight (we use 10% HF overnight - I am not sure if you can used much higher - you will have to research this before proceeding and also see if 10% is good enough for the material you have).  But if your samples have a lot of silicates, you may have to add fresh HF to your samples in the morning and put the centrifuge tubes in a hot water bath for 5 min anyways.  I have tried to eliminate HF by using heavy liquids, but it doesn't really cut down on time and it didn't seem to be as effective for samples high in silicates.  Spending extra time on processing samples for pollen can save you days in the lab counting pollen.  I actually spend a lot of time checking my samples in between steps to see if I need to repeat any steps.  Although this adds time I think it is worth spending more time in the lab and less time counting (because you have cleaner and better prepared samples).  Good luck Reza!

FYI - I checked with a colleague of mine and his lab uses undiluted 48-50% HF for treatment (including if you let your samples sit overnight in HF or do a hot water bath).  I have processed many of my samples in his lab; therefore, I was using HF full strength (48-50%) overnight and with 5min hot water bath.  I never had any issues re: pollen deterioration.

Salam Reza, Hale shoma Chetori ast? Khoobi? Che khabar? I assume that these samples are from lake late Quaternary Lakes in the desert regions of Iran where the preservation may or may not be good. You should have written me earlier....Acid reduction as discussed by others above is often very destructive of the already poorly preserved pollen. In order to maximize your preservation you will have to use flotation. It does increase the sediment  that you will have to scan through to count your pollen but the amount of pollen you will recover will be much more representative. Acid reduction leaves only the toughest of the poorly preserved pollen and your pollen counts will be unrepresentative. Here is the procedure that we use in the desert regions of the US. Again you should have asked....you have my email address...I may actually be in Tehran soon. I can help you if you can get the needed chemical....Detailed Pollen Extraction Methods

Samples, sized at approximately 150 cc, are collected at 2 cm intervals.

The following methods are standardized procedure for pollen extraction, as given by Dr. Peter Wigand via personal communication:

In the lab, each sample is transferred to a beaker with enough distilled water added to cover the sample. In a separate beaker, one to two Lycopodium tracer tablets are dissolved with hydrochloric acid then after the solution they are in is neutral add it to the beaker with the sample. The beaker is filled with distilled water until it is almost full and contents within the beaker are stirred or swirled until the sample disaggregates. In order to begin separating the sands stop stirring the sample and after 20 seconds the vortex will slow and sands will have settled at the bottom of the beaker.  Pour the suspended sediment and liquid quickly through a 100 micron screen into a second beaker. You might add more water to the first beaker and swirl one more time, let settle for 20 second and pour the suspension into the second beaker. This time the suspension should be relatively clear. The residue in the first beaker can be set aside for ostracode analysis. At this point the material in the second beaker can be stirred, and left to settle one to two hours....what is left in suspension at that time is clays. They can be poured off. You can repeat this step several times until the majority of the clays have been removed from the sample. What remains is primarily the silt sized fraction.

The sample can then be transferred to 40 ml test tubes with hot water and centrifuged. If you run the centrifuge at a low speed about 1900 rpm for three minutes or at even a lower speed, the clays will remain in suspension and can be poured off in the decant. It may take some time...but the investment in time to clean the samples results in cleaner samples that are easier to count. At this point make sure that you have centrifuged that sample well and decanted all the liquid from the test tube. Now this next step is important, and I hope that you can have access to the chemical. Sodium Polytungstate (polygee) is prepared to a specific gravity of 2.2 then is then added to each test tube until each is about half full. Mix the samples well with a vortex mixer, until the sample and the Sodium Polytungstate are well mixed. The centrifuge as before (1900 rpm for 3 minutes). Have clean, dry 12 ml test tubes prepared. When the centrifuge has stopped pour the suspension into the second set of test tubes. That suspension contains silt and pollen and also diatoms, and other organic material that is of the specific gravity of 2.2. The residue in the original 40 ml test tubes can be discarded.  Your 12 ml test tubes will be about half full. Add distilled water to the 12 ml test tubes, and mix thoroughly. That is important! No centrifuge the samples in the 12 ml test tubes for 10 minutes at highest speed. The pollen and other silt sized materials will settle to the bottom of the test tube. When the centrifuge stops....pour the liquid into a large beaker. It and be filtered and reused!!!!! If you use 10 micron glass filter paper and a beuchner funnel, you can pour that liquid through the filter paper....I do this twice....and then the filtered liquid can be evaporated and re-concentrated to a specific gravity of 2.2 and reused. This chemical is expensive. But the advantage is that it can be reused, and you do not have the chemical disposal problem that you have with HF, HCL, Sulfuric Acid, Acetic Anhydride, etc.  The residue in the 12 ml test tubes can be rinsed and centrifuged several times with hot distilled water to remove additional clays. You can experiment with the centrifuge speed, by trying different speeds and checking to see what remains at the bottom of the test tube. Select the ideal speed that leaves your samples the cleanest. Also check the residue at the bottom of the beaker containing the decanted liquid to make sure that you are not pouring off pollen. When your samples are clean...usually three to five hot water washes to also remove residual Sodium Polytungstate  which could crystalize in your sample slides after mounting....you can begin drying your samples. Add enough ethanol (ETOH) to each sample to cover...mix thoroughly...then add a single drop of HCL to each sample to acidify it so that it will take the stain more easily. Then add three to eight drops of safrinin stain and mix with the vortex mixer. Initially test the first sample adding safrinin stain and checking to see how the material is taking the stain. Add more drops as needed until you have determined the correct amount. The pollen should be pink and not red....you should be able to see the surface detail of the pollen grains. Then fill the test tube to the top with ETOH, centrifuge and decant into a large beakers. The adde ETOH again mix and centrifuge and decant. I save the ETOH, because I can also filter it and reuse it for the first ETOH treatment. No I have never had problems with cross contamination. Then treat each sample with tert-butyl alcohol adding a little initially, mixing with a vortex mixer, and then filling to the top. Centrifuge and decant (this decant can also be filtered and reused). Then transfer the samples by adding a little Tert-butyl alcohol to the test tube, vortexing it and pouring it into the vials. Repeat until all or most of the sample is transferred to vials. I let the samples settle until the sediment/pollen/diatom mixture is at the bottom. I pour about half of the Tert-butyl alcohol off, and then add 2000 cs silicone oil. Usually I try to have 1/3 sample, 1/3 Tert-butyl alcohol, and 1/3 silicone oil in each vial. Then put the cap on, and vortex mix each vial. Then place the vials with the cap off, and a clean covering over them to allow evaporation on a very low temperature hot plate....very low temperature....you can also simply let them evaporate in a protected area of your lab. Then after a day or so....smell the samples. If you don't smell Tert-butyl alcohol anymore you can cap the samples and mount them. Most importantly have labels prepared and ready to place on the vials. We mount the samples, about a drop of sample, on number 1 slides, mix the sample well and spread some so that the sample will disperse evenly over the slide, then cover with a cover slip, and seal all sides with a colorful nail polish so that if a leak occurs you can detect where the leak is. 

The really nice thing about this procedure is that you not only have pollen, but a great variety of algae, including diatoms to analyze. Acid reduction with HF of course destroys the diatoms. We are able to add a greater dimension to our analyses. For example, we have been analyzing pollen scrubs from ancient Native American milling stones for years and just identifying the pollen bearing plants that my have been milled on the stones...more recently we have been able to tell the archaeologist that aquatic plants were also being processed, from fresh-water springs, because we had fresh-water spring diatoms.

I hope this helps....Reza you should invite me to Tehran University when I am there this next time....I would love to see your lab and meet your colleagues. Check out the additional contributions that I have uploaded to ResearchGate now....there about 69 or so. Khoda hafez....Peter

Reza, the problem you deal with is poor initial preservation....samples from acid lake environments and bog can easily survive acid reduction. But from desert environments, basic or hyper saline environments are a real problem. Regarding the Acetolysis step...you don't need that...in fact is is helpful for you not to do it. First of all it is the most destructive step in the procedure, and second of all your samples, in most cases, because of their environments of deposition will no longer have cytoplasm to remove. The nice thing is, if you do see pollen with cytoplasm in them during your counting....you will usually see that they are modern contaminants....in fact it is a good test of how clean your lab is. Write me if you have more questions....you have my email...and I would like to see you this time....and meet your professor.

Reza, take a look on the pollen processing protocol from LacCore. If you pass by the heavy liquid separation step is fast.

Good luck

Diego

Hi Reza,

in our lab we usually need 2.5-3days for 18 samples as well. However, if you material is rich in sand I would also recommend, as mentioned above, to shortly vortex it to get the sediment into solution, let the heavy particles settle for 5-10 seconds and pour the still floating material into a new tube, repeat the step to make sure no desirable material still sticks to the first tube and gets lost. We use HF (45-48%) over night as well as the next day for up to two hours in a hot water bath with a half an hour cooling break. Depending on your samples you can also leave out the HF step completely or also the dehydration part and sieve you samples at the end of the cleaning with 7 microns in an ultrasonic bath, which might speed up the process as well, as most of the unwanted parts are already disposed of. Other than that I agree with Diana to rather spend more time with the preparation as it saves you a lot of time counting, especially if you pollen concentration is already low.

Good luck, Liv

Peter Ernest Wigand  : "Samples, sized at approximately 150 cc, are collected at 2 cm intervals."Wow, they're huge samples ! Our guys would normally do 30~50cc initial samples, which are loose aggregates so probably only about 15cc of bulk rock. If we're in relatively barren sediments (sands of Jurassic or older) they may have to go up in size by a factor of 2.

I'm really going to have to pick the brains of the next sample prep tech I work with.

Hi Aldan. I am sure that Reza is working with Late Quaternary samples. I have communicated with him via email So working in desert sediments as he is you have to concentrate your samples, because the preservation is so bad. If I were working with decent acid lake environments....I would use only a couple of cc. But over the years I have dealt mainly with samples from alluvium, open archaeological site, and other really poor environments for pollen preservation (even Tulare Lake in the southern Central Valley of California). In those cases I have to start with large samples and concentrate the silt sized fraction. It works. It is a standard protocol that I use. If I were doing Mesozoic or pre Quaternary pollen from rock....I obviously would have to use HF, but I am not dealing with coal or siltstone, or other indurated sediments. I am willing to plow through the extra silt to find the pollen. Cheers....I have actually developed a reputation in my consulting business for finding pollen where others won't even bother trying to run the samples.  

Hello Reza,,

it would be faster if you did first the acid attack and finish with the sieving.

Good luck.

Suzanne

All good answers.

One thing that you might be careful of...if you treat with HF first....if the samples have clays, and sands in them yet....they may be highly reactive. You may have boil over. Also you will use a lot more HF than if you sieve the samples first and concentrate them to the silt sized portion. One comment I will make is that the extra time you take to process your samples, you will save during analyses, because your samples will be cleaner, and more concentrated. A student of mine just found out that adding a few more water washes...cleaned up his samples significantly. He spends much less time counting now.

Thank you all for your valuable answers.

Dear Peter, Thank you so much for your answer

We have some sediment cores from different parts of Iran including arid, semi-arid Zagros Mountains and Caspian forest (Hyrcanian forest, N Iran). We use acid reduction procedure (e.g. Faegri & Iversen, 1975) for the sample preparation. This method seems to be suitable for the samples that are retrieved from humid and semi-arid regions. However, for the samples belong to the arid parts of Iran, using this method causes a very low concentration of the pollen.

Due to its nature, concentration of pollen is low in the desert areas because of poor vegetation and also preservation conditions. So, according to your explanation, using heavy liquid method instead of acid reduction could highly help us to overcome this issue.

I want to use the heavy liquid method for a core from desert; but unfortunately I found that the Sodium Polytungastae (SPT) and 7 micrometers filter are not available here. Could you please help us to find an easy way to buy those?

I have spoken with my professor Dr. Ghasem Azizi (https://www.researchgate.net/profile/Ghasem_Azizi) to hold a pollen analysis workshop in our faculty with your leadership. He is happy to arrange a workshop here in the faculty of Geography; so we appreciate if you inform us about your schedule?

Dear Reza, I would be most happy to participate in such a workshop perhaps with the Geological Survey as well. Right now we are still in the midst of setting the dates for the visa.  I will see you soon, Inshallah! I have been looking into sources of the sodium polytungstate as well, and the screen. I will send more information. Sincerely, Peter