Microtechnique

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Microtechnique is an aggregate of methods used to prepare micro-objects for studying. [1] It is currently being employed in many fields in life science. Two well-known branches of microtechnique are botanical (plant) microtechnique and zoological (animal) microtechnique.

Contents

With respect to both plant microtechnique and animal microtechnique, four types of methods are commonly used, which are whole mounts, smears, squashes, and sections, in recent micro experiments. [2] Plant microtechnique contains direct macroscopic examinations, freehand sections, clearing, maceration, embedding, and staining. [3] Moreover, three preparation ways used in zoological micro observations are paraffin method, celloidin method, and freezing method. [4]

History

The early development of microtechnique in botany is closely related to that in zoology. Zoological and botanical discoveries are adopted by both zoologists and botanists. [5]

The field of microtechnique lasted from at the end of the 1930s when the principle of dry preparation emerged. [6] The early development of microtechnique in botany is closely related to that in zoology. Zoological and botanical discoveries are adopted by both zoologists and botanists. [5] Since Hooke discovered cells, microtechnique had also developed with the emergence of early microscopes. Microtechnique then had advanced over the period of 1800-1875. [6] After 1875, modern micro methods have emerged. In recent years, both traditional methods and modern microtechnique have been in use in many experiments. [3]

Commonly used methods

Some general microtechnique can be used in both plant and animal micro observation. Whole mounts, smears, squashes, and sections are four commonly used methods when preparing plant and animal specimens for specific purposes. [2]

Whole mounts

Whole mounts are usually used when observers need to use a whole organism or do some detailed research on specific organ structure. [7] This method requires objects in which moisture can be removed, like seeds and micro fossils. [2]

According to different purposes, Whole-mounts can be divided into three categories, Temporary whole mounts, Semi-permanent whole mounts, and permanent whole mounts. Temporary whole mounts are usually used for teaching activities in class. [8] Semi-permanent whole mounts are prepared for longer using time, which is no more than fourteen days. In this preparation, Canada balsam is used to seal the specimens, and this method is used to observe unicellular and colonial algae, fungal spores, mosses protonemata, and prothalli. The third way is a permanent whole mount. [8] Two methods are usually used, which are hygrobutol method and glycerine-xylol method. [9]

Smear from solid medium and liquid medium Smear.svg
Smear from solid medium and liquid medium

Smears

Smears is an easy way for preparing slices. This method is used in many laboratories. [10] Smears can be employed when making slide specimens by spreading liquid or semi-liquid materials or lose tissues and cells of animals and plants evenly on the slide. [10] The steps and requirements for the application of the smear method are as follows: first, smear. When the solid material is smeared, the material should be placed on the glass slide and wiped away, then use the blade to press the material on one side. [11] The cells should be pressed out and distributed evenly on the glass slide in a thin layer, such as the anther smeared. [10]

Squashes

Squashes are methods, in which objects are crushed with force. This method is suitable for preparing both transparent and tender tissues. [12] When preparing squashes slides, specimens are supposed to be thin and transparent so that objects can be observed clearly under microscopes. [12]

This technique is to place the material on the glass slide and remove it with the scalpel or to dissect needle, then add a drop of dye solution. [2] After these steps, apply the second slide to cover the initial slide and apply pressure evenly to break the material and disperse the cells. [12] Furthermore, another possible way can be used to prepare slides. The specimens can also be extruded between the cover slide and the slide with equal pressure. [12]

Sections

Main article: Histology

Sections are known as thin slices need to be tested in all studies of cellular structures. [13] This technique can be used for the preparation of tissue of animals and plants. [14] For using under optical microscopy, the thickness of the material should be between above 2 and 25 micrometers. When observing under electron microscopy, sections should be from 20 to 30 nanometers. [2] Microtome can be used in sectioning of sufficiently thin slices. If the objects cannot satisfy the requirement of thickness, materials are required to be dehydrated using alcohol before section. [12] Three commonly used sectioning method are freehand section technique, paraffin method, and celloidin method.

Methods used in plant micro-experiments

Botanical microtechnique is an aggregate of methods providing micro visualization of gene and gene product in an entire plant. [15] Plant microtechnique is also a study providing valuable experimental information. [3] Plant microtechnique involves classical methods developed over a hundred years ago and new methods developed to expand our research scope and depth in botanical micro studies. [15] Both traditional and new micro technique is useful for experimental research, and some will have a significant influence on further study. [3] Different methods are used to prepare plant specimens, including direct macroscopic examinations, freehand sections, [16] clearing, maceration, embedding, and staining.

Direct microscopic examinations

The direct micro examination is a simple way prepared for observing micro-objects. Also, this method is useful to observe whether the mold grows on the surface of the specimens. This can be an initial step of the micro experiment. [17]

Freehand section

Freehand slicing is a method of making thin slices of fresh or fixed experimental materials with a hand-held blade. [18] Freehand slicing refers to the method of directly cutting fresh or fixed materials (generally plants with a low degree of lignification) into thin slices without special instruments or special chemical reagents. [16]

Clearing

Clearing technique provides translucent slides via removing part of cytoplasmic content and then applying high refractive index reagents to process the tissues. [2] This method is suitable for preparing whole mount slides. The clearing is a procedure of using clearing reagents for removal of alcohol and makes tissue translucent. [19] Xylene is the most popular clearing agent. [20] [21]

Maceration

Macerating tissues is the process of separating the constituent cells of tissues. This method enables observers to study the whole cell in third-dimensional detail. [8] Chemical maceration method means the using chemicals to process organs or part to soften tissue and dissolving the cells so that different cell can be identified. [8]

Tissue processing - Embedding station Tissue processing - Embedding station.jpg
Tissue processing - Embedding station

Embedding

Main article: Histology

Embedding technique is a medium stage when doing a sectioning process. [22] When preparing specimens, it is difficult to make uniform slices since the tissue is soft. [23] Therefore, it is necessary to soak the tissue with a certain substance to harden the whole tissue, to facilitate the slicing. This process is called embedding. [23] The substance used to embed tissue is embedding media, which is chosen depends on the category of the microscope, category of the micro tome, and category of tissue. [24] Paraffin wax, whose melting point is from 56 to 62℃, is commonly used for embedding. [25]

Tissue processing - Tissue sections on slides are stained on an automated stainer Tissue processing - Tissue sections on slides are stained on an automated stainer.jpg
Tissue processing - Tissue sections on slides are stained on an automated stainer

Staining

Main article: Histology

Since few plant tissues have a color, there is little chromatically difference between plant tissues makes it difficult to differentiate botanical structure. [26] Material is usually dyed before installation. This process is called staining, which can be used to prepare botanical specimens so that it is possible to distinguish one part of the sample from another in terms of color. [2] Acid dyes can be used when staining micro slides, for example, acid dyes are in use when coloring nuclei and other cellular components are stained using alkaline. [2] There are also staining machine used for staining, which allows tissue to be stained automatically. [27]

Microtechnique used for animal observation

The zoological microtechnique is the art of the preparation for microscopic animal observation. Although many microtechniques can be used in both plant and animal micro experiments. Some methods may differ from itself when employed in different field. Three commonly used preparation ways used in zoological micro observations can be concluded as paraffin method, celloidin method, freezing method, and miscellaneous techniques. [4]

Paraffin wax Paraffin.jpg
Paraffin wax

Paraffin method

Infiltration and embedding

This process usually consists of steps of infiltration, embedding, sectioning, affixing and processing the sections. [28] Followed by the initial stage, fixation, the next step is dehydration, which removes the water in the tissue using alcohol. [29] Then the tissue can be infiltrated and embedded with wax. A tissue specimen can keep for several years after finishing embedding this tissue into the wax. [29] Paraffin wax, which is soft and colorless, is the most commonly used reagent. [30]

Microtome-knife-profile Microtome-knife-profile.svg
Microtome-knife-profile

Sectioning

Sectioning a tissue can use either the micro tome knife or the razor blade as the cutting blade. [4]

The micro tome knife is used for handling sectioning. It is necessary to use a micro tome knife when preparing sections less than 1/1000 micrometers. [31] When using such a knife, the operators must be extremely careful. This instrument is impractical sometimes, so using the razor blade for general work to prepare sections above 9 microns (1 micron equals 1/1000 micrometers). [31] Furthermore, the razor blade works better than the micro tome knife when requiring thick sections with no less than 20 microns. [4]

Affixing and processing

After sectioning, the prepared slices are affixed on slides. There are two commonly used affixatives, Haupt’s and Mayer’s. [32] Haupt’s affixative contains 100 ccs (cubic centimeter) distilled water, 1gm gelatin, 2 gm phenol crystals, 15 cc glycerine. Mayer’s affixative is consist of 5 cc egg albumen, 50 cc glycerine, 1 gm sodium salicylate. [33] The general steps of affixing paraffin sections can be concluded as 1. Clean the required slides, 2. Mark the cleaned slides, 3. Drop affixative on each slide, 4. Put on another slide, 5. Spread the affixative, 6. Drop floating medium, 7. Divide the paraffin into required length, 8. Transfer the sections, 9. Add more floating medium if incomplete floating occurs, 10. Rise the temperature, 11. Remove slides and redundant floating medium, 12, drying the section. [4]

Processing paraffin sections include 1. Deparaffination, 2. Removing the deparaffing solution, 3. Hydration, 4. Staining, 5. Dehydration, 6. Dealcoholisation and clearing, 7. Mounting the cover slide. [4]

Celloidin method

Celloidin technique is the procedure of embedding a specimen in celloidin. [34] This method can be used for embedding large, hard objects. [35] Celloidin is a digestive fiber, which is flammable, and it is soluble in acetone, clove oil, and the mixture of anhydrous alcohol and ether. [36] Celloidin will turn into white emulsion turbid liquid when it meets water, so it is required to use a dry container to contain celloidin. [35]

The method of celloidin slicing is to fix and dehydrate the tissue, then treat it with the anhydrous alcohol-ether mixture. After this step, to impregnate, embed and slice the tissue with celloidin. [37] Moreover, this slicing method can slice large tissues and has the advantage that its heat allows the tissues does not shrink. However, this technique contains some shortcomings. For instance, the slices cannot be sliced very thin (more than 20 microns), and impregnation with celloidin is time-consuming. [38]

Freezing method

Freezing technique is the most commonly used sectioning method. [39] This method can preserve the immune activity of various antigens well. Both fresh tissue and fixed tissue can be frozen. Moreover, it is also a technique used for freezing sections of either fresh or fixed plant tissues. [40]

During the freezing procedure, the water in tissues is easy to form ice crystals, which often affects the antigen localization. [39] It is generally believed that when ice crystals are small, the effect is small, and when ice crystals are large, the damage to the tissue structure is large, and the above phenomenon is more likely to occur in tissues with more moisture components. [41] The size of an ice crystal is directly proportional to its growth rate and inversely proportional to the nucleation rate (formation rate), that is, the larger the number of ice crystal formation, the smaller it is, and the more serious the impact on the structure. [42] Therefore, the number of ice crystals should be minimized. The freezing method allows sectioning tissues rapidly and biopsy without using reagents. This procedure should be rapidly in case of the form of ice crystal. [41]

See also

Related Research Articles

Histology Study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

Microscope slide Thin, flat piece of glass onto which a sample is placed to be examined under a microscope

A microscope slide is a thin flat piece of glass, typically 75 by 26 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.

Staining Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

Histopathology Microscopic examination of tissue in order to study and diagnose disease

Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.

Cryostat

A cryostat is a device used to maintain low cryogenic temperatures of samples or devices mounted within the cryostat. Low temperatures may be maintained within a cryostat by using various refrigeration methods, most commonly using cryogenic fluid bath such as liquid helium. Hence it is usually assembled into a vessel, similar in construction to a vacuum flask or Dewar. Cryostats have numerous applications within science, engineering, and medicine.

Immunohistochemistry Common application of immunostaining

Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue. Albert Coons conceptualized and first implemented the procedure in 1941.

A microtome is a cutting tool used to produce extremely thin slices of material known as sections. Important in science, microtomes are used in microscopy, allowing for the preparation of samples for observation under transmitted light or electron radiation.

Tissue microarray

Tissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.

<i>In situ</i> hybridization

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

Ultramicrotomy is a method for cutting specimens into extremely thin slices, called ultra-thin sections, that can be studied and documented at different magnifications in a transmission electron microscope (TEM). It is used mostly for biological specimens, but sections of plastics and soft metals can also be prepared. Sections must be very thin because the 50 to 125 kV electrons of the standard electron microscope cannot pass through biological material much thicker than 150 nm. For best resolutions, sections should be from 30 to 60 nm. This is roughly the equivalent to splitting a 0.1 mm-thick human hair into 2,000 slices along its diameter, or cutting a single red blood cell into 100 slices.

H&E stain Histological stain method

Hematoxylin and eosin stain is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

Frozen section procedure Rapid histological sectioning procedure

The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection. The microtome device that cold cuts thin blocks of frozen tissue is called a cryotome.

Laser capture microdissection

Laser capture microdissection (LCM), also called microdissection, laser microdissection (LMD), or laser-assisted microdissection, is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms.

Immunocytochemistry

Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Fixation (histology) Preservation of biological tissue

In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules as proteins and nucleic acids.

Frozen tissue array

Frozen tissue array consists of fresh frozen tissues in which up to 50 separate tissue cores are assembled in array fashion to allow simultaneous histological analysis.

Automated tissue image analysis

Automated tissue image analysis or histopathology image analysis (HIMA) is a process by which computer-controlled automatic test equipment is used to evaluate tissue samples, using computations to derive quantitative measurements from an image to avoid subjective errors.

Vibratome

A vibratome is an instrument used to cut thin slices of material. It is similar to a microtome but uses a vibrating blade to cut through tissue. The vibration amplitude, the speed, and the angle of the blade can all be controlled. Fixed or fresh tissue pieces are embedded in low gelling temperature agarose. The resulting agarose block containing the tissue piece is then attached to a metal block and sectioned while submerged in a water or buffer bath. Individual sections are then collected with a fine brush and transferred to slides or multiwell plates for staining.

Bread loafing

Bread loafing is a common method of processing surgical specimens for histopathology. The process involves cutting the specimen into 3 or more sections. The cut sections are mounted by embedding in paraffin or frozen medium. The cut edge is then thinly sliced with a microtome or a cryostat. The thin slices are then mounted on a glass slide, stained, and covered with another layer of glass.

The conservation and restoration of herbaria includes the preventive care, repair, and restoration of herbarium specimens. Collections of dried plant specimens are collected from their native habitats, identified by experts, pressed, and mounted onto archival paper. Care is taken to make sure major morphological characteristics are visible. Herbaria documentation provides a record of botanical diversity.

References

  1. ↑ "microtechnique - Wiktionary". en.wiktionary.org. Retrieved 2019-05-19.
  2. 1 2 3 4 5 6 7 8 Peter, G (2014). "Microtechnique". Access Science. doi:10.1036/1097-8542.424010.
  3. 1 2 3 4 Yeung, E. C. T., Stasolla, C., Sumner, M. J., & Huang, B. Q. (Eds.) (2015). Plant microtechniques and protocols. Switzerland: Springer International Publishing.{{cite book}}: CS1 maint: multiple names: authors list (link)
  4. 1 2 3 4 5 6 Weesner., F.M. (1968). General zoological microtechniques. Maryland, U.S.A.: The Williams & Wilkins Company.
  5. 1 2 Smith, G. M. (1915). "The Development of Botanical Microtechnique". Transactions of the American Microscopical Society. 34 (2): 71–129. doi:10.2307/3221940. ISSN   0003-0023. JSTOR   3221940.
  6. 1 2 Apathy, S (1896). Die Mikrotechnik der thierischen Morphologie. Braunschweig.
  7. ↑ "wholemount - Wiktionary". en.wiktionary.org. 28 April 2018. Retrieved 2019-05-19.
  8. 1 2 3 4 Nandhagopalan (2013-04-06). "Whole mount preparation". world of nandha. Retrieved 2019-05-19.
  9. ↑ Hills, P. "SYLLABUS FOR POST-GRADUATE PROGRAMME IN" (PDF).
  10. 1 2 3 "SMEAR PREPARATION". coproweb.free.fr. Retrieved 2019-05-19.
  11. ↑ "Coverslip Smear Preparation Technique - LabCE.com, Laboratory Continuing Education". www.labce.com. Retrieved 2019-05-19.
  12. 1 2 3 4 5 "How to prepare squash specimen samples for microscopic observation – Microbehunter Microscopy". 17 July 2011. Retrieved 2019-05-19.
  13. ↑ "Sectioning of paraffin-embedded tissue protocol | Abcam". www.abcam.com. Retrieved 2019-05-19.
  14. ↑ "Advanced Sectioning Techniques: How to Section Difficult Tissues". Bitesize Bio. 2013-12-17. Retrieved 2019-05-19.
  15. 1 2 Ruzin, S.E. (1999). Plant microtechnique and microscopy. New York: Oxford University Press.
  16. 1 2 "lab3". www.cas.miamioh.edu. Retrieved 2019-05-19.
  17. ↑ Roberts, Glenn D.; Yu, Pauline K. W.; Washington, John A. (1981), Washington, John A. (ed.), "Direct Microscopic Examination of Specimens", Laboratory Procedures in Clinical Microbiology, Springer US, pp. 69–89, doi:10.1007/978-1-4684-0118-9_2, ISBN   9781468401189
  18. ↑ "Plant Anatomy_Free Hand Sectioning | Microscope | Plant Stem". Scribd. Retrieved 2019-05-19.
  19. ↑ "Tissue Clearing - LabCE.com, Laboratory Continuing Education". www.labce.com. Retrieved 2019-05-19.
  20. ↑ "Clearing Tissue Sections | National Diagnostics". www.nationaldiagnostics.com. Retrieved 2019-05-19.
  21. ↑ Rolls, Geoffrey (2019-04-15). "An Introduction to Specimen Processing". Leica Biosystems.
  22. ↑ "Sectioning of paraffin-embedded tissue protocol | Abcam". www.abcam.com. Retrieved 2019-05-19.
  23. 1 2 "Embedding | National Diagnostics". www.nationaldiagnostics.com. Retrieved 2019-05-19.
  24. ↑ "Embedding".
  25. ↑ "Embedding | National Diagnostics". www.nationaldiagnostics.com. Retrieved 2019-05-19.
  26. ↑ "Staining Techniques". www.cliffsnotes.com. Retrieved 2019-05-19.
  27. ↑ Wilkie, R. N., & Mooradian, A. (1978). Automatic slide stainer. Washington, DC: U.S.: Patent and Trademark Office.{{cite book}}: CS1 maint: multiple names: authors list (link)
  28. ↑ Kacena, M., Troiano, N. W., Wilson, K. M., Coady, C. E., & Horowitz, M. C. (2004). "Evaluation of two different methylmethacrylate processing, infiltration, and embedding techniques on the histological, histochemical, and immunohistochemical analysis of murine bone specimens". Journal of Histotechnology. 27 (2): 119–130. doi:10.1179/his.2004.27.1.15. S2CID   86700855.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  29. 1 2 "Paraffin Processing of Tissue". Protocols Online. 2010-06-24. Retrieved 2019-05-19.
  30. ↑ Brown, W (1915). "Studies in the Physiology of Parasitism: I. The Action of Botrytis cinerea". Annals of Botany. 29 (115): 313–348. doi:10.1093/oxfordjournals.aob.a089551.
  31. 1 2 Jacoby, J. G. W. (1953). Microtome knife. Patent and Trademark Office.
  32. ↑ Pappas, P. W. (1971). "The use of a chrome alum-gelatin (subbing) solution as a general adhesive for paraffin sections". Stain Technology. 46 (3): 121–124. doi:10.3109/10520297109067835. PMID   4105404.
  33. ↑ Haupt., A. W. (1930). "A gelatin fixative for paraffin sections". Stain Technology. 5 (3): 97–98. doi:10.3109/10520293009115555.
  34. ↑ Wetmore, E.H. (1932). "The use of celloidin in botanical technic". Stain Technology. 7 (2): 37–62. doi:10.3109/10520293209116071.
  35. 1 2 Baker, J. R. (1933). Cytological Technique. London: Methuen And Co. Ltd.
  36. ↑ "celloidin - Wiktionary". en.wiktionary.org. Retrieved 2019-05-19.
  37. ↑ Portmann, D., Fayad, J., Wackym, P. A., Shiroishi, H., Linthicum Jr, F. H., & Rask‐Andersen, H. (1990). "A technique for reembedding celloidin sections for electron microscopy". The Laryngoscope. 100 (2): 195–199. doi:10.1288/00005537-199002000-00017. PMID   2405230. S2CID   1645611.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  38. ↑ Plowman, A. B. (1904). "The celloidin method with hard tissues". Botanical Gazette. 37 (6): 456–461. doi: 10.1086/328510 .
  39. 1 2 "Tissue Freezing Methods for Cryostat Sectioning" (PDF).
  40. ↑ Knox, R. B. (1970). "Freeze-sectioning of plant tissues". Stain Technology. 45 (6): 265–272. doi:10.3109/10520297009067799. PMID   5490087.
  41. 1 2 "Freezing tissues for histology" (PDF).
  42. ↑ J. Byrwa-Neff, Kimberly; Cunningham, Miles (2012-07-12). "Freezing Biological Samples".{{cite journal}}: Cite journal requires |journal= (help)
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