Tuesday, May 28, 2019

Living the single life

Scientists are increasingly taking advantage of microfluidic technology for single cell applications to understand individual cells and their role in health and immunity. Here we look at latest advances in the technology and some examples of microfluidic approaches to single cell research…
Understanding how single cells within an individual person, tissue or organ differ throughout development or as a result of disease has great potential to help advance personalised medicine.
Since high throughput single cell applications based on microfluidic droplet technology came to the fore in 2015,1,2 researchers have taken advantage of single cell RNA-seq (scRNA-seq) methods to increase their knowledge of cell development, immunology and immunotherapy, as well as tumour heterogeneity, lineage analysis and clonal evolution.3,4,5,6
Individual immune cells can be profiled making it possible to identify tumour-associated macrophages that are crucial to determining tumour fate
The commercial availability of easy-to-use microfluidic instruments has helped drive the acceptance and widespread use of high throughput scRNA-seq, particularly in the materials science and chemistry sectors. Biologists were slower to embrace this technology, perhaps believing that it was necessary to become an expert in setting up microfluidic experiments. Everything changed with the advent of intuitive, application-orientated microfluidic instruments, enabling researchers to explore cell diversity and tissue heterogeneity, and quantify gene expression at the single cell level.
Many cells, few minutes…
Using these systems, tens of thousands of single cells can be encapsulated in a matter of minutes, capturing the mRNA of each cell on a solitary uniquely barcoded oligo bead before reverse transcription and sequencing. As each sequencing read can be associated with its original cell, it is possible to perform transcriptome analysis of thousands of cells. Scientists are also starting to use individual nuclei as a proxy for whole cells, allowing the investigation of gene expression in cells that are difficult to isolate – such as brain cells – or cells obtained from archived tissue, which has potential applications for clinical studies.7,8
Single cell techniques also have the potential to enable identification of the full range of cell types and states involved in immune responses, and to increase knowledge of immune cell development and differentiation. In the past, technical limitations – such as only being able to analyse a few RNA molecules at a time4 – have hindered immunology research, but these are being overcome by new scRNA-seq methods, offering improved sensitivity, reproducibility and throughput. Individual immune cells can be profiled with a higher degree of accuracy and in high numbers, making it possible to identify tumour-associated macrophages that are crucial to determining tumour fate. It is also possible to study cell states and cell activation to identify genes that act as drivers of immune responses.9
As well as transcriptomic analysis by scRNA-seq, other technologies are now available to aid the investigation of genomics, epigenetics and proteomics at the single cell level – including some commercial systems with standard protocols and applications for methods such as CITE-seq, CNV analysis or ATAC-seq – with more in the pipeline.10,11, 12
Mapping the human cell atlas
Single cell technologies have advanced rapidly, revolutionising scientists’ understanding of individual cells and their role in health and immunity.
The Human Cell Atlas (HCA) project – an international collaboration launched in 2016 – is one such example, aiming to create a comprehensive reference map of all human cells and their function within the body. This will further researchers’ understanding of human health to help improve the diagnosis, monitoring and treatment of disease.
It is an ambitious target, but one that is becoming increasingly achievable with the easy-to-use tools and instruments for high throughput single cell research available today. Novel cell types and markers can be identified and used to guide the development of new drugs and treatments and, in the long term, personalised healthcare.
scRNA-seq in practice
Although single cell technologies are comparatively new, researchers are already reaping the benefits of this approach. At the Centre for Neural Circuits and Behaviour at the University of Oxford, scientists with an interest in learning and memory are using the Drop-Seq1 technique and a single cell system to study cellular diversity in the Drosophila (fruit fly) midbrain.13 The midbrain of the fruit fly is ideal for single cell sequencing as it only contains 50,000 cells, allowing the whole brain to be analysed in one experiment. Different areas of the brain are then compared to identify and understand the neurons involved in memory and learning, typing them according to the neurotransmitter or neuromodulator that they produce.
The group is also performing scRNA-seq experiments aimed at discovering new genes that are important in the formation of these neurons, as well as identifying neuropeptides and exploring dopamine pathways.
Another notable project is underway in Denmark, where researchers at the Aarhus University Hospital are focusing on translational research to obtain a deeper understanding of bladder cancer, seeking to identify and validate molecular markers that could aid personalised medicine. In early stage bladder cancer, the main clinical challenge is to predict disease recurrence, aggressiveness and treatment response. Analysis of the normal tissue surrounding the tumour may allow the underlying disease mechanism forming the field cancerisation to be established, helping to predict disease outcomes.
In the past, the group has used next generation sequencing (NGS, DNA and RNA-seq) for its cancer genomics studies, but it is now moving into the single cell arena to delve further into cell heterogeneity, clonality and the tumour microenvironment. NGS-based analysis of bulk tumours identified three major molecular groups predicting different disease outcomes, and scRNA-seq technology is now being used to determine the specific cell type contribution of these molecular subgroups, investigating whether particular immune cells are over- or under-represented in the aggressive tumours. The characterisation of cell type composition in apparently normal bladder tissue may also contribute to the development of molecular tools and clinically-applicable pipelines to predict the likely aggressiveness of the disease and the frequency of recurrence. 
The single cell market is young and fast moving. As new protocols and applications are developed, the widespread availability of flexible, automated and easy-to-use microfluidics-based droplet systems will be crucial to scientists exploring fresh avenues for biology research. In this regard, open systems have an advantage over locked platforms, as they can be reconfigured to support an ever-growing list of applications, enabling further insight into cell diversity throughout development or disease.
The future of scRNA-seq is exciting, and biologists, whether they have microfluidics expertise or are novices, are well-placed to exploit the technique and reap the benefits of single cell droplet encapsulation.
CREDIT TO LABNEWS.CO.UK

Saturday, May 18, 2019

Clostridium botulinum toxin-one of the most lethal substances known(WELL EXPLAINED)

See the source image

Key facts

  • Clostridium botulinum is a bacterium that produces dangerous toxins (botulinum toxins) under low-oxygen conditions.
  • Botulinum toxins are one of the most lethal substances known.
  • Botulinum toxins block nerve functions and can lead to respiratory and muscular paralysis.
  • Human botulism may refer to foodborne botulism, infant botulism, wound botulism, and inhalation botulism or other types of intoxication.
  • Foodborne botulism, caused by consumption of improperly processed food, is a rare but potentially fatal disease if not diagnosed rapidly and treated with antitoxin.
  • Homemade canned, preserved or fermented foodstuffs are a common source of foodborne botulism and their preparation requires extra caution.

Foodborne botulism is a serious, potentially fatal disease. However, it is relatively rare. It is an intoxication usually caused by ingestion of potent neurotoxins, the botulinum toxins, formed in contaminated foods. Person to person transmission of botulism does not occur.
Spores produced by the bacteria Clostridium botulinum are heat-resistant and exist widely in the environment, and in the absence of oxygen they germinate, grow and then excrete toxins. There are 7 distinct forms of botulinum toxin, types A–G. Four of these (types A, B, E and rarely F) cause human botulism. Types C, D and E cause illness in other mammals, birds and fish.
Botulinum toxins are ingested through improperly processed food in which the bacteria or the spores survive, then grow and produce the toxins. Though mainly a foodborne intoxication, human botulism can also be caused by intestinal infection with C. botulinum in infants, wound infections, and by inhalation.

Symptoms of foodborne botulism

Botulinum toxins are neurotoxic and therefore affect the nervous system. Foodborne botulism is characterized by descending, flaccid paralysis that can cause respiratory failure. Early symptoms include marked fatigue, weakness and vertigo, usually followed by blurred vision, dry mouth and difficulty in swallowing and speaking. Vomiting, diarrhoea, constipation and abdominal swelling may also occur. The disease can progress to weakness in the neck and arms, after which the respiratory muscles and muscles of the lower body are affected. There is no fever and no loss of consciousness.
The symptoms are not caused by the bacterium itself, but by the toxin produced by the bacterium. Symptoms usually appear within 12 to 36 hours (within a minimum and maximum range of 4 hours to 8 days) after exposure. Incidence of botulism is low, but the mortality rate is high if prompt diagnosis and appropriate, immediate treatment (early administration of antitoxin and intensive respiratory care) are not given. The disease can be fatal in 5 to 10% of cases.

Exposure and transmission

Foodborne botulism

C. botulinum is an anaerobic bacterium, meaning it can only grow in the absence of oxygen. Foodborne botulism occurs when C. botulinum grows and produces toxins in food prior to consumption. C. botulinum produces spores and they exist widely in the environment including soil, river and sea water.
The growth of the bacteria and the formation of toxin occur in products with low oxygen content and certain combinations of storage temperature and preservative parameters. This happens most often in lightly preserved foods and in inadequately processed, home-canned or home-bottled foods.
C. botulinum will not grow in acidic conditions (pH less than 4.6), and therefore the toxin will not be formed in acidic foods (however, a low pH will not degrade any pre-formed toxin). Combinations of low storage temperature and salt contents and/or pH are also used to prevent the growth of the bacteria or the formation of the toxin.
The botulinum toxin has been found in a variety of foods, including low-acid preserved vegetables, such as green beans, spinach, mushrooms, and beets; fish, including canned tuna, fermented, salted and smoked fish; and meat products, such as ham and sausage. The food implicated differs between countries and reflects local eating habits and food preservation procedures. Occasionally, commercially prepared foods are involved.
Though spores of C. botulinum are heat-resistant, the toxin produced by bacteria growing out of the spores under anaerobic conditions is destroyed by boiling (for example, at internal temperature greater than 85 °C for 5 minutes or longer). Therefore, ready-to-eat foods in low oxygen-packaging are more frequently involved in cases of foodborne botulism.
Food samples associated with suspect cases must be obtained immediately, stored in properly sealed containers, and sent to laboratories in order to identify the cause and to prevent further cases.

Infant botulism

Infant botulism occurs mostly in infants under 6 months of age. Different from foodborne botulism caused by ingestion of pre-formed toxins in food, it occurs when infants ingest C. botulinum spores, which germinate into bacteria that colonize in the gut and release toxins. In most adults and children older than about 6 months, this would not happen because natural defences in intestines that develop over time prevent germination and growth of the bacterium.
C. botulinum in infants include constipation, loss of appetite, weakness, an altered cry and a striking loss of head control. Although there are several possible sources of infection for infant botulism, spore-contaminated honey has been associated with a number of cases. Parents and caregivers are therefore warned not to feed honey to the infants before the age of 1 year.

Wound botulism

Wound botulism is rare and occurs when the spores get into an open wound and are able to reproduce in an anaerobic environment. The symptoms are similar to the foodborne botulism, but may take up to 2 weeks to appear. This form of the disease has been associated with substance abuse, particularly when injecting black tar heroin.

Inhalation botulism

Inhalation botulism is rare and does not occur naturally, for example it is associated with accidental or intentional events (such as bioterrorism) which result in release of the toxins in aerosols. Inhalation botulism exhibits a similar clinical footprint to foodborne botulism. The median lethal dose for humans has been estimated at 2 nanograms of botulinum toxin per kilogram of bodyweight, which is approximately 3 times greater than in foodborne cases.
Following inhalation of the toxin, symptoms become visible between 1–3 days, with longer onset times for lower levels of intoxication. Symptoms proceed in a similar manner to ingestion of botulinum toxin and culminate in muscular paralysis and respiratory failure.
If exposure to the toxin via aerosol inhalation is suspected, additional exposure to the patient and others must be prevented. The patient's clothing must be removed and stored in plastic bags until it can be washed thoroughly with soap and water. The patient should shower and be decontaminated immediately.

Other types of intoxication

Waterborne botulism could theoretically result from the ingestion of the pre-formed toxin. However, as common water treatment processes (such as boiling, disinfection with 0.1% hypochlorite bleach solution) destroy the toxin, the risk is considered low.
Botulism of undetermined origin usually involves adult cases where no food or wound source can be identified. These cases are comparable to infant botulism and may occur when the normal gut flora has been altered as a result of surgical procedures or antibiotic therapy.
Adverse effects of the pure toxin have been reported as a result of its medical and/or cosmetic use in patients, see more on 'Botox' below.

'Botox'

The bacterium C. botulinum is the same bacterium that is used to produce Botox, a pharmaceutical product predominantly injected for clinical and cosmetic use. Botox treatments employ the purified and heavily diluted botulinum neurotoxin type A. Treatment is administered in the medical setting, tailored according to the needs of the patient and is usually well tolerated although occasional side effects are observed.

Diagnosis and treatment

Diagnosis is usually based on clinical history and clinical examination followed by laboratory confirmation including demonstrating the presence of botulinum toxin in serum, stool or food, or a culture of C. botulinum from stool, wound or food. Misdiagnosis of botulism sometimes occurs as it is often confused with stroke, Guillain-BarrĂ© syndrome, or myasthenia gravis.
Antitoxin should be administered as soon as possible after a clinical diagnosis. Early administration is effective in reducing mortality rates. Severe botulism cases require supportive treatment, especially mechanical ventilation, which may be required for weeks or even months. Antibiotics are not required (except in the case of wound botulism). A vaccine against botulism exists but it is rarely used as its effectiveness has not been fully evaluated and it has demonstrated negative side effects.

Prevention

Prevention of foodborne botulism is based on good practice in food preparation particularly during heating/sterilization and hygiene. Foodborne botulism may be prevented by the inactivation of the bacterium and its spores in heat-sterilized (for example, retorted) or canned products or by inhibiting bacterial growth and toxin production in other products. The vegetative forms of bacteria can be destroyed by boiling but the spores can remain viable after boiling even for several hours. However, the spores can be killed by very high temperature treatments such as commercial canning.
Commercial heat pasteurization (including vacuum packed pasteurized products and hot smoked products) may not be sufficient to kill all spores and therefore the safety of these products must be based on preventing bacterial growth and toxin production. Refrigeration temperatures combined with salt content and/or acidic conditions will prevent the growth of the bacteria and formation of toxin.
The WHO Five Keys to Safer Food serve as the basis for educational programmes to train food handlers and educate the consumers. They are especially important in preventing food poisoning.
The Five Keys are:
  • keep clean
  • separate raw and cooked
  • cook thoroughly
  • keep food at safe temperatures
  • use safe water and raw materials.

Diagnosis of EBOLA Virus

See the source image

Diagnosis

To determine whether Ebola virus infection is a possible diagnosis, there must be a combination of symptoms suggestive of EVD AND a possible exposure to EVD within 21 days before the onset of symptoms. An exposure may include contact with:
  • blood or body fluids from a person sick with or who died from EVD
  • objects contaminated with blood or body fluids of a person sick with or who died from EVD
  • infected fruit bats and primates (apes or monkeys)
  • semen from a man who has recovered from EVD
If a person shows early signs of EVD and has had a possible exposure, he or she should be isolated (separated from other people) and public health authorities notified. Blood samples from the patient should be collected and tested to confirm infection. Ebola virus can be detected in blood after onset of symptoms, most notably fever. It may take up to three days after symptoms start for the virus to reach detectable levels. A positive laboratory test means that Ebola infection is confirmed. Public health authorities will conduct a public health investigation, including tracing of all possibly exposed contacts.

Microscopical examination of faecal specimens simplified


Microscopical examination of faecal specimens
Examine immediately those specimens containing blood and mucus and those that are unformed because these may contain motile trophozoites of E. histolytica or G. lamblia

Examination of dysenteric and unformed specimens

1 Using a wire loop or piece of stick, place a small amount of specimen, to include blood and mucus on one end of a slide. Without adding saline, cover with a cover glass and using a tissue, press gently on the cover glass to make a thin preparation.
2 Place a drop of eosin reagent on the other end of the slide. Mix a small amount of the specimen with the eosin and cover with a cover glass.
Value of eosin: Eosin does not stain living trophozoites but provides a pink background which can make them easier to see.
3 Examine immediately the preparations microscopically, first using the 10 objective with the condenser iris closed sufficiently to give good contrast. Use the 40 objective to identify motile trophozoites, e.g. E. histolytica amoebae or G. lamblia flagellates
Note: The eggs of Schistosoma species and T. trichiura, and the trophozoites of B. coli can also result in specimens containing blood and mucus.

Examination of semi-formed and formed faeces
1 Place a drop of fresh physiological saline on one end of a slide and a drop of iodine on the other end. To avoid contaminating the fingers and stage of the microscope, do not use too large a drop of saline or iodine.
2 Using a wire loop or piece of stick, mix a small amount of specimen, about 2 mg, (matchstick head amount) with the saline and a similar amount with the iodine. Make smooth thin preparations. Cover each preparation with a cover glass.
Important: Sample from different areas in and on the specimen or preferably mix the faeces before sampling to distribute evenly any parasites in the specimen. Do not use too much specimen otherwise the preparations will be too thick, making it difficult to detect and identify parasites.
3 Examine systematically the entire saline preparation for larvae, ciliates, helminth eggs, cysts, and oocysts. Use the 10 objective with the condenser iris closed sufficiently to give good contrast. Use the 40 objective to assist in the detection and identification of eggs, cysts, and oocysts.Always examine several microscope fields with this objective before reporting ‘No parasites found’.
4 Use the iodine preparation to assist in the identification of cysts.
5 Report the number of larvae and each species of egg found in the entire saline preparation as follows:
Scanty . . . . . . . . . . . . . . . . 1–3 per preparation
Few . . . . . . . . . . . . . . . . . 4–10 per preparation
Moderate number . . . . 11–20 per preparation
Many . . . . . . . . . . . . . . . 21–40 per preparation
Very many . . . . . . . . . over 40 per preparation

Identification of larvae: In a fresh faecal specimen, S. stercoralis is the only larva that will be found. It can be easily detected in a saline preparation by its motility and large size. If the specimen is not fresh, S. stercoralis will require
differentiation from hookworm larvae
.

Identification of helminth eggs: Eggs are recognized by their:
– size,
– colour (colourless, pale yellow, brown),
– morphological features.
Note: Some helminths also have a limited geographical distribution.



Identification of intestinal flagellates: The trophozoite of G. lamblia and its differentiation from non-pathogenic flagellates that can be found in faeces
.

Identification of ciliates: B. coli is the only ciliate found in human faeces (rare infection). 

Identification of cysts and oocysts: In saline and eosin preparations, protozoan cysts and oocysts can be recognized as refractile bodies (shine brightly when focused). Cysts can be identified by their shape, size, nuclei, and inclusions as seen in an iodine preparation Iodine stains nuclei and glycogen but not chromatoid bodies. Burrows stain or Sargeaunt’s stain
can be used to stain chromatoid bodies
. The cysts of E. histolytica/E. dispar and those of G. lamblia and their differentiation from non-pathogenic or other species.

Faecal leukocytes: The presence of polymorphonuclear neutrophils (pus cells) in faeces is mainly associated with inflammatory diarrhoea caused by bacteria.

Non-parasitic structures found in faeces: Care must be taken not to report as parasites those structures that can be normally found in faeces such as muscle fibres, vegetable fibres, starch cells (stain blue-black with iodine), pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs. Large numbers of fat globules may be seen in faeces when there is
malabsorption. Charcot Leyden crystals (breakdown products of eosinophils) can sometimes be seen in faeces (also in sputum) in parasitic infections. They appear as slender crystals with pointed ends, about 30–40 m in length.

Surprising research result: All immature cells can develop into stem cells

A new study conducted at the University of Copenhagen challenges traditional knowledge of stem cell development. The study reveals that the destiny of intestinal cells is not predetermined, but instead determined by the cells' surroundings. The findings may make it easier to manipulate stem cells for stem cell therapy. Results have just been published in Nature.
All cells in the fetal gut have the potential to develop into stem cells, a new study conducted at the Faculty of Health and Medical Sciences at the University of Copenhagen concludes. The researchers behind the study have discovered that the development of immature intestinal cells -- contrary to previous assumptions -- is not predetermined, but affected by the cells' immediate surroundings in the intestines. This discovery may ease the path to effective stem cell therapy, says Associate Professor Kim Jensen from the Biotech Research & Innovation Centre (BRIC) and the Novo Nordisk Foundation Center for Stem Cell Biology (DanStem).
"We used to believe that a cell's potential for becoming a stem cell was predetermined, but our new results show that all immature cells have the same probability for becoming stem cells in the fully developed organ. In principle, it is simply a matter of being in the right place at the right time. Here signals from the cells' surroundings determine their fate. If we are able to identify the signals that are necessary for the immature cell to develop into a stem cell, it will be easier for us to manipulate cells in the wanted direction."
Throughout life the organs in the body are maintained by stem cells, which are also able to repair minor tissue damage. A better understanding of the factors that determine whether or not an immature cell develops into a stem cell may therefore be useful in the development of stem cells for therapy and transplantation.
'We have gained greater insight into the mechanisms through which cells in the immature intestines develop into stem cells. Hopefully we are able to use this knowledge to improve treatment of non-healing wounds, e.g. in the intestines. So far, though, all we can say for sure is that cells in the gastrointestinal tract have these characteristics. However, we do believe this is a general phenomenon in fetal organ development'.
Luminescent Cells and Mathematical Collaboration
The surprising findings are the result of a search for understanding of what controls the destiny of intestinal stem cells. Postdoc Jordi Guiu developed a method for monitoring the development of the individual intestinal cells. By introducing luminescent proteins into the cells he could, using advanced microscopy, monitor the development of the individual cells.
After the initial tests, the cells that researchers previously believed to be fetal stem cells were only able to explain a fraction of the growth of the intestines during fetal development. Therefore, they established a collaboration with mathematical experts at the University of Cambridge. And when they studied the data more closely together, they arrived at the surprising hypothesis that all intestinal cells may have the same chance of becoming stem cells. Subsequent tests were able to prove the hypothesis.
"The next step is to determine precisely which signals are necessary for immature cells to develop into the kind of stem cells we need. This is one of our research focuses," says Kim Jensen.
Stem cells and stem cell therapy facts
Throughout life stem cells help maintain the organs in the body and repair damaged tissue. However, the stem cells found in the body can only renew and repair minor tissue damage.
Using stem cell transplantation and therapy it is possible to supplement the body's own cells with new, healthy stem cells that can help repair or replace damaged tissue.
The project was funded by the European Research Council, the Horizon 2020 research programme, the Lundbeck Foundation, the Novo Nordisk Foundation, the Carlsberg Foundation and the Marie Curie fellowship programme.

Darwinian cancer drug project

A new drug discovery programme with a “Darwinian” approach to treating cancer will be opened at the Institute of Cancer Research in London.
The new Centre for Cancer Drug Discovery will use “evolutionary herding” to tackle cancer’s ability to evolve resistance to treatment.
Professor Paul Workman, Chief Executive of The Institute of Cancer Research, London, said: “Cancer’s ability to adapt, evolve and become drug resistant is the cause of the vast majority of deaths from the disease and the biggest challenge we face in overcoming it.”
Dr Andrea Sottoriva, Deputy Director of Cancer Evolution at the new centre, said: “By encouraging cancer to evolve resistance to a treatment of our choice, we can cause it to develop weaknesses against other drugs – and hopefully send it down dead ends and to its own destruction.”
Some ICR scientists believe powerful chemotherapy treatments fail because they help fuel competition and evolution of cancer cells that are able to withstand chemotherapy – which it calls “survival of the nastiest”.
Using AI and mathematical predictive methods, the centre’s approach will force cancer DNA to adapt to one treatment by developing weaknesses against others, making them highly susceptible to a second drug or pushing them to an evolutionary dead end.
The new centre will work to create the world’s first anti-evolution cancer drug to slow down the disease’s ability to evolve and so delay its resistance to treatment. It will also devise multi-drug combinations that block several different cancer genes at once or boost the immune system – as used to achieve long-term control for HIV and tuberculosis.
The Institute of Cancer Research will invest £75 million in creating a global centre of expertise in anti-evolution therapies. ICR is seeking a further £15 million in donations to complete and equip the building.

Wednesday, May 15, 2019

tissue processing part 2

continuation of part two

read tissue processing part one here 

Tissue Processing : Factors, Steps Of Tissue Processing, Types -part 1

3.  Embedding

  • It is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning.

Image result for tissue processingEmbedding

Properties of embedding media

Ideally an infiltrating and embedding medium should be

  • soluble in processing fluids
  • suitable for sectioning and ribboning
  • molten between 30°C and 60°C
  • translucent or transparent; colorless
  • Stable
  • Homogeneous
  • capable of flattening after ribboning
  • non-toxic
  • Odorless
  • easy to handle
  • inexpensive

Paraffin wax

  • most popular embedding medium in histopathology
  • It is a mixture of long chained hydrocarbons produced in the cracking of mineral oil.
  • Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled.
  • It has a wide range of melting points ranging from 47 to 64°C which signifies its use in the different climatic
  • Heating the paraffin wax to a high temperature alters the properties of the wax.
  • It is inexpensive and provides quality sections
  • Compatible with most routine and special stains

Paraffin wax additives

  • These additives helps to increase hardness
  • Substances added to paraffin wax include beeswax, rubber, ceresin, plastic polymers and diethylene glycol distearate.
  • Many of these additives had a higher melting point than paraffin wax and make the tissue more brittle.

MODIFIED PARAFFIN WAXES

  • The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:
  • improve ribboning: prolong heating of paraffin wax at high temperatures or use micro-crystalline wax
  • stearic acid :  increase hardness
  • spermaceti or phenanthrene : decrease melting point
  • 5% ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene : improve adhesion between specimen and wax (alter crystalline morphology)
  • Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax
  • Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity
  • Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

Image result for paraffin blocks


Paraffin Blocks

Alternative embedding media

Resin

  • Resin is used exclusively as the embedding medium for electron microscopy, ultra-thin sectioning for high resolution and also for undecalcified bone.

Agar

  • Agar alone does not provide sufficient support for sectioning tissues.
  • Its main use is as a cohesive agent for small friable pieces of tissue after fixation (double embedding)
  • Fragments of tissue are embedded in melted agar, allowed to solidify and trimmed for routine processing.

Gelatin

  • Gelatin is primarily used in the production of sections of whole organs using the Gough-Wentworth technique and in frozen sectioning.
  • It is rarely used.

Celloidin

  • The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements needed to house the processing reagents and the limited use these types of sections have in neuropathology.
  • It is rarely used.

Double embedding

  • It is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded.

Embedding tissues in paraffin wax

Requirements for embedding are as follows:
  • a supply of clean, filtered paraffin wax held at 2-4°C above its melting point.
  • a paraffin dispenser
  • a cold plate to rapidly cool the wax.
  • a supply of moulds in which to embed the tissues.
  • Paraffin wax is dispense automatically from a nozzle into a suitably sized mold.
  • The tissue is oriented in the mold, a cassette is attached.
  • The mold is placed on a small cooling area to allow the paraffin
Overnight processing schedule
Image result for histokinette

Reagents

Time

Temperature 

10% Formalin1 h38°C
10% Formalin1 h38°C
50% Alcohol/formalin1 h38°C
70% Alcohol1 h38°C
95% Alcohol1 h38°C
95% Alcohol40 min38°C
100% Alcohol1 h38°C
100% Alcohol40 min38°C
Xylene1 h38°C
Xylene30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C

Microtome cutting

The tissue block is attached to microtome and ribbons are cut.

Image result for microtomeMicrotome

Equipment required for Paraffin section cutting

  • Flotation (water) bath
  • Slide drying oven or hot plate
  • Fine pointed or curved forceps
  • Sable or camel haired brush
  • Scalpel
  • Slide rack
  • Clean slides
  • Chemical-resistant pencil or pen

Water Bath

  • water bath is used for floating out tissue ribbons after sectioning.
  • The trailing end of the ribbon making contact with the water first
  • The temperature of the water in the bath should be 10°C below the melting point of the paraffin.
  • Alcohol or a small drop of detergent may be added to the water allowing the section to flatten out with greater ease
  • 30 seconds are long enough for a ribbon to flatten

Image result for water bathWater Bath

Slides

  • For normal routine work 76 × 25 mm slides are universally used.
  • Those with thickness of 1.0–1.2 mm are preferred

Section adhesives


  • Albumen
  • gelatin
  • Starch
  • Poly-L-lysine (PLL)
  • 3-aminopropyltriethoxysilane (APES)