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Category Archives: histology

Gram-Twort Special Stain For Bacteria – Method and Tips


The Gram-Twort variation of the standard Gram stain is the most common variant used in histopathology laboratories for the demonstration of Gram-positive and Gram-negative bacteria in formalin-fixed sections.

The standard Gram stain was developed and reported in 1884 by Hans Christian Gram (therefore Gram stain should always be spelt with a capital). He developed the technique to distinguish a certain group of bacteria in lung tissue, but noted that Typhus Bacillus was not visualised. The standard Gram stain works on the premise that the peptidoglycan rich cell wall of Gram-positive bacteria is stained by the crystal violet and the Gram-negative bacteria take up the counterstain.

Below is the author’s method of choice.

Solutions

– Crystal Violet – 0.5% crystal violet in 25% alcohol

– Gram’s iodine – 1g iodine + 2g potassium iodide. Dissolve in a few mls of distilled water. Make up to 300ml with distilled water.

– Acetone

– Stock neutral red-fast green – 90ml of 0.2% neutral red in ethanol + 10 ml of 0.2% fast green in ethanol.

– 2% acetic acid in alcohol.

Method

1. Take sections to water.

2. Stain with filtered crystal violet for 2 minutes.

3. Wash well in tap water.

4. Treat sections with Gram’s iodine for 2 minutes.

5. Wash well in tap water.

6. Rinse sections with acetone until colour stops leeching from them (approx 5 seconds).

7. Counterstain with filtered neutral red-fast green (dilute stock 1 in 4 with distilled water) for 5 minutes.

8. Wash well in tap water.

9. Differentiate in 2% acetic acid in alcohol until red ceases to run from section (approx 5-10 seconds).

10. Rinse in alcohol.

11. Clear and mount in DPX-type mountant.

Tips

– Always run a positive control.

– If the Gram’s iodine step is missed it takes longer to differentiate and background staining is increased.

– If having problems with differentiation try completely drying section before differentiating. Blot dry and leave for 10 minutes to ensure complete dryness.

Thanks for reading and I welcome any comment.

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Pigmented Spindle Cell Naevus of Reed: A Controversial Diagnostic Entity In Australia


Interesting article in the May 2011 edition of the Australasian Journal of Dermatology by Scott Webber, Greg Siller and Peter Soyer entitled ‘Pigmented Spindle Cell Naevus of Reed: A Controversial Diagnostic Entity In Australia.’

Points of note include

– Pigmented spindle cell naevus of Reed (PSCN) is a recognized distinctly separate entity away from Spitz naevus.

– The clinical and dermatoscopic features of Spitz naevi and melanoma overlap with PSCN.

– Clinical diagnosing of PSCN by dermatology training groups remains difficult.

– Well defined dermatoscopic and physical criteria for lesional morphology and histopathological characteristics are available and have increased the accuracy in distinguishing PSCN from Spitz naevi and melanoma.

– An accurate diagnosis is best gained from clinicopathological correlation.

– PSCN typically occurs in young women, only 25% of cases in patients > 30 years old.

– Punch biopsy as initial management is not recommended as misdiagnosis as melanoma is more likely.

I recommend reading the entire article. Below is a link.

http://onlinelibrary.wiley.com/doi/10.1111/j.1440-0960.2011.00743.x/abstract

Thanks for reading and I welcome any comments

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Role of Genetics and Sex Steroid Hormones in Male Androgenetic Alopecia and Female Pattern Hair Loss: An Update of What We Know


An interesting review article in the May 2011 edition of ‘The Australasian Journal of Dermatology’ entitled ‘Role of Genetics and Sex Steroid Hormones in Male Androgenetic Alopecia and Female Pattern Hair Loss: An Update of What We Know’ authored by Leona Yip, Nick Rufaut and Rod Sinclair.

Points of note include:

– Male androgenetic alopecia (MAGA) and female pattern hair loss (FPHL) are identical in histological terms but are separate clinical entities. Differences include rates of prevalence, hair loss pattern and peak ages.

– MAGA spares the occipital area of the scalp even in severe cases.

– In FPHL complete areas of baldness are rare.

– The androgen dependency of MAGA is not reflected in FPHL.

– MAGA and FPHL both result in hair follicle miniaturisation and hair follicle cycling alteration.

– In MAGA and FPHL the growth (anagen) phase is shortened dramatically and the telogen phase remains the same or is lengthened. The telogen follicle proportion also goes up from 5-10% to 15-20%.

– Dihydrotestostetone (DHT) is thought to be the key androgen in the induction of MAGA.

I recommend reading the full article if it is available to you. Below is a link.

http://onlinelibrary.wiley.com/doi/10.1111/j.1440-0960.2011.00745.x/abstract

Thanks for reading and I welcome any comments

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Grocott Hexamine-Silver Special Stain For Fungus – Method and Tips


The Grocott Hexamine-Silver special stain is the method of choice for a large majority of histopathology laboratories for the demonstration of all fungi. The formalin-fixed sections are exposed to chromic acid which reacts with fungal cell wall polysaccharide components to form chromic acid-aldehydes. These then reduced by a hexamine-silver solution at an alkaline pH. This causes them to be selectively blackened.  It should be noted that this method is not specific for fungi but rarely fails to demonstrate any fungi within the test tissue.

Below is the author’s method of choice.

Solutions

5% aqueous chromic acid (chromium trioxide)

1% aqueous sodium metabisulphite

Stock hexamine-silver solution = 100ml 3% aqueous hexamine + 5% aqueous silver nitrate.

Working hexamine-silver solution = 2ml 5% aqueous sodium tetraborate (borax) + 25ml distilled water. Mix, then add 25ml stock hexamine-silver solution.

0.1% aqueous gold chloride

5% sodium thiosulphate

0.2% light green in 0.2% acetic acid

Method

1. Take sections to water.

2. Treat sections with chromic acid for 1 hour.

3. Wash thoroughly in tap water.

4. Treat with sodium metabisulphite solution for 1 minute.

5. Wash well in tap water.

6. Wash well in several changes of distilled water.

7. Treat sections with working hexamine solution (preheated in coplin jar at 56 degrees Celsius) at 56 degrees Celsius for 10-20 minutes. Check control sections to see if fungi are a dark brown colour, if not return to solution checking regularly at 3 minute intervals until correct colour achieved.

8. Wash in several changes of distilled water.

9. Treat sections with 0.1% aqueous gold chloride for 3 minutes.

10. Wash well in distilled water.

11. Treat sections with 5% sodium thiosulphate for 5 minutes.

12. Wash well with tap water.

13. Counterstain with 0.2% light green in 0.2% acetic acid for 1 minute.

14. Wash well in tap water.

15. Dehydrate, clear and mount.

 

Tips

– As with all other silver stains wash everything that you are going to use thoroughly with distilled water.

– Store the stock hexamine-silver solution at 4 degrees Celsius away from sunlight. It will keep for 1-2 months. If a white precipitate forms give it a good shake and it should redissolve.

– Do not extend the time in chromic acid too long as this can over oxidize the carbohydrates to carboxylic acid and therefore not take up the silver stain.

– Do not reduce the time in chromic acid as this will lead to under oxidation and therefore no take up of the silver stain.

– The chromic acid can be reused but its efficiency will decrease after each use.

– If the control sections are failing to stain even after extending the staining time in the heated hexamine-silver solution you have more than likely forgotten to add the borax. If so, you can just add it and continue with the stain.

– If you have a large amount of silver precipitate over your sections it is probably due to using low-grade silver.

– If you forget the sodium thiosulphate step you will not realise until after retrieving the slide from storage further in the future, as the remaining silver not removed will react with sunlight turning black.

– Try to keep your counterstain fairly light as dark counterstaining can mask fungal elements.

 

Thanks for reading and I welcome any comments

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Perls’ Technique For The Demonstration of Haemosiderin – Method and Tips


Iron is absorbed in the duodenum by cells called enterocytes. It is then stored or combined with a transport protein molecule. This iron-protein complex is then taken to the bone marrow where the iron is incorporated into the substance known as haemoglobin which is involved in oxygen transportation.

Iron can be stored in the bone marrow and spleen in its ferric state (Fe3+) as haemosiderin when combined with protein. When haemoglobin is broken down by tissues this results in the formation of haemosiderin.

When there is excess iron in the body haemosiderin can be found deposited in organs that are involved with iron storage such as the spleen, bone marrow and liver. This condition is known as haemosiderosis.

A condition called haemochromatosis exists where the body indiscriminately absorbs iron resulting in the deposition of copious amounts of haemosiderin in many tissues.

Haemosiderin founds in histology sections is usually derived from the breakdown of damaged erythrocytes and can also be found absorbed by macrophages (siderophages).

The method used by the wide majority of histology laboratories for the demonstration of haemosiderin is the Perls’ technique. This method works by the hydrochloric acid (HCL) splitting off the bound protein which then allows the potassium ferrocyanide to bind with the Fe3+ and form ferric ferrocyanide (Prussian blue).

Below is the author’s favoured method.

SOLUTIONS

2% hydrochloric acid (HCL)

2% aqueous potassium ferrocyanide

1% aqueous neutral red

METHOD

1. Take sections to water

2. Mix equals parts of HCL and potassium ferrocyanide and filter onto sections. Leave for 15 minutes.

3. Wash for 5 minutes in running tap water.

4. Counterstain with neutral red for 1 minute.

5. Dehydrate, clear and mount.

TIPS

– Always include a positive control.

– Stronger staining results can be found by carrying out step 2 at higher temperatures (e.g. 37-56°C). This can result in false positive results. This author has found room temperature to suffice.

– The washing step (step 3) should not be decreased below 5 minutes as thorough washing is required to prevent a heavy dye precipitate resulting from the neutral red counterstain.

– The author has found neutral red to be the best counterstain. Do not use safranin as this can stain the Prussian blue granules a dark purple colour.

– Always mount in a DPX-type mountant as other mounting media results in fading of the stain.

– A common artefact is the presence of blue granules on and around the section. This can be due to expired HCL or potassium ferrocyanide. It can also be due to iron contaminants in the tap water. This can be fixed by replacing all steps from the cutting of the sections to the mounting of the stained slide that involve tap water with distilled water.

Thanks for reading and I welcome any comments.

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Modified Ziehl–Neelsen Stain For Leprosy Bacilli – Method and Tips


Leprosy bacilli in comparison with tubercle bacilli are much less acid- and alcohol-fast. The leprosy bacilli’s lipid envelope is also much more affected by the fat solvents traditionally used to dewax sections (i.e. Xylene). Due to these factors a modification on the standard Ziehl-Neelsen technique is used for the demonstration of leprosy bacilli.

Below is the author’s preferred technique.

SOLUTIONS

Dewaxing solution – equal parts of liquid paraffin and rectified turpentine

Carbol Fuchsin – as per standard Ziehl-Neelsen technique

Methylene blue counterstain – as per standard Ziehl-Neelsen technique

10% sulphuric acid

METHOD

1. Dewax in ‘dewaxing solution’ described above for 30 minutes.

2. Blot dry and wash in running water for approximately 10 minutes.

3. Stain with filtered Carbol Fuchsin for 30 minutes at room temperature.

4. Wash well in tap water.

5. Differentiate in 10% sulphuric acid until section is pale pink.

6. Wash well in tap water.

7. Counterstain with Methylene Blue for 15 seconds.

8. Wash well in tap water.

9. Blot dry, clear and mount.

TIPS

– This author always puts sections on ‘sticky’ slides to prevent any floating off.

– There are many variations on the ‘softer dewaxing solution’ for the modified Ziehl-Neelsen technique for leprosy bacilli including:

– Two parts xylene to one part vegetable oil / clove oil / groundnut oil / olive oil / cottonseed oil.

– Residual oil on the section after washing prevents shrinkage of the section.

– Place slides directly from heater into dewaxing solution as this helps quicken the dewaxing

– Some methods use a weaker acid-alcohol solution for differentiation, but this author prefers 10% sulphuric acid as it is quicker.

– Don’t be alarmed when the section is placed into the sulphuric acid as it will turn a black colour. It will return to a pink colour when placed back in water.

– Ensure the counterstain colour isn’t too intense as this can mask some leprosy bacilli and even turn them a purple colour.

– This is author lets the sections dry after washing after counterstaining and then directly mounts them.

I welcome any other tips and comments.

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Lack of UV-A Protection In Daily Moisturising Creams


Came across another interesting article in the May edition of ‘The Archives of Dermatology’ 2011. The article is entitled ‘Lack of UV-A Protection In Daily Moisturising Creams’ on page 618.

Ultraviolet radiation (UV) contains UVA, UVB and UVC subtypes. The major source of UV exposure for humans is sunlight. The earths ozone layer blocks approximately 98% of all UV radiation and the 2% which reaches the earths surface 99% is of the UVA subtype. UVB can cause direct DNA damage whereas UVA causes indirect damage of DNA via the formation of free radicals. Therefore it is important any sunscreen solution contains both UVA and UVB filters.

This article reported the estimated long-range UVA1 protection of 29 creams.

Major points of note from the article include

– most daily facial creams contain ingredients known as UV filters claiming broad spectrum UV protection

– Sun protection factor (SPF) doesn’t reflect UV-A1 protection

– UVA penetrates window glass whereas UVB is blocked, therefore women working indoors need to protect themselves from UVA exposure

– of the 29 creams 6 didn’t contain any UVA1 filters

I recommend reading the full article as it is an interesting read. Below is a link to the article.

Lack of UV-A Protection in Daily Moisturizing Creams
Steven Q. Wang; Jacqueline M. Goulart; Henry W. Lim
Arch Dermatol. 2011;147(5):618-620

Thanks for reading and I welcome any comments.

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