Validating Your New Stainer According to CAP and CLIA Requirements

Validating Your New Stainer According to CAP and CLIA Requirements

Congratulations on purchasing a new autostainer! There are three parts to the start-up process:  Instrument Verification, Stain Protocol Optimization, and Validation of the Staining Protocols.

Instrument Verification see CAP All Common Checklist (06/04/2020) COM.40350

Optimally, the company you purchase the instrument from should provide an operator manual; provide verbal procedural instructions; and test the stainer to verify its proper operation. You should also be provided signed documentation confirming that it operates as intended.

If you are in a large hospital system, the Clinical Engineering department will do a safety, operational, and functional inspection. Obtain a copy of the Clinical Engineering records showing their approval of the instrument and include these documents with the other papers you are putting together for the Verification and Validation.

Stain Protocol Optimization

Prior to staining patient tissue, you will need to test the staining protocol(s) to make certain that the stain quality that the new instrument is providing is acceptable for diagnosis. This needs to be done even if the staining protocol is the same one as used on the old instrument – they are different machines, and will operate differently, thus requiring an independent optimization.

To verify what an acceptable H&E stain looks like, look at the photos in reputable Histotechnology reference books such as Carson, Bancroft and Brown. To improve your knowledge, read the differences between reference-quality H&E staining versus poor quality staining. If you ever found yourself in a legal predicament you really don’t have the excuse to say, “well, this is what my pathologist likes”. Consider what could happen if your lab was sued based on the quality of your work and your employer had to go to court. The prosecuting attorneys would show text book quality photos, as established by ASCP, NSH and/or HistoQIP (proficiency standard for US laboratories) to support their case. In turn, your employer would need to show high quality photos as evidence to win its case.

With this ideal in mind, do some test runs with several different types of tissues. Use tissues that you process frequently, such as breast, skin, GI biopsy, and placenta. Use tissues that are sensitive to the stain expressions of the eosin and hematoxylin chemicals, such as small and large intestine.

To assess the staining quality of eosin you may want to use epithelial, muscle and lymphoid tissue to better visualize nuclei. Use muscle, collagen, epithelial cells, and mucin cells to better visualize connective tissue. Use eosinophils and plasma cells to better visualize various WBCs. 

To document your results, you need to design an H&E Stain Optimization Worksheet. Start with a grid that lists all the elements of the protocol that is being used on your H&E stainer.

Document the following details on the H&E Stain Optimization worksheet:

  • Name of solution/reagent in each container
  • Time that slides spend in each container
  • Temperature, if appropriate, e.g., running water
  • pH of appropriate solutions, e.g., water, hematoxylin, eosin, acid rinse
  • Date and test run number
  • Signature of the Tech that oversaw the run
  • Signature of the Pathologist that assessed the results

Next, have a section on the optimization worksheet where you record your results. Some example questions might be:

Overall Stain Quality (observed using a 10x objective):

  • Is the staining even?Are the nuclei standing out darker than the background?
  • Is there an absence of splotches, e.g., water droplets?

Hematoxylin Staining (observed using a 40x objective):

  • Is the nuclear wall dark and crisp?
  • Is the chromatin pattern stippled, not smudgy?
  • Is the nucleolus, if present, a red to purple color?
  • Are other cells (plasma cells and pancreatic acinar cells) which are expected to stain bluish, doing so?
  • Are the mucin cells clear of color (often seen as a pale blue color if you are using a Gill hematoxylin and/or are not doing a regressive stain)?
  • Are the muscle and connective tissue cells free from a bluish color?

Eosin Staining (observed using a 40x objective):

  • Are RBCs the darkest red?
  • Are eosinophil granules, Paneth cell granules, and zymogen granules as dark, or nearly as dark, as RBCs. Note, if you are using a fixative with acetic acid, these organelles will be lysed and this question does not apply.
  • Is muscle tissue a medium shade of pink, and is collagen a light shade of pink?
  • Can muscle be differentiated from collagen? Observing medium size blood vessels should show this differentiation.

If the results are not satisfactory, follow up to find out what is causing the problem. Then make changes to the H&E protocol, run another rack of test slides, and then record the results. Continue doing this until you finally get good quality H&E staining. This is now your optimized H&E Stain Protocol.

Now, you are required to do a control run every day, which can tell you when you need to rotate or change solutions/reagents. Or, you may want to do a control run every 200 or 400 slides.

Validation of the Staining Protocol

Before the stainer is used for any patient slides, you should validate the staining program(s).

According to the new CAP standards which were published in June 2020, Validation of the staining protocols is no longer required! COM.40350 – see NOTE 8: This checklist requirement (validation) does not apply to LDTs that employ the following methods: Manual microscopy (eg, histopathologic and cytologic interpretation, microscopic examination of blood or body fluids, Gram stains)”

HOWEVER, all US labs are licensed by CLIA, and CLIA does require stain protocol validation. You could still be inspected by a CLIA inspector even if you are CAP accredited, and if you did not do validations you would be cited.

Each different H&E program must be separately validated. This means that if you use one program with more delicate staining for your biopsies, and a different program for routine surgical specimens, both staining programs must be separately validated. To do this, stain 20 different slides of differing common tissue types according to your optimized protocol. Twenty is the commonly accepted number of test runs for most laboratory validations.

Design your Staining Protocol Validation Worksheet with the following details:

  • A header that includes the make, model, and serial number of the instrument.
  • The body to record the following information:
  • Accession/ID number for the 20 slides
  • Tissue type
  • Review approval/non-approval
  • Comments
  • A footer with the following information:
  • A statement which says: “This protocol has been validated and is approved for patient use”.
  • A signature sign-off for the Medical Director and the date
  • The lab name and address

You are required to keep the records of the Instrument Verification and the Staining Protocol Validations for the years you own the instrument plus two years. Optimization records are not required to be archived.

Re-validation of the instrument is required if:

  • The staining protocols are changed
  • The solutions/reagents are changed
  • The instrument is moved to a different location, within or outside of your lab
  • The instrument has had any major repairs


  1. Peggy A. Wenk, BA, BS, HTL(ASCP)SLS, Former Program Director, Beaumont School of Histotechnology
  2. Beth A. Cox, HTL/SCT(ASCP)QIHC, 11/09/2020
  3. Robert G. Rankin, MSM, SM(ASCP), 11/09/2020
  4. CAP All Common Checklist COM.40350, 06.04.2020

The H&E Stain: Far From Routine Part 2

This blog is a followup to the previous article “The H&E Stain: Far From Routine”.  In that article, the basics of the routine hematoxylin and eosin (H&E) stain were discussed.  Now we shall discuss how to trouble shoot the routine H&E and how to ensure a high quality stain, once you have worked with your pathologist to determine the best stain result for your laboratory.


A standard H&E staining protocol is provided below.  It applies to either a manual or automated staining procedure.  While it may not be ideal for your laboratory, it can be used as a starting point.  The final color rendition of your H&E stain should be determined by working with your pathologists.  This will consequently make their job easier.  Each day, once the H&E stain set up is completed, you should run down one test slide to confirm that the staining is optimal.  This also will help you to document quality control procedures.


Hematoxylin and Eosin (H&E) Stain


PRINCIPLE: Hematoxylin stains nuclear material a dark blue, while the eosin stains cytoplasm and connective tissue varying shades of pink.



  1. Harris’ hematoxylin
  2. Alcoholic Eosin
  3. Clarifier (i.e. acid alcohol)
  4. Bluing reagent (i.e. ammonium hydroxide)



  1. Cut paraffin sections 4-5 microns.  Bake at 60 C for 45 minutes; allow to cool.
xylene 2 x 5 min Removes paraffin; this is a minimum time
100% alcohol 2 x 2 min Removes xylene
95% alcohol 1 x 2 min Begins hydration
Running water 2 min Hydrates the sections
Harris’ Hematoxylin 5 min Stains nuclei and tissue
Running water 2 min Removes excess hematoxylin
Clarifier 1 min Removes hematoxylin from tissue
Running water 2 min Stops clarifier action
Bluing reagent 1 min Changes hematoxylin from red to blue with positive ions
Running water 2 min Stops bluing action
95% alcohol 1 min Readies for eosin
Eosin 1 min Stains cytoplasm pink
95% alcohol 15 seconds Differentiates eosin into 3 shades
100% alcohol 3 x 2 min Dehydrates
Xylene 2 x 5 min Readies for coverslip

Each step in the H&E stain procedure is essential, and if not managed correctly, any of them can cause suboptimal slides.  We shall begin discussion here and continue through the next blog, identifying potential problems and solutions along the way.


  1. Deparaffinization and dehydration. Many H&E and special stain protocols begin with step #1 as simply “Deparaffinize and hydrate to water”. In fact, nothing could be more complicated.  The sections to be stained are surrounded by a matrix of paraffin wax, which is insoluble in water.  All of the paraffin must be removed to ensure optimal staining.  Two changes of xylene (or xylene substitute) for 5 minutes each may accomplish this in a low volume laboratory; however a high volume laboratory may need to use three changes of xylene / xylene substitute to completely remove all paraffin.  In automated H&E stainers, it is important to adjust the height of xylene (and all reagent) containers to ensure that the sections next to the label end of the slide are being deparaffinized sufficiently.


  1. Harris’ hematoxylin is recommended for histology. Gill’s hematoxylin is generally used for cytology, and as a counterstain in many special stains. Harris’ hematoxylin continues to oxidize as it sits in the original container, and in the stain containers; hence, it should be filtered through Whatman #1 filter paper prior to use for the days’ staining runs.  This will prevent precipitate from adhering to the microscope slides.


  1. Clarifier. Harris’ hematoxylin is a regressive stain.  A clarifier solution must be used to remove excess hematoxylin staining from both the nuclei and other tissue elements.  The original formulation  is 1% hydrochloric acid in 70% ethanol, for “a few quick dips”.  However, with the current use of automated stainers, “a few quick dips” needs to be quantified into seconds or minutes. This usually requires a dilution of the original clarifier solution, with some experiments to determine the exact time.  The recommended starting point for clarifier on an automated stainer is to use the following for 30 seconds:


100% ethanol                            700 ml

tap water                                  2600 ml

hydrochloric acid (concentrated)  2.0 ml


Once made, the resulting intensity of the nuclear stain can be adjusted by changing the time of the clarifier incubation.  Alternatively, one may adjust the amount of acid added to the alcohol (i.e. decrease from 2.0 ml to 1.5 ml).  These adjustments will allow you to obtain a final stain with the required nuclear staining intensity.


  1. Bluing reagent. The recommended starting point for bluing reagent is:


Tap water                                                    3500 ml

Ammonium hydroxide (concentrated)               1.5 ml


As with the clarifier solution, results can be obtained by adjusting the time of incubation and/or concentration of the solution.  Alternatively, one can use lithium carbonate as a bluing reagent, or simply use a running water step to “blue” the nuclei.


  1. Eosin.  Working eosin solution is the most stable reagent of the H&E stain.  Hundreds of slides can be stained with the same batch of eosin on board an automated stainer.  The incubation time is usually kept short, as eosin penetrates and stains rapidly and reproducibly (i.e. 1-2 minutes).  The variable step in eosin staining is the subsequent 95% alcohol incubation, which produces the final “three shades of pink” within the tissue sections.  The number of 95% stations (i.e. one or two), and the incubation times (i.e. 30 seconds to 2 minutes) will determine the final quality of eosin staining.  Finally, it is important that the final 100% alcohol stations after the eosin staining remain uncontaminated with water.  Any water left in the sections after the coverslip is applied may cause the eosin to “bleed out” of the section.


In summary, the information contained herein will hopefully help you to manage and maintain consistent high quality of the H&E slides produced in your laboratory.



  1. Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY.  Fourth edition. 1996
  1. Theory and Practice of Histotechnology.  DC Sheehan, BB Hrapchak.  CV Mosby Company, St. Louis. First edition. 1980.
  2. Luna L.  AFIP. Manual of Histologic Staining Methods.  Third Edition. McGraw-Hill.

p39. 1968.  As modified by CM Chapman

  1. Dermatopathology Laboratory Techniques.  CM Chapman, I Dimenstein.  In press.
  2. The H&E Stain: Far from Routine.  CM Chapman.  Advance for Laboratory Professionals.  April 8, 2002.  Vol. 14 No.8

The H&E Stain: Far From Routine Part 1

[Editor’s note:  Segments of following blog are taken directly from an original article “The H&E Stain: Far from Routine” published by the author in Advance for Medical Professionals in April 2002.]


What exactly is a routine “H&E”?  And what makes it routine?  The first question is easy.  “H” stands for ”hematoxylin” and “E” stands for “eosin”.  Both are dyes used to stain tissue sections in histology.  However, the procedure for correctly applying this combination of stains to tissue sections is far from routine.

Since both dyes are water soluble, the first step is to completely remove the paraffin that is present in the tissue sections from the microscope slides.  Soaking in xylene, or a xylene substitute, followed by 100% and 95% alcohol, allow the slides to then be immersed in running water, which hydrates the sections.

The next step is to place the slides in a solution of hematoxylin, a natural dye obtained from the logwood tree Haematoxylon campechianum that will stain the nuclei of cells blue/black.  The staining is enhanced by the addition of an aluminum, iron or lead salt, which acts as a mordant for the hematoxyin to bind to the tissue sites.  This type of hematoxylin is referred to as “Harris” type, and is a “regressive’ stain.  That is: the tissues are overstained with the hematoxylin, which stains all tissue elements, and then differentiated with an acid alcohol solution (i.e. “clarifier”) to remove excess hematoxylin, leaving only the nuclei of the cells stained.

The composition of the clarifier varies.  The original procedure written by Lee Luna in the Armed Forces Institute of Pathology (AFIP) Manual specifies differentiation of the Harris hematoxylin solution with a “1% acid alcohol” solution for a time duration of “a few quick dips”.  This solution is 1% hydrochloric acid in 70% ethanol.  However, with the current use of automated stainers, “a few quick dips” needs to be quantified into seconds or minutes. This usually requires a dilution of the original clarifier solution, with some experiments to determine the exact time.

Harris’ hematoxylin can be compared to “Gill’s” or “Mayer’s” hematoxylin, which are used “progressively”.  The longer the sections are left in these solutions, the darker the nuclei stain.

In either case, the resulting hematein-mordant stains nuclei a reddish color.  Subsequent treatment in a weak basic aqueous solution, using either lithium carbonate or ammonium hydroxide, changes the dye molecules from red to blue; hence the term “bluing reagent”.  The resulting blue color is dependent upon the freshness, type and age of the mordant.  It is important that the pH of the bluing reagent is not too high, as section loss may occur during staining.  Additionally, warm running tap water may be used as the “bluing reagent”, as it contains positive ions.  The final blue color will be decided upon by the pathologist and histologist, who should agree what is optimal for the laboratory.

Eosin, the second dye in the H&E, is used to demonstrate the general histology of the tissue architecture.  When used correctly, eosin should stain both cytoplasmic and tissue elements in three shades of pink.  There are several types of eosin dye available, however Eosin Y is widely used for routine staining, as it is soluble in both water and alcohol.  Usually a 0.5 % to 1.0 % solution of Eosin Y is made up in 80% alcohol, with a small amount of acetic acid added.  Additionally, phloxine may be added to provide a more intense reddish color.

After staining in eosin for one to three minutes, the final shades of pink are determined by the differentiation steps following this staining.  The differentiation can be carried out in running water, 95% or 100% alcohol.  In any case the slides must be dehydrated completely after differentiation in several changes of 100% alcohol and xylene prior to coverslipping.  Failure to do so may cause “bleeding” of the eosin dye in the final microscope slide and/or variable eosin staining.

Complete removal of paraffin is an essential first step in any H&E staining procedure.  If xylene substitutes are being used, it may be necessary to adjust and lengthen times to guarantee paraffin removal.  In addition, xylene substitutes do not tolerate traces of water, as xylene does.  Thus, it is important also in the dehydration steps prior to coverslipping that the slides move through fresh changes of alcohol and xylene substitute.  If any water remains in the sections during coverslipping, the final slides will appear cloudy.  Additionally, the eosin stain may bleed out of the sections, as mentioned above.

This article has provided the basics of the H&E “routine” stain.  Please keep an eye open for the next blog, which will provide additional information regarding how to maintain the quality of your H&E stain, once you have optimized it.



  1. Theory and Practice of Histological Techniques.  JD Bancroft, A Stevens ed.  Churchill   Livingstone, NY.  Fourth edition. 1996.
  2. Theory and Practice of Histotechnology.  DC Sheehan, BB Hrapchak.  CV Mosby Company, St.     Louis. First edition. 1980.
  3. Luna L.  AFIP. Manual of Histologic Staining Methods.  Third Edition. McGraw-Hill.

p39. 1968.  As modified by CM Chapman.

  1. Dermatopathology Laboratory Techniques.  CM Chapman, I Dimenstein.  In press.
  2. The H&E Stain: Far from Routine.  CM Chapman.  Advance for Laboratory Professionals.  April 8, 2002. Vol. 14 No.8.

Silver Stains

In the histology world, the mere mention of a “silver stain” may be the cause of panic and uncertainty with regard to the performance of the stain, and the quality of the final resulting microscope slide.  All other special stains, with few exceptions, are relatively easy and straightforward to perform; not so with silver stains.

Silver stains can be categorized into (a) stains to visualize substances, such as calcium, melanin and reticulin and (b) stains for microorganisms, such as fungi and spirochetes.  The goal of all silver stains is the same: to get metallic silver to precipitate out at the staining site, and then replace it with gold to provide the final, stabilized, black reaction product.

The specific procedures for these stains are outside of the scope of this blog.  However, the following stain procedure outline explains the steps used.

Oxidation enhances subsequent staining by the silver solution.  Oxidizers include phosphomolybdic acid, potassium permanganate and periodic acid.

Sensitization usually employs a metal salt to help bind silver from the silver solution.  The original sensitizer for Wilder’s silver technique and the Steiner and Steiner stain is 1% uranyl nitrate.  However, Margeson and Chapman pioneered the substitution of zinc formalin for the original radioactive uranyl nitrate solution, which is also a strong oxidizer.

Silver impregnation solution contains metallic silver in a solution.  The idea is to have the silver carrying solution composed such that the silver ions will move from the solution, bind to the tissue section and then precipitate out in metallic form.

The reduction step in the reaction involves providing electrons, in the form of substances such as hydroquinone and formaldehyde, to chemically make the silver ions precipitate out into visible metallic silver.  This allows the structures in question to be visualized in dark black staining.

Toning  of the sections in gold chloride is a chemical reaction whereby the metallic silver is replaced by metallic gold, which is very stable and maintains the black color product.

The use of sodium thiosulfate, or “hypo”, helps to remove any unbound silver remaining from the toning reaction.  This is followed by counterstaining, usually either with nuclear fast red or fast green, for a proper final color rendition.

Thus, with all of these simple, straight forward steps, what could possibly go wrong?  Let’s begin at the beginning and work it through.

Use acid clean glassware for all containers and Coplin jars.  Why?  This will prevent the presence of any unwanted ions on the glass surfaces (including the slides themselves) causing non- specific precipitation of the silver.  You will see this as a black or mirror precipitate on the inside of the Coplin jar, or on the surface of the microscope slide.  If this does happen, the unwanted silver can be removed (see procedure at the end of this article).

Use plastic forceps to handle all slides — not metal.  Metal present in metal forceps may cause the silver to precipitate out.  Back in the old days, before plastic forceps, we simply dipped the tines of metal forceps into paraffin, allowed it to cool, thereby preventing the metal from contacting any of the solutions.

Pay attention to the slides when they are in the silver solution – especially if the solution is a hot, heated solution.  The point at which the silver solution itself may precipitate out is a fine one.  The slides and staining solution should be monitored closely during these incubations.  Also, if you are monitoring the slide under a microscope, make sure to rinse the slide in distilled water before viewing, thereby preventing the foundation of black silver precipitate forming on the microscope stage or slide. Your coworkers especially will appreciate this.



  1. Sheehan and Hrapchak. Theory and Practice of Histotechnology. Second ed.  CV Mosby Co.  St. Louis. pp 181-183.  1980.
  2. Chapman CM. Dermatopathology: A Guide for the Histologist. Copyright 2003.
  3. Chapman CM and Dimenstein IB. Dermatopathology Laboratory Techniques. Copyright 2015.  In press.
  4. Margeson LM, Chapman CM: The use of zinc formalin as a sensitizer in silver stains for spirochetes. J Histotech, 19:135-138, 1996.
Stains for Microorganisms

Stains for Microorganisms

The staining of microorganisms in histology can be challenging. Filamentous fungi and associated conidia are more easily demonstrated as they are visible under light microscopy when stained with periodic acid Schiff’s (PAS) as in Figure 1. The diameter of fungi filaments is 5-10 microns, which is approximately the same as the diameter of a red blood cell, while their length may be hundreds of microns (Figure 2). Microorganisms are extremely small and are at the limit of resolution of the light microscope. Viruses are even smaller (Figure 3).

Bacteria exist in three different shapes. Cocci are round, bacilli appear as rods and spirochetes are corkscrew shaped. The first level of determination for bacteria is whether they are Gram positive or Gram negative. The original method of Gram’s technique published in 1884 is still used today. Tissue sections are first stained with a crystal violet solution, which stains all of the tissue section. This is followed by Gram’s iodine which locks the crystal violet stain into the cell wall of the bacteria. Subsequent decolorization with acetone removes the stain from everything – except the Gram “positive” bacteria, which remain a dark blue/purple color (Figure 4).

The second phase of the stain involves the application of fuchsin / neutral red solution which stains Gram “negative” bacteria pink/red (Figure 5). This can be the most variable step in the procedure and relies on the correct application and retention of the basic fuchsin within Gram negative bacteria.

There are two additional steps which may result in variable staining as well. The first is an over-decolorization with acetone, which may result in faint staining of Gram positive bacteria. The second is the use and timing of the final counterstain, which may be either picric acid/acetone or tartrazine. While picric acid/acetone results in a more reliable result, the picric acid is a dangerous chemical and requires specific disposal procedures. Tartrazine is safer, but can easily be leached out during the dehydration process prior to coverslipping. The histotechnician performing this stain must rely on experience and precise timing to produce consistent results for this stain.

Actinomycetes are a Gram positive, non acid fast, branching filamentous organism. However, care must be taken when performing the Gram positive stain sequence. It may be necessary to (a) increase the time in crystal violet solution and (b) decrease the time in the acetone decolorization step in order to demonstrate the organism.

A second determination of bacteria is based on an organisms “acid fastness”. The Kinyoun method for acid fast bacteria, the Fite method for leprosy bacilli and the Ziehl-Neelsen stain for tubercule bacilli are all based on the same stain theory. Tissue sections are first stained with a carbol fuchsin solution, which stains all tissue elements pink/red. Subsequent decolorization in an acid-alcohol solution removes stain from all the tissue elements, except acid fast bacteria. Methylene blue is usually used as a counterstain (Figure 7).

Spirochete bacteria are ubiquitous in nature. They are found everywhere in soil and water. The issue is that some are pathogenic to humans. TheTreponema pallidum spirochete is sexually transmitted and causes syphilis. The Borrelia burgdorferi spirochete is present in certain tick species, and may be transmitted to humans when the tick attaches to the skin to feed. The result is Lyme disease, which if left untreated, can be debilitating to the patient.

Silver stains were developed in the early 1900’s to stain spirochetes. The Dieterle (1927), Warthin-Starry (1920) and Steiner (1944) are still used in histology today to demonstrate spirochetes. Some of the methods have been modified to make them safer to use (Margeson and Chapman, 1996). The stain theory is the same in all methods: pre-treat the sections to make the spirochetes more readily able to pick up and bind the silver solution. The final result is to stain the spirochetes black, against a gray/ brown background (Figure 7). Those who have performed any of these stains are aware of the many staining procedure pitfalls, which can render the spirochetes either over or under stained. As a result, many laboratories currently use immunohistochemistry to stain spirochetes. While both methods can stain spirochetes, neither is able to demonstrate the exact species to which the stained organism belongs.

Both positive and negative control slides should be used when performing stains for microorganisms. The negative control slide ensures that there is no bacterial contamination present in source water or stain solutions.




  1. Theory and Practice of Histological Techniques. Chapter 10.       JD Bancroft, A Stevens ed.       Churchill Livingstone, NY.       Fourth edition. 1996
  2. Theory and Practice of Histotechnology. Chapter 9.   DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.
  3. Margeson LM, Chapman CM: The use of zinc formalin as a sensitizer in silver stains for spirochetes. J Histotech, 19:135-138, 1996.
Staining Fungi

Staining Fungi

Fungi include molds, yeasts and higher fungi. All fungi are eukaryotic and have sterols but not peptidoglycan in their cell membrane. Their cell walls are composed of cellulose; the same building blocks that plants use. Fungi may produce large, reproductive mycelium, called mushrooms, which may be edible, or poisonous. Other naturally occurring fungi may infect humans, one example being “athlete’s foot”.

Fungi are chemoheterotrophs which require organic nutrition and most are aerobic. Many fungi are also saprophytes which live off of dead organic matter, in soil and water and acquire their food by absorption. Fungi may produce sexual and asexual spores. There are over 100,000 species recognized, with over 100 of them known to be infectious agents in humans. Molds are composed of numerous, microscopic, branching hyphae known collectively as a mycelium.

Hyphae growth occurs from the apical tip, and apical vesicles contain materials and enzymes for the formation of new hyphal wall. Older hyphae are less biochemically active and contain many storage vacuoles. In most molds these hyphae have septa, which are cross-walled divisions, but in some there are none and the hyphae are aseptate. A septum is a cross-wall formation which divides one fungal hypha into two cells. These septa may add strength to the hyphae or serve to isolate adjacent parts to allow differentiation, such as during production of the reproductive structures.

Spores are formed from the reproductive mycelium. Asexual spores are produced by the aerial mycelium of a single organism, whereas sexual spores are formed by the fusion of cells and nuclei from opposite mating strains (Figures 1 and 2).

Fungal nail infections are common infections of the fingernails or toenails that can cause the nail to become discolored, thick, and more likely to crack and break. The technical name for a fungal nail infection is “onychomycosis.”

Fungal nail infections can be caused by many different types of fungi (yeasts or molds) that live in the environment. Small cracks in your nail or the surrounding skin can allow these germs to enter your nail and cause an infection. Onychomycosis can be diagnosed by microscopic examination or fungal culture of the nail clipping.

Your histology laboratory may receive skin and nail specimens to be evaluated for presence of fungi. The most common special stains used to visualize fungi are the periodic acid Schiff’s stain (PAS) and the Grocott’s methenamine stain for fungi (GMS).

Both stains are based on the chemistry of the fungal cell wall, which is made of cellulose. Cellulose is composed of glucose molecules, attached together very tightly. The PAS stain uses a primary step of oxidation of the glucose with periodic acid to form aldehyde groups. Once formed, the aldehydes are available to subsequently bind with the Schiff’s reagent, which results in the fungal cell walls being stained pink (Figure 3). Diastase digestion may, or may not be used, as it does not affect the fungal staining; it simply removes any glycogen which may be present.

Some dermatopathologists feel that the GMS stain is a more sensitive stain for detection of fungi in tissue sections. In this stain, chromic acid is used to oxidize the glucose molecules, leaving the aldehyde groups open to bind silver molecules, present in the methenamine silver solution. Subsequent development and toning of the sections renders the fungal cell walls black (Figure 4).

Either the PAS or the GMS method may be utilized to stain fungi in tissue sections.

Previous blogs (Hair Histology) described how to stain fungi that are present on hair shafts.




  2. Scher RK, Rich P, Pariser D, Elewski B. The epidemiology, etiology, and pathophysiology of onychomycosis. Semin Cutan Med Surg. 2013 Jun;32 (2 Suppl 1):S2-4.