In an ever changing, evolving and developing scientific community, spare a thought for a traditional product that is still a foundation in diagnostics….the Stain.
Staining techniques have long been used as an essential aid to diagnosis of disease, cellular differentiation and bacterial identification, and still have an irreplaceable role in many disciplines in hospital diagnostic pathology departments. Stains will be found in routine use in cytology, histology, haematology, microbiology and parasitology, for studying a range of specimens in both in-vitro and in-vivo conditions..
Ask a tourist what Delft is famous for, and it will more than likely be delicate blue collectable pottery, rather than Microscopes and staining technique origins.
Some of the most important origins of staining date back not to a scientist, but to a Dutch draper known as Antonie Van Leeuwenhoek (1632 – 1734), whose hobby was the grinding of glass, which he used to examine threads in fabrics and later a variety of biological samples. Born Thonis Philipszoon, he became known as Van Leeuwenhoek perhaps as he was born in a house on the corner of Lions Gate in Delft, Van Leeuwenhoek translating literally as From Lions Gate. Due to his skills in grinding glass lenses which at the time were no more than slivers of glass, Van Leeuwenhoek is today credited with the discovery of the microscope, and further to this was the first to observe blood cells and bacteria using this simple invention. He was the only one who made these observations and it would take many years before other people could confirm them.
The 17th and 18th centuries saw many advances in science, and competition between scientists was fierce as they strived for discoveries and acknowledgement. Van Leeuwenhoek avoided confrontation and carried on his work quietly. Had it not been for a learned friend Dr. Regnier de Graaf, he may never have been credited with his discovery, it being de Graaf who sent the first transcripts of his work to the Royal Society of London. Founded in 1660, The Royal Society, possibly the oldest scientific society, had many now legendary scientists and inventors as founder members including Robert Boyle, Christopher Wren, Isaac Newton and Robert Hooke.
It was Robert Hooke who then furthered the development of the microscope and is one of a number of scientists credited with the invention of the compound microscope (Figure 2) using more than one lens and allowing magnification up to x1000 for the first time.
Now that we had microscopes, we needed colours to differentiate cells. Working alone in Delft in 1714 Van Leeuwenhoek was examining the muscles of fat and lean cows, and found that the material was too transparent for clear observation. It is here that his work on the theory of staining began. He treated the muscle with an extract obtained by macerating Saffron in burnt wine. The Saffron, taken from the styles and stigmas of the crocus sativus flower, native to Spain, is still used to this day as a stain in Histology to show tissue structure. This method opened the door for many naturally occurring dyes to be experimented with for staining all manner of cells, structures and tissues.
In 1849, Goppert and Cohen, used Carmine with limited success, and in 1854 Hartig continued the work with Litmus, Black Ink and Copper Sulphate and is credited as a front runner in the process of Histological staining, later completed by Gerlach who actually made his most important discovery by accident that lead to a huge advance with the staining of nerve fibres and nerve cells using Amnoniacal Carmine. This spurred many researchers on, and today there are hundreds of differential staining techniques using numerous blends of pigments and dyes. These can be referenced in the International Colour Index first published in 1925 containing over 27,000 individual products offering a valuable reference for the scientific, textile, paint, printing and plastic industries. Pigments and dyes can be acidic, basic, dispersing, direct, fluorescent, mordant and reactive, and can be used in many staining techniques such as direct, simple, indirect, progressive, regressive, vital, intra-vital, supra-vital, negative, impregnative, polychromatic and metachromatic.
Perhaps one of the most widely used staining methods in Microbiology is the Gram Stain, and although one of the simplest staining methods used, it is one of the most fascinating polychromatic methods enabling initial differentiation based on colour and morphology.
Gram staining is named after the Danish microbiologist Hans Christian Gram, who initially researched the method in 1882 and published the first recommended method in 1884. It is still one of the most important staining techniques in microbiology being one of the first tests performed for the identification of bacteria offering valuable information for clinical diagnostics.
The principle stain of the Gram’s Method is crystal violet, sometimes substituted with methyl violet. The method allows for a crystal violet-iodine complex to form in the protoplast of all organisms stained. After decolorizing, those organisms that are able to retain this dye complex are classified as Gram positive; organisms that are decolorized and take up the counterstain are classified as Gram negative.
The Gram staining method is based on the ability of a bacterial cell wall to retain the crystal violet or methyl violet during solvent treatment. The cell wall of Gram positive microorganisms has a higher peptidoglycan and lower lipid content than Gram negative bacteria. Once the bacterial cell wall is stained, Iodine is used as a mordant to form the crystal violet-iodine complex so that the dye cannot be easily removed. The use of this mordant, derived from the Latin word to bite, Mordere, is an essential step in the method to distinguish Gram positive and Gram negative microorganisms.
Subsequent treatment with a decolourizer or differentiator dissolves the lipid layer from the gram-negative cells. This removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. Also the solvent dehydrates the thicker Gram positive cell walls, closing the pores as the cell wall shrinks during dehydration. This effectively blocks the diffusion of the violet-iodine complex, and the microorganisms remain stained. The length of the decolourization is critical in differentiating the Gram positive microorganisms from the Gram negative microorganisms. Prolonged exposure to the decolorizing agent will remove all the stain from both types of miroorganisms.
Finally, a counterstain such as safranin, neutral red, dilute carbol fuchsin or basic fuchsin is applied to give decolorised Gram negative bacteria a pink/red colour. The selection of the counterstain can be personal preference or application driven. Safranin and Neutral Red offer very similar results, dilute carbol fuchsin and basic fuchsin stains many Gram negative bacteria more intensely, making them easier to see.
Microorganisms which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are better stained by fuchsin counterstains than safranin.
Care must always be taken, Gram Staining is an art form, practiced over many years by all microbiologists, and results can vary if care is not taken.
Quality control is important in any laboratory method, and this also applies to staining. The age of the cultures and the pH of the medium in which the bacteria are grown can markedly affect their reaction to the Gram stain. It is recommended to use fresh cultures up to 24 hours old for quality control of the Gram staining method.
Recommended QC cultures; Escherichia coli NCTC 10418 (Pink to Red Gram Negative Bacilli), Oxford Staphylococcus aureus NCTC 6571 (Blue to Purple Gram Positive Cocci).
Around the same time that Gram introduced the Gram Stain, the acid fastness of some bacteria was noted by Paul Ehrlich in 1882, and a stain for Acid / Alcohol Fast Bacilli (AFB), primarily for Mycobacterium species was introduced by Ziehl – Neelsen in 1883, the ZN stain. This then became the classic method for staining Mycobactrium tuberculosis.This method requires heat to be applied to the specimen slide, although a “cold” alternative is available with the Kinyoun staining method.
Fluorescence microscopy was introduced early in the 20th century and initially observations were limited to specimens that fluoresce naturally, then fluorescent dyes for staining tissues and cells were investigated. During the 1940s fluorescence microscopy became popular, when Coons and Kaplan introduced a technique to label antibodies with a fluorescent dye to study antibody-antigen interactions. This lead to major changes and important developments in the field of immune histochemistry. Then in 1994, the discovery that really brought fluorescence microscopy to the forefront was when M. Chalfie et al. succeeded in expressing a naturally green fluorescent protein (GFP), in living organisms, leading to additional research for a whole new class of tagging methods.
The development of fluorescent microscopy also lead to the Auramine Phenol Stain becoming a popular choice for screening sample smears for AFB’s, although all positives still require checking with the traditional ZN staining method.
In 1931, Ernst Ruska and Maximilian Knoll were credited with the invention of the Electron Microscope and by 1964 resolution of 1nm was possible.
As workloads increase in busy laboratories we are presented with many challenges for more accurate results in a more timely fashion, and this inevitably leads to the question of automation. A time span of almost 360 years has seen major advances in Microscopy from its humble beginnings with the Van Leeuwenhoek Microscope, to the scanning electron microscope. Throughout this time, many staining techniques have been developed and tradition remains with some of the most tried and trusted methods still used today as mentioned above.
Many staining methods can, and have been automated offering accurate and consistent results, whilst freeing up valuable laboratory technician time. This automation can be achieved in all disciplines, and in Microbiology the Poly Stainer, manufactured by IUL Spain and available from Pro-Lab Diagnostics in the UK, offers the ultimate in automated staining for a variety of Microbiological staining methods, for example – Gram staining, TB staining, Cryptosporidium staining and Trichomonas staining.
With the development of the first oil immersion lens in 1880 by Carl Zeiss Jena, the use of immersion oil was also required. Magnification was improved with the use of oil immersion lenses and the image quality could be directly related to the optical properties of the immersion oil used. Therefore, the use of oil with the same refractive index as the glass used in the manufacture of glass microscopy slides, increases the resolution between the oil immersion lens and the slide by replacing the air and easing the spread of light at the same speed as in glass (1.513), hence avoiding image distortion.
Hazards can be associated with certain oils, and the correct choice is important. Preferably oils free of hazards such as Dibutyl Phthalate (DBP) should be used, for example, PL.396 Immersion Oil from Pro-Lab Diagnostics).
An extensive range of ready to use and concentrated microbiological staining solutions in a selection of convenient, economical sizes.
All stains are manufactured in our own purpose designed laboratories to the highest standards, in accordance with all current regulatory requirements and CE labelled. Using the finest raw materials, and adopting strict quality control and filtering techniques prior to release, all stains offer excellent performance characteristics. Also available are a range of convenient staining kits for Gram, TB and Cryptosporidium staining.
In addition to the standard range of products available routinely, Pro-Lab Diagnostics also offer a custom delivery and preparation service. To enquire or order, please contact our sales coordination desk (T: 0151 252 1613 / E: uksupport@pro-lab.co.uk)
