Staining is a valuable technique used in microscopy to enhance contrast in the microscopic image. Stains are used to highlight structures in clinical specimens, often when viewed with the aid of different microscopes. Stains have different affinities for different organisms and are used to differentiate types of organisms or to view specific parts of organisms.
Staining involves the sample preparation onto slides, fixation (which aims to preserve the shape of the cell), the staining with dyes, and the observation under the microscope.
Common Errors During Staining Procedure
The duration of each step may vary depending on the concentration and formulation of staining solutions and other reagents. Follow the manufacturer’s instructions where possible.
Rinsing step
The use of tap water is not recommended when making the smears or when
performing rinse steps in some staining protocols, for example, in the Ziehl-Neelsen protocol, Mycobacterium gordonae has been found in tap water and may interfere with the accurate assessment of the specimen to be stained. Deionized or distilled water is recommended.
Excess rinsing between steps could also cause error in a staining procedure.
Decolourising step
Many laboratories do not adhere to a fixed decolorizing time for staining protocols and so results may vary. In some laboratories, laboratory staff is taught to add the decolorizing reagent drop by drop until it runs clear.
Difficulties in interpreting stain results
The staining technique is one factor that affects results. This may be due to differences in applying the steps in the protocols which might warrant analysis if problems in interpretation persist. Standardization of the protocols will minimize variation in results.
Other issues that may affect results are:
• when cultures have not been sufficiently mixed to break up clumps of cells, the resulting smear can be difficult to read because individual cells are not discernible
• partially acid-fast bacteria may also contribute to confusion during a smear evaluation
• the type and quality of specimen/smear. Smears that are too thick will not be readable and those that are too thin may result in false negatives or result in the need to repeat the procedure
• expired reagents
• preparation of reagents – this includes confirming the expiration dates of
reagents and confirming protocols to ensure proper reagent concentrations.
Difficulty in reading stains can occur when reagents are not prepared to their right concentrations
• improper operation of the microscope
Bacteria Staining Procedures
1 Auramine-phenol stain – 1 (acid fast bacilli)
This staining technique is used to demonstrate the presence of acid-fast bacilli (Mycobacterium species). These organisms have waxy envelopes that make them difficult to stain and decolorize. A fluorescent stain is used in this method. Auramine stain show higher sensitivity and specificity than Ziehl-Neelsen’s method. It is a better method for screening samples from suspected cases of tuberculosis especially pulmonary and extrapulmonary cases where bacilli count is usually low.
Method
• prepare smear and heat to fix
• flood the slide with Auramine-phenol (1:10v/v) and leave for 10min
• gently rinse with water (ensure water is either deionized or distilled)
• decolorize with 1% acid alcohol for 3-5min
• gently rinse with water as above
• repeat acid alcohol step until no further stain seeps from the film
• counterstain with 0.1% potassium permanganate or thiazine red for 15sec (this ensures a dark background for the fluorescing alcohol and acid-fast bacilli (AAFB) which are easier to see). KMNO4 stains all epithelial cells making it more difficult to see AAFB
• gently rinse with water as above and air dry. Do not blot dry
• examine slides using ultraviolet epi-fluorescence microscopy at 25 x or 40 x magnification (the use of a 40 x magnification non-cover-glass (NCG) objective lens will avoid the need to apply a cover glass)
Interpretation
Positive result
Acid fast bacilli vary from 0.5-10µm in length and stain bright yellow-green against a dark background.
Negative result
No fluorescence observed. Non-acid-fast cells appear dark.
Quality control organisms
Positive control
Mycobacterium species.
Negative control
A proven negative smear may be used as a negative control.
2 Gram stain
The Gram stain is complex and differential staining technique that remains a useful test performed in microbiology laboratories. The staining procedure differentiates organisms of the domain bacteria according to the cell wall structure. Organisms are classified according to their Gram staining reaction – Gram-positive and Gram-negative. The name “Gram” comes from its inventor, Hans Christian Gram. Gram-positive bacteria have thicker and denser peptidoglycan layers in their cell walls.
Iodine penetrates the cell wall in these bacteria and alters the blue dye to inhibit its diffusion through the cell wall during decolorization. Gram-positive bacteria must have an intact cell wall to produce a positive reaction. Gram-negative cells which do not retain the methyl/crystal violet are stained by a counterstain. Neutral red, safranin, or carbol-fuchsin may be used as the counterstain. This technique has also been used for staining of certain fungi such as Candida and Cryptococcus which are observed as Gram-positive yeasts.
Method
Hucker’s modification of Gram staining technique for the examination of smears
• prepare a smear and heat gently to fix
• flood the slide with 0.5% crystal violet and leave for 30sec
• tilt the slide, and rinse slide gently with water
• flood on sufficient (1%) Lugol’s iodine (also known as Gram’s iodine) to rinse off excess water, cover with fresh iodine and allow to remain for 30sec
• tilt the slide and wash off the iodine with water
• decolorize with 95 – 100% ethanol or acetone until color ceases to run out of the smear
• rinse with water
• flood the slide 0.1% counterstain safranin and leave to act for about 30sec to 1min
Note: It can be counterstained for longer if using other dyes, for example,
neutral red for about 2min
• wash briefly with water and blot dry
• examine the slide using an oil immersion objective to observe cell morphology and Gram reaction
Interpretation
Positive result
Gram-positive organisms stain deep blue/purple.
Negative result
Gram-negative organisms stain pink/red.
Note: Other counterstains (such as carbol fuchsin) used may give more intense colours.
Quality control organisms
A culture containing Gram-positive and Gram-negative organisms may be used for quality control.
Common errors in the Gram staining procedure
These are the errors that arise depending on the method and techniques used and which could result in a Gram-positive organism staining Gram negatively. They include;
• smear preparations being too thick
• excessive heat during fixation
• low concentration of crystal violet
• excessive rinsing between steps during the staining procedure. This could
cause the step of the crystal violet or the dye-iodine complex to be washed off from the Gram-positive cells
• insufficient iodine exposure
• prolonged decolorization. Over-decolourising will lead to an erroneous result where Gram-positive cells may stain pink to red indicating a Gram-negative result, and under-decolorizing will lead to an erroneous result where Gram-negative cells may appear blue to purple indicating a Gram-positive result. The degree of decolorizing required is determined by the thickness of the smear
• excessive counterstaining
• uneven staining or decolorization due to insufficient reagent being used for staining
• decolorizing step missed or need to increase the time of decolorizing step
3 Kinyoun stain (Mycobacterium and Nocardia species)
Introduction
The Kinyoun stain is a method of staining acid-fast microorganisms, specifically Mycobacterium and Nocardia. The procedure for Kinyoun staining technique is similar to the Ziehl-Neelsen stain but does not involve heating the slides being stained. This method has become known as the “cold staining” method because the heating step was removed in favor of using a higher concentration of the carbol fuchsin primary stain1.
It is also less time-consuming and is easier to perform.
Method
• prepare a thin smear of the specimen or colony to be stained and fix in methanol
• flood slide with Kinyoun’s carbol fuchsin and allow staining for 5min at room temperature. No heat is required
• rinse gently with water until water flows off clear
• decolourise with acid- alcohol (3% HCl in ethanol) for 3min until all excess carbol fuchsin is removed and rinse with water
• repeat decolourising with acid-alcohol again for 1-2min or until no more red colour runs from the smear
• rinse gently with water and drain standing water from the slide surface by tipping the slide
• flood slide with Methylene blue counterstain and allow staining for 3-4min
• rinse gently with water and allow to air dry
• examine under high dry (400X) magnification, and confirm acid-fast structures under oil immersion (1000X)
Interpretation
Positive result
Acid-fast organisms appear red.
Negative result
Non-acid-fast organisms appear blue.
Quality control organisms
Positive control
Mycobacterium species
Nocardia asteroides
Negative control
A proven negative smear may be used as a negative control.
Technical information
Kinyoun carbol fuchsin has a greater concentration of phenol and basic fuchsin and does not require heating in order to stain properly.
Rinsing step
The use of tap water is not recommended when making the smears or when performing rinse steps in some staining protocols, for example, in the Ziehl-Neelsen protocol, Mycobacterium gordonae has been found in tap water and may interfere with the accurate assessment of the specimen to be stained. Deionized or distilled water is recommended.
Agar media
Organisms grown in media containing complex lipids will grow better and will typically stain better than growth on Blood Agar plates which provides only starvation level lipids and may limit the ability of the organisms to demonstrate the acid-fast property after staining.
Other factors that may affect results:
Some of the factors that could influence the results of microscopic examination of slides are the following;
• the type and quality of the specimen
• the number of mycobacteria present in the specimen
• the method of processing (direct or concentrated)
• the method of centrifugation
• the staining technique used
• the quality of the examination – this encompasses the training and competency of the trainer and the trainee
• the prevalence and severity of the disease
4 McFadyean stain
Introduction
The McFadyean stain is a modification of the methylene blue stain and is used for detecting Bacillus anthracis in clinical specimens.
Method
• prepare a smear of the specimen or colony to be stained and air dry
• cover the smear with absolute alcohol for approximately 3min and air dry
• flood the smeared slide with methylene blue solution (0.05mg/mL in 20mM potassium phosphate adjusted to pH 7.3) for 30-45sec
- wash the slide gently with water or as a safety precaution, wash slide using a 10% hypochlorite solution
- allow slide to dry and then examine under oil immersion
Interpretation
Positive result
Virulent B. anthracis rods will be surrounded by a clearly demarcated zone giving the appearance of a reddish pink capsule.
Negative result
N/A
Quality control organisms
Positive control
Bacillus anthracis
Negative control
A proven negative smear may be used as a negative control.
5 Modifications of the Kinyoun stain method
The modified kinyoun stain method involves the use of a solution of 1% sulphuric acid in place of 3% HCl solution as a decolourising reagent36. The sulphuric acid solution does not decolourise as strongly as the hydrochloric acid and this makes it useful for staining organisms that are weakly acid fast, such as Nocardia. It has also been used for staining species of Rhodococcus, Gordonia, Actinomadura and Tsukamurella. Malachite Green or Brilliant Green may be used instead of Methylene Blue as a counterstain, resulting in non-acid fast organisms appearing green rather than blue.
Another alternative modification is the use of 20% sulphuric acid for decolourising instead of HCl followed by 95% alcohol.
6 Sandiford’s modification of Gram stain
Introduction
Sandiford’s modification of Gram staining technique was originally used for demonstrating the presence of Gram negative diplococci intracellularly. This technique has been used successfully for Neisseria and Haemophilus species identification. The counterstain also enhances the appearance of Gram negative and Gram variable organisms.
Method
• spread a loop of clinical specimen thinly on a degreased slide. Air dry
• stain with crystal violet stain for 2min
• rinse in tap water
• counterstain with Lugol’s iodine solution for 2min
• rinse in tap water and blot dry
• decolourise in acetone-alcohol (mixture of 500mL of 95% ethyl alcohol and 500mL acetone) for 10-15sec
• wash in running tap water
• blot dry
• counterstain with Sandiford’s malachite green solution (mixture of 1.5g pyronin Y and 0.5g malachite green and 1000mL of distilled water) and leave for 3min
• flood the slide with water (do not wash) and air dry
Interpretation
Positive result
Gram positive organisms stain deep blue/purple.
Negative result
Gram negative or Gram variable organisms stain pink against a blue green background.
Background and cellular debris stain blue/green.
Quality control organisms
A culture containing Gram positive and Gram negative organisms may be used for quality control.
7 Spore stains
Introduction
Endospore production is an important characteristic of some bacteria (such as Bacillus and Clostridium species); this allows them to resist environmental conditions such as extreme heat, chemical exposure, etc.
The following methods below may be used for the demonstration of spores in Gram positive bacilli.
Safety considerations
Malachite green is hazardous when ingested and slightly hazardous in case of skin contact, eye contact and inhalation. Severe over-exposure can result in death.
Methods
Schaeffer and Fulton’s method (as modified by Ashby)
• prepare a smear and heat gently to fix
• place the slide over a beaker of boiling water, resting it across the rim with the bacterial smear uppermost
• when large droplets of water appear on the underside of the slide, flood it with the 5% malachite green solution and leave it to act for 1min while the water is still boiling
• rinse with cold water
• counterstain with 0.5% safranin or 0.05% basic fuchsin for 30sec
• rinse in cold water and air dry
• examine the slide under the oil immersion with a light microscope for the presence of endospores
Wirtz-Conklin’s method
• prepare a smear and heat gently to fix
• flood the slide with 5-10% malachite green solution
• leave the slide to stain for 45min or alternatively, the slide can be heated gently to steaming for 3-6min, reapplying stain if it begins to dry out
• rinse under running tap water
• counterstain with 0.5% safranin for 30sec
• rinse and dry
• view slide under oil immersion (magnification of 1000X) with a light microscope
Interpretation
Positive result
Bacterial spores stain green.
Lipid granules remain unstained.
Negative result
Vegetative cells stain red. Non-spore forming bacteria stain pink.
Quality control organisms
Positive control
Bacillus species.
Negative control
Non-spore producing organisms, for example, E. coli.
Technical information
It should be noted that any debris on the slide can also take up and hold the malachite green stain and so caution should be taken when interpreting slides.
Older culture
The age of the culture will affect sporulation. Young cultures (less than or a day old) may have only vegetative cells, whereas older cultures (5 to 7 days old) are excellent for good sporulation.
Fixing of slides
Heat fixing should be done with minimal flaming as excess heat will destroy the integrity of the cells, causing them to shrink and to aggregate together on the slide.
8 Vincent’s stain (oral bacteria)
Introduction
This staining technique is used to stain Borrelia vincentii (a spirochaete causing Vincent’s angina) from oral and throat swabs. The presence of large numbers of Borrelia vincentii in conjunction with barred fusiform bacilli and Gram-negative rods together with polymorphonuclear leucocytes indicates infection.
Method
The procedure for Vincent’s stain is similar to that of Gram stain except that the counterstain (1% carbol fuchsin) is applied for 30sec.
Interpretation
Positive result
Borrelia vincentii appears as pale pink staining spirals together with pink cigar-shaped fusiform.
Note: The presence of both organisms is needed for establishing the diagnosis of Vincent’s disease.
Negative result
N/A
Quality control organisms
Borrelia vincentii are large spirochaetes that vary between 10-30μm in length.
Positive result
Borrelia vincentii.
Negative result
A proven negative smear may be used as a negative control.
9 Ziehl-Neelsen stain (acid fast bacilli)
Introduction
This staining technique is used to demonstrate the presence of acid and alcohol fast bacilli (AAFB) which have waxy envelopes that make them difficult to stain and decolorize. In this method, heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells. The use of heat in this method is the reason that this technique is called the “hot staining” method.
Auramine-phenol staining is more sensitive than Ziehl-Neelsen and is thus more suitable for the assessment of smears from clinical specimens. Ziehl-Neelsen staining provides morphological details and is more useful for confirming the presence of AAFB in positive cultures.
Safety considerations
- All suspected Mycobacterium species must be processed in a Class 1 exhaust protective cabinet in a Containment Level 3 room.
- Disposable gloves must be worn when handling the reagents to avoid contact– Carbol fuchsin is carcinogenic while the acid-alcohol is corrosive.
- Phenol is a component of the carbol fuchsin reagent for the Ziehl-Neelsen and Kinyoun methods for acid-fast staining. Phenol is a dangerous chemical if not handled carefully. And so caution should be taken by laboratory staff as it is poisonous, corrosive and combustible.
- Smeared material should be fixed by placing the slides on an electric hotplate prior to staining (65-75°C). This procedure should be performed in a Class 1 exhaust protective cabinet until the smeared material is dried and fixed to the slide. They should then be placed in a rack or suitable holder.
Note: Heat fixing does not kill Mycobacterium species and slides should be handled with care.
Method
• flood the slide with strong carbol fuchsin (ie 100mL of 10x Concentrated carbol fuchsin which should be diluted in 900mL distilled water before use)
• heat the underside of the slide gently by placing on a hot plate until steam rises but not boiling
(Caution: overheating causes spattering of the stain and may crack the slide)
• leave for 3-5min keeping the slide moist with stain
• rinse the slide well in a gentle and indirect stream of deionized water until no color appears in the water
• decolorize for 10-20sec with a (3% v/v) acid-alcohol solution and then rinse well with water. Repeat the decolorizing and the washing until the stained smear appears faintly pink and the water washing off the slide runs clear
• counterstain with (1% w/v) methylene blue or malachite green for 20-30sec
• rinse with water and allow to dry
• apply immersion oil and view under a transmitted light microscope
Note: Follow manufacturer’s instructions, if commercial ready to use reagent kits are used.
Interpretation
Positive result
Acid-fast bacilli vary from 0.5-10μm in length and stain bright red. Some may appear beaded.
Negative result
All other organisms and background material stain green if malachite green counterstain is used or blue if methylene blue counterstain is used.
Quality control organisms
Positive control
Mycobacterium species
Negative control
A proven negative smear may be used as a negative control.