Auramine Staining: Introduction, Principle, Procedure, Result Interpretation, Limitations, Keynotes, and Related Footages

Introduction of Auramine Staining

Auramine is a fluorochrome stain used to visualize acid-fast structures of various microorganisms especially Mycobacterium tuberculosis and in modified form for Mycobacterium leprae, Nocardia species, Cryptosporidium parvum, Cyclospora cayetanensis , Isospora belli, and fungal spores. Ziehl-Neelsen (hot), and Kinyoun (cold) are still widely used methods to detect acid-fast structures in these organisms in developing countries but the sensitivity is high of fluorochrome stain. The acid fastness of Mycobacterium tuberculosis is due to having a thick cell wall composed of waxes and lipids that has a high content of mycolic acid.

Principle of Auramine Staining

The property of acid-fastness is based on the presence of mycolic acids in the microbes’ cell walls. Auramine (primary stain) binds cell-wall mycolic acids. Intense decolorization using strong acids-alcohol ties up the primary stain from the cell wall and the organisms retain the fluorescent bright yellow color of auramine. Potassium permanganate is applied to quench fluorescence in the background; however, it provides little contrast for focusing, and stains are thus sometimes preferred, of which blue ink may be the best. All mycobacteria are acid-fast, but very few other bacteria possess this property and then only weakly (e.g. Nocardia) while other than bacteria are Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and fungal spores.

Acid-fast bacilli (AFB) found in respiratory specimens of patients from countries with high tuberculosis prevalence are almost always tubercle bacilli. Non-TB mycobacteria are more commonly found in countries where tuberculosis prevalence is low. Some patients suspected of having MDR-TB in high-burden regions may really have an illness caused by non-TB mycobacteria. Extrapulmonary specimens containing AFB, such as gastric washings, feces, or urine, should never be assumed to contain tubercle bacilli.

Test Requirements for Auramine Staining

• Alcohol sand jar (only if a loop is used, not needed with disposable sticks).
• Bunsen burner or spirit lamp
• Diamond pencil or lead pencil (if frosted-end slides are available)
• Filter paper, appropriate for funnel size
• Funnels, small, for filtering solutions in use
• Forceps
• Lens paper or soft tissue paper
• Plastic bag for waste disposal
• Bamboo or wooden sticks or wire loops
• Fluorescence microscope with objectives of 20x or 25x, and 40x (ideally specific for fluorescence microscopy), and eyepieces of 10x
• Slide staining rack
• Slide boxes
• New, clean slides (rinse in alcohol and dry if necessary)
• Timer
• Test specimen-Sputa

1. Spontaneous sputa: Sputa from suspects should be rejected only if they are liquid and as clear as water, with no particles or streaks of mucous material. However, they should be accepted if the patient cannot produce a better specimen on a repeated attempt. Sputa from follow-up patients should be accepted and examined even if they look like saliva since these patients often cannot produce mucoid specimens.
2. Induced sputa: These specimens resemble saliva but have to be processed as adequate specimens.
3. Decontaminated sputa, concentrated by centrifugation.

• Other specimens

1. Laryngeal swabs, gastric lavages, bronchial washings, brushings, and transtracheal aspirates.
2. Urine.
3. Body fluids (spinal, pleural, pericardial, synovial, fluids from ascites, blood, pus, bone marrow).
4. Tissue biopsies.

• Staining reagents
• Staining bottles, 250 ml, with spout
• Beaker for rinsing water
• Sink and water supply
• Disinfectant solution
• Auramine staining solution, 0.1%
• Acid-alcohol decolourizing solution, 0.5%
• Counterstaining solution: Potassium permanganate, 0.5%, or blue ink, 10%

Smear Preparation for Auramine Staining

• Disinfect the working area.
• Label the slides properly using the laboratory register serial number marked on the sputum container.
• Place each slide on its corresponding container
• Proceed to smear, taking the labeled slides and opening containers one by one; do the smearing behind the flame of a Bunsen burner or spirit lamp.
• For a direct sputum smear, select a small portion of purulent or mucopurulent material with the stick/loop and transfer it to the slide.
• If a smear is prepared after specimen decontamination, the concentrated material must be transferred to the slide with a sterilized loop to avoid splashing.

• Spread the material carefully over an area equal to about 2–3 cm x 1–2 cm using repeated circular movements, without touching the edge of the slide. Make the smear as even as possible by continuing this process until no thick parts remain. The thickness of the smear should be such that a newspaper held under the slide can barely be read through the dried smear.
• Disinfect the working area after smear preparation
• Let the smears air-dry at room temperature; do not use heat to speed the drying. Where humidity is high, gentle warming will be needed on a slide warmer (or locally made box with a glass top under which there is a 20-watt light bulb).
• When dry, hold the slides in forceps and fix them by passing them three times slowly through the flame of a spirit lamp or quickly through that of a Bunsen burner, smear upwards; do not overheat or AFB staining will be poor.
• Always keep smears out of direct sunlight.

Staining Procedure

• Place the slides, smear upwards, on the staining rack over a sink, about 1 cm apart.
• Place a new filter paper in a small funnel, keep it over the first slide and fill it up with auramine staining solution.
• Let the solution filter through the paper, covering each slide completely. Do not heat. Leave for 20 minutes.
• Using forceps, tilt each slide to drain off the stain solution. Rinse the slides well with distilled water or clean tap water from a beaker (not directly from the tap).
• Pour the acid solution over the smears, covering them completely, and allow them to act for 3 minutes.
• Using forceps, tilt each slide to drain off the acid-alcohol solution. Gently rinse each slide again with distilled water or clean tap water from a beaker (not directly from the tap).
• Flood smears with potassium permanganate or blue ink solution for 1 minute. Time is critical because counterstaining for longer may quench the AFB fluorescence.
• Using forceps, tilt each slide to drain off the counterstain solution. Gently rinse each slide again with distilled water or clean tap water from a beaker (not directly from the tap).
• Using forceps, take each slide from the rack and let the water drain off. Stand the slide on the edge of the drying rack and allow it to air-dry.

Reading, Result, and Interpretation of Auramine Staining

• Keep stained smears in the dark (in a box or folder) and read them as soon as possible –fluorescence fades quickly when exposed to light.
• Switch on the fluorescent lamp 5 minutes before use; leave the lower ordinary lamp off.
• Rotate the nosepiece so that the 20x (or 25x) objective is in the light path.
• Select the filter set position suitable for auramine stain (see manufacturer’s manual)
• Check that there is a strong blue light; if not, open shutters and/or the fluorescent light beam diaphragm
• Load the positive control slide on the stage and move the stage to position the slide under the objective.
• Use the coarse adjustment first, and then the fine adjustment, to focus on the objective. If this fails (i.e. in thin negative smears), turn the filter set to transmitted light, switch on the lower normal lamp and focus as with a light microscope. Then switch off the lower lamp and return to the required filter position. The field should now be in focus.

• Keep stained smears in the dark (in a box or folder) and read them as soon as possible –fluorescence fades quickly when exposed to light.
• Switch on the fluorescent lamp 5 minutes before use; leave the lower ordinary lamp off.
• Rotate the nosepiece so that the 20x (or 25x) objective is in the light path.
• Select the filter set position suitable for auramine stain (see manufacturer’s manual)
• Check that there is a strong blue light; if not, open shutters and/or the fluorescent light beam diaphragm
• Load the positive control slide on the stage and move the stage to position the slide under the objective.
• Use the coarse adjustment first, and then the fine adjustment, to focus on the objective. If this fails (i.e. in thin negative smears), turn the filter set to transmitted light, switch on the lower normal lamp and focus as with a light microscope. Then switch off the lower lamp and return to the required filter position. The field should now be in focus.
• Because fluorochrome-stained smears are examined at magnifications of 200x to 400x, the number of AFB can roughly be divided by a factor of 10 or 5, respectively (depending on the objective) to make them equivalent to fields seen on examination of fuchsin-stained smears at 1000x.
• Test Positive: Acid-fast organisms fluoresce bright yellow or reddish-orange against a dark background.
• Negative Test: Non-acid-fast organisms will not fluoresce.

IUATLD/WHO scale (1000x field = HPF)	Microscopy system	Microscopy system	Microscopy system
Result	Bright-field (1000x magnification: 1 length = 2 cm = 100 HPF)	Fluorescence (200–250x magnification: 1 length = 30 fields = 300 HPF)	Fluorescence (400x magnification: 1 length = 40 fields = 200 HPF)
Negative	Zero AFB / 1 length	Zero AFB / 1 length	Zero AFB / 1 length
Scanty	1–9 AFB / 1 length or 100 HPF	1–29 AFB / 1 length	1–19 AFB / 1 length
1+	10–99 AFB / 1 length or 100 HPF	30–299 AFB / 1 length	20–199 AFB / 1 length
2+	1–10 AFB / 1 HPF on average	10–100 AFB / 1 field on average	5–50 AFB / 1 field on average
3+	>10 AFB / 1 HPF on average	>100 AFB / 1 field on average	>50 AFB / 1 field on average

Reporting

Results must be reported in a special register of TB laboratory examination. Use red ink for positive results. Reports must be provided as soon as possible.

• For a negative result report: “Acid-fast bacilli were not seen.”
• For a positive result: report quantification of AFB seen. (It should not be assumed that AFB are tubercle bacilli.)
• Never report “No TB” (or equivalent wording).

Limitations of Auramine Staining

• Among the possible reasons for false-positive results are:

1. re-use of containers or positive slides;
2. contaminated stain prepared with water containing environmental mycobacteria;
3. use of scratched slides;                   
4. AFB floated off one slide and became attached to another during the staining procedure because there was no space between adjacent slides;
5. Inadequate decolorization;
6. lack of experience, confusion with artifacts (especially if stains are not or poorly filtered);
7. microscope (lamp) in poor condition or poorly adjusted: interpreting glitter as AFB;
8. poor quality of staining solutions.

False-positive results

• Among the possible reasons for false-positive results are:

1. Re-use of containers or positive slides
2. contaminated stain prepared with water containing environmental mycobacteria
3. Use of scratched slides                  
4. AFB floated off one slide and became attached to another during the staining procedure because there was no space between adjacent slides.
5. Inadequate decolorization
6. Lack of experience, confusion with artifacts (especially if stains are not or poorly filtered)
7. Microscope (lamp) in poor condition or poorly adjusted: interpreting glitter as AFB
8. Poor quality of staining solutions.

• This type of stain needs a fluorescent microscope which is costly, and cumbersome to handle ( needs trained staff).
• A positive result only provides presumptive evidence of the presence of mycobacteria and a negative result does not indicate that the specimen will be culturally negative. Thus, cultural methods must be employed.
• Acid–alcohol, and potassium permanganate are also strong irritants to the skin, eyes, and respiratory system, and therefore precaution is required while handling and staining using such reagents.
• Most strains of rapid growers may not appear fluorescent.
•  All negative fluorescent smears should be confirmed with the Ziehl-Neelsen stain.
• Excessive exposure to the counter (potassium permanganate) stain may result in a loss of the brilliance of the fluorescing organism.
• Stained smears slides should be observed within 24 hours of staining because of the possibility of fluorescence fading.

Keynotes on Auramine Staining

1. Fluorescence microscopy allows smears to be examined more rapidly than is possible with the basic fuchsin procedures and is particularly indicated for high-volume laboratories. It may also be more sensitive for paucibacillary specimens since it allows the examination of more fields with less effort. However, it requires a stable power supply, greater expertise in reading and microscope adjustment, and a regular supply of costly and short-lived bulbs. Cheaper systems using halogen lamps have less stringent requirements, but performance does not entirely match that of the standard mercury vapor lamps.
2. Newly developed blue LED light sources adjusted to fluorescence microscopes may overcome these difficulties in the near future because a 5-W lamp is sufficient, can be operated with simple batteries, and has a life of at least 15 000 hours.
3. Here the preparation of staining solutions and/or (if staining solutions are provided centrally) method used or recommended by the NTP should be inserted or described.
4. If a loop is used, it must be sterilized before use by heating it until red-hot within the glass chimney of the Bunsen burner. After use, plunge the loop into the alcohol sand jar, moving it up and down to remove any remaining material, then heat it again until red-hot.
5. If blue ink was used as the counterstain, the stained smear should show a light blue color. A dark blue color usually indicates that the smear is too thick.
6. If blue ink was used as the counterstain, the stained smear should show a light blue color. A dark blue color usually indicates that the smear is too thick.
7. Nearly 10% more sensitive than Z-N stain
8. It does not require the use of oil immersion fields and thus no need for cedarwood oil.
9. Heat is not required for auramine-phenol staining.

Related Footage for Auramine Staining

An auramine-stained smear of sputum ready for observation in a fluorescence microscope

Numerous acid-fast bacilli of Mycobacterium tuberculosis in Auramine stained smear of sputum fluorescence microscopy at 40X objective

Mycobacterium tuberculosis in Auramine stained smear of sputum fluorescence microscopy at a high magnification

Nocardia in Auramine Staining

Bibliography

• https://universe84a.com/auramine-phenol-fluorochrome-staining/
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• Laboratory services in tuberculosis control. Part II: Microscopy. Geneva, World Health Organization, 1998.
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• https://core.ac.uk/download/pdf/82107117.pdf
• https://extranet.who.int/lqsi/content/tb-sop-auramine-staining