Acridine Orange Staining – Principle, Procedure, Uses

Acridine orange staining is a fluorescent staining method used for the detection of microorganisms and examination of nucleic acids present in cells. It uses acridine orange (AO), a fluorochrome which binds to the nucleic acids of bacterial cells and other cells. In this method, a smear is prepared from the specimen and stained with acridine orange solution. The stained smear is then examined under a fluorescence microscope.

Microbiological specimens may contain bacteria along with host cells and cellular debris. In certain clinical smears, an acidic staining solution is used to distinguish the bacterial cells from other materials present in the specimen. The fluorescence of the stained material depends upon the pH and the staining conditions used.

Acridine orange staining is usually carried out on fixed microbiological smears. The dye can also be used for staining living cells in certain laboratory assays. These assays require different methods of specimen preparation and staining.

Purpose of Acridine Orange Staining

The following are the purposes of acridine orange staining:

• It is used to detect the presence of bacteria and other microorganisms in clinical specimens.
• Acridine orange staining is used for screening microbiological smears, especially when microorganisms are difficult to detect by Gram staining.
• It helps to distinguish bacterial cells from host cells and cellular debris present in the specimen. This is usually carried out under acidic staining conditions.
• The dye is used to stain nucleic acids such as DNA and RNA present in bacterial cells and other cells.
• It is used to examine nucleic-acid-containing cellular material under a fluorescence microscope.
• Acridine orange staining can detect microorganisms but cannot identify their species or determine their Gram reaction. Other tests are required for their identification.

Principle of Acridine Orange Staining

Acridine orange is a positively charged, metachromatic fluorochrome. It binds to the negatively charged DNA and RNA present in cells and produces fluorescence when exposed to suitable blue light.

Acridine orange binds to double-stranded DNA by intercalation, in which the dye enters between the base pairs. The dye molecules remain mainly in the form of single molecules and give green fluorescence. It also binds to single-stranded DNA and RNA by electrostatic attraction. At suitable dye concentrations, the molecules form aggregates which give orange to red fluorescence.

The fluorescence colour is affected by the dye concentration, pH of the staining solution and preparation of the specimen. Green and orange-red fluorescence are not always specific to DNA and RNA.

An acidic staining solution is used for certain clinical smears. Under these conditions, bacterial cells may appear orange, while host cells and tissue debris give green to yellow fluorescence. The low pH reduces the fluorescence of some background material.

In living cells, acridine orange may also accumulate in acidic cellular compartments and give orange-red fluorescence.

Materials Required for Acridine Orange Staining

The following are the materials required for acridine orange staining:

Reagents and Staining Solution

1. Acridine Orange Staining Solution– A 0.01% acridine orange solution (approximately 100 mg/L) is used for staining fixed clinical smears. It is prepared by dissolving 20 mg of acridine orange in 190 mL of acetate buffer.
2. Acetate Buffer– The buffer is prepared using 100 mL of 1 M sodium acetate solution and 90 mL of 1 M hydrochloric acid. The pH is adjusted to 3.5–4.0. The acidic pH helps to differentiate bacterial cells from the surrounding host cells and tissue debris.
3. Methanol– Absolute methanol is used for fixation of the air-dried smear.
4. Distilled Water– It is used for the preparation of reagents and washing the stained slides. Approved laboratory rinse water may also be used.
5. Commercial Acridine Orange Reagent– Ready-to-use staining solutions are also available. Their concentration and pH may differ depending upon the manufacturer. The 0.01% acidic solution described here is used for fixed clinical smears and should not be confused with the staining solutions used in live-cell assays and flow cytometry.

Instruments and Other Materials

1. Glass Slides– Clean, grease-free microscope slides are required for the preparation of smears.
2. Inoculating Loop or Sterile Pipette– Used to transfer the specimen onto the glass slide.
3. Staining Rack or Staining Trough– It is used to hold the slides during staining.
4. Dropper or Pasteur Pipette– Used for adding the staining solution to the smear.
5. Measuring Cylinder and Beakers– Required for measuring and preparing the staining reagents.
6. pH Meter or pH Paper– Used to check the pH of the staining solution.
7. Amber-Coloured Bottle– The prepared acridine orange staining solution is stored in a light-protected bottle.
8. Fluorescence Microscope– An epifluorescence microscope with suitable blue-light excitation and emission filters is required. The filter combination should allow the examination of green and orange-red fluorescence.
9. Slide Drying Materials– A clean slide rack and absorbent paper are required for drying the slides.
10. Positive Control Slides– A known bacterial smear is used to check the staining solution and fluorescence microscope before examining clinical specimens.
11. Protective Equipment– Laboratory coat, gloves and protective eyewear are required while handling acridine orange, methanol and clinical specimens.

Procedure of Acridine Orange Staining

The procedure of acridine orange staining is given below:

1. Take a suitable microbiological specimen and place a small amount of the material on a clean glass slide.
2. Spread the specimen to prepare a thin smear and allow it to air dry completely.
3. Fix the dried smear with methanol for 1 to 2 minutes. Allow the slide to dry.
4. Flood the smear with the acidic acridine orange working solution. One clinical method uses 100 mg/L acridine orange at pH 3.5.
5. Allow the stain to remain on the smear for 2 minutes.
6. Rinse the slide with tap water or distilled water and allow it to air dry.
7. Examine the dried smear under a fluorescence microscope using a filter suitable for acridine orange. Blue excitation in the range of about 470–500 nm is used in documented methods.
8. For the pH 3.5 clinical method, examine the smear at 600× magnification. Suspicious fluorescent areas can be examined under oil immersion at 1,500× magnification.
9. Bacteria and other microorganisms appear as bright orange fluorescent cells against a dark to yellow-green background.
10. Stain a known positive bacterial control along with the test smear. The control should show the expected bright orange fluorescence.
11. This procedure is used for fixed microbiological smears. Different procedures are required for live-cell staining, acridine orange flow cytometry, and phenolic acridine orange acid-fast staining.

Results and Interpretation of Acridine Orange Staining

The stained smear is examined under a fluorescence microscope. In clinical smears stained with an acidic acridine orange solution at pH 3–4, bacterial cells usually give bright orange to orange-red fluorescence. The host cells and tissue debris appear green to yellow.

The following are the results and their interpretation:

The fluorescence colour depends upon the concentration of acridine orange, pH of the staining solution and preparation of the specimen. Green fluorescence is not always specific to DNA, and orange-red fluorescence does not always indicate RNA. In fixed microbiological smears, the fluorescence is mainly used for the detection of microorganisms. Live-cell staining and flow cytometry use different conditions. In living cells, acridine orange may also accumulate in acidic cellular compartments and give orange-red fluorescence.

Acridine orange stains nucleic acids present in both living and dead cells. Therefore, the fluorescence of a bacterial cell does not confirm its viability. It also cannot determine the bacterial species or Gram reaction. Other microbiological tests are required for identification.

Applications of Acridine Orange Staining

The following are the applications of acridine orange staining:

• Acridine orange staining is used for the detection of bacteria in clinical specimens such as cerebrospinal fluid, wound exudates and other body fluids.
• It is used to examine clinical smears in which bacteria are difficult to detect by Gram staining.
• The staining method is used for the early detection of microorganisms in blood cultures, even before visible growth is observed.
• It can also be used to detect certain fungi and bacteria such as Mycoplasma and Ureaplasma, which are difficult to examine by conventional Gram staining.
• Acridine orange staining is used to count bacterial cells in freshwater, seawater and other environmental samples by epifluorescence microscopy. Both living and dead bacteria may be included in the count.
• It is used for the differential staining of DNA and RNA present in cells under suitable staining conditions.
• In flow cytometry, acridine orange is used to measure the DNA and RNA content of individual cells. It is also used to study the different stages of the cell cycle.
• Acridine orange is used to examine acidic cellular compartments such as lysosomes and autolysosomes in living cells. It also helps to study changes in acidic vesicles during autophagy.
• It is used in sperm chromatin studies to examine DNA denaturation and certain changes in chromatin structure.
• Acridine orange is used along with ethidium bromide for the examination of apoptotic and necrotic cells. The combined staining method helps to distinguish living and dead cells by fluorescence microscopy.

Advantages of Acridine Orange Staining

The following are the advantages of acridine orange staining:

• Acridine orange staining is a rapid method used for the detection of microorganisms in clinical specimens. The staining procedure takes only a few minutes.
• It is more sensitive than Gram staining for detecting bacteria in certain clinical specimens, especially when the bacterial cells are present in small numbers.
• Under acidic staining conditions, the bacterial cells give bright orange to orange-red fluorescence while the host cells and tissue debris appear green to yellow. This helps in distinguishing bacteria from the surrounding materials.
• Bacteria present in bloody fluids and thick exudates can be detected even when their examination by Gram staining is difficult.
• It helps in the early detection of bacteria and yeasts in blood cultures before visible growth is observed.
• The staining method is simple and requires only a few reagents. The stained smear is examined under a fluorescence microscope.
• Microorganisms present in clinical specimens can be detected directly without waiting for their growth on culture media.
• It can be used to count bacterial cells in environmental water samples by fluorescence microscopy. Both living and dead bacterial cells are included in the count.
• Acridine orange binds to both DNA and RNA present in the cells. Under suitable staining conditions, the difference in fluorescence colour helps in the examination of cellular nucleic acids.

Limitations of Acridine Orange Staining

The following are the limitations of acridine orange staining:

• Acridine orange stains nucleic acids present in both microorganisms and host cells. Therefore, it is not specific for bacterial cells.
• It cannot differentiate between Gram-positive and Gram-negative bacteria.
• The staining method does not identify the bacterial species. Other microbiological tests are required for identification.
• Both living and dead bacterial cells can take up acridine orange. Hence, the viability of bacteria cannot be determined by this staining method alone.
• Cellular debris, leukocyte granules and dye precipitates may also show fluorescence. These particles can sometimes be mistaken for bacteria.
• Intracellular microorganisms may be difficult to detect due to the fluorescence of the surrounding host cell nuclei.
• The staining results depend upon the concentration of acridine orange, pH of the staining solution and preparation of the specimen. Improper staining conditions may produce high background fluorescence.
• The fluorescence of acridine orange may fade during prolonged exposure to excitation light. Therefore, the stained slides should be examined promptly.
• Bacteria present in very small numbers may remain undetected. A negative staining result does not exclude the presence of microorganisms in the specimen.
• Acridine orange staining requires a fluorescence microscope with suitable filters, which is more expensive than a conventional light microscope and may not be available in all laboratories.

Precautions of Acridine Orange Staining

The following are the precautions of acridine orange staining:

• Suitable gloves, eye protection and a laboratory coat should be used while handling acridine orange. The stain should not come in contact with the skin and eyes.
• Acridine orange powder should not be inhaled. The staining solution should be prepared in a well-ventilated area.
• The acridine orange solution should be protected from direct light. It should be stored in a dark bottle.
• A clean glass slide should be used for preparing the smear. The smear should be thin.
• Staining solution containing precipitate should not be used.
• Wash the slide properly after staining to remove the excess stain.
• Correct fluorescence filter and objective should be used during examination.
• A known positive control should be stained along with the test smear.
• Acridine orange waste should be discarded as laboratory chemical waste.

References

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