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Acid-fast stains and mycobacterial culture

16 min

Skills you'll build3 skills
  • Grade an acid-fast smear on the correct scale and report it without a species
  • Match mycobacterial decontamination to the specimen and its contamination rate
  • Distinguish M. tuberculosis complex from nontuberculous mycobacteria

At the bench: Read an organism stain and communicate a significant preliminary finding

Try first

Try first

An auramine-rhodamine smear of a concentrated sputum is read at 250× and averages 20 acid-fast bacilli (AFB) per field. What grade is reported?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Grading AFB smears

Acid-fast bacilli keep carbol fuchsin or auramine after acid-alcohol decolorization because of their mycolic-acid wall. They appear as red rods on a blue background with Ziehl-Neelsen or Kinyoun at 1,000×. With a fluorochrome stain they fluoresce yellow-orange on a dark field at 250× or 450×.1 A field at low magnification covers more smear, so the same count per field means a lower density. The report uses the 1+ to 4+ scale in the column for the stain and magnification used.1,2

ReportCarbol fuchsin, 1,000×Fluorochrome, 250×Fluorochrome, 450×
Exact count, repeat specimen1–2 per 300 fields1–2 per 30 fields1–2 per 70 fields
1+1–9 per 100 fields1–9 per 10 fields2–18 per 50 fields
2+1–9 per 10 fields1–9 per field4–36 per 10 fields
3+1–9 per field10–90 per field4–36 per field
4+More than 9 per fieldMore than 90 per fieldMore than 36 per field

A positive smear shows acid-fast bacilli. It cannot distinguish Mycobacterium tuberculosis complex (MTBC) from nontuberculous mycobacteria (NTM), so the report names no species.1

Specimen processing

Sputum and other specimens with normal flora are digested and decontaminated with N-acetyl-L-cysteine and sodium hydroxide (NALC-NaOH) for 15 minutes, then neutralized with phosphate buffer and concentrated. The sodium hydroxide also kills some mycobacteria, and longer contact kills more. Specimens from sterile sites are inoculated directly.3,4 The contamination rate shows whether decontamination is balanced. About 2% to 5% of solid media and 7% to 8% of liquid media are expected to contaminate. A lower rate with more smear-positive, culture-negative specimens suggests over-decontamination.3,5

Species identification

Broth and solid media are both inoculated. A culture is called negative only after 42 days in liquid systems and 8 weeks on solid media.3 Growth rate and pigment place mycobacteria into groups and can be misleading. Species identification uses a nucleic acid amplification test (NAAT), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with a mycobacterial library, or sequencing.4,6

Follow the bench demonstration

Sources for this step
References
  1. Centers for Disease Control and Prevention. Core Curriculum on Tuberculosis: What the Clinician Should Know. 7th ed. CDC; 2021. Accessed September 27, 2026. https://www.cdc.gov/tb/media/Core_Curriculum_TB_eBook.pdf
  2. Kent PT, Kubica GP. Public Health Mycobacteriology: A Guide for the Level III Laboratory. Centers for Disease Control; 1985.
  3. Association of Public Health Laboratories. Mycobacterium tuberculosis: Assessing Your Laboratory. APHL; April 2019. Accessed September 27, 2026. https://aphl.org/docs/default-source/technical/ID-2019Apr-TB-Toolkit.pdf
  4. Martin I, Pfyffer GE, Parrish N. Mycobacterium: general characteristics, laboratory detection, and staining procedures. In: Carroll KC, Pfaller MA, Landry ML, et al, eds. Manual of Clinical Microbiology. 12th ed. ASM Press; 2019:558-575.
  5. Forbes BA, Hall GS, Miller MB, et al. Practical guidance for clinical microbiology laboratories: mycobacteria. Clin Microbiol Rev. 2018;31(2):e00038-17. doi:10.1128/CMR.00038-17
  6. Daley CL, Iaccarino JM, Lange C, et al. Treatment of nontuberculous mycobacterial pulmonary disease: an official ATS/ERS/ESCMID/IDSA clinical practice guideline. Clin Infect Dis. 2020;71(4):e1-e36. Accessed September 27, 2026. https://pmc.ncbi.nlm.nih.gov/articles/PMC7768748/

Watch one

Read each decision and its explanation.

A Kinyoun-stained smear of a concentrated sputum is read under oil immersion at 1,000×. The laboratory professional counts 25 acid-fast bacilli across 100 fields. How is the smear graded and reported?

  1. Confirm the read: Kinyoun is a carbol fuchsin stain, read at 1,000×, so the carbol fuchsin column applies.

    The grading column depends on the stain and the magnification.

  2. Convert the count to the column's units: 25 AFB in 100 fields is 2.5 AFB per 10 fields.

    The rows of that column are written per 100 fields, per 10 fields and per field.

  3. Match the row: 1 to 9 per 10 fields is 2+.

    Each row covers a range of counts.

  4. Word the report: acid-fast bacilli seen, 2+, with culture and identification to follow.

    The stain shows acid-fastness and cannot separate MTBC from NTM.

Report "Acid-fast bacilli seen, 2+." The report names no species until culture, NAAT or another identification method gives one.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

An auramine-rhodamine smear read at 250× averages 5 acid-fast bacilli per field. What is the reported grade?

Incorrect. At 250×, 1+ is 1 to 9 organisms per 10 fields, far fewer than 5 per field.

Correct. In the 250× fluorochrome column, 1 to 9 per field is 2+. The grade comes from the column that matches the stain and magnification in use.

Incorrect. At 250×, 3+ is 10 to 90 per field. A count of 5 per field would read 3+ in the 1,000× carbol fuchsin or 450× fluorochrome column, where each field covers less of the smear.

Hint
  1. Find the column for a fluorochrome stain at 250×.
  2. In that column, find the row for a count of a few per field.

Review Acid-fast staining

Problem 2 of 3 · Hints available

A mycobacteriology bench records a contamination rate of 0.5% on solid media over the quarter, with a rise in smear-positive, culture-negative specimens. What does this pattern suggest?

Incorrect. Some contamination is expected, about 2% to 5% of solid media, so 0.5% with more smear-positive, culture-negative specimens indicates a decontamination problem.

Correct. The decontaminant must be mild enough for mycobacteria to survive, so about 2% to 5% of solid media are expected to contaminate. A rate below that range, with more smear-positive, culture-negative specimens, indicates excessive NaOH exposure, and the bench investigates its digestion time and digestant preparation.

Incorrect. Delayed transport and contaminated reagents raise the contamination rate above the expected range.

Hint
  1. Compare 0.5% with the rate expected on solid media.
  2. Ask what else the decontaminant can kill besides the contaminating flora.

Review Specimen collection and decontamination

Problem 3 of 3 · On your own

A mycobacterial broth from a bronchoalveolar lavage signals positive, and its smear shows acid-fast bacilli. At 3 weeks the subculture on Middlebrook 7H11 grows rough, buff, nonpigmented colonies. What does the laboratory do next?

Rough, buff colonies fit M. tuberculosis, and several NTM can look the same. Colony features only suggest a group.

Named a mycobacterium from growth rate and pigment

Growth rate and pigment only sort mycobacteria into groups. M. kansasii has rare nonchromogenic strains, and some M. avium complex (MAC) strains develop pigment late. A species named from those features can be wrong. MALDI-TOF with a mycobacterial library, probes, or sequencing gives the identification.

Many mycobacteria are nonpigmented, and some M. avium complex strains pigment late. Pigment cannot identify the species.

Named a mycobacterium from growth rate and pigment

Growth rate and pigment only sort mycobacteria into groups. M. kansasii has rare nonchromogenic strains, and some M. avium complex (MAC) strains develop pigment late. A species named from those features can be wrong. MALDI-TOF with a mycobacterial library, probes, or sequencing gives the identification.

Runyon groups describe growth and pigment and are obsolete for identification. Clinically significant mycobacteria are identified to species.

These methods identify the species or complex. Until then, the report says mycobacteria are growing with identification to follow.

Review Identification methods

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Tobias Parham, 58, a retired ferry mechanic, has had a cough for 6 weeks and has lost weight.
  • Two specimens arrive for AFB smear and culture: an expectorated sputum, 6 mL, and a pleural fluid, 20 mL, collected by thoracentesis.
  • The sputum is processed first. Its auramine-rhodamine smear, read at 250×, averages 3 AFB per field.
  • The pleural fluid smear shows no AFB.
Decision 1 of 3

How is each specimen prepared for culture?

Pleural fluid comes from a sterile site and has no flora to remove. Decontaminating it kills some of the mycobacteria it contains.

Decontaminated every specimen whatever its source

Sterile-site specimens can be inoculated directly, and specimens with normal flora need NALC-NaOH digestion and decontamination. Even the standard 15-minute exposure kills some mycobacteria, and each extra minute kills more, so unnecessary or prolonged decontamination lowers recovery.

Sputum contains oral flora that would overgrow the culture. The pleural fluid is sterile, so it goes to culture without decontamination.

Oral flora in the sputum would overgrow the media and mask slow-growing mycobacteria.

The standard exposure is 15 minutes. Longer contact with sodium hydroxide kills more mycobacteria and lowers recovery.

Review Specimen collection and decontamination

Decision 2 of 3

What grade does the sputum smear get?

Three per field is 3+ in the 1,000× carbol fuchsin column. This smear was read with a fluorochrome at 250×.

Graded a smear on the scale for another magnification

The same count means different densities at different magnifications, so 5 AFB per field is 2+ at 250× fluorochrome and 3+ at 450× fluorochrome or 1,000× carbol fuchsin. Using the scale for another stain or magnification misstates the grade by one level.

At 250×, 1+ is 1 to 9 AFB per 10 fields. This smear has 3 in every field.

In the 250× fluorochrome column, 1 to 9 AFB per field is 2+.

Review Acid-fast staining

Decision 3 of 3

How is the sputum smear reported?

This reports what the stain showed. The organism is named after culture and identification.

The smear shows acid-fastness only. NTM and other acid-fast organisms look the same on it.

Reported M. tuberculosis from an acid-fast smear

A positive smear shows acid-fast bacilli without separating M. tuberculosis complex (MTBC) from nontuberculous mycobacteria or environmental acid-fast organisms. Reporting M. tuberculosis from the smear names a species the stain cannot identify. Species identification needs culture, NAAT, or another identification method.

Other acid-fast organisms stain the same way, so the stain cannot confirm mycobacteria. The report also leaves out the grade, which shows how many were seen.

Review Acid-fast staining

The findings that matter are the source of each specimen and how its smear was read. The sputum contains flora and needs decontamination, and the pleural fluid does not. The 250× fluorochrome column grades the sputum 2+, and the report names acid-fast bacilli without a species.

For reference

Sources checked