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Wounds, tissue and anaerobes

15 min

Skills you'll build2 skills
  • Select a wound specimen that represents the infected tissue
  • Recognize bite wounds and deep abscesses that need anaerobic culture

At the bench: Select specimen preparation, media, and incubation for a culture

Try first

Try first

A surgeon wants to know which organism is invading a chronic diabetic foot ulcer. Which specimen answers that question best?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Choosing the wound specimen

Tissue and aspirates recover more organisms than swabs, and they come from the infected site. A surface swab of a chronic ulcer picks up skin flora and colonizers. For a chronic ulcer, collect deep tissue or an aspirate after debridement.1 When anaerobic culture is requested, tissue or aspirate from a deep wound goes into an anaerobic transport device.1

Interpreting the smear and the culture

Report the wound Gram stain with quantitated neutrophils and squamous epithelial cells. Many squamous cells indicate surface contamination, and abundant neutrophils support active infection.2

Significance depends on the source.1,2

  • Staphylococcus aureus and beta-hemolytic streptococci get a full workup from any wound.
  • Pseudomonas aeruginosa and the Enterobacterales are significant from deep tissue. On the surface of a chronic wound they are common colonizers.
  • A polymicrobial superficial culture is reported as mixed flora, with a note that full identification and susceptibility testing are not indicated.

Gram-positive cocci in chains in tissue, or large boxcar gram-positive rods without neutrophils, are called to the provider at once.2

When anaerobes belong in the workup

Anaerobes take part in mixed infections after mucosa or tissue is disrupted. Bite wounds and deep abscesses are the usual examples. Infected dog and cat bites grow a median of 5 organisms, aerobes and anaerobes together.2 An aerobic culture cannot grow anaerobes, so these specimens need anaerobic transport and anaerobic culture.

Obligate anaerobes form no colonies on plates incubated in air or in 10% CO2. The anaerobic setup follows a few rules.3

  1. Plate an enriched blood agar with vitamin K and hemin, selective agars and a broth.
  2. Incubate at 35 to 37 °C in an oxygen-free atmosphere checked by an indicator.
  3. Keep jars and pouches closed for at least 48 hours, because exposure to air during early growth kills slow-growing species.
  4. Incubate plates 5 to 7 days when slow growers are possible.

Actinomyces grows slowly, and its cultures are kept for up to 2 to 3 weeks. Crushed sulfur granules show branching gram-positive filaments.2 Stool is not a specimen for general anaerobic culture, because anaerobes dominate normal fecal flora.3

Follow the bench demonstration

Sources for this step
References
  1. Miller JM, Binnicker MJ, Campbell S, et al. Guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2024 update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. Published online March 5, 2024. doi:10.1093/cid/ciae104
  2. Tille PM. Bailey & Scott's Diagnostic Microbiology. 16th ed. Elsevier; 2025.
  3. Nagy E, Boyanova L, Justesen US. How to isolate, identify and determine antimicrobial susceptibility of anaerobic bacteria in routine laboratories. Clin Microbiol Infect. 2018;24(11):1139-1148. doi:10.1016/j.cmi.2018.02.008

Watch one

Read each decision and its explanation.

A surface swab of a chronic venous leg ulcer is sent for aerobic culture.

  • Gram stain: many squamous epithelial cells, few neutrophils, mixed gram-positive cocci and gram-negative rods.
  • At 24 hours: S. aureus, P. aeruginosa, a lactose-fermenting gram-negative rod and mixed skin flora.

What do you work up, and how do you report the rest?

  1. Read the source: a surface swab of a chronic ulcer, where colonizers are expected.

    The source determines what each organism can mean.

  2. Read the Gram stain: many squamous cells and few neutrophils indicate surface contamination.

    The smear shows how much of the specimen is surface material.

  3. Look first for organisms that get a full workup from any wound: S. aureus is present, so it gets identification and susceptibility testing.

    S. aureus is an important potential pathogen in any wound.

  4. Consider the gram-negative rods: P. aeruginosa and the lactose fermenter are significant from deep tissue, and these came from the surface.

    Gram-negative rods on the surface of a chronic wound are usually colonizers.

  5. Report the rest as mixed flora, with a comment that deep tissue after debridement can show whether the gram-negative rods are invading.

    Full identification of surface colonizers cannot show which one, if any, is invading.

Identify S. aureus and test its susceptibility. Report the gram-negative rods and skin flora as mixed flora, with a comment that deep tissue culture after debridement can show whether they are invading.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A surface swab of a chronic leg ulcer grows Pseudomonas aeruginosa, two lactose-fermenting gram-negative rods, and mixed skin flora, with no Staphylococcus aureus or beta-hemolytic streptococci. How is the culture reported?

Incorrect. A polymicrobial surface culture of a chronic ulcer reflects colonizers, and working up every isolate cannot show which organism, if any, is invading.

Correct. A surface culture of a chronic ulcer reflects colonizers. The report states mixed flora, with a note that full identification and susceptibility testing is not indicated and a request for deep tissue or an aspirate taken after debridement. P. aeruginosa and the Enterobacterales are significant from deep tissue.

Incorrect. P. aeruginosa is a common colonizer of superficial chronic wounds, so a surface swab cannot show that it is invading, and its significance has to be judged from a deeper specimen.

Hint
  1. Begin with the specimen source.
  2. Check whether any organism on the plate gets a full workup from every wound.

Review Gram stain interpretation and organism significance

Problem 2 of 3 · Hints available

Pus aspirated from a deep abscess shows many neutrophils, gram-positive cocci and slender gram-negative rods with pointed ends on Gram stain. At 48 hours the aerobic plates show no growth. Only aerobic culture was set up. What does the result mean?

Organisms seen with many neutrophils in an abscess are the likely cause. The plates were incubated in air, where obligate anaerobes form no colonies.

An aerobic culture cannot grow anaerobes. A negative aerobic result leaves the anaerobes seen on the smear untested.

Excluded anaerobes after an aerobic-only culture

Anaerobes are expected in mixed infection after mucosal or tissue disruption, as in bite wounds and deep abscesses, and they need anaerobic transport and culture. An aerobic-only culture cannot grow them, so a negative aerobic result leaves an anaerobic infection unexamined.

A deep abscess with organisms on the smear and no aerobic growth fits a mixed anaerobic infection. Anaerobic culture on the remaining specimen, or a new specimen in anaerobic transport, is needed.

Hint
  1. Compare what the smear shows with what the plates could grow.
  2. Consider the atmosphere the plates were incubated in.

Review Gram stain interpretation and organism significance

Problem 3 of 3 · On your own

Pus from a draining sinus of the jaw contains yellow grains about 1 mm across. A crushed grain shows branching gram-positive filaments radiating from a center. How is the culture set up?

The filaments fit Actinomyces, an anaerobe. Aerobic plates read at 48 hours miss it.

Sulfur granules with branching gram-positive filaments suggest Actinomyces. It needs enriched anaerobic culture and grows slowly, so plates are kept for weeks.

Nocardia grows in air and is modified acid-fast positive. Actinomyces grows anaerobically and is modified acid-fast negative, and the granules fit Actinomyces.

Review Gram stain interpretation and organism significance

Use it

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

  • Theo Miller, 34, a veterinary technician, was bitten on the hand by a cat 2 days ago.
  • The hand is swollen, and the surgeon drains an abscess.
  • The surgeon sends two specimens: pus in an anaerobic transport vial and a swab of the skin around the bite.
  • The order reads "wound culture".
Decision 1 of 3

Which specimen is cultured as the wound specimen?

The skin swab contains skin flora from around the bite. It cannot show what is growing in the abscess.

Worked up surface-swab flora as the deep-wound pathogen

A surface swab of a chronic wound grows colonizers such as Pseudomonas aeruginosa and Enterobacterales, whose significance can be judged only from deep tissue. Working up the surface isolates as pathogens can report the wrong organism. Tissue or an aspirate collected after debridement shows what is invading.

Working up skin flora alongside the abscess organisms reports organisms that are not part of the infection.

Pus drained from the abscess comes from the infected site. It is also in an anaerobic transport vial.

Review Specimen strategy by lesion type

Decision 2 of 3

How is the pus set up?

An infected cat bite is polymicrobial, with aerobes and anaerobes together. Both setups are needed to recover them.

Cat-bite organisms that grow in air are only part of the mix. Anaerobes from the cat's mouth need anaerobic culture.

Excluded anaerobes after an aerobic-only culture

Anaerobes are expected in mixed infection after mucosal or tissue disruption, as in bite wounds and deep abscesses, and they need anaerobic transport and culture. An aerobic-only culture cannot grow them, so a negative aerobic result leaves an anaerobic infection unexamined.

The aerobic organisms of a bite wound are part of the infection too. The vial preserves anaerobes and does not prevent aerobic culture.

Review Gram stain interpretation and organism significance

Decision 3 of 3

At 24 hours the aerobic plates grow a gram-negative rod on blood and chocolate agar, with no growth on MacConkey. The clinician asks for the final report today. What do you send?

A final report now would close the culture before the anaerobes had time to grow. Anaerobic jars stay closed for at least 48 hours.

Released a preliminary report as final

A preliminary report releases verified findings before the planned work is done, and its status must be clear. Presenting it as final can end follow-up on pending identification and susceptibility results. A later error needs a corrected report, and new information goes in an addendum.

The aerobic growth can be reported now as a preliminary result. The anaerobic plates stay closed for at least 48 hours and are kept until the culture is complete.

Withholding the aerobic result delays useful information. A preliminary report releases it while the culture continues.

Review Preliminary, final, added, and corrected reports

The workup follows from the bite. A bite drives mouth flora deep into the tissue, so the abscess pus is the specimen, and it needs anaerobic culture along with the aerobic plates.

For reference

Sources checked