Tissue and Biopsy Specimens: Collection, Transport, and Processing

Tissue is the best specimen microbiology can receive for a deep or serious infection. A biopsy samples the organisms actually invading the tissue, not the flora on the surface, which is why it beats pus, wound, and sterile-fluid in terms of sensitivity. And yet tissue is one of the easiest specimens to waste, because the errors that destroy it happen in the first few minutes, at the bedside or on the cutting bench, before the sample ever reaches a culture plate. This article is about handling tissue so that its advantage is not thrown away.

Tissue is collected by a surgeon, an interventional radiologist, or a clinician performing a biopsy. Microbiology does not collect it, but what happens to the sample in the moments after it is collected decides whether the culture can work at all.

The one rule that outranks everything: split for microbiology first

A biopsy is often small, and it is often wanted for more than one purpose. The instinct in operation theatre is to drop the whole piece into the formalin pot for the pathologist. If that happens, microbiology gets nothing usable, because formalin kills every organism and makes culture impossible.

So the governing rule is: the portion for microbiology is separated first, into a sterile container, before any part of the specimen touches fixative. Once tissue is in formalin, it is a histopathology specimen only. There is no way to recover it for culture. The same logic applies to bone and decalcification.

One tissue, many tests: how the sample is divided

A single biopsy may need to answer several questions at once: routine bacteria, mycobacteria, fungi, and histopathology. Each has different handling, and some of the requirements conflict, so the division must be deliberate.

The workflow that protects every test: take the fresh microbiology portions first (dividing further for bacterial, AFB, and fungal as needed), keep them moist in a sterile container, and only then place the remaining piece in formalin for histology. When volume is very limited, the clinician and laboratory prioritize together based on the most likely diagnosis.

Grinding versus mincing: the mold exception

To release organisms trapped inside tissue, the laboratory normally homogenizes (grinds) the specimen before culture. There is one important exception.

If a mucormycosis (a Mucorales or zygomycete mold) infection is suspected, the tissue is minced or cut into small pieces, not ground. The hyphae of these molds are broad, non-septate, and fragile, and grinding shatters them, so the culture turns falsely negative even when the fungus is clearly present on histology. Cutting the tissue into small fragments and pressing them onto the media preserves viable hyphae. This is why suspected mucormycosis must be mentioned on the request form.

Bone and osteomyelitis: the decalcification trap

For osteomyelitis, bone is the specimen, and the split-first rule becomes even more important because of an extra hazard.

Bone sent for histopathology is decalcified, usually in an acid solution, so that it can be sectioned and stained. That acid decalcification also kills any organisms present. So a piece of bone that goes straight to histology cannot then be cultured, for the same reason formalin-fixed tissue cannot: the processing has sterilized it.

The rule: the microbiology portion of a bone specimen is split off before the bone is fixed or decalcified. Send the culture portion fresh, in a sterile container, kept moist, and let histology decalcify only its own share. As with soft tissue, once bone has been through fixation or decalcification, it is a histopathology specimen only.

Keep it moist, but do not drown it

Small tissue pieces dry out quickly, and a desiccated specimen loses viable organisms. To prevent this, a few drops of sterile saline are added to keep the tissue moist during transport. Use enough saline to stop drying, not so much that it dilutes an already small specimen.

Transport

• Deliver tissue to the laboratory as soon as possible.
• Keep it in a sterile, leak-proof container with a few drops of sterile saline to prevent drying.
• Never place tissue intended for culture in formalin. Formalin is for histopathology only.
• If anaerobes are suspected (deep, necrotic, or abscess-associated tissue), use an anaerobic transport system.
• Refrigeration is acceptable for a short delay for routine bacterial culture, but not when fastidious organisms are suspected; keep those at room temperature and transport quickly.
• Flag suspected mycobacterial, fungal, or Mucorales infection on the request, because it changes how the lab processes the sample.

How Microbiology Lab Process Tissue and Biopsy Specimen

Once tissue reaches the bench, the microbiology portion is processed as follows.

Direct examination. A portion is used for immediate microscopy, and this often gives the first clue before any culture grows:

• Gram stain for bacteria and yeasts.
• A KOH or calcofluor-white preparation when a fungus is suspected, which shows hyphae or yeast forms. For a suspected Mucorales mold, the broad, non-septate (aseptate) hyphae branching at wide angles are the giveaway.
• A Ziehl-Neelsen or auramine-rhodamine stain when mycobacteria are suspected.
• A portion is sent in parallel for histopathology, which complements culture by showing the organism in the tissue and the host reaction around it.

Homogenization, with the mold exception. For routine bacterial and fungal culture, the tissue is ground to release organisms trapped within it. As covered above, tissue from a suspected Mucorales infection is minced, not ground, to protect the fragile hyphae.

Culture media. The prepared tissue is inoculated onto a range of media chosen to cover the likely pathogens, because tissue is often taken precisely when the cause is unknown:

• Blood agar and chocolate agar for common aerobes and fastidious organisms.
• MacConkey agar for gram-negative bacilli.
• An anaerobic medium (and an enrichment broth such as thioglycollate) for deep or necrotic tissue, where anaerobes are common.
• Sabouraud agar when a fungus is suspected.
• Lowenstein-Jensen or a liquid mycobacterial medium when tuberculosis or another mycobacterial infection is suspected.

Incubation. Bacterial plates are incubated at 35 to 37 degrees C, with chocolate agar and fastidious cultures in 5% CO2, and read over 24 to 48 hours and beyond. Fungal and mycobacterial cultures are held far longer, weeks, because these organisms grow slowly.

Reading and interpretation. Tissue comes from a normally sterile site, so any organism recovered is potentially significant.

Growth is read against the direct smear and the histopathology: an organism seen on the Gram stain or in the tissue section and then grown in culture is far more convincing than a single colony with no supporting evidence, which may reflect contamination at collection.

Identification and antimicrobial susceptibility testing

Once an organism is isolated, it is identified and, where relevant, tested for antimicrobial susceptibility. Tissue is taken when the cause is often unknown, so a wide range of organisms may grow. The most commonly isolated, and the first approach to each:

• Staphylococcus aureus: gram-positive cocci in clusters, catalase positive, coagulase positive. The commonest cause of bone, joint, and deep soft-tissue infection. See the Staphylococcus aureus article for full identification and susceptibility, including methicillin resistance.
• Streptococcus and Enterococcus species: gram-positive cocci in chains or pairs, catalase negative; distinguished by hemolysis and further reactions.
• Gram-negative bacilli (Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Proteus): common in diabetic foot, chronic, and post-surgical tissue infections; separated on MacConkey by lactose fermentation, with Pseudomonas as a non-lactose fermenter with a characteristic look and smell.
• Anaerobes (Bacteroides, Clostridium, anaerobic cocci): common in deep, necrotic, and abscess-associated tissue; grow on anaerobic media and are identified from there.
• Mycobacteria (Mycobacterium tuberculosis and others): acid-fast on Ziehl-Neelsen; slow-growing on selective mycobacterial media. See the tuberculosis diagnosis page.
• Fungi (Aspergillus, Mucorales molds, Histoplasma, Candida): shown on KOH or calcofluor-white and grown on Sabouraud agar; Mucorales handled by mincing, not grinding, as above.

How to Remember

Split for micro before anything is fixed. Formalin and acid decalcification both kill organisms, so the culture portion comes off first, into saline, before histology gets its share.

Tissue beats swab because it holds the invaders. A swab samples the surface; tissue samples the organisms inside. That is why for deep infection, tissue is the best specimen.

Grind routine tissue, mince a suspected mold. Grinding releases organisms from ordinary tissue, but it shatters the fragile hyphae of Mucorales and gives a false-negative culture. Suspected mucormycosis is minced, not ground.

Bone adds the decalcification trap. Bone for histology is dissolved in acid, which also kills organisms. So the culture portion of bone should be taken off before any fixing or decalcifying, exactly like the formalin rule for soft tissue.

Moist, not drowned. A few drops of saline stop the tissue drying; a pool of saline dilutes a small sample. Aim for damp.

Key exam facts in one table

Where Students Get Confused

"Why not just send the whole biopsy in formalin and let each lab take what it needs?" Because formalin kills every organism, so once tissue is fixed, culture is impossible. The microbiology portion must be separated into a sterile container before anything touches formalin. Fixed tissue is a histopathology specimen only.

"Tissue is the best specimen, so how can it give a false-negative culture?" Most often through handling. Formalin fixation, acid decalcification of bone, and grinding a fragile mold all destroy the organisms before they can grow. The specimen was excellent; the processing wasted it.

"Why mince rather than grind when mucormycosis is suspected?" The hyphae of Mucorales molds are broad, non-septate, and easily shattered. Grinding fragments them and the culture comes back negative even when histology clearly shows the fungus. Cutting the tissue into small pieces preserves viable hyphae, so suspected mucormycosis must be reported so the lab minces instead of grinds.

"Bone went to histology and grew nothing on culture, why?" Bone for histology is decalcified in acid, which kills organisms just as formalin does. The culture portion of bone must be split off fresh before any fixation or decalcification, or there is nothing viable left to grow.

"How much saline should go in with the tissue?" Just a few drops, enough to keep it moist. Too much saline dilutes an already small specimen. The goal is damp tissue, not tissue floating in fluid.

References and further reading

1. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
2. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. DOI: 10.1128/9781683670438.CMPH
3. Procop GW, Church DL, Hall GS, et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.