Any tissue specimen acquired surgically should be considered a sterile specimens and the isolation of any organism(s) considered significant. These are generally collected in the operating room or in radiology and are priority specimens. Setup and work up for all types of bacteria and fungi is indicated.
Some specimens identified as tissue are wound debridement tissue (in which case the tissue has been exposed for some time to the outside world) and are much “dirtier” in nature. A more limited setup (no fungi unless specifically asked for) and workup is appropriate for these types of specimens.
Lung biopsy tissue specimens are processed somewhat differently than other tissues. Refer to Lung Biopsy.
Direct Examination
Prepare 3 touch preparation slides using fresh cut surfaces of representative material. Gram stain, KOH Calcofluor stain and one unstained for potential future use.
Culture
Macerate the tissue using a grinder. Bone should be inoculated directly into thioglycolate broth and is not macerated.
Fungus culture is NOT set up for wound debridement tissue, unless specifically requested.
| Media | Incubation |
| Blood Agar (BA) | CO2, 35°C x 48 hours |
| Chocolate Agar (CHOC) | CO2, 35°C x 48 hours |
| MacConkey Agar (MAC) | O2, 35°C x 48 hours |
| Wilkins Chalgren Agar (WC) | AnO2, 35°C x 48 hours |
| WC Nalidixic acid – Tween 80 (NAT) | AnO2, 35°C x 48 hours |
| WC Nalidixic acid – Vancomycin (NAV) | AnO2, 35°C x 48 hours |
| Thioglycolate broth (THIO) | O2, 35°C x 7 days |
If surgical tissue specimens (organs etc., obtained in operating room), or specifically requested on other specimens, set up:
| Sabouraud’s Heart Infusion Agar (SABHI) | O2, 30°C x 4 weeks |
| SABHI with Chloramphenicol (SABHI-C) | O2, 30°C x 4 weeks |
Culture
Examine the BA, MAC and CHOC after 24 and 48 hours incubation and the WC, NAT and NAV after 48 hours incubation. Examine the THIO daily for evidence of growth. If no growth on culture plates but evidence of growth in THIO, then perform Gram stain and subculture THIO onto BA, MAC, CHOC and WC (as appropriate) and incubate and process as above.
If organisms were seen in direct Gram stain and cultures yield no corresponding growth after 48 hours of incubation, check direct Gram stain (if discrepant compared to original report, check with the Charge technologist), and re-incubate all aerobic plates and broth for a total of 7 days.
Identify all isolates.
Gram stain
Report with quantitation the presence of WBCs and organisms.
Telephone all positive Gram stain results to ordering physician or ward / unit.
Culture
Negative Report
“No growth”
Positive Report
Report all isolates with quantitation and appropriate susceptibilities.
Telephone all positive culture results to ordering physician or ward / unit.
H.D. Isenberg, 2004. Wound Cultures – Wound and Soft Tissue Cultures, p. 3.13.1.1 – 3.13.1.16. In Clinical Microbiology Procedures Handbook, 2nd Edition, Vol 1 ASM Press, Washington, D.C.
Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988
H.D. Isenberg. 2004. Microbiological Assessment of Orthopedic Surgery Sites p. 13.14.1 – 13.14.6. In Clinical Microbiology Procedures Handbook, 2nd Edition, Vol 1 ASM Press, Washington, D.C.
H.D. Isenberg. 2004. Culture for anaerobes p. 4.3.1 – 4.3.9 In Clinical Microbiology Procedures Handbook, 2nd Edition, Vol 1 ASM Press, Washington, D.C.
H.D. Isenberg. 2004. Examination of Primary Culture plates for anaerobic bacteria. p. 4.4.1 – 4.4.6. In Clinical Microbiology Procedures Handbook, 2nd Edition, Vol 1 ASM Press, Washington, D.C.
H.D. Isenberg. 2004. Incubation techniques for anaerobic bacteriology specimens. p. 4.5.1 – 4.5.4 In Clinical Microbiology Procedures Handbook, 2nd Edition, Vol 1 ASM Press, Washington, D.C.
Cumitech 5A Practical anaerobic bacteriology December 1991