Beth Israel Deaconess Medical Center Laboratory Manual

The Microbiology Laboratory offers a broad range of services for the diagnosis of infectious diseases including bacteriology, virology, mycology, mycobacteriology, parasitology, serology, and molecular microbiology.

Appropriate diagnostic techniques are applied as described in BIDMC Panels, Descriptions, and References , for most routine specimen types. For instance, if a brain abscess sample is received, Gram stain, aerobic and anaerobic bacteriologic cultures are performed routinely. However, certain organisms require special procedures for collection or processing. Examples are: Legionella, Brucella, Nocardia, viruses and many molds. Therefore, please notify the laboratory if a specific pathogen is suspected.

The technologists in the laboratory are available to assist clinicians who wish to review slides or other results.  In addition, the Microbiology Resident and Laboratory Director are available to provide consultation concerning diagnostic or interpretive problems.

A number of pathogens can be detected directly by staining smears made from infected patient specimens.

Bacterial and fungal (yeast) pathogens are most commonly visualized by the Gram stain, which is routinely performed on sterile tissue and body fluid specimens, sputum, and deep wound specimens and may be specifically ordered on other types of specimens. (Gram stains on unspun urine should be limited to critically ill patients.)

Specimens for mycobacterial detection are stained (fluorochrome and/or Kinyoun) and cultured, and positive results are phoned STAT to the requesting physician.

Wet mounts for fungi, cryptococcal antigen testing, Legionella urinary antigen testing, C. difficile toxin testing, and stains for Pneumocystis carinii, Nocardia, parasites and viruses are available as described in the alphabetical listing of tests. Molecular testing for Chlamydia trachomatis, Neisseria gonorrhoeae by PCR, DNA quantitation for HBV (viral load), RNA quantitation for HIV and HCV (viral loads), and HCV genotyping are also available.

Significant aerobic isolates will be identified and tested for susceptibility to a variety of oral and parenteral antibiotics. The antibiotics tested and the method of susceptibility testing will depend on the species and the specimen type.  Susceptibility testing for drugs other than those reported may be requested when necessary.

Specimens for anaerobic culture must be transported anaerobically. Specimens transported anaerobically also are acceptable for aerobic bacteria, fungi and mycobacteria, provided that sufficient material is submitted. Tissue and syringe aspirates are far superior to swabs for recovery of anaerobes, fungi and mycobacteria.

A full work-up of all organisms from a specimen in which more than two to three organisms are present is rarely clinically useful. When this "mixture" of microorganisms resembles the normal flora expected from that specimen type, they will be reported as such (i.e., sparse respiratory flora). If you have any questions about how the specimen should be worked up, contact the Clinical Manager, the Microbiology resident or the Laboratory Director on call.

Direct and concentrated smears and culture for detection of AFB are performed at BIDMC. Positive cultures are referred to the Massachusetts State Laboratory for identification by molecular methods. All M. tuberculosis are tested for susceptibility at the State Laboratory. Direct molecular amplification for M. tuberculosis is available from the State Laboratory on smear-positive samples only, since sensitivity for smear-negative samples is not sufficiently high to rule out M. tuberculosis infection with confidence. All requests for direct molecular amplification must be accompanied by culture requests. First positive AFB smear, first positive mycobacterial culture, and identification of M. Tb are called.

Yeast and mold pathogens are detected directly (Calcofluor White stained wet mounts or cryptococcal antigen) or by culture in the Mycology section. Most clinically encountered yeasts and opportunistic molds will be isolated within 3 to 5 days of culture. Dimorphic molds causing systemic disease may take up to 4 weeks for isolation. Yeast isolates may require 3 to 5 days and molds 7 to 14 days for identification after isolation. Pneumocystis carinii is detected from respiratory samples or tissue by a fluorescein labeled monoclonal antibody stain.

Stool parasites are detected by examination of concentrates and by stained smears of stool material, including fluorescein labeled monoclonal antibody detection of Giardia and Cryptosporidium by DFA. This section also identifies adult helminths and arthropods of medical importance, as well as providing consultation for blood parasite identification.

Commonly ordered tests include antibody to CMV, EBV, VZV, Helicobacter pylori, Lyme, Rubella, Syphilis, Toxoplasma, and measles, as well as Legionella urinary antigen, Cryptococcal antigen, and C. difficile A toxin immunoassay. Results for the above tests will be found in the computer in the Microbiology System. Serologic testing for unusual pathogens may require acute and convalescent serum samples.

Specimens for Human Immunodeficiency Virus (HIV) antibody testing must be accompanied by a properly executed Informed Consent form and test results will be available in the computer in the Chemistry System. Hepatitis (A, B, and C) antibody testing results will also be found in the computer under the Chemistry System.

The following viruses can be cultured by the Virology section: herpes viruses (HSV, CMV, VZV), enteroviruses (ECHO, coxsackie, polio), adenovirus, influenza and parainfluenza virus, RSV, measles and mumps. A rapid test for direct detection of influenza is available during influenza testing as well as a rapid respiratory viral panel for direct detection of influenza A/B, RSV, parainfluenza, and adenovirus.  The use of the latter test is intended specifically for severely immunocompromised patients and those with life threatening penumonia.  Direct detection of HSV and VZV from skin or mucsoal lesions is available by immunofluorescence methods. This section also performs molecular amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae, quantitation of HIV, HCV, CMV, and HBV viral loads and HCV genotyping.

• For direct examinations and serologic diagnosis, look under the name of the procedure (e.g., Gram stain) or under either the name of the organism or type of organism.
 
ex: Legionella Antibody

ex: Parasite (Blood) Screen
 

• For culture, look under "C" for "culture". These have been listed in several different ways:
 
according to the way in which the specimen was obtained
  ex: Culture, Aspirate

ex: Culture, Respiratory (Bronchial Washings)
 

according to the organism or type of organism suspected  
ex: Culture, Campylobacter

ex: Culture, Fungal
 

according to the site being sampled  
ex: Culture, Genital

ex: Culture, Respiratory (Sputum)
 

according to the specimen received  
ex: Culture, Catheter or Line

Whenever possible, the relevant information has been included in the individual test listings. General information about how to obtain or transport specimens for Microbiology is listed in the Collection Guidelines Appendix.