Answering your questions

Jan. 1, 2004

Answering your questions

Lab concerns elicit expert opinions

Incubator controls 

Q I run a CLIA-waived lab in a small, suburban clinic. To perform controls on our incubator for strep cultures, I use 5% sheeps blood agar plates,
S.agalactiae and S.pyogenes swabs to inoculate, and bacitracin discs. The incubator stays steady between 35C and 37C. For the control to be accurate, the
S.agalactiae must show negative, and the S.pyogenes must show positive. I use the swabs the first week and inoculate from the plates each of the next two weeks. Approximately three times per year, for reasons I cannot explain, my
S.agalactiae will show positive on the first plate inoculating directly from the swab. If I inoculate the plates the next week using the positive plate, however, it will read negative at the end of the three-day growth period. I have tried taxo A discs, a different brand of bacitracin disc, and the same outcome has occurred. Can you offer any explanation as to why this is happening? 

A Streptococcus pyogenes (group
A streptococcus) and Streptococcus agalactiae (group B streptococcus) will both grow on 5% sheeps blood agar (SBA). A test for bacitracin susceptibility can be helpful in differentiating
S.pyogenes from S.agalactiae. After overnight incubation at 35C and 37C on an SBA plate that has been heavily inoculated with three to four colonies of a pure culture of the organism, any zone of growth inhibition around the disk is interpreted as indicating susceptibility. Most stains of
S.pyogenes will show susceptibility to bacitracin, where as most strains of
S.agalactiae will be

It would be prudent to subculture the organisms prior to performing your test. The NCCLS document M22 on the quality control (QC) of microbiology media indicates that subcultures should be prepared from lyophilized, frozen, or stock cultures prior to performing QC
tests.2 When taken directly from transport swabs or other such transport/storage conditions, organisms may not give the appropriate reactions when performing some types of QC. They often need to adapt to their new environment prior to being tested. Subculturing the organism from the swab onto a SBA plate without the bacitracin disk one day prior to performing your QC should alleviate this problem. This would explain why the test works when a previously plated organism is used.

Assuring that the temperature of your incubator is between 35C and 37C is very important; the QC described does not necessarily assure this. Monitor your incubator temperature with a standardized thermometer, and keep a record that it is actually heating the incubator to between 35C and 37C whenever you have patient or QC samples testing inside. What you are actually controlling when testing the above organisms with a bacitracin disk is the bacitracin disk itself. Although it is correct that your incubator temperature must be accurate for your organisms to grow optimally, this QC actually tests that the disk you are using contains the appropriate amount of bacitracin so that you get the correct results with the
S.pyogenes and S.agalactiae. If you were to get the same results for both organisms (e.g., both organisms showed no zone of growth inhibition), the content of the bacitracin disk could be in error.

Susan E. Sharp, PhD DABMM
Director of Microbiology
Kaiser Permanente;
Associate Professor
Pathology Regional Laboratory
Oregon Health Science Univ.
Portland, OR


  1. Ruoff KL, Whiley RA, Beighton D. Streptococcus. In: Manual of Clinical Microbiology. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. 8th ed. ASM Press. 2003:405-421.
  2. Krisher K, Callihan DR, Jones RN, Luper DC, Miller JM, Sharp SE, Shively RG. NCCLS. Quality control for commercially prepared microbiological culture media; proposed standard 2nd ed. NCCLS document M22-P2 (ISBN 1-56238-504-506). 2003.

Environmental cultures

Should a clinical microbiology lab in a community hospital be doing environmental cultures on specimens collected in the hospital? I am referring specifically to culturing of deionized water, swabs of cleaned whirlpools in physical therapy, water samples from cleaned endoscopes, water from dialysis tubing, and sterilized spore strips from the operating room and central processing. If it is appropriate, are there any references to use as guidelines for procedures and interpretation of results? 

The routine microbiologic sampling of the hospital environment is not useful or cost effective unless it is performed for a specific epidemiologic purpose (e.g., investigate a cluster of nosocomial respiratory infections), to monitor sterilization processes, or to detect contamination of water used in the dialysis unit and the laboratory. Therefore, the need to perform environmental cultures should be determined by the members of the infection- control committee and to include, as a minimum, a representative from the microbiology laboratory, the hospital epidemiologist, and the infection control practitioner. The specimens should be sent to an appropriate reference laboratory in the event that the hospital laboratory cannot perform the cultures.

After approval by the infection control committee, the microbiology laboratory must apply the appropriate procedure and interpretive guideline for the myriad of potential specimens that may be submitted for culture. The problem confronting the microbiology laboratory is that the procedures for culturing potential environmental specimens are scattered among numerous publications, associations, societies, governmental agencies, and organizations. For example, NCCLS
( publishes a procedure for the evaluation of laboratory
water.1 The Association for the Advancement of Medical Instrumentation
( publishes standards for the water used to prepare dialysate used in dialysis units. These standards include the procedures and interpretive guidelines for endotoxin contamination and total viable microbial counts in the water. Often, the dialysis unit has a copy of these standards. The American Society for Microbiology
( publishes a handbook containing procedures for evaluating strilization processes and culture of water and other environmental
specimens.2 Additional information can be obtained from the Centers for Disease Control and Prevention
( on healthcare guidelines; the American Public Health Association
( on standards for the examination of water and wastewater; and other selected
publications.3,4 Also, the members of the infection control committee are a valuable resource for identifying a source for a specific procedure.

David Sewell, PhD, ABMM
Director of Microbiology
Veterans Affairs Medical Center
Portland, OR


  1. NCCLS. Preparation and testing of reagent water in the clinical laboratory. 1997.
  2. Gilchrist MRJ, ed. Epidemiologic and infection control microbiology. in: Clinical Microbiology Procedures Handbook, Isenberg HD, ed. American Society for Microbiology. 1992;pp:11.0.1-11.17.4.
  3. Mietzner SM, Stout JE. Laboratory Detection of Legionella in Environmental Samples.
    Clin Microbiol Newsl. 2002;24:81-85.
  4. Wenzel R. Prevention and Control of Nosocomial Infections. Lippincott, Williams and Wilkens. 1993. new edition 2003.

Daniel M. Baer is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLOs editorial advisory board.

January 2004: Vol.
36, No. 1

2004 Nelson Publishing, Inc. All rights reserved.

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