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Chapter 3.3 : Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures

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Abstract:

The initial processing of clinical specimens for bacteriology is a multifaceted endeavor involving a number of decision-making steps, including the need for processing the specimen for anaerobic bacteriology, mycology, virology, and parasitology, depending on the nature of the specimen. The need for direct tests, such as Gram stains, must also be considered. These issues will determine whether the specimen requires any pretreatment before inoculation.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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Figures

Image of Figure 3.3.1-1
Figure 3.3.1-1

Appropriate method to streak plate for isolation of bacteria. Inoculate first quadrant with a few drops or pieces of specimen or by rolling a swab on a small area.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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Image of Figure 3.3.1-2
Figure 3.3.1-2

Inoculation of biplate, often used for specimens from sterile sites when blood agar and CHOC are needed. To avoid cross contamination of media with plate contaminants, always use a separate loop or flame loop between inoculation of each side of plate.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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Image of Figure 3.3.1-3
Figure 3.3.1-3

Optional method of streaking plate for throat cultures.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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Image of Figure 3.3.1-4
Figure 3.3.1-4

Alternative for detection of BAP (e.g., respiratory specimens).

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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Image of Figure 3.3.1-5
Figure 3.3.1-5

GasPak envelopes in airtight jar, used to produce 5 to 7% CO atmosphere.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
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References

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1.Clinical and Laboratory Standards Institute. 2004. Quality Assurance for Commercially Prepared Microbiological Culture Media, 3rd ed. Approved standard M22-A3. Clinical and Laboratory Standards Institute, Wayne, PA.
2. Meredith, F. T.,, H. K. Phillips,, and L. B. Reller. 1997. Clinical utility of broth cultures of cerebrospinal fluid from patients at risk for shunt infections. J.Clin.Microbiol. 35:31093111.
3. Morris, A. J.,, S. J. Wilson,, C. E. Marx,, M. L. Wilson,, S. Mirrett,, and L. B. Reller. 1995. Clinical impact of bacteria and fungi recovered only from broth cultures. J.Clin.Microbiol.33:161165.
4. Scythes, K. D.,, M. Louis,, and A. E. Simor. 1996. Evaluation of nutritive capacities of 10 broth media. J.Clin.Microbiol. 34:18041807.
5. Thomsen, R. B.,, Jr. 2007. Specimen collection, transport, and processing: bacteriology, p. 291333. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.), Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
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8. MacFaddin, J. F. 1985. Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, vol. 1. The Williams & Wilkins Co., Baltimore, MD.
9. Miller, J. M. 1999. A Guide to Specimen Managementin Clinical Microbiology, 2nd ed. American Society for Microbiology, Washington, DC.
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11. Ray, C. G.,, J. A. Smith,, B. L. Wasilauskas,, and R. J. Zabransky. 1993. Cumitech 14A, Laboratory Diagnosis of Central Nervous System Infections. Coordinating ed., A. J. Smith. American Society for Microbiology, Washington, DC.
12. Runyon, B. A.,, M. R. Antillon,, E. A. Akriviadis,, and J. G. McHutchison. 1990. Bedside inoculation of blood culture bottles with ascitic fluid is superior to delayed inoculation in the detection of spontaneous bacterial peritonitis. J.Clin.Microbiol.28:28112812.
13. Sharp, S. E.,, A. Robinson,, M. Saubolle,, M. Santa Cruz,, K. Carroll,, and B. Baselski. 2003. Cumitech 7B, Lower Respiratory Tract Infections. Coordinating ed., S. E. Sharp. American Society for Microbiology, Washington, DC.
14. Silletti, R. P.,, E. Ailey,, S. Sun,, and D. Tang. 1997. Microbiologic and clinical value of primary broth cultures of wound specimens collected with swabs. J.Clin.Microbiol. 35:20032006.
15. Simor, A. E.,, F. J. Roberts,, and J. A. Smith. 1988. Cumitech 23, Infection of the Skinand Subcutaneous Tissues. Coordinating ed., J. A. Smith. American Society for Microbiology, Washington, DC.
16. Waites, K. B.,, M. A. Saubolle,, D. F. Talkington,, S. A. Moser,, and V. Baselski. 2005. Cumitech 10A, Laboratory Diagnosis of Upper Respiratory Tract Infections. Coordinating ed., S. E. Sharp. American Society for Microbiology, Washington, DC.
17. Wilhelmus, K. R.,, T. J. Liesegang,, M. S. Osato,, and D. B. Jones. 1994. Cumitech 13A, Laboratory Diagnosis of Ocular Infections. Coordinating ed., S. C. Specter. American Society for Microbiology, Washington, DC.
1. Breakwell, D.,, R. Robison,, C. Adams,, K. Smith,, and R. MacDonald. 2007. Colony Morphology. http://microbelibrary.org.
2.Clinical and Laboratory Standards Institute. 2006. Methods for Dilution Antimicro-bial Susceptibility Tests for Bacteria That Grow Aerobically, 7th ed. Approved standard M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.
3.Clinical and Laboratory Standards Institute. 2006. Performance Standards for Anti-microbial Disk Susceptibility Tests, 9th ed. Approved standard M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA.
4. Meredith, F. T.,, H. K. Phillips,, and L. B. Reller. 1997. Clinical utility of broth cultures of cerebrospinal fluid from patients at risk for shunt infections. J. Clin. Microbiol. 35:3109-3111.
5. Morris, A. J.,, S. J. Wilson,, C. E. Marx,, M. L. Wilson,, S. Mirrett,, and L. B. Reller. 1995. Clinical impact of bacteria and fungi recovered only from broth cultures. J. Clin. Microbiol. 33:161-165.
6.NCCLS. 2002. Abbreviated Identification of Bacteria and Yeast. Approved guideline M35 A. NCCLS, Wayne, PA.
7.U.S. Department of Health and Human Services (Public Health Service), Centers for Disease Control and Prevention, and National Institutes of Health. 2007. Biosafety in Microbiological and Biomedical Laboratories, 5th ed. U.S. Government Printing Office, Washington, DC.
25. Barrow, G. I.,, and K. A. Feltham (ed.). 2007. Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge University Press, New York, NY.
26. Forbes, B. A.,, D. F. Sahm,, and A. S. Weissfeld. 2007. Bailey and Scott's Diagnostic Microbiology, 12th ed. Mosby, St. Louis, MO.
27. Garrity, G. (ed.). 2001. Bergey's Manual of Systematic Bacteriology, vol. 1. Williams & Wilkins, Baltimore, MD.
28. Garrity, G. (ed.). 2005. Bergey's Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD.
29. Garrity, G. (ed.). 2009. Bergey's Manual of Systematic Bacteriology, vol. 4. Williams & Wilkins, Baltimore, MD.
30. Garrity, G. (ed.). 2009. Bergey's Manual of Systematic Bacteriology, vol. 5. Williams & Wilkins, Baltimore, MD.
31. Holt, J. G.,, N. R. Krieg,, P. H. A. Sneath,, J. T. Staley,, and S. T. Williams. 1994. Bergey's Manual of Determinative Bacteriology, 9th ed. Williams & Wilkins, Baltimore, MD.
32. Horvath, R. S.,, and M. E. Ropp. 1974. Mechanism of action of eosin-methylene blue agar in the differentiation of Escherichia coli and Enterobacter aerogenes. Int. J. Syst. Bacteriol. 24:221-224.
33. MacFaddin, J. (ed.). 1985. Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria. Williams & Wilkins, Baltimore, MD.
34. Murray, P. R.,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.). 2007. Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
35. Parks, L. C. (ed.). 2004. Handbook of Microbiological Media by Ronald Atlas, 3rd ed. CRC Press, Boca Raton, FL.
36. Thomsen, R. B., Jr., 2007. Specimen collection, transport, and processing: bacteriology, p. 291-333. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.), Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
37. Truant, A. L. (ed.). 2002. Manual of Commercial Methods in Microbiology. ASM Press, Washington, DC.
38. Weyant, R. S.,, C. W. Moss,, R. E. Weaver,, D. G. Hollis,, J. G. Jordan,, E. C. Cook,, and M. I. Daneshvar. 1996. Identification of Unusual Pathogenic Gram-Negative Aerobic and Facultatively Anaerobic Bacteria, 2nd ed. Williams & Wilkins, Baltimore, MD.
39. Winn, W. C., Jr.,, S. D. Allen,, W. Janda,, E. Koneman,, G. Procop,, P. Schreckenberger,, and G. Woods (ed.). 2005. Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. J. B. Lippincott, Philadelphia, PA.

Tables

Generic image for table
Table 3.3.1-1a

Common laboratory media used for aerobic cultures

For enteric selective and differential media used for stool cultures (e.g., Hektoen, XLD, and chromogenic agars), refer to procedure 3.8.1.

N, nutrient; D, differential; S, selective; E, enrichment.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.1-1b

Common laboratory media used for aerobic cultures

For enteric selective and differential media used for stool cultures (e.g., Hektoen, XLD, and chromogenic agars), refer to procedure 3.8.1.

N, nutrient; D, differential; S, selective; E, enrichment.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.1

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.1-2

Order of specimen processing for bacteriology when multiple specimens are received at the same time

BALs, bronchoalveolar lavage samples.

sp. viability is compromised unless the specimen is placed in transport medium.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.1-3a

Recommended aerobic culture media for inoculation of common clinical specimens,

See reference for detailed recommendations for Gram stain and aerobic and anaerobic plating media for bacteriology specimens or organisms.

Abbreviations: BAL, bronchoalveolar lavage; BW, bronchial wash; ET, endotracheal; CF, cystic fibrosis; GC, gonococci; TM, Thayer Martin; MTM, modified Thayer Martin.

Clinical specimens that should have anaerobic culture also performed provided the samples have been properly collected and transported to ensure anaerobiasis.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.1-3b

Recommended aerobic culture media for inoculation of common clinical specimens,

See reference for detailed recommendations for Gram stain and aerobic and anaerobic plating media for bacteriology specimens or organisms.

Abbreviations: BAL, bronchoalveolar lavage; BW, bronchial wash; ET, endotracheal; CF, cystic fibrosis; GC, gonococci; TM, Thayer Martin; MTM, modified Thayer Martin.

Clinical specimens that should have anaerobic culture also performed provided the samples have been properly collected and transported to ensure anaerobiasis.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-1

Terms to describe gross colonial morphology

See reference .

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-2

Enumeration guidelines

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-3

Colonial morphology on primary media

See reference .

CNA, Columbia colistin-nalidixic acid agar.

EMB reaction is listed after MAC reaction separated by a slash, if they differ.

Inhibited on CNA.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-4

Commonly used primary plating media

Abbreviations: L, lactose; Su, sucrose; TM, Thayer-Martin; MTM, modified Thayer-Martin. For other abbreviations, refer to Table 3.3.1-1 .

Chromogenic substrates within the media produce unique colony changes that allow direct identification of the pathogen of interest after minimal additional testing from the primary plate (http://www.chromagar.com).

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-5a

Common pathogens and rapid and conventional methods to identify them when suspected from colony morphology listed in Table 3.3.2-3

For procedures on biochemical testing, refer to procedure 3.17; for information on multitest biochemical kit tests, refer to procedure 3.18.

All testing requirements for identifications are found in NCCLS document M35 (6), except those noted by this footnote, which are taken from the references listed in Supplemental Reading and in procedures 3.18.1 and 3.18.2. The identifications from the NCCLS document are greater than 95% accurate without further testing.

CNA, Columbia colistin-nalidixic acid agar; SPS, sodium polyanethol sulfonate.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-5b

Common pathogens and rapid and conventional methods to identify them when suspected from colony morphology listed in Table 3.3.2-3

For procedures on biochemical testing, refer to procedure 3.17; for information on multitest biochemical kit tests, refer to procedure 3.18.

All testing requirements for identifications are found in NCCLS document M35 (6), except those noted by this footnote, which are taken from the references listed in Supplemental Reading and in procedures 3.18.1 and 3.18.2. The identifications from the NCCLS document are greater than 95% accurate without further testing.

CNA, Columbia colistin-nalidixic acid agar; SPS, sodium polyanethol sulfonate.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3
Generic image for table
Table 3.3.2-5c

Common pathogens and rapid and conventional methods to identify them when suspected from colony morphology listed in Table 3.3.2-3

For procedures on biochemical testing, refer to procedure 3.17; for information on multitest biochemical kit tests, refer to procedure 3.18.

All testing requirements for identifications are found in NCCLS document M35 (6), except those noted by this footnote, which are taken from the references listed in Supplemental Reading and in procedures 3.18.1 and 3.18.2. The identifications from the NCCLS document are greater than 95% accurate without further testing.

CNA, Columbia colistin-nalidixic acid agar; SPS, sodium polyanethol sulfonate.

Citation: Garcia L. 2010. Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures, p 124-150. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.3

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