6 © Traditional Culture Images RF/GettyCavan Images and Identification Methods Maria E. Delost, PhD, MT(ASCP) Chapter Outline Introduction Automated Identification Systems Colonial Morphology Matrix-Assisted Laser Desorption Ionization Time of Preliminary Biochemical Tests Flight Mass Spectrometry (MALDI-TOF MS) Multitest Systems Blood Culture Systems Detection of Metabolic Activity Key Terms α hemolytic Colorimetry MALDI-TOF MS Nonhemolytic β hemolytic Fluorometry Nephelometry Phenotypic characteristics Colonial characteristics Learning Objectives Upon successful study and review of this chapter, the learner should be able to: 1. Describe the common bacterial streaking techniques. 7. Discuss the use of manual multitest systems in the 2. Explain the importance of colonial morphology in microbiology laboratory. clinical microbiology. 8. State the principle of the following detection 3. Describe the major phenotypic characteristics used methods and give an application of each: colorim- to evaluate colonial morphology. etry, nephelometry, and fluorometry. 4. Identify and describe the types of hemolysis 9. Describe the principle of MALDI-TOF MS and its observed on sheep blood agar. applications in clinical microbiology. 5. Discuss how the following tests can be used in 10. State the principle of operation and capabilities of the preliminary identification of bacteria: catalase, automated microbiology systems, including identi- cytochrome oxidase, coagulase, PYR hydrolysis, fication and antimicrobial testing. and carbohydrate utilization. 11. Discuss manual and automated blood culture 6. Explain the three methods to detect bacterial systems. metabolism. 81 Copyright © 2022 by Jones & Bartlett Learning, LLC, an Ascend Learning Company 82 Part I Introduction to Clinical Microbiology Introduction primary plates, the microbiologist evaluates the growth to determine if the colonies represent pathogens, normal Traditional methods of identification usingphenotypic microbiota, or contaminants. The importance of colo- characteristics to identify microorganisms include nial morphology is summarized in Box 6-2. microscopic properties, such as the Gram stain reaction Because clinical specimens often contain more and morphology, and macroscopic properties, such as than one organism, all primary plates are streaked colonial morphology on artificial media. Chapter 6 will for isolation. Isolated colonies provide pure growth discuss the importance of colonial morphology or iso- of the organism that is needed for all subsequent lates recovered from clinical specimens in preliminary laboratory testing. Methods for obtaining isolated identification and how these phenotypic characteristics colonies are illustrated in Figure 6-1A-C and are are used to identify microorganisms. This includes colo- summarized below. After streaking plates for isola- nial morphology, preliminary tests, and confirmatory tion, the inoculated media are incubated in ambient methods. Manual identification methods and instru- air at 35ºC to 37oC; certain primary plates are incu- mentation will be discussed. An outline of identification bated in increased carbon dioxide (CO2). If anaerobic methods is found in Box 6-1. cultures are requested, anaerobic media are set up and incubated in 85% nitrogen gas, 10% hydrogen gas, and 5% CO2 gas using a glove box or anaerobic Colonial Morphology jar. For some specimens, when fungi or mycobacteria are suspected, a second set of plates may be inoculated Clinical specimens are cultivated on artificial media to and incubated at room temperature (20ºC to 25oC). determine if bacteria are present in the specimen, and Method A can be used on either liquid specimens or to identify any pathogens. Media selected for primary swabs. inoculation depend on the site of the specimen and 1. Transfer a drop of the liquid specimen using a sterile which microorganisms are most likely associated with pipette to a corner of the agar plate. Swabs are plated infection in that site. Media should support the growth directly by rolling over an area in the corner of the of those bacteria most often known to cause infection plate. in that site. Colonial morphology or colony charac- teristics of bacteria are important observations in the preliminary identification of bacteria. Thesecriteria are used to differentiate closely related species and genera. BOX 6-2 Colonial morphology can provide a preliminary diagno- sis and guides the identification scheme. By examining THE VALUE OF COLONIAL MORPHOLOGY 1. Provides a presumptive identification to guide BOX 6-1 the diagnostic identification procedures used in the work-up of pathogenic organisms. Prevents CHARACTERISTICS OF unnecessary, costly, time-consuming work-up of MICROORGANISMS normal microbiota and contaminants. 2. Communicating preliminary results to the Phenotypic Characteristics healthcare provider can provide information on Microscopic morphology and staining characteristics: a possible potential pathogen, which can assist Gram stain reaction in the patient’s diagnosis and treatment. In some Colonial morphology: appearance on artificial plating cases, antimicrobial therapy can be initiated on media preliminary findings. Reporting the isolation of normal microbiota or contaminants and not a Environmental requirements for growth: oxygen, potential pathogen prevents unnecessary patient capnophilic treatment. Nutritional needs: enriched or supplemented media 3. The early identification of an organism associated Antimicrobial susceptibility: resistance or sensitive to with healthcare-associated infections or specific antibiotic(s) community-acquired infections so that infection Genetic Characteristics control and public health officials can be notified, and appropriate measures taken. Molecular techniques to identify bacterial genome 4. Antimicrobial susceptibility testing can be Identification of specific gene that identifies the performed based on preliminary findings, including organism identification of antimicrobial resistance to guide Identification of specific gene products patient therapy. Copyright © 2022 by Jones & Bartlett Learning, LLC, an Ascend Learning Company Chapter 6 Traditional Culture and Identification Methods 83 12 12 3 4 34 (A) (B) Figure 6-1. Agar plate with streaks. 2. Sterilize the wire loop and pass it through the initial Method C is a method preferred by some microbiolo- inoculum several times, streaking the top quarter of gists for broth cultures. the plate into the second quadrant. This is streak area 1. 3. Rotate the plate 90° and repeat the streaking from the second quadrant into the third quadrant. This is streak area 2. 4. Rotate the plate 90° again and continue the streak- ing from the third quadrant into the fourth quad- rant. This is streak area 3. Flame between quadrants unless inoculum is light. 12 This method can be semi-quantitated by using the following scale: Growth rating Quadrant 2 3 4 1+ Less than 10 3 2+ More than 10 Less than 5 (C) 3+ More than 10 More than 5 Less than 5 4+ More than 10 More than 5 More than 5 1. Apply a loopful of inoculum near periphery of plate. Cover approximately one-fourth of the plate with close parallel streaks. Sterilize loop. Method B can be used for cultures in heavy broth or 2. Make one light sweep through lower portion of solid media. streaked area. Turn plate 90° and streak approxi- 1. Streak specimen or inoculum in top quarter of plate. mately half of remaining plate. 2. With sterile loop, make a light sweep through inocu- 3. Turn plate 180° and streak remainder of plate. Avoid lated area and streak entire top quarter of plate with any previously streaked areas. parallel strokes. Sterilize loop. After incubation in the appropriate atmospheric con- 3. Turn plate 90° and make a light sweep into lower ditions, primary plates are observed for colonial mor- portion of area streaked in step 2. phology at 18 to 24 hours. This time may differ based on Streak as in step 2, covering approximately half of when the specimen was received and processed in the remaining plate. laboratory. Some microbes require 48 hours to grow, so 4. Turn plate 180° and streak remaining plate with ster- primary plates are incubated for another 24 hours after ilized loop. Avoid any previously streaked areas. the initial assessment. Copyright © 2022 by Jones & Bartlett Learning, LLC, an Ascend Learning Company 84 Part I Introduction to Clinical Microbiology This process is known as plate reading and includes the following assessments: • The set of primary plates (enriched, differential, selective) are evaluated together for a particular site. Compare the growth characteristics on each plate, including the amount of growth and description. • Determine the type and pattern of hemolysis on the blood agar plate. Evaluate both surface and subsur- face hemolysis as described in Box 6-3 and illustrated in Figures 6-2, 6-3 and 6-4. • Assessment of atmospheric requirements is made. For example, if an abscess grows only in ambient air and not anaerobically, the isolate is most likely an aerobe or a facultative anaerobe. If growth is enhanced under Figure 6-2. α hemolytic colonies of Streptococcus pneumoniae on sheep blood agar, showing greening of agar and also mucoid increased CO2, the organism may be capnophilic. consistency. • Assessment of optimal growth temperature when a Courtesy of Maria Delost. second set of plates is incubated at a temperature other than 35ºC to 37oC. • Correlate the primary plate assessment with the direct Gram stain morphology. Hemolysis Hemolytic patterns