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6 c a v traditional culture an i and identification methods mages rf g maria e delost phd mt ascp ett y i mages chapter outline introduction automated identification systems ...

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                         6
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                                                                                                                                          a
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                  Traditional Culture                                                                                                     an I
                  and Identification Methods                                                                                              mages RF/G
                  Maria E. Delost, PhD, MT(ASCP)                                                                                          ett
                                                                                                                                          y I
                                                                                                                                          mages
                  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 using phenotypic                        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% CO  gas using a glove box or anaerobic 
                                                                                                                        2
                   Colonial Morphology                                                           jar. For some specimens, when fungi or mycobacteria 
                                                                                                 are suspected, a second set of plates may be inoculated 
                                                                                                                                                                 o
                   Clinical specimens are cultivated on artificial media to                      and incubated at room temperature (20ºC to 25 C).
                   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. These criteria ar             e 
                   used to differentiate closely related species and genera.                         BOX 6-2
                    Colonial morphology can provide a  preliminary diagno-                           THE VALUE OF COLONIAL 
                   sis and guides the identification scheme. By examining                            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.
                       Phenotypic Characteristics                                                     2.  Communicating preliminary results to the 
                                                                                                          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 
                       Environmental requirements for growth: oxygen,                                     normal microbiota or contaminants and not a 
                          capnophilic                                                                     potential pathogen prevents unnecessary patient 
                       Nutritional needs: enriched or supplemented media                                  treatment.
                       Antimicrobial susceptibility: resistance or sensitive to                       3.  The early identification of an organism associated 
                          specific antibiotic(s)                                                          with healthcare-associated infections or 
                                                                                                          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
                    1212
                    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
                     2+              More than 10    Less than 5                        3
                     3+              More than 10    More than 5     Less than 5       (C)
                     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 
                  increased CO the organism may be capnophilic.              on sheep blood agar, showing greening of agar and also mucoid 
                               2,                                            consistency.
               • Assessment of optimal growth temperature when a             Courtesy of Maria Delost.
                  second set of plates is incubated at a temperature 
                                        o
                  other than 35ºC to 37 C.
               •  Correlate the primary plate assessment with the direct 
                  Gram stain morphology.
               Hemolysis
               Hemolytic patterns are best observed by passing a bright 
               light through the bottom of the blood agar plate and 
               looking through the top of the agar plate. The patterns 
               are described as one of the following:
                hemolytic: incomplete; greening or browning of the 
                  medium as shown in Figure 6-2
                hemolytic: complete clearing of the medium; should 
                  be observed at the surface and below the surface of 
                  the agar (Figure 6-3)
               Nonhemolytic: no change in the color of the medium;           Figure 6-3.  β hemolytic colonies of Group A streptococcus on 
                  may be referred to as gamma hemolysis (Figure 6-4)         sheep blood agar. Colonies are pinpoint and translucent, and agar 
                  Figure 6-5 illustrates colonial characteristics which      is completely cleared.
               are described below.                                          Courtesy of Maria Delost.
                  BOX 6-3
                  HEMOLYTIC PATTERNS
                  After streaking the blood agar plate for isolated, “stab” 
                  the plate by forcing a small amount of the colony into 
                  the agar in two to three areas without cutting through 
                  the media. Hemolytic patterns are best observed 
                  by passing a bright light through the bottom of the 
                  blood agar plate and looking through the top of the 
                  agar plate.
                   hemolytic: incomplete; greening or browning of the 
                    medium
                   hemolytic: complete; total clearing of the medium        Figure 6-4. Nonhemolytic colonies of Enterococus faecalis on 
                  Nonhemolytic: no change in the color of the medium;        sheep blood agar. Colonies are grayish with no change in appear-
                    may be referred to as gamma hemolysis                    ance of the blood agar.
                                                                             Courtesy of Maria Delost.
               Copyright © 2022 by Jones & Bartlett Learning, LLC, an Ascend Learning Company
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...C a v traditional culture an i and identification methods mages rf g maria e delost phd mt ascp ett y chapter outline introduction automated systems colonial morphology matrix assisted laser desorption ionization time of preliminary biochemical tests flight mass spectrometry maldi tof ms multitest blood detection metabolic activity key terms hemolytic colorimetry nonhemolytic fluorometry nephelometry phenotypic characteristics learning objectives upon successful study review this the learner should be able to describe common bacterial streaking techniques discuss use manual in explain importance microbiology laboratory clinical state principle following major used give application each colorim evaluate etry identify types hemolysis its observed on sheep agar applications how can operation capabilities bacteria catalase including identi cytochrome oxidase coagulase pyr hydrolysis fication antimicrobial testing carbohydrate utilization three detect metabolism copyright by jones bartlett ...

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