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6
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Traditional Culture an I
and Identification Methods mages RF/G
Maria E. Delost, PhD, MT(ASCP) ett
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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.
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