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Mikrobiologi Klinik FK UNUD CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms George Manuselis, Connie R. Mahon* CHAPTER OUTLINE ■ IMPORTANCE OF COLONIAL MORPHOLOGY AS A Form or Margin DIAGNOSTIC TOOL Elevation ■ INITIAL OBSERVATION AND INTERPRETATION OF Density CULTURES Color ■ GROSS COLONY CHARACTERISTICS USED TO Consistency DIFFERENTIATE AND IDENTIFY MICROORGANISMS Pigment PRESUMPTIVELY Odor Hemolysis ■ COLONIES WITH MULTIPLE CHARACTERISTICS Size ■ GROWTH OF ORGANISMS IN LIQUID MEDIA OBJECTIVES After reading and studying this chapter, you should be able to: 1. Describe how growth on blood, chocolate, and MacConkey agars is 4. Using colonial morphology, differentiate among the following used in the preliminary identification of isolates. microorganisms: 2. Differentiate α-hemolysis from β-hemolysis on blood agar culture • Staphylococci and streptococci medium. • Streptococcus agalactiae and Streptococcus pyogenes 3. Associate the colony characteristics shown on blood, chocolate, and • Neisseria spp. and staphylococci MacConkey agars with the microscopic findings on direct smear, and • Yeast and staphylococci use the information in the presumptive identification of • “Diphtheroids” and staphylococci microorganisms. • Lactose fermenters from lactose nonfermenters • “Swarming” Proteus species from other Enterobacteriaceae Case in Point with a surrounding pink precipitate and a few clear nonlactose fermenting colonies. Based on the Gram stain results and colo- An exudate from a sacral decubital ulcer on a 65-year-old hos- nial characteristics of the isolates, appropriate biochemical tests pital inpatient was cultured on blood agar plate (BAP), chocolate and antibiotic susceptibilities were performed to identify the (CHOC), and MacConkey (MAC) agars. Direct smear examina- causative agents of the ulcer. tion showed many white blood cells, a moderate number of gram-positive cocci in pairs and clusters, and a few gram- negative bacilli. After overnight incubation, three colony mor- Issues to Consider photypes were visible on the BAP. The first was a moderate After reading the patient’s case history, consider: growth of a medium-sized β-hemolytic, which was yellowish ■ How the colony morphology of isolates is used to identify white and creamy-buttery looking. The second colony was microorganisms presumptively also β-hemolytic but larger, mucoid, and gray. The third type ■ How to correlate the direct smear examination findings Mikrobiologiof colony was large, gray, and mucoid similar to the second Klinik FK UNUD with the colony morphology of isolates on each culture but was nonhemolytic. The MAC agar showed two colony medium morphotypes—a light growth of dark pink, dry-looking colonies ■ How the colony morphology of each isolate can dif- *My comments are my own and do not represent the view of Health Resources ferentiate between pathogenic and nonpathogenic and Services Administration of the Department of Health and Human Services. microorganisms 169 170 PART I Introduction to Clinical Microbiology Key Terms • Enhance the quality of patient care through rapid report- ing of results and by increasing the cost-effectiveness of α-Hemolysis Lactose fermenter laboratory testing. This may best be illustrated by using β-Hemolysis Margin sputum cultures as an example. The upper respiratory tract Brittle Nonlactose fermenter contains many indigenous organisms, and to identify every Butyrous Opaque organism in culture would be a time-consuming, cost- Colonial morphology Pigment prohibitive, and insurmountable task. Microbiologists must be Consistency Rhizoid able to differentiate potential pathogens from the “usual” Creamy Smooth inhabitants of the upper respiratory tract and direct the diag- Density Streamers nostic workup toward only potential pathogens. Potential Elevation Swarming pathogens are presumptively identifed by colonial character- Escherichia/Citrobacter-like Transillumination istics, and preliminary reporting initiates immediate therapy. organisms Translucent Fastidious Transparent • Play a signifcant role in quality control, especially of auto- Filamentous Turbidity mated procedures and other commercially available iden- Form Umbilicate tifcation systems. When commercial and automated systems Hemolysis Umbonate are used, a mixed inoculum (polymicrobic/containing more Klebsiella/Enterobacter-like than one genus and species or both) produces a biochemical organisms test result or erroneous interpretation of reactions that signif- cantly alters the identifcation (see Chapter 9). The ability of the microbiologist to determine whether the inoculum is mixed and to ascertain whether the results generated by a commercial or automated system correlate with the suspected he importance of mastery of colonial morphology (colony identifcation of the organism is an important component of characteristics and form) and interpretation of Gram- quality control that is accomplished by recognizing organisms Tstained smears from clinical specimens and microbial by their colonial characteristics. colonies cannot be overemphasized. Although Gram-stained smears provide initial identifcation of microorganisms by micro- scopic characterization, description of the physical growth char- Initial Observation and Interpretation acteristics of microorganisms on laboratory media facilitates the initial identifcation processes. of Cultures Close your eyes and imagine the physical characteristics of a Generally, microbiologists observe the colonial morphology person you know well. The person’s height, weight, shape, color of organisms isolated on primary culture after 18 to 24 hours and style of hair, eyes, freckles, and color of skin as well as voice of incubation. Incubation time may vary according to when or laugh may make that person distinctive in a crowd or when the specimen is received and processed in the laboratory, his or her back is facing you. In the same manner, many micro- which may affect the “typical” morphology of a certain isolate. organisms have specifc characteristics that distinguish them in a For example, young cultures of Staphylococcus aureus may crowd of other genera or species. appear smaller and may not show the distinct β-hemolysis This chapter explains how the characterization of colonies on that older cultures produce. In addition, the microbiologist culture media and the fndings on stained direct smears facilitate must be aware of factors that may signifcantly alter the colo- presumptive identifcation of commonly isolated organisms. The nial morphology of growing microorganisms. These factors characteristics that are used to describe colony morphology of include the ingredients present in the medium, the inhibitory certain groups of organisms and how these characteristics are nature of these ingredients, and antibiotics that may be present used to differentiate one species from a closely related species in the medium. and one genus from another are discussed. The interpretation of primary cultures, commonly referred to as plate reading, is actually a comparative examination of the colony morphology of microorganisms growing on various Importance of Colonial Morphology culture media. Many specimens, such as sputum and wounds that arrive in the clinical laboratory, are plated on various culture as a Diagnostic Tool media such as BAP, CHOC, and MAC. Each type of agar plate In many ways, the usefulness of colonial morphology extends is examined in relationship to the other. As a set of culture media, the capabilities of the microbiologist and, ultimately, the clinical comparative colonial examination of growth from a specimen laboratory. The ability to provide a presumptive identifcation by occurs. colonial morphology may include the following: The ability to determine which organisms grow on selective Mikrobiologi• Provide a presumptive identifcation to the physician. Klinik and nonselective media FK aids the microbiologist UNUD in making an Even in this age of rapid identifcation systems, incubation initial distinction between gram-positive and gram-negative iso- times and procedures can be protracted. In a critical situation, lates. BAP and CHOC support the growth of various fastidious the microbiologist makes an educated judgment about the (hard to grow, requires additional growth factors) and nonfas- presumptive identity before performing diagnostic identifca- tidious organisms, gram-positive bacteria, and gram-negative tion procedures. bacteria. CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 171 presumptive identifcation and determine how to proceed in iden- ✓ Case Check 8-1 tifying the isolated organisms. As illustrated in the Case in Point at the beginning of the chapter, three While MAC supports most gram-negative rods, especially the colony morphotypes were observed on BAP. Because the Gram-stained Enterobacteriaceae, it inhibits growth of gram-positive organ- smear showed both gram-positive and gram-negative bacteria, three isms and some fastidious gram-negative organisms, such as Hae- types of organisms should be observed on a nonselective medium such mophilus and Neisseria spp. Growth on BAP and CHOC but not as BAP. on MAC is indicative of a gram-positive isolate or of a fastidious gram-negative bacillus or coccus. Generally, organisms that grow on BAP also grow on CHOC, Gram-negative rods are better described on MAC agar because but not all organisms that grow on CHOC grow on BAP. Although these organisms produce similar-looking colonies on BAP and BAP supports fastidious
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