Download - 3 b chapter8
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Use of Colonial Morphology for the Presumptive Identification of
Microorganisms
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Objectives
• Describe how growth on blood, chocolate, and MacConkey agars is used in the preliminary identification of isolates.
• Differentiate α-hemolysis from β-hemolysis.
• Describe how gross colony characteristics are used in the presumptive identification of microorganisms.
• Using colonial morphology to differentiate microorganisms.
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Importance of Colonial Morphology as a Diagnostic Tool
• provide a presumptive identification to the physician.
• enhance the quality of patient care through rapid reporting of results and may be increasing this cost-effectivenesss of laboratory testing
• play a significant role in quality control, especially of automated procedure and other commercially available identification system
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Initial Observation and Interpretation of Cultures
• Microbiologists observe colonial morphology of organisms isolated on primary culture after 18 to 24 hours of incubation.
• Incubation time vary according to when the specimen is received and processed in laboratory.
• There are factors that may significantly alter the colonial morphology of growing organisms such as the medium's ingredients, inhibitory nature, and antibiotics present in the medium.
• Interpretation of primary cultures, commonly referred to as plate reading, is a comparative examination of microorganisms growing on a variety of culture media.
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• Many specimens, such as sputum and wounds that arrive in the clinical laboratory are plated on A. Blood agar (BAP), B. Chocolate Agar (CHOC), C. MacConkey Agar (MAC).
• These three culture media illustrates the comparative colonial examination of plate reading.
• A microbiologist must know the ability to determine which organisms grow on selective and nonselective media that aids in making an initial distinction between gram-positive and gram-negative isolates.
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BAP AGARand
CHOC Agar
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• BAP and CHOC support the growth of a variety of fastidious (hard to grow, requires additional growth factors) and nonfastidious organisms, gram-positive and, gram-negative bacteria.
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• An example of a blood agar showing three types of morphotypes. It is because the gram stained smear showed both positive and gram-negative bacteria that three types of organism should be observed on a nonselective medium such as BAP.
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• Generally, organisms that grow on BAP will also grow on CHOC, but not all organisms that grow on CHOC will grow on BAP.
• CHOC agar provides nutritional growth requirements to support highly fastidious species such as Haemophilus species and Neisseria gonorrhoeae.
• Therefore, a gram negative bacillus that grows on CHOC but not on BAP or MAC will be suspected to be Haemophilus species, whereas gram-negative diplococci with the same pattern will be suspected N. gonorrhoeae.
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• The large colonies growing in these plates are gram-negative rods (enterics). These gram negative rods grow larger, gray, and mucoid on BAP and CHOC. Notice the smaller grayish-brown fastidious colonies of Haemophilus organisms growing on CHOC , which are not growing on BAP or MAC.
CHOC AGAR BAP AGAR
The microbiologist then is able to provide a presumptive identification and determine how to proceed in identifying isolated organism.
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MAC AGAR
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• inhibits gram positive organisms and some fastidious gram-negative organism, such as Haemophilus and Neisseria spp.
• supports most gram-negative rods, especially the Enterobacteriaceae.
• growth on BAP and CHOC but not on MAC, therefore is indicative of a gram positive isolate or of a fastidious gram-negative bacillus or coccus.
• gram-negative rods are better described on MAC agar.
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MAC is best used to differentiate lactose fermenters from nonlactose fermenter.
A. Example of nonlactose-fermenting gram-negative rods producing colorless colonies on MAC. B. Example of lactose-fermenting gram-negative rods producing pink colonies on MacConkey agar.
This differentiation in particularly important in screening for enteric pathogens from stool cultures. Most enteric pathogens do not ferment lactose.
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• Certain enteric pathogens produce a characteristic colony on MAC that is helpful in presumptive identification.
Escherichia/Citrobacter-like organism growing on Macconkey
Agar. Notice the dry appearance of the colony and the pink precipitate of bile salts extending beyond the
peripheryof the colonies.
Klebsiella/Enterobacter-like lactose fermenters growing on
MacConkey Agar. Notice the pink, heaped, mucoid appearance.
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GROSS COLONY CHARACTERISTICS
USED TO DIFFERENTIATE AND
PRESUMPTIVELY IDENTIFY
MICROORGANISMS
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• By observing the colonial characteristics of the colonial organism that have been isolated, the microbiologist is able to make an educated guess regarding the identification of the isolation.
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Hemolysis
• Greek word:
Lysis: dissolution or break apart
Hemo: pertaining to red blood cells
• a reaction caused especially by enzymatic or toxin activity of the bacteria, observed in the media immediately surrounding or underneath the colony.
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Hemolysis in Blood Agar• Helpful in the presumptive identification,
particularly of streptococci. • Can be variable for streptococci and
Enterococcus.
Transillumination • The passing of bright light through the
bottom of the plate.
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The use of transillumination to determine whether the colonies are hemolytic. The technique can be used for MacConkey agar also to see slight color differences in nonlactose fermenters.
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Gamma (γ)-hemolytic or nonhemolytic
• Organism has no lytic effect on the RBC’s in the BAP.
α – Hemolysis• Partial lysing of erythrocytes in a BAP
around and under the colony that result in the green discoloration of the medium.
Example:
Streptococcus pneumoniae and certain viridans of streptococci
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β-hemolysis• Complete clearing of erythrocytes in a
BAP around or under the colonies because of the complete lysis of RBCs.
• There are two groups of β-hemolytic streptococci.
• A β-hemolytic streptococci- produce a wide, deep, clear zone of β-hemolysis.
• B β-hemolytic streptococci- produce a narrow, diffuse zone of β-hemolysis close to the colony.
These features are helpful hints in the identification of certain species of bacteria.
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• Organisms that are hemolytic or hemolytic on BAP usually show a green coloration around the colony on CHOC. This coloration, however should not be mistaken for a hemolytic characteristic.
Size• Colonies are described as large,
medium, small or pinpoint.• Generally a visual comparison between
genera or species.
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Gram positive bacteria, in general, produce smaller
colonies than gram-negative bacteria
+ -
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Form or MarginDescribed as:
• Smooth• Filamentous• Rough or Rhizoid• Irregular
Bacillus anthracis
Described as “Medusa Heads” because of the filamentous
appearance
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Swarming colonies of Proteus spp. This
organism was inoculated in the blood
agar plate.
Swarming is a hazy blanket of growth on the surface that extends well beyond the streak lines.
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FORM OR MARGIN
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Elevation
-is determined by tilting the culture plate and looking at the side of colony.
It may be:• Raised• Convex• Flat• Umbilicate(depressed center, concave,
an “innie”)• Umbonate(raised or bulging center,
convex, an “outie”)
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Elevation
Illustration of elevations to describe colonial morphology
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Density
Density colony can be:• Transparent• Translucent – allow some light to
pass through the colony• Opaque – organisms are
concentrated at the center of the colony described as a bull’s-eye colony.(Staphylococci, gram+ & gram-)
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Density
Transparent colony
Translucent colony
Opaque colony
• To see the difference of the density of the colonies it is useful to look through the colony while using
transillumination.
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Color
• In contrast to pigmentation• Is a term used to describe in general a
particular genus• Colonies maybe:• White: Coagulase-negative Staphylococci• Gray: Enterococcus spp.• Yellow or off white: Micrococcus
species and Neisseria species• Buff: “Diphtheroids”
Example of whiteColonies of coagulase-
Negative staphylococci on Blood agar.
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• Is determined by touching the colony with a sterile loop
• Colony consistency maybe:• brittle (splinters): Nocardia spp.• creamy (butyrous): S. aureaus• dry or waxy: Diphtheroid colonies• *Most β-hemolytic streptococci are dry
Consistency
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Pigment
• Is an inherent characteristics of a specific organisms confined generally to the colony.
• Organisms that produce pigment:– P. aeruginosa- green, sometimes a metallic
sheen– Serratiamarcescens- brick-red, specially at
room temperature– Kluyvera spp. – blue– Chromobacteriumviolaceum- purple– Prevotellamelaninogenica- brown-black
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Odor• Should be determined when the lid of the culture
plate is removed and its odor dissipates into the surrounding environment.
• Never inhale directly from the plate • Microorganisms the produced odors:
– S. aureus- old sock– P. aeruginosa- fruity or grape-like– P. mirabilis – putrid– Haemophilus spp. – musty basement, “mousy”
or “mouse nest” smell– Nocardia spp.- freshly plowed field
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Colonies with Multiple
Characteristics
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• Bacillus cereus- forms large, rough, greenish, hemolytic colonies on BAP.
• Eikenellacorrodens- forms a small, fuzzy edge colony with an umbonate center on BAP.
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Growth of organisms in liquid media
• Important clues to an organisms identification can also be detected by observing the growth of the organism in liquid media such as thioglycollate.
• Streamers – or vines and puffballs are associated with certain species of streptococci.
• Turbidity – refers to as cloudiness of the medium resulting from growth, is produced by
• manyEnterobacteriaceae• Yeast and Pseudomonas species- produce scum at
the side of the tube.• Yeast- occasionally grows below the surface, in the
Microaerophilic area of the media.
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*Gram-staining and biochemical reaction occur in microorganisms that produce characteristic features.
An agar plate -- an example of a bacterial growth medium. Specifically, it is a streak plate; the orange lines and dots are formed by bacterial colonies
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“Differentation of Streptococcus pneumoniae, α-hemolytic viridans
streptococci, Enterococcus by colonial morphology”
•Streptococcus pneumonia – translucent, may resemble a water droplet; umbilicate or flat with “penny” edge; entire margin, wide and strong zone of a-hemolysis •α-hemolytic viridans streptococci – translucent, grayer, rough, margin, umbonate center•Enterococcus – it does not have an umbilicate or umbonate center, have larger colonies, smooth and darker margin
Enterococcus
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“Differentation of Streptococcus pygones and Streptococcus
agalactiae by colonial morphology”
• Streptococcus pygones- pinpoint, brittle, gray that may turn brownish on continued incubation, large and deep zone of B-hemolysis in comparison to colony size.
• Streptococcus agalactiae- medium size colony copared with Streptococcus pygones, creamy texture, gray, small and diffuse zone of B-hemolysis compared with colony size
Streptococcus agalactiae
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“Differentation of Staphylococci and Candida albicans by colonial
morphology”
Staphylococci
Candida albicans
• Staphylococci- large, flat or convex or possesses an umbonate center after 24 hours of incubation, shiny, moist, creamy, white to yellowish
• Candida albicans(a yeast) – smaller than staphylococci, convex, grows upward more than outward, creamy, white, dull surface, usually displays tiny projections at the base of the colony after 24 hours of incubation.
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Group 2
• Calinawan, Mary Faith• Calubad, Chloetylle Faye Calubad• Casten, Roland• Castillo, Vhea• Castillo, Vher• Dalupan, Eliza Mae• Diaz, Ryz Kezzer• Dignadice, Maricar• Dizon, Sushmita