Use of Colonial Morphology for the Presumptive Identification of

Microorganisms

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.

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

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.

• 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.

BAP AGARand

CHOC Agar

• 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.

• 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.

• 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.

• 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.

MAC AGAR

• 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.

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.

• 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.

GROSS COLONY CHARACTERISTICS

USED TO DIFFERENTIATE AND

PRESUMPTIVELY IDENTIFY

MICROORGANISMS

• 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.

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.

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.

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.

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

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.

• 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.

Gram positive bacteria, in general, produce smaller

colonies than gram-negative bacteria

+ -

Form or MarginDescribed as:

• Smooth• Filamentous• Rough or Rhizoid• Irregular

Bacillus anthracis

Described as “Medusa Heads” because of the filamentous

appearance

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.

FORM OR MARGIN

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”)

Elevation

Illustration of elevations to describe colonial morphology

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-)

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.

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.

• 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

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

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

Colonies with Multiple

Characteristics

• Bacillus cereus- forms large, rough, greenish, hemolytic colonies on BAP.

• Eikenellacorrodens- forms a small, fuzzy edge colony with an umbonate center on BAP.

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.

*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

“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

“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

“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.

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