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 AGAR

and

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 willalso grow on CHOC, but not all organismsthat grow on CHOC will grow on BAP.

• CHOC agar provides nutritional growthrequirements to support highly fastidiousspecies such as Haemophilus species andNeisseria gonorrhoeae.

• Therefore, a gram negative bacillus thatgrows on CHOC but not on BAP or MACwill be suspected to be Haemophilusspecies, whereas gram-negative diplococciwith the same pattern will be suspected N.gonorrhoeae.

• The large colonies growing in these plates aregram-negative rods (enterics). These gramnegative rods grow larger, gray, and mucoidon BAP and CHOC. Notice the smaller grayish-brown fastidious colonies of Haemophilusorganisms growing on CHOC , which are notgrowing 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 andsome fastidious gram-negativeorganism, such as Haemophilus andNeisseria spp.

• supports most gram-negativerods, especially theEnterobacteriaceae.

• growth on BAP and CHOC but not onMAC, therefore is indicative of a grampositive isolate or of a fastidious gram-negative bacillus or coccus.

• gram-negative rods are betterdescribed 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 Margin

Described 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

colonyTranslucent

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 white

Colonies 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 zoneof a-hemolysis•α-hemolytic viridans streptococci – translucent, grayer, rough, margin,umbonate center•Enterococcus – it does not have an umbilicate or umbonate center, havelarger 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