1. Use of Colonial Morphology for
the Presumptive Identification
of Microorganisms

2. 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.

3. 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

4. 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.

5. • 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.

6. BAP AGAR
and
CHOC Agar

7. • 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.

8. • 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.

9. • 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.

10. • 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.

12. MAC AGAR

13. • 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.

14. 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.

15. • 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.

17. GROSS COLONY
CHARACTERISTICS
USED TO
DIFFERENTIATE AND
PRESUMPTIVELY
IDENTIFY
MICROORGANISMS

18. • 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.

19. 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.

20. 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.

21. 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.

22. 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

23. 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.

24. • 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.

25. Gram positive bacteria, in
general, produce smaller
colonies than gram-negative
bacteria
+ -

27. Form or Margin
Described as:
• Smooth
• Filamentous
• Rough or Rhizoid
• Irregular
Bacillus anthracis
Described as “Medusa Heads”
because of the filamentous
appearance

28. 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.

29. FORM OR MARGIN

30. 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”)

31. Elevation
Illustration of elevations to describe
colonial morphology

32. 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-)

33. 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.

34. 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.

35. • 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

36. 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

37. 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

38. Colonies with
Multiple
Characteristics

39. • Bacillus cereus- forms large, rough,
greenish, hemolytic colonies on
BAP.
• Eikenellacorrodens- forms a small,
fuzzy edge colony with an umbonate
center on BAP.

40. 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.

41. *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

42. “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

43. “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

44. “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.

45. 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