ALCANTARA S.

 Incubation can also vary according to when the specimen is

received and processed in the
Chapter 8 : Use of Colony Morphology for the  laboratory. (For example, young cultures of Staphylococcus
Presumptive Identification of Microorganism aureus may appear smaller and may not show the distinct B-
hemolysis that other cultures produce)
I. Introduction  Factors affecting growth and colony morphology:
 Culture medium
 Colony  Environmental conditions - oxygen, carbon
 Macroscopically visible growth of a microorganism on a dioxide availability, temperature, pH, and
solid culture medium. moisture (details discussed in chapter 6)
 Composed of microorganisms originating from single  There are factors that may significantly alter the colonial
mother cells morphology of growing microorganisms:
 Constitutes clone of bacteria that genetically alike  Ingredients present in medium
 Inhibitory nature of the ingredients
 Colonial Morphology  Antibiotics present in the medium
 Observing colonial characteristics and form
III. Interpretation of Culture

 The interpretation of primary cultures, commonly referred to

as plate-reading, is actually a comparative examination of
colony morphology of microorganisms growing on various
culture media
 Many specimens, such as sputum and wounds that arrive in
clinical laboratory, are plated on various culture media such
as:
a) BAP (Blood Agar Plate) - grows most
organisms
b) CHOC (Chocolate Agar) - most organisms
Figure 1. Agar plate that is exposed in air, colony is significantly
and fastidious organisms
numbered from 1-9 signifying the 9 different species of bacteria.
c) MAC (Mac Conkey Agar) - gram negative
Colony 3 and 4 are two different colonies that tends to overlap in
organisms
this case it might be difficult to characterize such colony. Which is
 BAP and CHOC plate it allows growth of most bacteria,
why sub-culturing is necessary to have a distinct characterization.
while MAC agar is selective specifically for members of
Enterbacteriaceae.
Colony differs from pigment, size, shape form and consistency

Figure 2. You can see large colonies of gram negative organisms

To Subculture - a portion of an isolated colonies is taken and
(Red arrow) growing on all the three plates. But notice also that the
transferred using a sterile loop to the surface of a suitable
very small colonies (Yellow arrow) of Haemophilus spp. are
enrichment medium. It will be incubated under conditions optimal
growing of CHOC but not in MAC and BAP.
for the growth of the organism. Streak plate method may be
performed.
What does this signifies?
- This signifies that CHOC provides nutritional growth
 Streak Plate Technique - A simple method of mechanically
requirements to support highly fastidious organisms such as
diluting a relatively large concentration of microorganisms
Haemophilus spp. and N. gonorrhoeae
to a small scattered population of cells.
II. Initial Observation of Culture  Differentiation using plated media
 MAC is best used to differentiates bacteria that can
 Observation of colony morphology is done after 18-24 and cannot ferment the sugar lactose.
hours incubation
 Lactose fermenters are easily detected by color
 But incubation time may still vary depending on the type of change they produce on the medium; as the pH
bacteria trying to grow for an example mycobacterium spp. changes when lactose is fermented, the organisms
slow growing bacteria may take longer as 7 days to produce produce pink, dark pink, or red colonies.
colonies.
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Non lactose fermenters Clear and Colorless  Certain yeast - stars or colonies with feet or pedicles
Lactose fermenters Pink  Certain genera such as Proteus spp. (especially
Proteus mirabilis and Proteus vulgaris) may swarm
on nonselective agar such as blood or chocolate.
 Swarming - hazy blanket of growth on the surface
that extends well beyond the streak lines.

Figure
3. (A) Lactose Fermenters (B) Non Lactose Fermenters seen in Figure 5. Bacillus anthracis with filamentous edges or "medusa
MAC agar plate heads" and Diptheroid with rough edges

III. Colony Characteristics

A. Size
 It is usually measure in millimeters (mm) or maybe
describe in relative terms such as pintpoint/punctiform,
small, medium or large.
 Size is generally a visual comparison between genera or
species.
 For example, gram-positive bacteria generally produce
smalle colonies than gram-negative bacteria.
 Staphylococcus spp. are usually larger than Streptococcus
spp.

Figure 6. Illustration of form or margin to describe colonial

morphology.

C. Elevation
 It should be determined by tilting the culture plate and
looking at the side of the colony (Figure 7)
 It should determine how much does the colony rise above
the agar
 Elevation
may be
raised,
convex,
flat,

Figure 4. Left portion: Neisseria gonorrhea has relative small

colonies compared to the large colonies of Bacillus cereus.
Right portion: There is small colonies of gram-positive cocci;
large mucoid colonies of gram-negative enteric rod.
umbilicate (depressed center convex-an "innie"), or
B. Form/Margin umbonate (raised or bulging center, convex-an "outie")
 The edge of the colonies must be observed and the
form, or margin, described as smooth, filamentous,
rough or rhizoid, or iregullar. (Refer to figure 6)
 Examples: Bacillus anthracis - filamentous
(commonly described as "medusa head"
 Diphtheroid colonies - rough edges
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 The density of the colony can be transparent,

translucent, or opaque.
 To see the differences in the density of colonies, it is
useful to look through the colony while using
transilluminination.
 Translucent colonies will allow some light to pass
Figure 7. Illustration of elevations to describe colonial through the colony and opaque colonies do not.
morphology

 Examples: Umbilicate: Cnvex with depressed center

(pitting); S. Pneumoniae (if no capsule)
 Umbonate: Convex with protruding nipple
diphtheoids

Figure 11. The

use of transillumination to determine whether colonies are
hemolytic. The technique can be used for MAC also to see slight
color differences in nonlactose fermenters.

Figure 8. "Diphtheroid" colonies with rough edges colonies,

dry appearance, and umbonate center growing on blood agar
plate

Figure 12. B-hemolytic streptococci having translucent colonies;

Staphylococcus aureus having opaque colonies.

E. Color
 In contrast to pigmentatipn, color is a term used to
describe a particular genus in general.
 Colonies may be white, gray, yellow or buff.

Figure 13. (L) White colonies of coagulase-negative

Figure 9. S. pneumoniae with umbilicate colonies staphylococci on blood agar plate. (R) Yellow colonies of
certain nonpathogenic species of Neisseria organisms on
blood agar plate. Diphtheroids are buff. Most gram negative

Figure 10. S. aureus that produce convex colonies rods are gray on BAP
D. Density
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F. Consistency picture notice the slightly cream-colored center after 48 hours'

 It is determined by touching the colony with sterile growth.
loop.
 Colony consistency may be brittle (splinters), G. Pigment
creamy (butyrous). dry, or waxy  Inherent characteristic of na specific organism confined
 Brittle (splinters) - Friable if it is dry and generally to the colony.
breaks apart  Examples of organisms that produce pigments:
 Creamy (butyrous) - Sticks to the loop and  P.aeruginosa - green, sometimes metallic sheen *
hard to get off  Serratia marcescens - brick red, specially at room
 Mucoidal - if its sticky and mucous like temperature *
 Ocassionally, the entire colony adheres (sticks) to  Kluyvera spp. - blue
the loop.  Chromobacterium violaceum - purple
 Examples:  Prevotella melaninogenica - brown-black
 S. aureus - creamy (anaerobic) pigment production for these
 Neisseria - sticky organisims is variable.
 Norcadia - brittle
 Streptococci - dry
 Diphtheroid - dry and waxy

Figure 17. (A)

Pseudomonas aeruginosa illustrating the metallic sheen and green

Figure 14. Creamy consistency of Staphylococcus aureus and pigmentation of colonies on blood agar plate (BAP). (B) Not all
Brittle consistency of Norcadia spp. strains of the same organism have the same colonial appearance.
This mucoid strain of P.aeruginosa on BAP.

Figure 15. (A) Lactose-fermenting Figue 18.

Brick-red pigment of Serratia marcescens, which is evident on
MAC (Right). Additional incubation at room temperature enhances
the brick-red pigmentation.

H. Odor
Escheria/Citrobacter-like organisms growing on MAC.  Odor should be determined when the lid of the culture plate
NOTICE: Dry appearance and pink precipitate of bile salts is removed and the odor dissipates into surrounding
extending beyond the periphery of the colonies. (B) Close-up environment.
of dry flat Escheria/Citrobacter-like organisms growing on  THE MICROBIOLOGIST SHOULD NEVER INHALE
MAC. DIRECTLY FROM THE PLATE!
Figure 16. Klebsiella/Enterobacter-like lactose fermenters growing  Examples of microorganisms that produce distinctive odors:
on MAC. NOTICE: Pink, heaped, mucoid appearance. Left
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 S. aureus - old sock; evident when growing on Gamma hemolysis

mannitol salt agar  Referred to organisms that does not produce
 P. aeruginosa - fruity or grapelike hemolysins and does not breakdown the red blood
 P. mirabilis - putrid cells; no clearing will occur.
 Haemophilus spp. - musty basement, "mousy" or  Nonhemolytic colonies
"mouse nest" smell
 Nocardia spp. - freshly plowed field

IV. Other Colony Characteristics

Hemolysis on Blood Agar Plate (BAP)

 Observed in the media immediately surrounding or

underneath the colony is caused by enzymatic or toxin
activity of bacteria.
 Some bacteria produce exoenzymes that lies on red blood
cells on great hemoglobin and these are called as
hemolysins.
 Hemolysis on BAP is helpful in presumptive identification,
particularly of streptococci and enterococci (Figure 19)
 Proper technique requires the passing of bright light through
the bottom of the plate (Transillumination) to determine
whether the organism is hemolytic

Beta hemolysins
 Break down the red blood cells (RBCs) and
hemoglobin completely and it leaves a clearer zone
around the bacterial growth. Such results are
referred to as beta-hemolysis.
Figure 18. Staphylococcus aureus growing on blood agar plate.
Beta hemolysis (-hemolysis) Take note: shiny, moist, creamy, white to yellowish color colonies
 Complete clearing of blood cells around the exhibiting beta-hemolysis
colonies
 Complete clearing of erythrocytes in BAP around or
under the colonies because of complete lysis of
RBCs.
 Group A -hemolytic streptococci (Streptococcus
pyogenes), produce a wide, deep, clear zone of -
hemolysis.
 Group B -hemolytic streptococci (Streptococcus
agalactiae) and Listeria monocytogenes (a short,
gram-positive rod) produce a narrow, diffuse zone
of -hemolysis close to the colony.
 This is a helpful hint in comparing colonial
characteristic of group A and group B of
streptococci. Figure 19. Hemolytic pattern of Streptococcus pyogenes and
Streptococcus agalactiae, both of this species produce beta-
hemolysis on BAP. But there are differences on their colonies
characteristics and hemolysis produced.
Alpha hemolysin
 Partially breaks down the red blood cells (RBCs) Streptococcus pyogenes: pinpoint, grayish and translucent colonies
and leaves a greenish color behind, and it is referred exhibiting large deep zone of beta hemolysis
to as alpha-hemolysis. The greenish color is Streptococcus agalactiae: medium size, grayish colonies with
caused by the presence of biliverdin, which is a by small and diffuse zone of beta hemolysis.
product of the breakdown of hemoglobin.
V. Colonies with Multiple Characteristics
Alpha-hemolysis
 Partial lysing of erythrocytes in BAP around and  Organisms that fit into multiple descriptive categories of
under the colony that results to green discoloration colonial morphology
of the medium.
 Examples of organisms that produce a-hemolysis Bacillus cereus
include Streptococcus pneumoniae and certain  Forms large, rough, greenish, hemolytic colonies on BAP
viridans streptococci.
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Eikenella corrodens
 Forms a small, "fuzzy-edged" colony with umbonate
center on BAP or CHOC

VI. Growth of Organisms in Liquid Media

 Turbidity
 Refers to cloudiness of the medium resulting from
growth (and usually gas if the medium contains
glucose).
 Turbidity produced by enterics growing in
thioglycollate
 In broth media, bacterial growth is indicated by a
change in broth's appearance. Take note: more
growth indicate a higher cell density and in effect
will elicit greater turbidity. (Refer to Figure 20)
  Growth =  Cell density  Greater Turbidity

Figure 20. Results from a Broth Media