[BACTERIOLOGY] CHAPTER 8: USE OF COLONIAL MORPHOLOGY FOR THE PRESUMPTIVE IDENTIFICATION OF MICROORGANISMS

INITIAL OBSERVATION AND INTERPRETATION OF CULTURES GROWTH OF ORGANISMS IN LIQUID MEDIA

• General incubation time: 18-24 hours • Observation of the growth of organism in liquid media such as
• Plate Reading: interpretation of primary cultures thioglycolate can give important clues to ID
• Blood Agar Plate (BAP or BA) • Turbidity: cloudiness of the medium resulting from bacterial growth
o Contains 5-10% mammalian blood (sheep or horse) • Gas is usually present if the medium contains glucose
o Used to isolate fastidious organisms and detect hemolysis • THIOGLYCOLLATE BROTH
• Chocolate Agar Plate (CHOC or CA) o Vine or Streamer effect: exhibited by certain species of streptocci
o Contains lysed RBCs (slow heating to 80°C o Puffed balls: exhibited by certain streptococcal species or gram-
o Used for growing fastidious bacteria (e.g., Haemophilus positive cocci
influenzae) o Turbidity: produced by enterics (formation gas bubbles at the
• In general, organisms on BA would also grow on CA but not all organism surface and in the middle of the broth)
that grow on CA grow on BA o Scum (at the surface): produced by yeast (grows in the
o Highly fastidious organisms: Haemophilus spp. And Neisseria microaerophilic area of the broth)
gonorrhoeae o Scum (at the sides): produced by Pseudomonas spp. (occasionally,
o Factor X (hemin): present in both BA and CA a diffusible green pigment and a metallic sheen can be seen at the
o Factor V (NAD): present only in CA surface)
• MacConkey Agar (MAC or MC)
o Supports most gram-negative rods especially Enterobacteriaceae
o Inhibits growth of gram-positive organisms
o Inhibits growth of fastidious organisms (e.g., Haemophilus and
Neisseria spp.)
o Best used to characterize gram-negative rods because lactose
fermenters (LF) can be differentiated from nonlactose fermenters
(NLF)
o LF: produce pink colonies
▪ Escherichia or Citrobacter: appears dry and flat; pink
precipitates of bile salts extend beyond the periphery of the
colonies
▪ Klebsiella or Enterobacter: pink, heaped, mucoid
appearance; slightly cream-colored center after 48 hour’s
growth
o NLF: colorless colonies
[BACTERIOLOGY] CHAPTER 8: USE OF COLONIAL MORPHOLOGY FOR THE PRESUMPTIVE IDENTIFICATION OF MICROORGANISMS

GROSS COLONY CHARACTERISTICS

HEMOLYSIS • Observed in the media underneath the colony
• Caused by enzymatic or toxin activity of bacteria
• Colony may be removed with a loop to visualize hemolytic
pattern and passing the bottom of the plate through a bright
light (transillumination)
• TYPES:
o α-hemolysis
▪ partial lysing of erythrocytes around and under the colony
▪ green discoloration of the medium
o β-hemolysis
▪ complete clearing around the or under the colonies
▪ complete lysis of erythrocytes
o γ-hemolysis/nonhemolytic
▪ no hemolysis, no green discoloration, no complete clearing
• α-hemolysis
o Streptococcus pneumoniae
▪ Strong zone of α-hemolysis, umbilicate center or flat with
penny edge, wet (mucoid) appearance of colonies
▪ Translucent, may resemble a water droplet
o Certain Viridans streptococci
▪ Translucent, grayer, rough margin, umbonate layer
o Enterococcus
▪ No umbilicate or umbonate center; larger colonies,
smooth, darker margin
▪ More heaped and gray-appearing
• β-hemolysis
o Streptococcus pyogenes: wide, deep, clear zone
o Streptococcus agalactiae: narrow, diffuse close, zone
o Listeria monocytogenes: narrow, diffuse, close zone

SIZE • Visual comparison between genera or species • Gram-positive cocci

• Described as large, medium, small, or pinpoint o Small, white colonies
• Gram-negative rods
o Large, gray, mucoid colonies
FORM/MARGIN • Observed from the edge of the colonies • Irregular margins
• Described as smooth, filamentous, rough or rhizoid, or irregular o Proteus spp. (swarms)
• Irregular margins: likely to be motile • Rough edges
o Diphtheroid
ELEVATION • Determined by tilting the culture plate and looking at the side of
the colony
• Described as raised, convex, flat, umbilicate (depressed center),
or umbonate (raised or bulging center)
DENSITY • Can be transparent, translucent, or opaque
• Visualize the colony while using transillumination
COLOR • May be white, gray, grayish white, yellow, or buff • White: coagulase negative staphylococci
• Gray: Enterococcus spp.
• Yellow/Off-white: Micrococcus spp. and nonpathogenic
Neisseria spp.
[BACTERIOLOGY] CHAPTER 8: USE OF COLONIAL MORPHOLOGY FOR THE PRESUMPTIVE IDENTIFICATION OF MICROORGANISMS

CONSISTENCY • Determined by touching the colony with a sterile loop
• May be brittle (splinters), creamy (butyrous), dry, waxy, or sticky
PIGMENT • Inherent characteristic of a specific organism
ODOR • Should be determined when the lid of the culture plate is
removed and the odor dissipates into the surrounding
environment
• NEVER INHALE DIRECTLY from the plate
Colonies with Multiple Characteristics
• Bacillus cereus: colonies appear large and rough on BA
• Eikenella corrodens: small, fuzzy-edged, umbonate center-appearing colony on CA; has a tendency to “pit” the agar
• Buff: Diphteriods
• Creamy: S. aureus
• Sticky: certain Neisseria spp.
• Brittle, crumbly, and wrinkled: Nocardia spp.
• Dry and Waxy: Diptheroids
• Green (sometimes metallic sheen): Pseudomonas aeruginosa
• Brick red (esp at RT): Serratia marcescens
• Blue: Kluyvera spp.
• Purple: Chromobacterium violaceum
• Brown-black: Prevotella melaninogenica
• Old sock: S. aureus (evident on MSA)
• Fruity/Grapelike: P. aeruginosa
• Putrid: P. mirabilis
• Musty basement/Mousy: Haemophilus spp.
• Freshly plowed field: Nocardia spp.

DIFFERENCES BETWEEN VARIOUS ORGANISMS BY COLONIAL MORPHOLOGY

Differentiation of Streptococcus pyogenes and Streptococcus agalactiae
[BACTERIOLOGY] CHAPTER 8: USE OF COLONIAL MORPHOLOGY FOR THE PRESUMPTIVE IDENTIFICATION OF MICROORGANISMS

Differentiation of Staphylococci and yeast

[BACTERIOLOGY] CHAPTER 9: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA
[BACTERIOLOGY] CHAPTER 9: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA

• CARBOHYDRATE UTILIZATION
o Oxidation-Fermentation Tests
o Triple Sugar Iron (TSI) Agar
o Ortho-Nitrophenyl-β-D-Galactopyranoside Test

• GLUCOSE METABOLISM AND ITS METABOLIC PRODUCTS

o Methyl Red (MR) Test
o Vogues-Proskauer (VP) Test

• AMINO ACID UTILIZATION

o Decarboxylase and Dihydrolase Tests
o Deaminase Test

• MISCELLANEOUS TEST
o Citrate Utilization
o DNase
o Gelatin Liquefaction
o Indole Production
o Malonate Utilization
o Motility
o Nitrate and Nitrite Reduction
o Oxidase
o Urease
o Lysine Iron Agar (LIA)
o Motility-Indole-Ornithine (MIO) Agar
o Sulfide-Indole-Motility (SIM) Agar