MICROBIOLOGY

063-1-MI

INTRODUCTION TO THE LABORATORY IDENTIFICATION OF COCCI

THE MICHENER INSTITUTE for Applied Health Sciences

1
 MICROBIOLOGY
063-1-MI

INTRODUCTION TO THE
LABORATORY IDENTIFICATION OF COCCI

Acknowledgements
Author
John TarBush, B Sc., A.R.T
Contributors And Reviewers
Christine Moore, B Sc., A.R.T

2000 by THE MICHENER lNSTITUTE for Applied Health Sciences

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 MICROBIOLOGY
063-1-MI

INTRODUCTION TO THE LABORATORY IDENTIFICATION OF COCCI

Table of Contents

OBJECTIVES 1

RESOURCE MATERIAL 5

MODULES/SUPPLEMENTS DEVELOPED BY THE MICHENER 5

INSTITUTE

REFERENCES 5
INTRODUCTION 7
I. Overview of Aerobic Gram-Positive COCCI 8
A. STAPHYLOCOCCUS-L1KElCATALASE-POSITIVE GROUP 10
Staphylococcus 10
Micrococcus 11
Stomatococcus 11

B. STAPHYLOCOCCUS-LiKEICATALASE-NEGATIVE GROUP 12
Aerococcus 13
Gameffa 13
C. STREPTOCOCCUS-LiKEICATALASE-NEGATIVE GROUP
13
Streptococcus
14
Enterococcus 14
Abiotrophla 16
Globicatella 16
Leuconostoc 16
II. Identification of Staphylococcus Species 17
A. IDENTIFICATION OF S. aureus 17
Colonial Characteristics 17
Coagulase 17
Commercial Rapid Slide Agglutination 19
Auxiliary Tests 20
B. IDENTIFICATION OF S. saprophyticus 22
III. Identification of Streptococcus Species 23
A. IDENTIFICATION OF BETA-HEMOLYTIC STREPTOCOCCI 25
Colonial Characteristics 25
Lancefield Grouping 25
VP Test 27
Presumptive Identification 27
B. IDENTIFICATION OF ALPHA-HEMOLYTIC STREPTOCOCCI 28
Cellular Characteristics 28
3
 Colonial Characteristics 28
Lancefield Grouping 29
Optochin Test 29
Bile Solubility 29
Bile Esculin 30
Latex Co-agglutination 30
C. IDENTIFICATION OF NON-HEMOLYTIC STREPTOCOCCI 32
IV Identification of Enterococcus Species 33
A. IDENTIFICATION OF ENTEROCOCCI FROM NON- 34
SURVEILLANCE SPECIMENS
Colonial Characteristics 34
Preliminary Grouping 34
Final Identification 35
B. IDENTIFICATION OF VRE IN SURVEILLANCE SPECIMENS 37
Gram Stain 37
PYR 37
Pigment 37
Rapid Xylose Fermentation 37
Arabinose Fermentation 38
Pyruvate Fermentation 38
Confirmation of Vancomycin ReSistance 38
Molecular Techniques 38
V Identification of Neisseria and MoraxeJla Catarrhalis 40
A. IDENTIFICATION OF N. gonorrhoeae 41
Colonial Characteristics 41
Cellular Characteristics 42
Oxidase 42
Superoxol (Catalase) 43
Growth Tests 43
Acid Production 43
Chromogenic Enzyme Substrate Tests 44
Multi-test Systems 45
Serologic Tests 45
DNA Probe 46
v Identification of Nelssena And Moraxella Catarrhalis (cont'd)
B. IDENTIFICATION OF N.
menmgitidis 46
Cellular Charactenstics 46
Colomal Characteristics 46
Other CharactensticsfTests 47
C. IDENTIFICATION OF M. catarrhalis 48
Colomal Characteristics 48
Other Characteristics/Tests 48

IDENTIFICATION TESTS 51

SELF-ASSESSMENT QUESTIONS 53

4
16. Describe the species-specific characteristics of S. aureus.
SELF-ASSESSMENT ANSWERS 62
17. Describe the principle, procedural details and limitations of the coagulase test (tube and slide
MICROBIOLOGY
versions) for identification of Staphylococcus species.
063-1-MI
LABORATORY IDENTIFICATION OF THE COCCI
18. Describe the principle, procedural details and limitations of commercial rapid slide agglutination
tests for identification of Staphylococcus aureus.
OBJECTIVES

19The
Sketch a flow chart
Objectives describing
indicate theshould
what you use of know,
coagulase tests and
understand commercial
and slide
be prepared agglutination
to explain upon tests for
identification of Staphylococcus aureus.
completion of this module. The self-assessment questions and answers will enable you to Judge
your understanding of the material. Upon completion of this module, the student should be able to:
I. State the two criteria used to categorize bacteria that appear coccoid in shape.
20. Name the test used for identification of Staphylococcus saprophytticus isolated in urine cultures.
2. State which genera of aerobic gram-positive cocci have "true" pathogenic species.
21. State 3 criteria used to place streptococci into broad categories to facilitate further identification.
3. Be able to state what Gram stain cellular morphology and arrangement can divide gram
positive cocci Into two broad groups.
22.4.BrieflyDescribe
describeclassic
the principle and value of Lancefield
"staphylococcus-like" grouping in the identification
and "streptococcus-like" of streptococci.
Gram stain morphology

23.5.State which
State the value of the catalase
characteristics/test test
results areInused
the categorization of gram-positive
to separate Groups A, C, and G cocci.
streptococci
from beta-hemolytic S. milleri species.
6. List the genera that are typically "staphylococcus-like" and catalase-positive.
24. State the use and principle of the CAMP test for identification of streptococci.
7. State the tests and results required to place an organism into the genus Staphylococcus.
25. State the tests used for identification of alpha-hemolytic streptococci.
8. State one distinguishing characteristic that separates the genera Micrococcus and Stomatococcus
from members of the genus Staphylococcus.
26. Describe the cellular and colonial characteristics of S. pneumoniae which differentiate it from
9viridans streptococci.
List the genera that are typically "staphylococcus-like" and catalase-negative.

27.10.
BrieflyList
describe the principle
the genera and value"streptococcus-like"
that are typically of the optochin and and
bile catalase-negative.
solubility tests in the
identification of ococci.
11. Describe the defining characteristics of the genus Enterococcus.
28. List the streptococcal species that can appear non-hemolytic on sheep blood agar. Describe the
tests used
12. Statetothe
differentiate
new genusthese
nameorganisms.
for nutritionally variant streptococci.

2913.
State the testsone
State anddistinguishing
results that differentiate members
characteristic for eachofofthe
thegenus Enterococcus
following from
genera: Ailotrophia, Globicatella,
streptococci.
and Leuconostoc.

30.14. State
Explain whythe two primary
enterococci arepathogenic
on the rise species Inof
as agents theinfectlon.
genus Staphylococcus.

31.15.
OutlineDescribe
the significance of vancomycin-resistant
the principles enterococci
and circumstances which guide (VRE) from aintreatment
laboratories and
the identification of
infection control point of view.
Staphylococcus species.

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063-1-Ml 3 2762

32. State two enterococcal species that can be vancomycin-resistant.

33. Explain the circumstances when speciation of enterococci is warranted.

34. State which two species of enterococci account for most enterococcal infections.

35. Describe the problems (and solutions to problems) with commercial systems In the identification of
enterococci.

36. Describe the tests used to detect VRE In surveillance cultures.

37 Describe the genera and/or species that occur as gram-negative cocci.

38. Describe the defining characteristics of the genus Neisseria.

39 List the commonly used tests for differentiation of Neisseria-like organisms.

40. Explain the importance of accurate identification when reporting a culture positive for Neisseria
gonorrhoeae (GC).

41. Outline the limitations of acid production and enzyme substrate tests for the identification of GC.

42. What test results provide a presumptive identification of GC In symptomatic males with urethritis and
discharge.

43. Describe the test and results for a definitive identification of GC.

44. Describe the test and results for a definitive identification of meningococci.

45. Describe the test and results for a definitive identification of Moraxella catarrhalis.

46. Correctly spell and transcribe the names of the following:

a) Staphylococcus aureus
b) Staphylococcus epidermidis
c) Staphylococcus saprophyticus
d) Streptococcus pyogenes

e) Streptococcus pneumoniae
f) Streptococcus agalactiae
g) Enterococcus faecalis
h) Neisseria gonorrhoeae
i) Neisseria menizngitidis
J) Neisseria lactamica.

If you have studied this subject previously, you may test your ability using the self-assessment questions. If you are
able to obtain 90% or greater, you may choose not to do the unit and merely review the sections, or parts of sections,
where weakness may exist. If you obtain less than 90%, it Iis recommended that the module be done in its entirety,
stressing areas where more review is needed.

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063-I-MI 5 2762
RESOURCE MATERIAL
This module includes the information necessary to meet the objectives. For the references listed below:
[P] designates sources of prerequisite information.
All others contain additional information.

MODULES/SUPPLEMENTS DEVELOPED BY THE MICHENER INSTITUTE

026-I-MI Colonial Appearance of Bacteria
024-I-MI An Overview of Culture Media
060-I-MI Staphylococcus
061-I-MI Streptococcus
062-I-MI Neisseria and Moraxella catarrhalis

REFERENCES

lh
1. Murray, P R., Editor m Chief, Manual of Clinical Microbiology, 7 Edition, ASM Press,
Washington D.C., 1999

2. Barrow, G. 1., (Editor), Cowan and Steel's Manual for the IdentificatIOn of Medical
rd
Bactena, 3 EditIOn, Cambndge Umverslty Press, U.K., 1993.

3. Isenberg H. (Editor), Clinical Microbiology Procedures Handbook, Vol. I, ASM

Publications, 1992.

4. Janda W M., Aerobic & Facultative Gram-Positive Bacteria, Self Study Course #3,
CAMLE, 1993.

5. Koontz F., Is There a Clinical Necessity to Identify Coagulase-Negative Staphylococci to

the Species Level? -Okay, So I was Wrong!, Clinical Microbiology Newsletter, No. 20, Vol. 9,
pages 78-80, 1998.

6. Bannerman T., New Staphylococcus species, Clinical Microbiology Newsletter, No. 18,
Vol. 10, pages 73-76, May 15, 1996.

7. Araj G. F., Reliability of Rapid Kits for Staphylococcus aureus Identification, Laboratory
Medicine, Vol. 28, No.2, pages 126-129, February 1997

8. McGeer A., The Rapid Emergence of a New Strain of MRSA m Ontario: Laboratory and
Infection Control Implications. LPTP Newsletter, No. 190, October 29, 1996.

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063-1-MI 6

9. Gardam M. A., Optochin Revisited: Defining the Optimal Type of Blood Agar for
Presumptive Identification of Streptococcus pneumoniae, J. Clin. Micro., pages 833-834, March
1998.

10. Tenover F C., Laboratory Methods for Surveillance of Vancomycin-Resistant Enterococci,

Vol. 20, No. I, pages 1-4, January 1, 1998.
nd
11. MacFaddin J.F., Biochemical Tests for Identification of Medical Bacteria, 2 Edition, The
Williams & Wilkins Co., Baltimore, Md, 1980.

12. Gold H. S., Antimicrobial-Drug Resistance, The New England Journal of Medicine, Vol.
335, No. 19, November 7,1996.
th
13. Koneman E.W., Color Atlas and Textbook of Diagnostic Microbiology, 5 Edition,
Lippincott, Philadelphia, 1997

14. YTI' Supplementary Committee Comments, Survey B-9802-1, Volume 3, Section 2.2,
pages 408-418, September 8, 1998.

15. Nelssena gonorrhoeae Gonorrhea Home Page, Centers for Disease Control and Prevention,
Atlanta, Georgia, U.S.A., http://www.cdc.gov/ncldod/dastlr/gcdir/gono.htrnl

16. Knapp, J.S., Historical Perspectives and Identification of Neisseria and Related Species,
Clinical Microbiology Reviews, Vol. 1, Pages 415-428.

17. Chernesky, M.A., Standards of Laboratory Practice for the Diagnosis of Neisseria
gonorrhoeae and Chlamydia trachomatis Infections, Standards of Practice Workshop,
CACMID,1997

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 2762
063-I-MI 7

INTRODUCTION

The technologist In bacteriology is faced with the challenge of Identifying significant c1imcal isolates to
genus or species level. This is a two-part process. The first phase is to categorize the isolate in broad
terms, using basic test results or characteristics. This crude form of Identification permits the selection of
appropriate bacterial identification algorithms, which lead to genus/species-level Identification (the second
phase).

Bacteria can be classified according to their atmospheric requirements. Some grow only In
ambient air (aerobes), in the absence of air (anaerobes), or in both air and in the absence of air
(facultative anaerobes). Other species require low oxygen levels for growth (microaerophiles).
The Gram stain can be employed to further categorize bacteria (for a detailed discussion on Gram staining,
refer to module 840-279). Bacteria can be categorized according to their cellular shape or morphology
Round bacteria are called cocci (Singular: coccus). Rod-shaped bacteria are called bacilli (singular:
bacillus) or rods. Many bacteria occur naturally In shapes somewhere In between a coccus or bacillus;
appearing as elongated cocci or coccobacilli. Some bacteria will take on the blue colour of the primary
stain (crystal Violet) and are classified as gram-positive. Other bacteria maintain the pink/red colour of the
counterstain (e.g. safranin) and are termed gram-negative.

This module focuses on the identification of aerobic cocci -both gram-positive and gram negative. Refer to
module 070-I-MI for a discussion of anaerobic cocci.

The module is split into 4 sections, as follows:

I OVERVIEW OF AEROBIC GRAM-POSITIVE COCCI

II IDENTIFICATION OF STAPHYLOCOCCUS SPECIES
III IDENTIFICATION OF STREPTOCOCCUS SPECIES
IV IDENTIFICATION OF ENTEROCOCCUS SPECIES
V IDENTIFICATION OF NEISSERIA AND MORAXELLA CATARRHALIS

Specimen selection and processing, isolation media, and antimicrobial susceptibility testing are not
discussed in this module. At the end of the module there is a section "IDENTiFICATION TESTS" which
describes some of the key Identification tests used to Identify these organisms.

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063-I-MI 8 2762

Overview of Aerobic Gram-Positive Cocci

There are 14 genera that fall into the gram-positive cocci group (Table I). Only 3 genera, Staphylococcus,
Streptococcus and Enterococcus have species, which are considered "true" pathogens and are recovered
with regularly from clinical samples. Other genera/species occur in clinical specimens as opportunistic
pathogens. The list of genera m this group will continue to grow and change, as more opportunists are
recognized and as taxonomic designations are refined. These uncommon organisms are being recovered
more often from clinical specimens.

It is important in normally sterile sites (blood or CSF) to provide a definitive identification to

species level for Isolates of aerobic gram-positive cocci.

Phase-one of Identification (broad categorization) involves the use of Gram stain

reaction/morphology/arrangement characteristics, atmospheric requirements, and the catalase test (as
shown m Table 1).

Cellular shape and arrangement can be used to divide these organisms into two broad groups:

1. Those With "staphylococcus-like" Gram stain characteristics -spherical to ovoid

cocci in pairs, tetrads (packets of four) or clusters
-Cellular morphology and arrangement characteristic of Staphylococcus species

2. Those with "streptococcus-like" Gram stain characteristics -more ovoid cocci or

coccobacilli in pairs and chains.
-Cellular morphology and arrangement characteristic of Streptococcus species

Gram stains performed on organisms grown m broth culture yield the most characteristic morphology

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063-1-MI 9 2762
Table 1. Characteristics of aerobic gram-positive cocci.· (Derived from 7'h Edition, Manual of Clinical Microbiology).

a Abbreviations and symbols: +, positive; -, negative; w, weak; C, cocci; Cb, coccobacilli; Pr, pairs; Tet, tetrads; CI, clusters; Ch,
chains
GROUP b GramSUB·GROUP FREQUENTLY
stains of organisms INFREQUENLY
grown in broth provide GRAM
most characteristic morphology CATALASE OXYGEN NEEDS
ISOLATED GENERA ISOLATED MORPHOLOGY •
C AlloiococclIs strains may be catalase-negative

d Gamella haemolysans may appear gram-variable GENERA

or gram-negative
"STAPH C G. haemolysans prefers aerobic incubation while G. morbillorum prefers anaerobic incubation
LIKE"
fI G. haemolysans usually occur as diplococci with adjacent sides flattened, while G. morbil!orllm cells are found in pairs and chains
CATALASE
POSITIVE
STAPHYLOCCOCCUS C, Cl (Pr, Tetl + Facultative anaerobe
Micrococcus C,Tel, CI + Strict Aerobe
Stomatacoccus C,Pr,CI ~. +. or w Facultative anaerobe
Alloiococcus Cb, Pr, Tet +' Strict Aerobe
Aerococcus C, Pr, Tel, Cl -or w MicroaeroDhile
CATALASE·
NEGATIVE
Gamella C", Pr, Ch, CI . Aerobe or Facultative
anaerobee
Helcococcus C, Pr,Ch,Cl - Facultative anaerobe
Pediococcus C, Pr, Tet, CI - Facultative anaerobe

"STREP·
LIKE"
CATALASE
NEGATIVE
STREPTOCOCCUS Cor Cb, Pr, Ch - Facultative anaerobe
ENTEROCOCCUS Cor Cb, Pr, Ch - Facultative anaerobe
Abiotrobhia C or Cb, rods . Facultative anaerobe
Globicatella Cb, Pro Ch - Facultative anaerobe
Lactococcus 'Cb, Pr, Ch - Facultative anaerobe
Leuconostoc Cb,Pr, Ch - Facultative anaerobe
063-1-MI 10 2762

A. STAPHYLOCOCCUS-LIKE I CATALASE-POSITIVE GROUP

Genera that are catalase-positive and have a "Staph-like" cellular morphology and
arrangement by Gram stain include: Staphylococcus, Micrococcus, Stomatococcus,
Alloiococcus, and Aerococcus (occasional strains).

Genus-level identification IS possible using a few basic tests (Table 2). The test/result pathway to genus-
level identification is shown for Staphylococcus, Micrococcus, and Stomatococcus. Alloiococci are not
usually recovered in clinical specimens. Aerococci will be discussed later.

Table 2. Differentiation of Staphylococcus-like I Catalase-positive genera

GENUS Strict Oxidase Growth

Aerobe a in NaCl Other Characteristics
Staphylococcus No - +b BaCItraCIn-resIstant
Micrococcus Yes + +b Bacitracin-Sensitive;
yellow colony
Alloiococcus Yes - +c Large coccobacilli
Stomatococcus No - -b
Bacitracin-sensitive;
sticky, adherent colony
Aerococcus d
Noe +c Colonies can resemble
viridans group
streptococci

a Modified oxidase test; 6% tetramethyl-phenylene-diamine hydrochloride

In dimethyl sulfoxide; reagent is dropped onto growth smeared on filter
paper; dark blue colour In 2 minutes is positive reaction
b Growth on nutrient agar supplemented with 5% NaCl
C Growth In 6.5% NaCl broth medium (salt tolerance medium for gram

positive cocci
d Most Aeroccoci are catalase-negative
e
Microaerophilic

Staphylococcus:
Staphylococcus is the most clinically significant genus in this group. S. aureus is the most virulent species.
For a detailed discussion of the taxonomy, natural habitat, clinical significance and other characteristics of
S. aureus and other staphylococci, refer to the module -060-1-ML

TestlResult
063-I-Ml Catalase-positive (strong) 11 gram-positive cocci in clusters and 2762
tetrads
Test/Result
facultative anaerobe tolerate 5% NaCI
Micrococcus:
Pathway'
Staphylococcus
After recent taxonomic changes, two species remain in the genus; M. luteus being the
 more common species m clinical samples. Micrococci are transient members of the
normal flora of human skin. M. luteus has been implicated as the causative agent m
infections (e.g. meningitis) of immunocompromised patients.

Stomatococccus:
S. mucilaginosus is part of the upper respiratory tract flora m healthy humans. It has been implicated as a causative
agent m infections (e.g. endocarditis) of debilitated patients. It can be either catalase-positive or negative (for
identification of catalase negative isolates -see next section). Colonies on blood agar are transparent to white with a
Sticky or rubbery consistency. They have a tendency to adhere to the agar.

TestlResult Catalase-positive (strong) gram-positive cocci in clusters and tetrads yellow

Pathway' colony (often) strict aerobe oxidase-positive or bacitracin-sensitive
Micrococcus

TestlResult Catalase-poslt!ve (weak) gram-pOSItive COCCI In clusters and tetrads rubbery,

Pathway' adherent colony no growth In 5 % NaCI
Stomatococcus

063-1-MI 12 2762

B. STAPHYLOCOCCUS-LIKE I CATALASE-NEGATIVE GROUP

Genera that are catalase-negative and have a "Staph-like" cellular morphology and
arrangement by Gram stain include: Stomatococcus. Aerococcus. Gemella.
Helcococcus. and Pediococcus.
Genus-level identification is possible using a few basic tests (Table 3). Stomatococcus has been discussed
in the previous section. The clinical significance of Pediococcus and Helcococcus species remains to be
defined, therefore, they will not be discussed here. It should be noted that pediococci are vancomycin-
resistant and must be distinguished from vancomycm-resistant enterococci (VRE).

Table 3. Differentiation of Staphylococcus-like I Catalase-negative Group (Excerpt

from 7thEd., Manual of Clinical Microbiology) a
GENUS PYR VAN LAP GAS ESCULIN Growth in 6.5
% Nacl
Stomatococcu +b S + -- + --
s
Aerococcus: S
viridans + -- -- V +
-- + -- V +
urinae
Gemelia + S V -- -- --
Helcococcus + S -- -- + V
Pediococcus - R + -- Vb V

a Abbreviations and symbols: PYR: detection of pyrrolidonyl

aminopeptidase; VAN: susceptibility to vancomycin; LAP: detection of
peptidase; GAS, gas production from glucose; ESCULIN:
hydrolysis of esculin; +, positive; -, negative; V, variable; S, susceptible;
R, resistant
b Most strains are positive

2762
063-I-MI 13

Aerococcus:
Aerococcl are found in the environment (i.e. water, air, dust, vegetation and on hospital
surfaces). There are two species in the genus. A. viridans, although usually an
opportunist, can be the cause of endocarditis and bacterermia. A. urinae is a new species
associated with urinary tract infections in debilitated patients. A. viridans forms alpha
hemolytic colonies on blood agar under aerobic conditions. As a result, this species is
often mistaken for viridans streptococci. A. unnae forms small alpha-hemolytic,
convex, shiny colonies on blood agar under aerobic conditions.

Gamella

This genus includes two species: G. morbillorum and G. haemolysans. G. morbillorum is a resident of the
respiratory and gastrointestinal tracts of humans and has been recovered from blood, respiratory,
genitourinary and abscess specimens. G.haemolysans is a member of the upper respiratory tract flora and
has been reported to cause endocarditis and meningitis.
Both species occur as small colonies on blood agar, very similar in appearance to colonies produced by
viridans streptococci. G. morbillorum prefers anaerobic incubation while G. haemolysans prefers aerobic
incubation. The cells of G. morbillorum are found in pairs and chains while G. haemolysans usually
occurs as diplococci with adjacent sides flattened. The latter species is easily decolorized by the Gram
staining method, leaving cells that resemble those of Neisseria species (i.e. gram negative diplococci).

C. STREPTOCOCCUS-LIKE I CATALASE-NEGATIVE GROUP

Genera that are catalase-negative and have a "Strep-like" cellular morphology and arrangement by Gram
stain include: Streptococcus, Enterococcus, Abiotrophia (or nutritionally variant streptococci).
Globicatella. Lactococcus, and Leuconostoc. Since there are very few reports of Lactococcus being
recovered from human sources, it will not be discussed in detail.

Genus-level identification can be achieved using many of the same tests used for the staphylococcus-likel
catalase-negatlve group (Table 4). The test/result pathway to genus level Identification is shown for:
Enterococcus Abiotrophia, Globicatella, and Leuconostoc (Table 4). The genus, Streptococcus, comprises
a diverse group of species. A test/result pathway is difficult to establish for this genus.

063-I-MI 14 2762

th
Table 4. Differentiation of Streptococcus-like I Catalase-negative Group (Excerpt from 7
 Ed., Manual of Clinical Microbiology).

GENUS HEM VANCO PYR LAP BE 6.5 % GROWTH

30 µg NaCl AT
100C 450C
Streptococcu A,B,N S --a + --b --c -- V
s
Enterococcus A,B,N Vd + + + + + +
Abiotrophia A,N S + + -- -- V --
Globicatella A S + -- -- + --- --
Lactococcus A,N S + + + V + V
Leuconostoc A,N R -- -- V V + V

Abbreviations and symbols:

HEM, hemolysIs on blood agar + 5% sheep blood; VANCO, susceptibility to vancomycm; PYR, production of
pyrrolidonyl aminopeptidase; LAP, production of leucine aminopeptide: BE, reaction on bile-esculin medium; 6.5%
NaCl, growth m broth containing 6.5% NaCl; A, alpha-hemolysis; B, beta- hemolysis; N, no hemolysis;
S, susceptible; R, resistant; +, positive; -, negative; V, variable.

Streptococcus:

A number of species in this genus are common pathogens m humans. The most significant members include
a
S. pyogenesGroup A Streptococci
(or Group are PYR-positive;
A streptococci), all others
S. agalactiae Streptococcus
(or Group spp are
B streptococci), PYR-negative
viridans streptococci, and
5 to 10% of viridans streptococci are weakly positive
S. pneumoniae. For a detailed discussion of the taxonomy, natural habitat, clinical significance and other
b

c
Someof
characteristics beta-hemolytic
Streptococcus streptococci cantogrow
species refer m 6.5%-061-I-MI.
the module NaCl broth
d Some strains are vancomycin-resistant .
Enterococcus:

Enterococci are normal inhabitants of the gastrointestinal tract and the genitourinary tract of humans. They are
involved m many different infections.

Enterococci are most commonly implicated m urinary tract infections. Enterococci have been increasingly more
common m abdominal or pelvic infections, which are often polymicrobial in nature. Enterococci are a leading cause
of nosocomial bacteremia.

063-1-MI 15 2762

Between 5 and 20% of bacterial endocarditis cases are caused by enterococci. Enterococcal infections of the
respiratory tract or the central nervous system are rare.

E. faecalis is the most common species of the genus, accounting for most of the human enterococcal infections. E.
faecium is the second most common species, responsible for 10 to 15% of enterococcal infections. This species is
usually more resistant to antibiotics than E. faecalis. Other Enterococcal species are rarely encountered m human
c1imcal specimens and normally reside in the gastrointestinal tracts of various animals.
Members of the genus Enterococcus form spherical (coccal) or ovoid cells (less than 2 µm in diameter) that are
gram-positive. Cells may appear coccobacillary when taken from an agar plate. As is the case with streptococci,
they tend to divide in one plane forming pairs and short and long chains.

The defining characteristics of the genus Enterococcus are as follows:

Gram-positive cocci in pairs and chains

Catalase-negative
Facultative anaerobes (better growth under aerobic rather than anaerobiC conditions)
Carbohydrates (e.g. glucose) are fermented
Growth In 6.5% NaCl
Hydrolyze esculin In the presence of 40% bile salts (bile esculin positive)
Positive for the enzyme leucine ammopeptidase (LAP}
Positive for the enzyme pyrrolidonyl aminopeptidase (PYR)
Usually vancomycin-susceptible

Note that the presumptive identification of Enterococcus (to genus-level) based upon a positive bile-esculin
reaction and growth In 6.5% salt broth is inaccurate. Other genera that share the same reactions (Lactococcus and
Leuconostoc) can occur in clinical specimens also.
Only approximately 80% of Enterococcus species test positively with Lancefield group D antiserum. Also, most
strains of Pediococcus and about half of Leuconostoc strains isolated from human infections have group D
antigens. As a result, determination of Lancefield group D antigen is not a definitive tool for genus-level
identification.

Molecular methods are emerging to permit identification to the genus-level in one step.
(e.g. GenProbe)

TestlResult Catalase-negative => gram-positive cocci in pairs and chains => facultative anaerobe
Pathway- => Vancomycin-susceptible (except rare strains) => LAP-positive => PYR-positlve =>
Enterococcus growth In 6.5% NaCl => growth at1645oC
063-1-MI 2762

Abiotrophia:

These organisms were formerly known as nutritionally variant streptococci. They are
normal residents of the oral cavity of humans. They are proven agents of endocarditis.
Members of the genus Abiotrophia require thiol compounds, cysteine or the active form
of vitamin B6 for growth. Consequently, they grow on chocolate agar but not on
Trypticase soy agar (TSA) with 5 % sheep blood.

A simple test to presumptively identify this species involves streaking a plate of TSA With 5% sheep blood for
confluent growth and applying a cross streak of Staphylococcus aureus (ATCC 25923). The plate is then incubated
 at 35°C in increased CO2• Abiotrophia strains will grow only near the "staph streak", a phenomenon called
satellitism.

Globicatella:

G. sanguis is the sole species m this genus. The natural habitat of this species is not yet. defined. It has been
isolated from patients with bacteremia, meningitis and urinary tract infection. Although both the cellular and
colonial morphology of this species can resemble members of the viridans group streptococci, they are PYR-
positive and salt tolerant (viridans group streptococci are PYR-negative and salt intolerant).

Leuconostoc:

A number of species of Leuconstoc have been described. These organisms are widespread m the environment and
are routinely recovered from a number of food products. Leuconstocs have been isolated from blood cultures of
patients with underlying malignancies and indwelling intravenous catheters. Leuconostocs produce small, alpha-
TestlResult Catalase-negative => gram-positive COCCI m pairs and chains => facultative anaerobe
hemolytic or non-hemolytic colonies that can be confused for viridans streptococci.
Pathway' => alpha or non-hemolytic => Vancomycin-susceptible => LAP-positive => PYR-
It should be noted that Leuconostocs (along with pediococci) are vancomycin-resistant and must be distinguished
AbwtrophUl positive => no growth m 6.5% NaCl => satelliting-positive
from vancomycin-resistant enterococci(VRE).

063-I-Ml 17
Identification of Staphylococcus Species

Generally, laboratories perform limited identification testing of staphylococci. Laboratories need to determine:

1. If a Staphylococcus isolate is S. aureus (Figure I)

2. If a urinary tract isolate of staphylococci is S. saprophyticus (Figure 2)
3. Species-level identity of isolates recovered from normally sterile body sites (e.g. blood, CSF),
which are deemed to be clinically significant.

Accurate speciation of staphylococci is a challenge. Many commercial identification systems can successfully
identify common species but are incapable of reliable identification of many of the more uncommon ones. Many of
these systems fail to yield a reproducible identification when challenged repetitively with the same isolate.
Fortunately, there are Simple, reliable and inexpensive conventional tests that can be used to determine If an isolate
TestlResult
is either S. aureus Catalase-negatlve => gram-posltlve COCCI m Parrs and chams => facultatlve anaerobe
or S. saprophyucus.
Pathway'
=> alpha-hemolytlc => Vancomycmsusceptible => LAP-nej1;atlve => PYR-pOSltlve
Globzcatella
Each laboratory should establish which isolates of staphylococci (beSides S. aureus and S. saprophytlcus) must
be identified to species level. Referral to a reference laboratory may be the best option for identification of
uncommon species.

This section will focus on the identification of S. aureus and S. saprophytlcus. For identification of other
staphylococci please refer to the references.

A. IDENTIFICATION OF S. aureus

Colonial Characteristics:

Most staphylococci grow readily on non-selective blood, nutrient, tryptic soy,

and brain heart infusion agars. Colonies usually range from I to 3 mm In diameter after 24 hours of incubation at
o
3S C, depending upon the species.
Typically, colonies of S. aureus are golden-coloured, beta-hemolytic on blood agar, and approach 2-3 mm in
diameter at 24 hours of incubation. Also, they have a smooth surface with a regular edge and low convex profile.
Unfortunately, colonial characteristics are not consistent and are no longer regarded as a reliable identification tool.
Some strains of S. aureus are white to gray in colour. Often these stains are methicillin-resistant (MRSA).

Coagulase:

The detection of coagulase production continues to be the most Widely used and accepted test for the preliminary
identification of staphylococci. The only coagulase-positive species recovered from human infection is S. aureus.
Other

063-I-MI 18 2762

coagulase-positive staphylococcal species exist in nature (S. intermedius and S. hyicus) but they are rarely
isolated from c1inical specimens.

It is common practice to categorize and report non-urinary strains of

staphylococci based upon results of the coagulase test. For example, coagulase
positive isolates can be reported as "S. aureus" Coagulase-negative. Isolates can
be reported as "Coagulase-negative staphylococci" (CNS). Isolates of CNS
from urine cultures must be tested to determine If they belong to the species, S. saprophyticus.

There are two different methods used to detect coagulase: the slide test and the tube test. Both tests
involve the use of plasma. Rabbit plasma containing EDTA is widely used. For a detailed discussion of the
principle of these tests consult the latest edition of Biochemical Tests for Identification of Medical
Bacteria by MacFaddin.

The slide coagulase test detects only "clumping factor" or coagulase bound to the surface of the cell wall.
This factor reacts directly with the fibrinogen in plasma making the bacterial cells "sticky" This process is
seen macroscopically as rapid clumping of bacteria Into visible aggregates.
One drop of plasma is added to a heavy, even suspension of the test organism in distilled water. Clumping
or agglutination within 10 seconds is considered to be a positive reaction. A smooth suspension at 10
seconds is considered to be a negative reaction. Some strains of staphylococci are inherently sticky and can
auto-agglutinate when combined with water. All Isolates should be checked for auto-agglutination while
making the original suspension m water. Strains that auto-agglutinate should not be tested using the slide
method. These strains can be tested reliably using the tube test.
TestlResult Catalase-negatlve => gram-pOSItive COCCI m p31rs and ch31ns => facultatlve
Pathway' anaerobe => alpha or non-hemolytlc => vancomycm-reslstant => LAP-negatlve =>
Leuconostoc PYR'-negatlve
Approximately 10 to 15% of S. aureus strains may have negative slide coagulase results because they lack
bound coagulase. Consequently, all slide negatives must be confirmed by the tube test (Figure I). Strains
deficient in clumping factor will usually produce free coagulase resulting in a positive tube test.

Note the following when performing the slide coagulase test:

 False-positive reactions may occur if the mixture is read beyond the recommended 10
seconds or if the inoculum is taken from a medium with a high salt concentration (e.g. mannitol-
salt agar)
 Two relatively uncommon Staphylococcus species -S. schleiferi and S. lugdunensis can
yield a positive slide test.
 False-negative reactions occur with some MRSA strains

063-l-MI 19 2762

The tube coagulase test is capable of detecting free coagulase (enzyme released into the environment)
and clumping factor. To perform this test, a large colony from a non-Inhibitory agar is suspended In a
small volume of plasma contained in a test tube. The mixture is incubated at 37°C for 4 hours. The
observation of a fibrin clot, upon tilting the tube 90° from vertical, is considered a positive reaction. A tube
test that is negative at 4 hours should be incubated at room temperature and read again at 24 hours.
The tube test is the most widely used method for the definitive identification of S. aureus.

Note the following when performing the tube test:

 Some strains of S. aureus will yield false-negative results at 24 hours due to the production
of high quantities of staphylokinase, which can lyse fibrin clots

 Any degree of clotting is considered a positive reaction

 Weak-positive or false-negative reactions have been reported for some MRSA strains,
especially after the initial 4 hour Incubation

Commercial Rapid Slide Agglutination:

There are a number of commercial kits that are designed to rapidly identify S. aureus. Some use latex
particles (carrier molecules), coated with plasma containing immunoglobulin G (IgG) and fibrinogen, to
detect both clumping factor and protein A on the surface of S. aureus cells. Others use erythrocytes
sensitized with fibrinogen as a carrier molecule for detection of clumping factor alone. Manufacturers
have recently introduced latex kits which are designed to detect protein A, clumping factor and specific
capsular antigen regions of the bacterial cell.

A portion of a suspect colony is mixed in a drop of the test reagent. Rapid agglutination is considered to be
a positive reaction.

These kits readily identify most methicillin-sensitive isolates of S. aureus. However, kits which target only
clumping factor and Protein A may fail to identify certain strains of MRSA. Kits that detect all three of the
above determinants seem to have the most promise for the identification of MRSAs.

063-I-MI 20 2762

Although these kits have of a higher specificity and sensitivity than the traditional slide coagulase test, they can
have significant limitations:

 Some strains of S. saprophyticus, S. lugdunensis, S. schleiferi, and other Staphylococcus

species plus Micrococcus species may produce a positive reaction with some kits and remain negative with
the conventional slide coagulase test
 Some MRSA strains may not yield a positive reaction

A properly controlled tube coagulase is more reliable than most if not all of these rapid slide agglutination tests.

Auxiliary Tests:

There are occasions when auxiliary tests are required to identify problematic Isolates or strains of S. aureus that do
not exhibit typical species characteristics. Some laboratories have even incorporated some of these tests Into routine
identification algorithms.
S. aureus and a few uncommon staphylococcal species (S. schleiferi, S. intermedius and S. hyicus) produce a
heat-stable nuclease (DNAse) that can cleave DNA. Virtually every strain of S. aureus, including MRSA, is
positive for this enzyme.
Accuprobe™, a DNA probe manufactured by Gen-Probe, demonstrates 100% specificity (no false positives) and
100% sensitivity (no false negatives) m most studies. It detects a specific gene sequence found only m the ribosomal
RNA of S. aureus.

063-1-MI 21 2762

Figure 1 Basic Identification algorithm for S. aureus.

Note: Modifications may have to be made to this algorithm based upon the performance
characteristics of the commercial slide agglutination product chosen.

Test for auto-agglutination

Positive Negative
S aureus Coagulase
-Negative
Staphylococci
Negative Positive

Negativ
Slide Coagulase
Positive Slide Coagulase
S aureus Negative

TUBE COAGULASE TEST

 Positive 063-1-MI Negative 22
S aureus 2762 Coagulase Negative
B. IDENTIFICATION OF S. saprophyticus
Staphylococci

In young women, S. saprophyticus is the second most common cause of cystitis (inflammation of
the bladder) after Escherichia coli. _This species can also cause infection In men and older women.
As a result, all urinary isolates of staphylococci are routinely tested to determine if they belong to
this species.

Susceptibility to novobiocin (an antibiotic) is the most widely used screen test for S. saprophytzcus
(Figure 2). S. saprophyticus is usually the only Staphylococcus species found in urine that is
novobiocin-resistant. Two other species found in humans, S. cohnii and S. xylosus, are also novobiocin-
resistant but they are encountered rarely.

In one method, a suspension of the test organism is adjusted to a 0.5 McFarland turbidity then swabbed
onto Mueller-Hinton agar (or other approved medium) for confluent growth.. A 5 µg novobiocin disk is
0
then placed on the inoculum. The plate is incubated at 35 C for 18 to 20 hours. A zone of clearing or
inhibition around the disk with a diameter of ≤16 mm is considered evidence of novobiocin resistance.
Such an isolate can be reported out as S. saprophyticus.

Figure 2. Screening algorithm for S. saprophyticus

COAGULASE-NEGATIVE
STAPHYLOCOCCI
IN URINE CULTURE

NOVOBIOOCIN
5 µG

Zone of Inhibition Zone of Inhibition

≤ 16 mm ≥ 16 mm

S saprophyticus Coagulase-negative
Staphylococcus – NOT
S saprophyticus
063-1-MI 23 2762

III Identification of Streptococcus Species

Hemolytic reactions, colonial morphology and Lancefield serological testing or serogrouping are tools
used routinely as first steps in the identification of clinical isolates.

Streptococci are initially differentiated into 3 groups based upon their hemolytic reactions on blood agar.
Briefly, these reactions are as follows:

1) Beta-hemolytic -zone of complete clearing around the colony

2) Alpha-hemolytic -zone of partial clearing (greenish colour) around the colony
3) Non-hemolytic -blood agar around the colony remains unchanged

Hemolytic reactions may vary with the source of the blood (type of animal) or the type of basal medium used
in the blood agar. The hemolytic reactions for streptococci in the following section are based upon studies
using sheep blood agar. Beta-hemolysis of Groups A, C, and G streptococci is enhanced by incubating blood
agar plates anaerobically or by stabbing the Inoculum into the agar, creating a relatively anaerobic
environment. Both techniques ensure that the oxygen labile hemolysin (streptolysin O) is not inactivated by
oxygen.

Streptococci can be arranged In categories according to cell-wall antigens using the Lancefield grouping
system. Many beta-hemolytic streptococci can be assigned to Lancefield groups A, B, C, F, and G. The
same system can be used to categorize some non-hemolytic isolates as members of groups B and D
streptOCOCCI. The system, however, has linuted value. Some genetically dissmIilar speCIes/groups may
share the same Lancefield group antigens. For example, S. pyogenes and some anginosus group viridans
streptococci may share the "A” antigen.

The Initial categorization of streptococci according to hemolysis, colonial morphology and Lancefield
antigens is shown In Table 5.

For a more detailed discussion of taxonomy, see Module 061-1-MI (Streptococcus).

063-1-MI 24 2762

Table 5 Initial categorization of streptococci

HEMOLYSISa COLONYb LANCEFIELD MEMBERS

ANTIGEN
Beta-Hemolytic Large Colony A Group A streptococci (S pyogenes)
 0.5 mm B Group B streptococci (S agalactiae)
C Group C streptococci
G Group G streptococci
Small Colony A,, C, F or G Viridans (group) streptococci
< 0.5 mm Anginosus group or S milleri group
S anginosus
S intermedius
S Constellatus

Not-groupedc S. iniae (rare)

S. porcinus (rare)
Viridans group streptococci
Mutans group (rare)
Alpha-Hemolytic Mucoid or Not-groupedc S peumoniae
dimpled colony
Pin-point Not-groupedc Viridans (group) streptococci
Mitis group
Mutans group
Salivarius group
Anginosus group (S milleri group)-
non-groupable
Non-Hemolytic Pin-point Not-groupedc Viridans (group) streptococci
Other types B Rare Group B streptococci (S agalactiae)
D S. bovis

Abbreviations and symbols:

a
Hemolytic reactions for sheep blood agar incubated aerobically in CO2 [are Enterococci can be beta-
hemolytic
b
Colonial morphology at 24 hours, "large colony" beta-hemolytic colonies are> 0 'j mm in diameter, "small
colony" beta-hemolytic colonies are < 0 5 mm in diameter ,
c
Strains are not grouped for identification purposes in routine laboratories
063-I-MI 25 2762

A. IDENTIFICATION OF BETA-HEMOLYTIC STREPTOCOCCI

Colonial Characteristics:

Some inexperienced technologists may confuse the colonial appearance of beta-hemolytic streptococci with beta-
hemolytic strains of staphylococci. Most beta-hemolytic streptococci have a high convex colony (i.e. very raised)
and a larger zone of hemolysis as compared to beta-hemolytic staphylococci. Remember· perform a gram stain
and catalase test when in doubt!

Beta-hemolytic groups A, C and G form large colonies (> 0.5 mm m diameter) after 24 hours of incubation. Small-
colony-forming beta-hemolytic strains of the S. milleri group, which may share common Lancefield antigens with
the above groups, are much smaller or pinpomt (< 0.5 mm m diameter). These small-colony species usually
produce a distinct carmel-like odour. Colonies of Group B streptococci tend to be large and have a smaller zone of
beta-hemolysis. Occasional Group B strains are non-hemolytic.

There are other more infrequently isolated streptococci that can produce beta-hemolytic colonies. S. iniae colonies
often exhibit a narrow zone of beta-hemolysis surrounded by a larger alpha-hemolytic zone. S. porcinus and
occasional strains of S. mutans can also be beta-hemolytic.

Lancefield Grouping:

Numerous commercial kits, using various antigen extraction methods and agglutination techniques, exist for
Lancefield grouping of beta-hemolytic streptococci. Streptococcal Isolates that are positive for the group B antigen
can be reported reliably as S. agalactiae (or Group B streptococcus). Small-colony beta-hemolytic streptococci that
test positive for the F antigen can be classified as viridans streptococci-S. milleri group. Isolates that possess
Lancefield antigens of groups A, C, and G may require additional testing (Table 6) when colony size can not be used
as an identifying characteristic.
Table 6 Identification of streptococci (Key tests and results are shaded)

Abbreviations and symbols:

GROUP I HEMOLYSIS COLONY SERO VP PYR ODOUR OPTO BII,E BILE 65% SATELL PIG
SPECIES GROUP CHIN SOLUB ESCULIN NRCI ITING MENT
ILITY

Group A (S
pyogelles)
bela large A +
GroupB (S bela none large B
aga/aefae) ace
RED

Group C beta large C

Group G beta large G

S milleri pin point CARMEL

ACGP or
beta alpha none
none
+

S iniae small
narrow beta &
outer alph3 +

S pneumoniae alpha often mucoid S soluble

Viridans alpha none nan mucoid R insoluble

S bovis none alpha non mucoid

, R insoluble +

insoluble V
Abiolrophia· alpha none non mucoid + R +
VP, Voges-Proskauer test, PYR, test for pyrrolidonyl aminopeptidase, (+) = Positive; (-) = Negative; V =
Variable; S = susceptible, R = resistant, • Abiatrophia is a new genus for organisms formerly known as
nutritionally variant streptococci
3-1-MI 27 2762

VP test:

The Voges-Proskauer (VP) test for acetoin production will differentiate large-colony beta hemolytic isolates
With Lancefield group A, C, or G antigens (VP-negative) from small-colony anginosus ("S. milleri”) group
strains with the same antigens (VP-positive). A VP method for testing Streptococci is described in the
IDENTIFICATION TESTS section.

Presumptive Identification:

Many laboratories use a battery of cheaper or more rapid tests rather than the more expensive serological
methods (Lancefield grouping). These tests can yield a presumptive identification that correlates With
serological results. Presumptive identification may be sufficient for isolates from non-sterile body sites.
Definitive identification should be carried out for those isolates recovered from sterile body sites (i.e.
serogrouping in association with hemolysis and colonial size plus VP ( required).

The PYR test which detects the enzyme pyrrolidonyl aminopeptidase is often used to presumptively identify
S. pyogenes, a PYR-positive species. The test is negative for all other beta· hemolytic streptococci, with
exception of two rarely encountered species -S. iniae and S. porcinu. Laboratories must exercise caution If
they rely exclusively on the PYR test for Group A streptococcal Identification. Occasional enterococcal
strains appear beta-hemolytic on sheep blood agar and they are PYR-positive as well.

The CAMP test IS used to presumptively identify S. agalactiae (or Group B streptococci). A streak of
beta-hemolysin producing S. aureus (ATCC 25923) is inoculated in a straight line on a blood agar plate. The
test organism is streaked perpendicular to the "staph-streak" -but not touching. The plate is incubated at 35°C
for 18-24 hours. Most strains of group B streptococci liberate a protein called CAMP factor. When CAMP
factor combines with the beta-hemolysin produced by specific strain of S. aureus, enhanced beta-hemolysis
occurs. An "arrowhead" of intense hemolysis is displayed where the CAMP factor overlaps with the beta-
hemolysin of the S. aureus (Figure 3), is considered a positive reaction or CAMP test. It should be noted that
a small percentage of group, streptococci can also be CAMP test positive.

Figure 3. Positive CAMP test for S. agalactiae

S. agalactiae
S. aureus

Blood Agar
53-Hill 28 2762

Some laboratories prefer to use other presumptive tests. Susceptibility to Trimethoprim/sulfamethoxazole

(TMP-SMX) and bacitracin are sometimes tested together to distinguish group A from groups C and G
streptococci. Also, the hippurate hydrolysis test is an excellent alternative to the CAMP test for presumptive
identification of group B Streptococci. Consult a reliable textbook for details of these tests.

C. IDENTIFICATION OF ALPHA-HEMOLYTIC STREPTOCOCCI

Refer to Table 5 for the list of species that appear alpha-hemolytic on sheep blood agar. Consult Table 6 for
the appropriate identification tests and expected results for each group or species. Figure 4 isa presentation of
a basic identification algorithm for this group of streptococci.

Vindans Streptococci are difficult to identify to species level. The traditional identification scheme
introduced by Facklam In 1977, which used a battery of physiological and carbohydrate fermentation tests, is
error-prone due to the extensive taxonomic reorganization of this group of streptococci over the past twenty
years. There are a number of commercial identification systems on the market which demonstrate variable
performance. Some of these systems do not have reliable testing algorithms for all members of this group.
Speciation of viridans streptococci may be best suited to large reference laboratories that have the necessary
resources. Modem identification schemes have emerged In the last few years which can be found In
microbiology textbooks.

Cellular Characteristics:

Often, S. pneumoniae cells are ovoid or "Lanceolate-shaped" and occur in pairs (diplococci). Pneumococcal
cells, when recovered in fresh specimens (i.e. sputum, CSF, and Synoval fluid), are often surrounded by a
capsule. In gram stained smears, the capsule may appear as a pink ha or as a non-staining area surrounding
the cells. The cells of viridans streptococci and S. bovis are often elongated.

Colonial Characteristics:

Alpha-hemolytic colonies with a central depression are suggestive of pneumococci. This colonial appearance
has been likened to a "checker" or "dime" in that the colony edges are raised above the central region. Often
pneumococci produce convex colonies in the early stages of growth and "checker-like" colonies later on. The
collapse of the central region is due to the death of Individual cells (autolysis) as the colony ages.
Pneumococci also produce varying amounts of capsular material. Some heavily encapsulated strains may
produce large mucoid colonies that look like droplets of oil. Colonies of pneumococci are usually > 1mm in
diameter at 24 hours on blood agar. Colonies with a large zone of alpha-hemolysis are common when
cultures are incubated in increased CO 2.

Many members of viridans streptococci produce alpha-hemolytic, convex or domed colonies that vary In

Size (from 0.1 to 0.5 mm In diameter) and texture depending upon the species. They may appear mucoid and
translucent or glossy and non-translucent. It should be noted that some uncommon species, belonging to
Globicatella, Leuconostoc, Aerococcus, and Pediococcus, produce colonies that can be mistaken for
viridans streptococci.
,3-l-MI 29 2762

Some strains of S. bovis produce convex, alpha-hemolytic colonies that resemble viridans
streptococci.

Small alpha hemolytic colonies that grow only on chocolate agar but not on blood agar may belong
to the genus Abiotrophia (formerly known as nutritionally variant streptococci).

Lancefield Grouping:

Lancefield grouping is not a useful identification tool for viridans streptococci and S.
pneumoniae. Testing for Lancefield group D antigen may aid in the identification of some
strains of S. bovis. However, other strains of this species may not express the D antigen
consistently

Optochin Test:

Susceptibility to optochin (ethyl hydrocupreine hydrochloride) is the most widely used method for
differentiating S. pneumoniae (optochin-susceptible) from other alpha-hemolytic streptococci
(optochin-resistant).

A few colonies of the test organism are transferred to blood agar plate then streaked for confluent
growth. An optochin disk (available from a number of suppliers) is applied to the inoculum, and
the plate is incubated overnight at 35°C under Increased CO2 (5%). Zones of inhibition with a
diameter > 14 mm with the 6 mm disk or > 16 mrn with the 10 mm disk are interpreted as
optochin-susceptible; enough evidence to call the isolate S. pneumoniae. For zone diameters that are
> 6 mm but ≤ 14 mm (for the 6 mm disk), additional testing such as bile solubility must be
performed. No zone is consistent with an alpha-hemolytic streptococcus other than S. pneumoniae.
Rare pneumococcal isolates are optochin-resistant under properly controlled test conditions.
One recent study (Gardam et al) indicates that tryptic soy agar (TSA)-sheep blood is an excellent
medium for reliable results. Fewer intermediate zone diameters (requiring further testing) were
noted for S. pneumoniae isolates when tested on this medium as compared to the other media tested.

Bile Solubility:

Bile solubility is another method for differentiating S. pneumoniae (soluble in bile) from other
alpha-hemolytic streptococci(insoluble in bile). It has been shown that bile triggers enzymes in
pneumococci, which hasten the autolysis of cells. The test can be carried out on a broth or saline
suspension of the test organism or directly on plate-growth.

For the broth test, 0.5 ml of a 1.0 McFarland saline (or broth) suspension of the test organism is
aliquotted into two tubes. An equal amount of 2% sodium deoxycholate (bile) is added to one tube.
An equal volume of saline is added to the second tube as a control. Both tubes are incubated at 35°C
for up to 2 hours. Clearing in the tube with bile and absence of clearing in the control tube indicates
that the organism is bile soluble.
 53-1-MI 30 2762

In the plate method, a drop of 10% sodium deoxycholate is placed directly on a colony of the
test organism. The plate is then held a room temperature or placed into an aerobic incubator at 350(_
for approximately 15 minutes or until the reagent dries. Pneumococcal colonies will disappear or
become flattened. The colonies of bile-resistant streptococci (e.g. viridans group) will be unaffected.

Bile Esculin:

The bile esculin test determines if the organism is able to hydrolyze the glycoside esculin in the
presence of 10 to 40% bile. Bile esculin medium, either in a tube or plate, is inoculated with one
to three colonies of the test organism. Inoculated media are incubated for up to 48 hours at 35°1
m ambient air. Blackening of at least half of the slant (tube test) or any blackening of the agar
near the colony (plate test) is considered a positive reaction.

S. boils (Group D streptococci) are bile esculin-positive and all other alpha-hemolytic streptococci
are typically negative. A few strains of viridans streptococci can produce a weak positive reaction. A
positive bile esculin result is not sufficient to definitively identify S. bovis. Isolates from serious
infections should be subjected to a full battery of tests for definitive identification.

Latex and Coagglutination:

There are a number of commercial latex and coagglutination kits available that provide definitive
identification of S. pneumoniae. Results are usually available m 30 seconds. Routine use of _these
products, as opposed to optochin and/or bile solubility, can be expensive. Consult a bacteriology
textbook for details.

53-1-MI 31 2762
Figure 4. Basic Identification algorithm for alpha-hemolytic streptococcI.

STREPTOCOCCUS
ALPHA-HEMOLYTIC

Zone > 14 mm Zone > 6 mm No Zone

But ≤ 14 mm

Report
S pneumoniae

I BILE ESCULIN (BE)

BILE SOLUBILITY (BS)

BE Positive BE Negative BE Negative

BS Insoluble BS Soluble BS Insoluble

? S bovis S pneumoniae Viridans

Streptococci

Identify to species level if Isolate IS

from a normally sterile stte (e.g.
blood)

Vindans streptococci can be classified as biotype I or II based upon mannitol fermentation and glucan
production
63-I-MI 32 2762

C. IDENTIFICATION OF NON-HEMOLYTIC STREPTOCOCCI

Some strains of viridans and group D streptococci (S. bovls) and occasional strains of group B
streptococci occur on sheep blood agar as non-hemolytic colonies _(Table 6). Figure 5 is an
Identification algorithm for tins group of streptococci.

Figure 5. BaSIC identification algorithm for non-hemolytic streptococcI.

STREPTOCOCCUS
NON-HEMOLYTIC

BILE ESCULIN

POSITIVE NEGATIVE

Viridans group
Or Group B
GROWTH IN
6.5 % NaCl
GROUP B SEROLOGY

POSITIVE NEGATIVE
Enterococcus sp S bovis

POSITIVE NEGATIVE

GROUP B STREPTOCOCCI VIRIDANS GROUP

Identify to specIes level if Isolate
IS from a normally sterile slle (e.g.
blood)