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CHAPTER 2: MICROCOCCACEAE

1.) IDENTIFICATION: STAPHYLOCOCCUS AUREUS

TEST PROCEDURE MORE INFO RESULT

• 37C incubation, 18 • micrococcus - no

BACITRACIN colony -> TSA/broth
hours growth
SUSCEPTIBILITY -> MH agar, add 0.04
• observe zone of • staphylococcus -
TEST bacitracin disc
inhibition has growth
• unlike bacitracin, s.
POLYMYXIN
aureus is
SUSCEPTIBILITY
susceptible to
TEST
polymyxin
• accurate, but false
• rapid differentiation
(-) with methicillin
• tube coagulase - • rapid commercial
resistant strains
confirms methicillin test
• methicillin
resistance and • latex particles:
PARTICLE resistant s.
confirms coagulase staphylatex, sero
AGGLUTINATION aureus - coagulase
test (-) stat, staph latex,
TEST (-), cell wall
• slide test - same accu staph
contributes to:
results as • sensitized RBC:
clumping factor and
commercial ones hemastaph,
methicillin
staphiloslide
resistance
• epidemiologic
PHAGE TYPING study
• not done routinely
• 85-90% s. aureus
are PCN resistant,
due to:
SUSCEPTIBILITY - B-lactamase
production
- extrachromosom
al plasmids
• staphylococcus
• RIA tests don't aid in Dx
• ELISA • detection of
NON-CULTURE • agar gel diffusion teichoic acid, useful • almost all human
in: sera contain
METHODS • immunoprecipitatio
(detection of n - monitor infection antibodies against
staphylococcal
antibodies) • crossed - differentiate teichoic acid
immunoelectrophores long-standing
is and deep seated
infections

• coagulase + • coagulase (+) -

COAGULASE TEST plasma fibrinogen most distinguishing
= organism characteristic of s.

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TEST PROCEDURE MORE INFO RESULT

agglutinates,
plasma clots
aureus
• SLIDE TEST -
• other
clumping factor
staphylococcus do
(cell bound
produce coagulase
coagulase, 95% of
s. aureus)

TEST PROCEDURE MORE INFO RESULT

• TUBE TEST - non-

clump factor, test
for ability to
produce
extracellular see: table below
coagulase (4 hrs,
37C)
- citrate: false (+)
• toluidine blue DNA • halo of pink,
agar -> incubate 1 surrounding the
hr before the test inoculum
• (+) halo - presence
THERMOSTABLE
• colony in 5% BA -> of thermostable
DEOXYRIBONUCLEAS
E TEST microcapillary tube deoxyribonuclease
-> water bath (15 • (+) - s. delphini,
mins) s. schleiferi, s.
-> place on DNA intermedius, s.
agar hyicus
• blood culture ->
lysostaphin -> 37C
-> centrifuge -> • observe for lysis of
LYSOSTAPHIN
sediment on slide cocci
TEST
• (+) for s. aureus
• air dry, heat fix,
gram stain
• oxidase reagent:
• colony in BA -> • (+) test - dark blue
tetramethyl
MODIFIED smeared to filter color (2 minutes)
phenylene diamine
OXIDASE TEST paper -> drop of • (+) for - micrococci,
dihydrochloride in
oxidase reagent s. sciuri
dimethyl sulfate

1.1) COAGULASE TEST: DIFFERENTIATION OF STAPHYLOCOCCI, MICROCOCCI AND

STOMATOCOCCUS

CATALASE (+) VS CATALASE (-) MICROCOCCUS STAPHYLOCOCCUS

coagulase (-) resemble lysostaphin resistant sensitive to

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CATALASE (+) VS CATALASE (-) MICROCOCCUS STAPHYLOCOCCUS

staphylococci by
lysostaphin
gram stain
morphology: (+) to modified (-) to modified
micrococci staphylococcus oxidase test and oxidase test and
mucilaginosus benzidine benzidine
sensitive to
staphylococci* resists bacitracin
bacitracin

***cocci are catalase (+)

2.) SUMMARY OF MICROCOCCEAE

ORGANISM DSS SITE OTHER INFO

• invade bloodstream • peptidoglycan +

• metastasize and teichoic acid -
appear in urine protect from lysis
STAPHYLOCOCCUS • source of food • abscess in body and for attachment
: S. AUREUS poisoning
organs • polysaccharide
• variety of human
• produce septic capsule + protein A
*** see book for infection
shock - protect from
morphology, cultural • carrier state -
characteristics,
phagocytosis by
reservoir of
enzymes/toxins • from anterior nares, PMN
infection
perineum, skin
sites (10-15% • protein A = Fc +
healthy people) IgG
MICROCOCCUS CHECK P. 39 OF MICROBIOLOGY BOOK
• cardiac valves, CNS
shunt insertion,
total hip
replacement
• salt tolerant like s.
- create viscous aureus
COAGULASE (-) extracellular • thermonuclease (-)
• saprophytic than substance that • pale transparent
STAPHYLOCOCCI: pathogenic colonizes white (also smooth
S. EPIDERMIDIS smooth and circular like s.
surfaces: aureus)
plastic/metal

• skin and mucus

membrane

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ORGANISM DSS SITE OTHER INFO

• resembles s.
epidermidis
COAGULASE (-) - resists
• in normal skin, peri- novobiocin
• common cause of
STAPHYLOCOCCI: urethral and
S.
UTI
urethral flora
- resists nalidixic
SAPROPHYTICUS acid
- doesn't grow on
MacConkey
• like staphylococci
but adhere to agar
due to capsule
• catalase (-)
• vancomycin
• oral flora
STOMATOCOCCUS: resistant
• endocarditis
S. • inability to grow on
• septicemia • compromised
MUCILAGINOSUS 5% NaCl -
patients, drug users
distinguishes from
other
micrococcaceae
• PCN - drug of
choice

CHAPTER 3: STREPTOCOCCI AND RELATED GENERA

1.) STAPHYLOCOCCUS, MICROCOCCUS, STREPTOCOCCUS

STAPHYLOCOCCUS MICROCOCCUS STREPTOCOCCUS

acid from glucose

- facultatively anaerobic
(anaerobically)
acid from glycerol lactic acid from CHO
-
(erythromycin) metabolism

lysis by lysostaphin -

catalase (+) catalase (+) catalase (-)

sheep's BA / horse blood

- preliminary grouping of
isolates from hemolysis of
colonies on BAP
- sheep's BA: cultures of
inflamed throats, inhibits
haemophilus species

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STAPHYLOCOCCUS MICROCOCCUS STREPTOCOCCUS

brown's classification /
lancefield classification

- see book

*** stomatococcus mucilaginosus: catalase (-), see book

2.) RELATED SPECIES: VIRIDANS AND GEMELLA

AEROCOCCUS VIRIDANS AEROCOCCUS GEMELLA

gram stain from culture/broth: tetrads, no

opportunistic** pathogen
chains
bacteremia, endocarditis, meningitis,
wounds, endocarditis, abscess
osteomyelitis

s. morbillorum

3.) IDENTIFICATION: PNEUMOCOCCI

TEST PROCEDURE MORE INFO RESULT

• Na deoxycholate • autolytic amidase - • not absolute - not

• Na taurocholate cleaves alanine and all pneumococci
• Na dodacyl sulfate muramic acid in are lysed
BILE SOLUBILITY peptidoglycan • a-hemolytic are
- lyse the • amidase - activated lysed
pneumococci by surface active • pneumococci are
• BAP agents bile soluble
OPTOCHIN • ethylhydrocupreine • most widely used • pneumococci -
SUSCEPTIBILITY (optochin) on BAP test to differentiate zone of inhibition
TEST • candle jar or CO2 pneumococci and (14-16mm for
incubation a-hemolytic 10mm disc, 6-
streptococci 14mm for 6mm)

• 10-16mm for
10mm disc -

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TEST PROCEDURE MORE INFO RESULT

pneumococci,
only if bile soluble
• capsular antisera • capsular swelling -
(available not actual swelling,
commercially) just change in
- omnivalent • rapid, identifies
refractive property
NEUFELD serum type
• precipitin reaction -
QUELLUNG • most accurate
REACTION
- react with all specific ID for
makes capsule
pneumococci visible
pneumococci
- for sputum, CSF,
pleural, synovial, • produce
(+) blood culture agglutination

3.1) ADDITIONAL TESTS

TESTS PROCEDURE MORE INFO RESULT

• differentiate
• detect
pneumococcal
pneumococcal • sputum gram stain
pneumonia from
CIE antigen in and CIE = same
other pneumonia
sputum/nasopharyn results
• not sensitive in
geal in children
non-pneumonia
CAPILLARY • rapid antigen ID in • like neufeld but
PRECIPITIN TYPING sputum and blood simpler
• more accurate than
DIRECT
gram stain (when
QUELLUNG/OMNI • on sputum smears
correlated with
TEST
culture results)
• coagulation test for
PHADEBACT • commercial test
ID of pneumococci

3.2) LABORATORY DIAGNOSIS

TEST PROCEDURE MORE INFO RESULT

• direct gram stain

on sputum • gram stain result -
lanceolate
DIRECT SPUTUM
- determine diplococci =
EXAMINATION probable cause presumptive Dx of
of infection pneumococcal
• direct examination pneumonia
by quellung

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TEST PROCEDURE MORE INFO RESULT

• presumptive ID
• pour plates with
- appearance of a-
• BHIA/TSA hemolytic
• broth - 5% blood blood
colonies that are
and thioglycollate - provide info on bile sensitive
CULTURE
• blood culture - TSB magnitude of and optochin
and thio broth (1:10 bacteremia sensitive
blood and culture) - prognosis - ferment inulin
- (+) quellung
• PCN is the drug of
choice
• resistant strains will
SUSCEPTIBILITY TO cause infections
ANTIMICROBIAL - meningitis
AGENTS
- bacteremia
- (in addition to
pneumonia
• high seasonal rate
• isolation from blood • recovery is difficult
or CSF to interpret
SIGNIFICANCE OF • clinically significant
THE RECOVERY OF
- definite
evidence of if
PNEUMOCOCCI
pneumococcal - type 1, 3, 4, 7, 8,
pneumonia 12
- lobar pneumonia

*** see: book for intro

*** know the difference between streptococcal pneumonia, pneumococcal pneumonia b-hemolytic and
a-hemolytic

TAKE NOTE:

CATALASE (+) STREPTOCOCCI BENZIDINE (+) contains cytochrome enzymes

(+) - micrococcaceae
differentiated by BENZIDINE TEST
(-) - streptococcaceae

*** usually, if it's coagulase (-), it's catalase (+)

4.) IDENTIFICATION: STREPTOCOCCUS PYOGENES *** see: book for pathogenicity

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TEST PROCEDURE OTHER INFO RESULT

• 18 to 24 hrs -
pinpoint, domed,
• primary culture: • differentiate from
LABORATORY opalescent, 0.5mm,
streak plate / pour staphylococci -
IDENTIFICATION zone of hemolysis
plate catalase (-)
bigger than the
colony
• predicts 95%
• based on sensitivity accuracy the
• for pharyngeal
of group a presumptive ID of
cultures
streptococci to pharyngeal isolate
BACITRACIN TEST • distinguish b-
bacitracin • any zone of
hemolytic
• selective inhibition inhibition is (+)
streptococci
of group a on BAP (either b or a but
usually a)
• observe for red
color - presence of
• colony -> filter • more specific than b-napthylamine (by
paper w/ pyr -> add bacitracin hydrolysis)
PYR TEST N,N- • detects hydrolysis • (+) for s.
dimethylaminocinn of pyyrolidonyl-b- pyogenes, l.
amaldehyde naphthylamide garviae,
enterococci
• light orange is (-)
• direct culture of
LABORATORY • BA, FAT, broth
posterior pharynx
DIAGNOSIS • bacitracin, pyr
and tonsils

5.) IDENTIFICATION: STREPTOCOCCUS AGALACTIAE *** see: book for antigenic

structures

TEST PROCEDURE MORE INFO RESULT

(+) CAMP TEST • accentuated zone • ability to hydrolyze • arrow-head pattern

of hemolysis when Na hippurate of hemolysis
group b • camp factor: heat adjacent to
streptococci is stable extracellular staphylococcal
inoculated protein act with b- streak
perpendicular to a hemolysin on
streak of s. aureus sheep/ox blood • small percentage
are sensitive to
• streak strain of • majority can grow bacitracin (falsely
staphylococi on in 6.5% NaCl, few identified as group
BAP -> in 40% bile a)
streptococcus is • esculin is not
streaked hydrolyzed -
perpendicular to differentiates from
staphylococci (w/o group d
touching)
• include group a/b

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TEST PROCEDURE MORE INFO RESULT

control

6.) SUMMARY OF STREPTOCOCCI

ORGANISM DSS SITE OTHER INFO

• nasopharyngeal/oro
pharyngeal flora
(15% children, 5%
adults)
• bacterial
• respiratory
meningitis,
secretions
preceded by
STREPTOCOCCUS • URT, lungs,
pneumonia (2nd
PNEUMONIAE systemic • bile sensitive
most common
• endocardium, • a-hemolytic
cause)
*** see book for history, pericardium, • 80 types have been
• lobar pneumonia
morphology, cultural meninges, joints described
characteristics • community
• middle ear,
acquired
mastoid, eyes
pneumonia
• 1/4 - bacteremia
• from peritoneal
fluid, urine, vaginal
secretions, wound
exudates
• sputum, spinal fluid
RESULT
PROCEDURE
• produce
• BA pour plate ->
HEMOLYTIC surface/subsurface
subculture ->
STREPTOCOCCI of colonies
microscopic
surrounded by clear
examination
zones of hemolysis
• streptococcal sore
throat
GROUP A: • pharyngitis, scarlet
STREPTOCOCCUS fever • Group B, C, G -
• oxidase (-)
PYOGENES • erysipelas found in pharynx
• killed in 30 mins at
• childbed and other tissue
60C
*** see: book for fever/puerperal sites
cultural characteristics sepsis: maternal
mortality in
preantibiotic era

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ORGANISM DSS SITE OTHER INFO

• destroyed by
• neonatal sepsis pasteurization
- infants acquire • only streptococci
GROUP B: such as they with specific
• flora - pharynx,
STREPTOCOCCUS pass through morphological
vagina, GIT
AGALACTIAE vagina criteria
• normal flora of the
- transmission is GUT of
• 15-20% pregnant
*** see: book for prevented by women are carriers
morphology
humans/mammals
ampicillin before • onset of infection -
delivery within 10 days of
• meningitis delivery, neonates
mortality 50%

*** make a table between a-hemolytic and b-hemolytic

ORGANISM DSS SITE OTHER INFO

• s. equisimilis -
source of
streptokinase
• severe pharyngitis
GROUP C: (thrombolytic Rx)
• followed by
STREPTOCOCCI • s. zooepidemicus
bacteremia and
*** see: book for table - postreptococcal
metastatic infection
and antigenic structure GMN after drinking
• hemolytic-b
milk
• s. dysgalactiae -
not hemolytic-b
• clin. significance of
enterococci in
blood cultures:
- people with
septicemia, more
(+) sets of blood
cultures, 2 (+)
• bacterial
bottles in a single
endocarditis
set
GROUP D: • s. bovis - px with bacteremia
STREPTOCOCCI septicemia is grew cultures with
*** see: book for shorter detection
categories
associated with
colon CA or times (90% yield)
intestinal • accurate
malignancy presumptive test -
BILE ESCULIN
MEDIUM
(eneterobacteriace
ae)
• useful biochemical
test - BHIB growth
(6.5% NaCl)

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ORGANISM DSS SITE OTHER INFO

• a-hemolytic
• catalase (-)
• no lancefield b and
d antigen
VIRIDANS • opportunistic • oral • not bile soluble
STREPTOCOCCI • low virulence • GIT • not sensitive to
*** see: book for table • facultative (some • RT optochin
on presumptive are obligatory - humans and • inulin not
identification anaerobe) animals fermented
• can't grow on 6.5%
NaCl
• green zone
hemolysis on BAP
• normal flora -
pyridoxal (B6)
• endocarditis
NUTRITIONALLY • oral dependent, thiol
• transferred to
VARIANT • GIT requiring, satellite
GENUS
STREPTOCOCCI • UGT • will not grow on 5%
ABIOTROPHIA
sheep's BAP unless
presence of B6

6.1) GROUP D STREPTOCOCCI: CHARACTERISTICS

ORGANISM DESCRIPTION CULTURE/TESTS

• NaCl plate medium - false

• a-hemolytic/non-hemolytic
• s. faecalis/zymogenes -
GROUP D: b-hemolytic
STREPTOCOCCI • buttery odor
*** see: table for BEM salt • differentiated from
tolerance streptococci through ability
to grow in 45C and 60C for
30 mins
(-) due to paraffin
• grow at: 45C
• withstand: above 60C
• grow in 40% bile
• hydrolyze esculin
• urinary isolates + thymine
= fail to grow on MH
- grow well on BA
• thymine dependent resist
clotrimazole
• listeria monocytogenes -
hydrolyze esculin to 6,7-
dihydroxycoumarin

7.) RELATED GENERA

GENERA DSS SITE OTHER INFO

LEUCONOSTOC • septicemia • sterile body fluids • gram (+)

• meningitis vancomycin
• UTI resistant cocci

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GENERA DSS SITE OTHER INFO

• resemble
streptococci
morphologically
and biochemically
• normal flora
• vancomycin
PEDIOCOCCUS • but also in human
resistant
infection
• VANCOMYCIN
SUSCEPTIBLE
• streptococci
lancefield group N
is part
• l. garviae - human
LACTOCOCCUS • nonmotile
disease
• catalase (-), pyr (-) -
except for l.
garviae
• a-hemolytic/non-
hemolytic
• virulence
mechanisms not • catalase (-)
AEROCOCCUS AND
known • blood • gram (+) cocci in
GEMELLA
• UTI tetrads
• wound infections

CHAPTER 4: FAMILY NEISSERIACEAE

1.) IDENTIFICATION: NEISSERIA GONORRHOEAE

TEST PROCEDURE MORE INFO RESULT

• indophenol oxidase • all neisseria -

+ redox dye = • if obtained from oxidase (+)
bacterial color GUT • color change from
change - oxidase (+) pink, violet to black
• 1% sol'n - gram (-) • slow change is not
tetramethyl-p- diplococci neisseria
phenylenediamine
IDENTIFICATION • oxidase (+)
dihydrochloride • if obtained from
(fresh/refrigerated) pharyngeal, or colonies are gram
• platinum wire loop medico-legal stained
• nichrome wire - situations, have a - observe gram (-)
false (+) more definitive diplococci
• repeat on 18 hr procedure - moraxella
culture BA (medium osloensis -

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TEST PROCEDURE MORE INFO RESULT

shouldn't contain
CHO)
gram (-)
• flood with oxidase
diplobacilli
rgt to detect
inapparent colonies
• glucose only, no
gas
• cystine trypticase
agar pH 7.6
- supports • less reliable • production of acid -
CHO
DEGRADATION
gonococci and - correct with rgt turbidity and yellow
meningococci grade CHO color of the upper
CONFIRMATORY
• from subcultured (gmsl) 10% conc layer
CA plate with
saline/TSB,
inoculate bacteria
to CTA
• paper disc with
MINITEK SYSTEM • identification of
high conc CHO and • results in 4 hrs
CONFIRMATORY neisseria
organism
• metabolized CHO
releases
BACTEC NEISSERIA
• 3 vials - dextrose, radioactive CO2
DIFFERENTIATION • exceed threshold -
maltose, fructose into supernatant
TEST incubate 3 hrs
(DMF) gas
CONFIRMATORY
• analyze gas for
radioactivity
• 97% accurate, 48
CTA
hrs incubation
• boil organism and
• slide coagulation • control test (-),
PHADEBACT mix with rgt
• small % false (-) confirms
GONOCOCCUS • correct n.
• false (+) w/ n. gonococci
TEST lactamica w/
lactamica presence
phadebact + onpg
LECTIN SLIDE • lectin as agglutinin • confirm n.
AGGLUTINATION • 6-8 minutes result gonorrhoeae
• confirm gonococci
• cross-reacts w/
DIRECT FAT group B
meningococci and
staphylococci

1.1) TREATMENT AND DIAGNOSIS: NEISSERIA GONORRHOEAE

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TREATMENT LABORATORY DIAGNOSIS

• IM aq procaine PCN G • gram stain of purulent material

• oral probenecid
• ampicillin by mouth, w/ probenecid
• for those allergic to PCN, tetracycline and
spectinomycin
• from - urethral discharge (male),
conjunctival discharge and purulent
synovial fluid
• culture - blood culture
• serology - RIA (pilus), ELISA (anti-pilus)

2.) IDENTIFICATION: NEISSERIA MENINGITIDIS

TEST PROCEDURE MORE INFO RESULT

• BA/CA
• oxidase test
• gram stain • glucose and
CULTURE
• pure culture for maltose (+)
biochemical test
• sugar fermentation
• A and C - capsular
swelling
• B - identified by • A and C - has
SEROLOGY slide agglutination capsule
- polyvalent sera
- monovalent sera

2.) SUMMARY OF NEISSERIACEAE

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ORGANISM DSS SITE OTHER INFO

• obligate human
parasite
• urethritis,
prostatitis,
epididymitis,
cervicitis,
salpingitis,
proctitis, • transmitted
pharyngitis, through sexual
perihepatitis, septic contact • IM aq PCN G
arthritis, dermatitis
- acute purulent • oral probenecid
NEISSERIA • vulvovaginitis
urethritis in men • ampicillin (mouth)
GONORRHOEAE (children)
- purulent with probenecid
*** see: book for • opthalmia
cervicitis in • for those allergic to
cultural characteristics neonatorum
women PCN - tetracycline,
(infants) -
spectinomycin
prevented by
crede's prophylaxis • joints, rectal crypts,
• vulvovaginitis blood
(women) - criminal
assault
• gonococcal
bacteremia -
women, 1%
• asymptomatic
urethritis

ORGANISM DSS SITE MORE INFO

NEISSERIA • bacterial meningitis • human • same morphology

MENINGITIDIS • waterhouse nasopharynx as gonorrhoeae
*** see: book for friedricksen • human carriers
cultural characteristics syndrome - septic
arthritis / • bacterial meningitis
endocarditis - bloodstream ->
CNS ->
meninges

• bloodstream ->
petechiae -> fatal

• CSF
• nasopharynx
• joint fluid
• blood
• transtracheal
aspirate
• skin petechiae

• eye, urethra,

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ORGANISM DSS SITE MORE INFO

endocervix, anal
canal
(homosexuals)

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