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135

MICROBIOLOGY I Bacteriology by Mary Lux

Bacterial Growth Requirements

l. Temperature ! Contain Na2C03 and sodium


••

a. Psychrophiles -cold loving; optimum borohydride


temperature = l5'C •!• Add water - produces H2 and C02
b iVIesophiles -moderate temperature; (aids in growth)
optimum temperature= 37 C; (most •!• Palladium pellets catalyze the
pathogenic organisms) reaction
c.. Thermophiles -heat loving; optimum c. Some require hemin, Vitamin K, and
temperature= 50-60'C yeast extract

2 .. pH 5 Incubation
a. . Bacteria 6 5-7,5 a., 5-.JO% C02 (incubator or candle jar)
b Fungi 5 .. 0-6.. 0 b. 35-37'C
C, Phosphate salts used to buffer media c. 50-70% humidity

3. Osmotic pressure 6 May be selective and/or differential


a Isotonic
b Halophiles (Vibrio)
·· - prefer higher
salt concentration Stationary
Phase
4. Oxygen Death
a., Aerobes -require 02 Phase

b. Facultative anaerobes -can gr·ow with


or without 02
c. Obligate anaerobes -harmed by 02
-� d.. Formation of superoxide radicals,
J
Time in Houts
toxic; neutralized by catalase and _ ...
superoxide dismutase (possessed by
GROWTH CURVE
aerobes andfacultatives)
a.. Lag phase -enzyme synthesis and cell
5,. Other atmospheric requirements elongation
a iVIicroaerophiles -prefer lower 02 b. Log phase -active reproduction
than in air c., Stationary p hase exhaustion of

; b ap_nophiles -prefer higher C02 than


In au·
nutrients, +waste, tpH, less
viability
d., Death phase -dead cells exceed new
CULTURE MEDIA cells
L Must meet growth requirements
STERILIZATION/INHIBITION TECHNIQUES
2 Agar -polysaccharide derived from L Heat - denatures protein
marine algae a, Moist - autoclave (steam under
a.. Melts at lOO'C pressure)
•!•
b. . Solidifies at approximately 45'C 15 lbs pressure/sq. in.,, 12l'C,
15 minutes
3.. Complex media -most common; made of •!• QC Bacillus stearothermophilus

peptones and extracts and R subtilis


b. Dry heat
4.. Anaerobic media -contain reducing t Flame, incinerator, hot air oven
••
agents which bind with dissolved 02
! 170'C, 2 hrs
••
( thioglycollate, cysteine) c, Pasteurization, ultra high
a., Broth tubes should be heated prior to
temperature
use to drive out 02
0:• 1 4 0'C, 3 seconds
b., Gas pak envelopes •!• NOT sterilization
136
2. Filtration c.. Alcohols -denature protein and
a Pore size 0.22 p. - 0.45 p. dissolve lipids
b. Used for sugar solutions, urea media,
vaccines STERiliZATION DISINFECTION

� �
Kills All Microorganisms Inactivation or Inhibition of
3. Refrigeration -slows growth (/nclud Spores and Microor anisms (May Not
/rUses) A ect Spores)
4. Dessication ···no multiplication, but
organisms remain viable (lyophilization) Examples: Example:
• Autoclave {121 'Cat 15psi • Bleach {1: 10 Hypochlorite)
for 15 min)
5.. Osmotic pressure hypertonic solution
• Inc ineration
a.. Causes plasmolysis • Filtration {Physically
b.. " ·Cure
· d" meat, fruit preserves Removes Microorganisms)

6. Radiation
a.. Forms hydroxyl radicals
b. Damages DNA

7. Disinfection
a.. Phenol damages cytoplasmic
·-

membrane, denatures protein


b.. Halogens (iodine and chlorine) -

oxidizers

Antibiotics/Susceptibility Testing

AhtiBi6tk:s.ctndi''the1iS4{:Ji6Hs:-·
ANTIBIOTIC EXAMPLES ACTION NOTES
B-lactams Penicillins Inhibits cell wall synthesis Watch for methicilin resistant
Cephalosporins Staphylococcus aureus (MRSA)
Carbapenams (lmipenam)
Monobactams (Azotreonam)
13-!actamase Inhibiting Combin-
ations (Augmentin, etc.)
-

Glycopeptides Vancomycin Inhibits cell wall synthesis Drug of choice for Clostridium
difficile and for methicillin··
resistant staph (MRSA)
-- - .. ··-
:......
Aminoglycosides Gentamicin Inhibits protein synthesis Acts on 308 subunit; not active
Tobramycin against anaerobes; used with a
Amikacin penicillin for Enterococcus
-
- --
-
Tetracyclines Tetracycline Inhibits protein synthesis Acts on 308 subunit; affects
Doxycycline bone and teeth in children; may
lead to superinfection of yeast
-

Chloramphenicol Chloramphenicol Inhibits protein synthesis Ac;ts �m 50S subunit; can cause
aplastic anemia
-

Macrolides Erythromycin Inhibits protein synthesis Acts on 508 subunit;


Clindamycin clindamycin for gram + and
gram -anaerobes
- -
--�- -

Quinolones Ciprofloxacin Inhibits nucleic acid For Pseudomonas aeruginosa


Norfloxacin synthesis and other aerobes
- -

Sulfa Drugs {Sulfonomides) Su!famethoxazole Analogue of PABA For UTI, enteric infections; used
(intermediate in folic acid with trimethoprim (Bactrim, etc.)
synthesis)

l
131
ANTIMJCROBIAL THERAPY · SOurces of Error: DiskDiffusioll ·

L Nanow spectrum - only certain groups


covered ABNORMAL RESULT PROBABLE CAUSE ··-
Tetracycline Zone Too Large pH of agar too low
2 Broad spectrum - gram pos and gram neg and Clindamycin Too
coverage Small with E coli or
5. aureus Controls
3. Selective toxicity - action against only
Tetracycline Zone Too Small pH of agar too high
microbial structures (70s ribosome, cell and Clindamycin Too
wall, etc) large with E coli or
5. aureus Controls
4. Bactericidal action -
host inunune help
kills bacteria without TI
Amino Iycoside Zone Too (a++ and/or Mg++
Sma with P. aeruginosa too high in agar
Control
5. Bacteriostatic action - reversible
Aminoglycoside Zone Too Ca++ and/or Mg++
inhibition (ultimate destruction depends large with P. aeruginosa too I ow in agar
on host defenses) Control
"

Zones Universally Too Large Inoculum too light


6.. Drug combination
r,
on Control Plates Nutritionally poor medium
a Synergism - combined better than the Slow·growing or nism
sum: 1 + 2 4= (not seen wit controls)
b.. Antagonism - one decreases activity of Agar depth too thin

other: 1 + 2 =I Zones Universally Too Small Inoculum too heavy


on Control Plates Agar depth too thick

SUSCEPTIBILITY TESTING �
. c-

Methicillin Zone Decreasin Methicillin degrading during


1 Kirby-Bauer Method over Days or Weeks wit refrigerator storage
Control Organisms
a Mueller-Hinton agar
b Depth=4mm t--- Methicillin Zone Methicillin being degraded
Indeterminate in Disk Test by strong &-lactamase
c. pH= 7.2-74
producing Staphylococci
d Physiologic concentration of Ca++
and JVIg++ Colonies within Zone of Mixed culture
Inhibition Resistant mutants within
e. 35oC, ambient air zone
f.. 108 organisms (McFarland 0. 5) .

"Zone within a Zone" A swarming Proteus


g. QC weekly and with each new lot of
Phenomenon Feather edges of zones
agar or discs (E. coli, S . aureus,
R
around enicillin or
P aeruginosa) ampicil in disks usually
occur with &-lactmase
2. Broth methods neg. strains of 5 aureus
&-lactamase pas.
a. MIC (minimum inhibitory concen­ H. inl/uenzae with
tration) penicillin or ampicillin
•!• Lowest concentration of drug that
prevents in vitro growth
•!• First dilution tube with no visible
growth
b. MBC (minimum bacteriostatic
concentration)
• !• Lowest concentration that results
in >99.9% killing
•!• Subculture tubes near MIC to find
.first plate with no growth
c. Schlichter test
•!• Serum bactericidal assay
•!• Tests patient serum (containing
antibiotic) against the infecting
orgamsm
f

138
Media I
t
· Special Media f
MEDIA PURPOSE MEDIA PURPOSE I
Blood Agar
IBA, BAP)
Most Bacteria;
Determines Hemolytic Reactions
Bordet-Gengou Agar 8 pertussis
1

l
Buffered Charcoal Legionella sp
Chocolate Agar �
Haemophilus and Nei seria sp �- Yeast Extract (BCYE)
Enriched with Hemoglobin or
lsoVitalex Cystine-Glucose Agar F tularensis

Phenylethyl Alcohol Selects for Gram Negative Anaerobic Fletcher's Medium Leptospira
Agar (PEA) Bacilli and Gram Positive Cocci;
Inhibits Growth of Facultative Gram Kelly's Medium Borrelia burgdorferi
Negative Organisms
··-

Skirrow Agar Helicobacter pylori


Columbia Colistin- Selects for Gram Positive Cocci
Nalidixic Acid (CNA) T hiosulfate Citrate-Bile Vibrio sp
Salts ITCBS)

Thayer-Martin Agar N. gonorrhoeae and N. meningitidis Vaginalis Agar IV-Agar) Gardnerella vagina/is
(human blood)
CAMPY-Blood Agar Campylobacter sp. ----
Cystine-Tellurite Blood C diphtheriae (Black Colonies)
Thioglycollate Broth "Back-Up" for Anaerobes __
(Timda/e)

Lowenstein-Jensen Mycobacterium sp Loeffler's Medium C diphtheriae (Enhances Grouping


Agar and Metachromatic Granules)

Middlebrook 7H1 0 Mycobacterium sp


Agar Confusing Tests
-
TEST DETECTS:
Anaerobic Media r--·
Schick Corynebacterium diphtheriae
-
c.___.MEDIA PURPOSE Dick Susceptibility to Scarlet Fever
---
/Streptococcus pyogenes)
Bacteroides Bile Selects for 8 fragilis Group
Schlichter Serum Bactericidal level
Esculin Agar IBBE) (Black Colonies) .
--
Schultz-Charlton Antiserum Injected into Rash; Blanches


Kanamycin- Bacteroides sp. if Scarlet Fever
Vanco cin Laked (Enhances Pigment Production) ---
Blo (KVLB) Elek Confirms Toxin Production for
Corynebacterium drphtheriae lin vitro)
Cycloserine-Cefoxitin C difficile
Fructose Agar
(CCFA)
1TEST.AlerU
--
CDC Anaerobic Blood Anaerobes (Enriched with Hemin,
Agar Cystine, and Vitamin K)
--· ·-

Cooked or Chopped Anaerobes


Meat Medium

;EliRiCHMENT.
• Listeria monocytogehes .
..· ·Yersinia ·
.:· _,
iliiif:P;,;., �lCoi tftis;).i!rJJii '
,, _

. . ·.
-
··
.. ,......•.... . .,·. ... :· .
139

Specimen Collection
and Handling
GENERAL.
L Material from infection site
2. Optimal time (ex . .: Salmonella typhi­
culture blood first week, culture stool
second and third weeks)
Laboratory Diagnostic
3 Snfficient quantity Methods
4. Appropriate collection devices MICROSCOPY

5.. NEVER refrigerate spinal fluids, L Light microscopy


anaerobic or GC specimens a.. Resolving power 0.2 pm
-

b . Ocular lens lOX; oil immension lens


6. Collect prior to antibiotic therapy � lOOX

7.. Set up within 2 hours of collection 2.. Darkfield - for spirochetes; reflected light
CRITERIA FOR REJECTION 3.. Fluorescence- near UV range; auranrine
l. Preservatives used rhodamine, acridine orange and calco­
fluor white stains
2. Insufficient quantity
4.. Electron microscopy - can resolve
3. Dry swab particles 0 001 pm apart; useful in viral
I. D.
4. Leaky containers -- contaminated
specimen as well as biohazard CULTURE CONDITIONS
l. Plates incubated at 35-3TC
BLOOD CULTURE COLLECTION
a.. Campylobacter- 42•c
L Must prep skin properly with alcohol b.. Yersinia - 25-so·c
(70%) and iodine c 5-10% C02 (Campy- microaerophilic)
d. 50-70% humidity
2. Best time to draw is just prior to fever
spike 2. Anaerobic Conditions
a.. Broths with thioglycollate or cysteine
3.. Draw at least 2 cultures, but no more b.. Pre-reduced media
than 3 in a 24 hr period •:• Gas pak jars or anaerobic
4.. May use antibiotic removal device (ARD) chamber
'.• Environment; 10% H2, 5% C02,
or resin bottles if patient on antibiotics
85% N2, palladium crystals
5.. Isolator® best for fungi and acid fast
MEDIA
organisms
L l-2% agar
6.. Must have 1:10 dilution of blood to broth;
on adults draw at least 10 ml if possible 2. . Non-selective
a. Supports most organisms
b Blood agar, chocolate agar, trypticase
agar
3. Selective agar
a.. Contains chemicals, dyes, antibiotics
to inhibit certain organisms (EMB,
MAC, CNA, Campy-blood)
b. May also be differential (HE, SS,
XLD, EMB, MAC)
140

INOCULATION for urine colony count plates)


L Streak for· isolation with nichrome,
3.. Number of colonies x 100 (01 loop) or
platinum, or disposable loops
number of colonies x 1000 (001 loop)
2. Calibrated 0 01 ml or 0 .. 001 ml (001 ml
4. Read and report after 18 -24 hrs

PRIMARY DECOLORIZER COUNTERSTAIN RESULTS PRINCIPLE


f-"'� �.=.,_, ;, ;=:---1
STAIN POS NEG

Gram Stain Crystal Violet Alcohol/Acetone Safran in Purple Pink • Iodine mordant
• Methanol or heat fix
• Violet dye & iodine form
complex in cell; washes
out of gram neg cells

Kinyoun & Carbo! Fuchsin Acid Alcohol Methylene Blue Pink Blue • Acid fast
Ziehl·-Nielson • For Mycobacteria

Auramine-- Auramine and Acid Alcohol Potassium Orange No • For Mycobacteria


Rhodamine Rhodamine Permanganate Fluoresc Fluoresc
{Fluorescent
Stain)
!--------+-- ---- -+-- ·-- -+-- ·---· -!------ -- --+---------1
Calcofluor Calcofluor White Bluish· No • For yeast and fungi
White + 10% KOH white Fluoresc • KOH to break down
Fluoresc. debris and mucous

Gram Positive Cocci


STAPHYLOCOCCUS !
•• Some sensitive to penicillinase­
l. ''·Grape-like" clusters resistant penicillins (PRP�)
(methicillin, oxacillin, etc); if
2. S.. aureu.s methicillin·-resistant S .. aureus
a.. Coagulase positive (MRSA), vancomycin is drug of
b. Most common pathogen of genus choice
c., Common infections g Laboratory diagnosis
•!• Furuncles (boils) and carbuncles •!• BAP - soft, opaque, regular
•!• Bullous impetigo (blisters) colonies 2-B mm in diam; some are
•} Paronychia (nails) beta hemolytic and some have
•!• Post surgical wounds and pale golden color
bacteremia •!• Growth in 7.5% NaCl and
d. Intoxications ferment mannitol
•!• Scalded skin syndrome •!• Catalase positive
(exfoliatin - neonates) •!• Phage typing and susceptibility
•!• Toxic shock syndrome profile for epidemiologic studies
(TSST-1) - women ages 12-52
•!• Food poisoning (enter otoxin) -
symptoms in 1-5 hrs after
ingestion (potato salad, cream
dishes)
e. Exotoxins -- hemolysins ., leukocidins,,
coagulase and hyalm onidase
(spreadingfactor) , nuclease, protease
l
I
and lipase
f. Resistance/sensitivity
I
•!• Most resistant to penicillin due to 3, Coagulase negative Staphylococcus
plasmid mediated B-lactamase a.. Opportunist in immunocompromised
hosts and patients with prosthetic \
valves and devices I
\
L
141
Biochemical Tests COAGULASE TEST
L Reagent -EDTA rabbit plasma
CATALASE TEST
1 Reagent: 3% H202 2, Bound coagulase -clumping on slide
(plasma and colony)
2 Add one drop to colony on slide
3, Free coagulase -gels in tube test (0,5 ml
3, If catalase present, H202 is broken down plasma and colony; 35-37'C 4-12 hrs)
to water and 02 (which bubbles off!
4, Agglutination tests -detect coagulase and
4, Positive: Staph; negative _, Strep protein A

5, QC each day of use 5, Postitive: S, aureus; negative: other


Staph (human pathogens)

ORGANISM COAGULASE INFECTIONS INTOXICATIONS NOTES


-"'

S. aureus + Carbuncles, ·furunc!es, Scalded Skin Syndrome, Most beta lactamase +:


paronychia, wounds, Toxic Shock Syndrome, many MRSA
and bacteremia and Gastritis (enterotoxin,
1-5 hours after eating)
"'" ,__ - --
S. epidermidis -
Endocarditis, prosthe- i
Most methic llin resistant;
tic device infections sensitive to novobiocin
-
-t-- --

S saprophyticus --
UTI in young women Resistant to novobiocin

Coag Negative Staph:


Opportunistic Infections in lmmunocompromised
Patients and Those with Prosthetic Devices

STREPTOCOCCUS
L Spherical or oval; chains or pairs

2 Lancefield grouping based on C


carbohydrate

3, S, pyogenes (Group A)
a,, Beta hemolytic
b,, Streptolysin S -stable in 02; non­
antigenic
c,, Streptolysin 0 _,oxygen labile;
antigenic
d, Erythrogenic toxin -rash of scarlet
fever
!
•• Dick test-inject toxin to detect
antibody
•!• Schultz -Charlton test - antiserum
injected into rash; blanches if
scarlet fever
e, Highly sensitive to penicillin
f Infections
(• Pharyngitis (Strep-most common
cause)
! Impetigo
••

•!• Erysipelas
•:• Wounds, burns
142
•!• Rheumatic fever (autoimmune
sequelae to infection with
-· Str�ptococ'cus
Streptococcus group A) pyogenes
•!• Lab diagnosis
I& Sensitive to 0 . 0 4 units
bacitracin disc
llF Typing i I Will kill
.with ease.
4. S . agalactiae (Group B) But Protein M is
a Narrow zone of beta hemolysis the biggest trick
b. Neonatal sepsis and meningitis; UTI; To make throat
vaginal infections and muscle sick
c Laboratory diagnosis
•!• Serotyping Antistreptolysin b
•:+ CAMP reaction (with S . aureus) Will laythe heart and kidneyslow
•!• Na hippurate positive Bacitracin is my sign
Zone of inhibition every time.
5. Streptococcus (Group D)
a.. Enterococcus See wha t fda on B·A-P
•!• Growth in 40% bile and 6 .5% salt Des1roy the sheep R-B-C.
•!• Alpha, beta or gamma hemolysis
b. Non-enterococcal - n o growth in

c.
6.5% salt
Laboratory diagnosis - bile esculin REMEMBER!
positive
Staph and Strep
6. S . pneumomae
a. Alpha hemolytic crater-like colonies
STREP:
or muco1'd , " water drop " co1orues
. • Pairs or Chains
b. Lancet-shaped diplococci • catalase '- '
c . Check sensitivity to penicillin using • Growth in 6 5% NaCI
OX (oxacillin) disc(> 20 mm = (Enterococcus)
sensitive)
d. Causes
•!• Primary lobar pneumonia (rusty
sputum)
•!• Meningitis
•!• Bacteremia
•!• Otitis media
e, Laboratory diagnosis
!
•• Typical colony morphology
•!• Quellung reaction
<t• Sensitive to optochin
! Bile soluble
••

7. Other alpha Streptococcus (viridans


group) - subacute bacterial endocarrlitis
(See Streptococci Chart on next page.)
,
.,
'"')';' ,..... --�,.•,._._.,..'• ,,, " ... !"·""".;:•• "·•:· """' , ..,, .,. . .
'<'; •>:l:';i' 'I!•<1�'-1•'!).•'')''' "-'�·· •,;;!.'! ' --" '.].',:<.:'",�11'1"' ''1�I'J:I,..IO'·'' ' '• (.•>,,: l'•ll t;' ''",',!•'r'{'O)Nl�'l<�����·.�·l,"'' , "-
�:''i:'J;·)},:l",'f�f<l·l��'�)lll:,'i:'I�I.�',W-'''"''' ! '•-i·\'J'"''/'il''f'ijlf\!11�1��"' "1':-:',1. '
·'
''
"

Streptococci (Cata/asf:! · 1\/egative) � -


-,

Hemolysis Bacitracin Na Optochin Bile Bile Esculin 6.5% NaCI lntections


Hippurate Solubility

S.pyogenes B s Pharyngitis, wounds, scarlet


(gr oup A) fever, impetigo,
sequelae�rheumatic tever

S.agalactiae I B, a I R I ...
Neonatal septicemia and

I
(group B) menmgitis, UTI (CAMP+)

Entercoccus
(group D)
I a,B,a R UTI, endocarditis (treat with
amJnoglycoside+pentcillin)

Non-enterococcus
(group D)
I a, a I R Endocarditis (rare)

Pneumonia, memngitis.
a ("water drop"/ R "+
S. pneumomae
I I
-
. ' + bacteremta (screen tor
I
(S)

I
crater coiomes) I
penicillin sensitive with Ox;
I > 20 mm =sensitive)
I
I
S. viridans I a,3 I R Endocarditis (rare)

a hemolysis = greening around colony B hemolysis = complete clearmg around colony


6 hemolysis = no hemolysis Shaded areas = Key Reactions

�; �c���;!!� the M. Qst :re;r&everm


,,,
. g.
" .- - -- ·..-_, - '
to

-
"""
'""
y
i

I
144
Gram Negative Cocci !
•• Waterhouse - Friderichsen
syndrome (scattered petechiae) =
NEISSERIA AND MORAXELLA meningiococcemia
!

1
1. Key characteristics •• Mainly caused by types A, B, C, Y
a.. Diplococci (kidney bean shape) and W

-�
b. Oxidase positive -

.
4.. Moraxella (Branhamella) catarrhalis
J

ha�··-
2. N. gonorrhoeae a.. Respiratory infections
a.. Grows on chocolate and Thayer­ b. Grows well on chocolate and BAP but
Martin (contains vancomycin, not on MacConkey's ,
colistin, nystatin, hemoglobin and c. Colony hard and "moves over";
isovitalex) asaccharolytic /-
b.. Requires 5--10% COz; may take 48 hrs d.. Usually beta lactamase positive -

c..
for growth
Ferments glucose
5 . Bacterial meningitis I
d .. Gonorrhea
a. +neutrophils I
b. tglucose ·i
••! Sexually transmitted :1
_

c.. +protein

.•t
•!• May be asymptomatic; may be !
;If

'REMEMBER! ·•
mixed with Chlamydia l


.

'

(ci0 '\
.·�
•:• Gra.m stain sensitive for males, .i _
_ ···

. I _
_

but NOT for females

· DifferentiatingN�issepa I
. -- _
-

0:• May be confused with Moraxella


.

or Acinetobacter ·

Maltose Lacfose I
i
•!• Glucose
Do NOT refrigerate prior to ·· ·-·

-
culture
•!• Penicillin or spectinomycin
.N. menhgitidis t +
I
I
sensitivity; perform beta �
N,, Jqcfa"mica
• ,• •, --- -

lactamase test to determine + + . + F


.
fl"l I
_ ._

penicillin sensitivity
Arlfer ent glucose: iJOnorrHoeae: only .glucose: (

·meni(Jgitidis: glucose and maltose; and

I
Neisseria lactamica: glucose, maltose and lactose,
gonorrhoeae
Gram Positive Rods
Gonorrhea, the · ·· CORYNEBACTERIUM DIPHTHERIAE
clap,GC
man old, 1 . Key Characteristics
fashioned .STD a. Small pleomorphic rods with clubbed
ends i>;/'1•1
I need C02 and ,.a b Palisade or "chinese letter"
chocolate agar· 1£:? arrangement
And may not grow c.. Metachromatic granules (stain
for 48 hours · red-purple with methylene blue)
d Tinsdale agar - black colonies due to
I fermentglucose, the only sugar forme; tellurite hyd,rolysis
Do an oxidase and purple you'll see e.. Elek test - determines toxin
production by the isolate in vitro

3. N. meningitidis 2. Loeffler's - enhances development of


a Grows on blood agar, chocolate and metachromatic granules
Thayer-Martin a.. Palisade arrangement may be seen
b. 5-10% COz enhances growth
c. Ferments glucose and maltose 3. Exotoxin production by only lysogenic
d Transmitted by respiratory droplets organsims carrying a B phage
and requires close contact
4. Produces pseudomembrane on tonsils,
e. Meningitis
uvula or soft palate
!
•• Seen mostly in children under 3
5. Causes diphtheria
!
1
145
LISTERIA MONOCYTOGENES ERYSIPELOTHRIX
L Key Characteristics 1. Key Characteristics
a, Small colonies with narrow zone of a Non-motile
beta hemolysis b, Catalase negative
b,. Catalase positive c.. "Test tube brush" growth in gelatin;
c, Tumbling motility; "umbrella" HzS positive in TSI
motility in SIM at room temperature
but NOT 3TC 2.. Occupational infection for fishermen,
d, Bile esculin positive butchers, veterinarians, rose growers

2, Causes neonatal meningitis and sepsis; BACILLUS SP. (Sporeformers)


sepsis in immunocompromised hosts 1 Key Characteristics
a Large, ground glass colonies
b. Beta hemolysis (EXCEPT B .
Listeiia
,,.
· -
-_
anthracis)
monocytogenes c, Catalase positive
d.. Large grm
a positive to variable rods
I'm Listeria -
in chains with spores
told temp's
my game 2,, B anthracis
Umbrella a. Very long chains ("bamboo shoots")
motility brings b, "Medusa head" colonies
mefame · c., Non-motile, non-·hemolytic
d. Anthrax- cutaneous, pulmonary, or
I'm agram positive gastrointestinal
rod· and hemolytic,
Kills babe in uteroaftermomgets sick 3, B cereus - Food poisoning due to
preformed toxin - "fried rice"
Frbm eating dairy orprocessedrneat
·Or veggies not exposed to heat

Gram Positive Rods


ORGANISM CATALASE ESCULIN H2S !TSI B HEMOLYSIS NOTES
-

Corynebacterium + - -
v "Chinese Letter" Arrangement;
Metachromatic Granules (Loeffler's
A� Slants); TeUurite Hydrolysis

17 �
(Tinsdale Agar); Elek Test
Determines Toxin Production
-

Listeria + + -
+ Tumbling Motility at 25C, but Not


37C; Cold Enrichment; Neonatal

E�a;x
Meningitis and Sepsis; Sepsis in
lmmunocompramised Hosts
--

- -
+
- ''Test Tube Brush" Growth in
Gelatin; Infection in Fishermen,
' Butchers, Veterinarians
'

v "Ground Glass"
Bacillus (Spore Formers) + v -

Hemolytic Colonies;
" 8,. anthracis (Non-Hemolytic,
il!!f,c!!\'IIo>IP''M''''i!!l Non-Motile, "Medusa" Head
Colonies); long, bamboo shoots
·· B. cereus (Food Poisoning,
Enterotoxin) B hemolytic
146
Gram Negative Rods •!+ Shigello-like toxin
•!• Food poisoning associated with
ENTEROBACTERIACEAE undercooked meat (hamburger)

I
l , General Characteristics •!• Hemolytic uremic syndrome
a, Peritrichous flagella when motile (HUS)
b. Fement glucose
4, Kl strains can cause neonatal meningitis
c.. Reduce N03 to N02

I
d Oxidase negative Shigella
e.. Antigens used in typing: l, Key Characteristics

II
•!• Flagella H Ag
=
a Lactose negative
•!• Envelope = K Ag b Non-motile
•!• Cell wall LPS c, Anaerogenic
(lipopolysaccharide) = 0 Ag
•!+ All possess LPS endotoxin; some 2. Bacillary dysentery - penetrate epithelial

t
produce exotoxins cells in small intestine
Escherichia coli 3..
S . dysenteriae (Group A) - most severe
L Key Characteristics
a . Indole and lactose positive 4, S . flexneri (Group B)

2.
. b.. IMViC = ++- -
5., S . boydii (Group C) I
!

t
Most common cause of UTI in females
6, S. sonnei (Group D) -most common
3
Intestinal infections
7 ,.
. organisms
< 200 needed for disease
,

a, Enterotoxigenic E, coli (ETEC)


•!• LT toxin (heat labile)
· · · · ·. . - ·
•!• ST toxin (heat stable) (EIEC)
b Enteroinvasive E.. coli
. •.·
·••.•··@
·
_ CJ."i)� REMEMBER!
. . · ·.·. ···. ·- ··. ·.•-- •._ .-·-.· · ··- · · · . .
· . . L&-�_- - · .- - _, · . shii�1la
-.· • • .

•!• Penetrate epithelial cells in large "• . _ , _- - - ' --- -- · ·


.>··- �- . �-· : ::_ :·:, : _ , ·
·· '

intestine
.

. .
•!• May be lactose negative · ··•· · $· dyiel)'teriae " GroppA,(fstOlphC1betic;aJ!y)·
·
- . '· ·
c . Enterohemorrhagic E.. coli (EHEC) ­ <•·S, te l xrierl;,GrourS.BJnot"B'oydiO __ .. .- ·_.
strain Ol57:H7
:,g: pc!yciii Groupp(bpyd,eee�· "'·.:�b·;.
·•

- _, : - :D'-_ (lasti::tlph -- ' - -_ ,_ ' ·._,-


__ ,- -"'- -_ .abetically)
=

,-. '"tfQei=
· · ·:;··-: so
i- -•:S,·• - -_ -'- - - Group
-_-:-:-: / , :_
'
'

AGAR DIFFERENTIATING SELECTIVE H2S LACTOSE LACTOSE


AGENT AGENT INDICATOR POSITIVE NEGATIVE

MAC Lactose Crystal Violet, None Red Transparent


MacConkey Bile Salts

EMB Sucrose Eosin Y None Green Sheen, Transparent


(Eosin, Methylene Blue) Lactose Methylene Blue Purplish and
Brownish Amber

HE Lactose Bile Salts Sodium Thiosulfate Salmon Green to Blue


(Hektoen-Enteric) Sucrose (11zS+ =Black)
Salicin

ss Lactose Brilliant Green Sodium Thiosulfate Red Transparent


(Salmonella .. Shigella) Bile Salts {HzS+=
Black Center)

XLD (Xylose " Lysine Lactose Bile Salts Sodium Thiosulfate Yellow Transparent on
Deoxycholate) Sucrose Red Medium
Xylose (H2S+ =Black)

Enrichment Broths = Selenite and G'N Broth

l
)i�-�-1��(W����f.W#"'"-'11W�l�IC,. ;,,�i'll·"�:1c:1T'!.''ie<"r,J;..W�.·: W
/! ',\'1'�,<'1�1·1;:·�-�·:
. ." ,, o •i.• ,.•;., \•:,,'"" "··:;e'ii•'" '"· "!�,·;,<�
. I'•• . •I"). ·�<".1'"'· 1.'; .'•>'>:<c.. ;. , ., ·�<·�;w:r"·'''.r""'"'"'"'"'''"f"l·,.-'"W\",'.";;";Il '''�'"-:'fW'<"<'\'.1°·". ··"·'·""·'"-'\< "\"?''"'l"O'"hfl"'"""••<
·
'•"'"1 -.'"' . ..
. ,," '" " .,,,..,••.., . ,... .,\�1

�.::·\;;'L.. :;;;��:.;.::):.:;�.;'·; t'· .·:. ·· :;:;;.x:;.:tJJfi#Ji!lf4f¢i(ftl�tlfl��g,�;�1�1:1i:fi9rx t!Jffifi��rittf1Jrtl9.:r��t�. . ,: .. ..


·
.

-'· . -· ·
. . . ·. · ·• ··••
.
··.··-

1
. .

TSI Ornithine VP Urease Lysine Motility H,S Deaminase DNase

.t I
. . <
v + v

Citrobacter K(A)/A, Gas · ;
· ·.:ot•

- -
>
,I +
Enterobacter AlA, Gas v
-
Escherichia A(K)/A, Gas + v

Klebsiella

MorganeJ/a
AlA, Gas

KIA, Gas +

II
- i
i
I
++ +

Proteus KIA, Gas v I - I + +


I

I
I v + v
II
Providencia KIA, Gas - -

I
Salmonella KIA, Gas +

Serratia K(A)/A

Shigella KIA v

· v -vanable
Shaded areas = Key reactions differentiating similar genera
=

>-'
"'"
_,
148

Klebsiellae 3. Similar to Salmonella biochemically


l. Opportunist; UTI, pneumonia; Proteus
ampicillin-resistant
l. Key Characteristics
2. Klebsiella a.. Urea positive
a. Non-motile b . Deaminase positive
b. Has capsule
2 .. P mirabilis
c Urea positive
a.. Most sensitive to penicillins
d. Ornithine negative
b .. Indole negative
e. Can cause lobar pneumonia
3. P. vulgaris
3 Enterobacter
a . Indole positive
a Motile
b . H2S positive
b Ornithine positive
Yersinia
4 .. Serratia
l. Y enterocolitica
a.. May produce red pigment
b . DNase, gelatinase positive a .. Optimal growth= RT; cold enrichment
h. Invasive and toxigenic

2. Y. pseudotuberculosis - Acute mesenteric


lymphadenitis and "pseudotubercules"

3. Y pestis - bubonic plague


BIOCHEMICAL TESTS
l. Oxidase test
a.. Reagent tetramethyl
p-phenylenediamine dihydrochloride
b.. Positive= purple

2. Nitrate test
a . Reagents - 0:.--uaphthylamine,
sulfanilic acid i
b.. Positive= pink (use zinc powder to
confirm) I
3 .. ONPG test
Salmonella a. Detects B-d--galactosidase
L Large number needed for infection b.. Reagent - 0-nitrophenyl-B-d­
(> 100,000) galactopyranoside
c . Positive = yellow
2. Biochemical reactions
a . H2S positive 4. TSI (triple sugar iron agar) slant
b Lysine positive a . 0 .. 1% glucose, l% sucrose, lo/o lactose
c. Indole negative h. Yellow butt - glucose fermented
d . Urea negative c.. Yellow slant -lactose or sucrose
fermented
3.. S cholerasuis - may cause septicemia d.. Red slant - neither lactose nor sucrose
fermented
4 . S. typhi - typhoid fever e. Black butt - H2S produced
a Blood positive early - lst week
b Stool positive in 2nd to 3rd week 5 .. KIA - same as TSI but with only glucose,
and lactose, no sucrose
5. S. arizona- ONPG pos (others neg)
Citrobacter 6. Citrate test
a . Media green
l. Opportunist b. Positive= blue
2 . Lysine negative
149
7.. Decarboxylase tests 3. Indole test
a.. Measures ability to decarboxylate a. Indole split from tryptophan
amino acids b. Reagent (Kovac�) -
•!• Lysine .,_ Cadaverine p-dimethyl-aminobenzaldehyde
•!• Ornithine � Putrescine c.. Positive =pink
•!• Arginine � Putrescine
•!• Indicator dye= bromcresol purple 9. Urease test
b.. Lysine iron agar (LIA) a. Urea hydolyzed to ammonia and C02
•!• Has H2S indicator b Phenol red indicator turns pink if
•!• 0..1% glucose and 1 % lysine positive
•!• Positive =purple butt 10 Voges-Proskauer (VP)
•!• Slant of LIA turns red for lysine a. Detects acetylmethylcarbinol (acetoin)
deaminase
b. Reagents = KOH and 0.-naphthol
c. Motility-indole-ornithine (MIO)
•!•
c. Positive= pink
0..1 % glucose and 1% ornithine
•!• Positive =purple butt 11. Phenylalanine deaminase
•!• Also tests for motility and indole a. Reagent = ferric chloride
(Kovac�) b. Positive= green
150

· '<·'·;;,:_.;.
.--·-.- -..' ',C.::�)-'''· :-. . .
;
:���-
---,. _,_,,
·-
.
-__,
-::::·:c,-··-.
;-__,,.-.,,_,
c
- __ :---:- -
··- .

A/A
Escherichia
A/A, H2S+
P roteus
·
KIA, H2S+
Salmonella
KIA
g
Shi ella
. KIK
Pseudomonas
(indole+) (ufea+, deam+) (mal-, ONPG-) (citrate·-.. ·_non-mOtile) (ox+, blue-green pigment,
growth at 42" C, growth in
-
- .·

Enterobacter Arizona Citrobacter Providencia cetdmide)


{ODC+, sugars) {LDC+} (LOC) (deam+)

Klebsiella CitrobEicter Edwardsiella :Citroba'cte�·


(ODC-) (LDC) (indole+ LDC+) (citrate+)
:
Serratia Proteus
(sugars) (urea+, deam+) __

Yersinia

_:c.
(small colonies, iJre8.+,
deam-) - .· I
II
I
(f) 0

Urea
I (f)
I
Indole 0

I I
Phe deaminase 0
(f)

I -, Citrate 0 LDC 0
(f)

I
Indole 0 (f)

I l
LDC 0
I I -l
I Citrobacter E. coli (f) ODC 0

I I
Proteus Proteus Arizona Citrobacter
vulgaris mirabilis (H�+)
Enterobacter Enterobacter
cloacae agglomerans

I
(f) ODC 0 I
I l
Klebsiella
I
I
(f) Gelatin 0

I
Serratia
l Lactose
Citrate

I i I
I
Enterobacter Enterobacter
aerogenes hafniae

\
'

-\
"'
-L.'--
151

Ell ------ Urea or Phenylalanine deam -----­ 8

I I
(PDA)

Indole Ell Indole

I
Proteus vulgaris
I
Proteus mirabilis Edwa L iella

1-J
Ell LDC 0

freundii

8 ODC Ell

I
Salmonella sp
I
Salmonella typhi
(most subgroups)

GRAM NEGATIVE NON-FERMENTERS W Pyoverdin (blue-white under


l. Opportunists UV)
!
•• Mucoid strains found in cystic
2. Characteristics fibrosis patients
a.. Glucose NOT fermented •!> Treated with aminoglycosides, 3rd
(T SI=alk/alk); lower acid production generation cephalosporins and
( 0F media = lower peptide content) extended spectrum penicillins
b.. Oxidase positive (some exceptions) •!• Infections - burns, pneumonia,
c.. May not grow on MacConkey's agar swimmer� ear; eye infections, UTI
3 Pseudomonads
a.. Oxidase positive (except Burkholderia
cepacia and Stenotrophomonas)
b . Motile by polar flagella
c . Pseudomonas aeruginosa
! 1Vlost common
••

! Lactose negative on MAC


••

•!• May produce.:


W Pyocyanin (blue-green)
W Pyorubin (rust)

Gram Negative
Oxidase Positive Fermenters

VIBRIO a.. Gastroenteritis - rapid onset 3-10


hrs.; profuse diarrhea
L Characteristics
b . Need high concentration of organism
a.. Curved rods with polar flagella
unless hypochlorohydric
b . TCBS selective for Vibrio
c . Stools contain mucus flecks (described
c.. BAP - hemolytic colonies
as "rice water" stools)
d.. Lactose negative (differs from
d .. Yellow on TCBS
enterics)
e. N03 positive (NOs to N02) 3.. V parahemolyticus
a Green on TCBS
2. V cholera (Serogroup Ol) b. Enteritis
152
4.. V vulnificus 2. Helicobacter pylori
a. Green on TCBS a. Associated with gastric and duodenal
b. Septicemia - can kill ulcers
imrnunocompromised or diabetics b . Produces large amounts of urease

OTHER ORGANISMS 3. . Aeromonas


L Campylobacter jejuni a.. Motile by polar flagella
a. Small curved rods, "seagull b Most are indole positive
appearance" (light staining) c.. Growth on MAC
b Microaerophilic, 2-4 days for growth d.. Cellulitis, wound infections and
c Growth on CAMPY agar diarrhea
d. Found in raw poultry and
4.. Chromobacterium violaceum
contaminated water (like Salmonella)
a . Grows on MAC
e. Erythromycin or tetracycline for
b.. Motile by single polar and lateral
treatment
flagellum
f Biochemical reactions
c.. Produces violet color (seen best at

It
•!• Catalase positive room temperature)
•:• Oxidase positive
•!• Hippurate positive

fI

l
l
I

Zoonotic Diseases
I
(Acquired Directly or Indirectly from Animals)

ORGANISM DESCRIPTION DISEASE NOTES


I

I!
Brucella Gram Neg Coccobacillus Brucellosis Blood Culture Pas in First Two
Weeks (Hold 21 days)

Thionine Basic Fuschin


B. abortus (cows) +/- +
B

B
suis (pigs)
me/entensis (goats)
+/-- +
+ t
I
Brucella
Cows eat Basic Fuschin
Pigs do NOT eat Basic Fuschin
Goats eat Anything.

L
153

ORGANISM DESCRIPTION DISEASE NOTES

Francisella tularensis Gram Neg Coccobacillus Tularemia Infected by Tick Bite

I
Pinpoint Colonies ("mercury drop''} "Rabbit Fever'' High Risk to Lab Personnel
Cystine-Glucose Media (Wear gloves and
Francis
H2S + with Lead Acetate work under hood)
the Rabbit
Uses the
"
Hood -

{i
� �

Yersinia pestis Gram Neg Plague Transmitted by Fleas, Rats,


Bi-Polar Staining (resembles .safety pin) Other Mammal Reservoir's

Yersin the
Safety Pin

·-

Pasturella muftocida Gram Neg Rod Contracted from �Mousy" Odor


Bi-Polar Staining Cat and Dog Bites
Oxidase and Indole Positive

MJ y
Ferments Glucose and Sucrose

I--· .. ·------
r--· --

StreptobaciJ/us monilitormis Long Filamentous Gram Neg Rods with "Rat Bite Fever'' Acitic Fluid Sample Needed;
Swellings (pleomorphic); "Puffball" or Haverhill Fever SPS Inhibits

J,
"String of Pearls" Colonies in
Thioglycollate Broth

Fastidious Gram Negative Rods


GENERAL INFORMATION Capnocytophaga)
1 Source - mouth flora b.. Bacterial vaginosis (Gardnerella)
c Bacteremia (Capnocytophaga,
2 Pathogenic in immunocompromised Cardiobacterium)
hosts; causes:
a.. Peridental and jaw abscesses 4 Grow slowly and require 5 - 10% C02;
(Eikenella, 1ictinobacillus, NO growth on MAC

ORGANISM OXIDASE CATALASE NITRATE INDOLE NOTES

Haemophilus aphrophilus -/weak + -·


+ - Colonies Similar to Actinobacillus;
Endocarditis
·---- ... -

Actinobacillus + I- + + - Peridental & Jaw Abscesses;


Center of colony has 4-6
Pointed Star
-·---

Cardiobacterium + - - + Endocarditis

r-- -

- + - "Bieachy" Odor; Pits Agar;


Eikenel!a +
Peridental & Jaw Abscesses
----

+ I- - Endocarditis
Kingella + -

..

Capnocytophaga - - + I--- - Needs 5-10% C02 or Anaerobic


Conditions; Gliding Motility;
Fusiform Shape; Bacteremia
154
GARDNERELLA VAGINALIS a , X factor hemin
=

l Bacterial vaginosis b , V factor= NAD


c, Both factors found in blood., but need
2, "Clue cells" -· high number of squamous heat to break down red cells and
epithelial cells colonized with gram release factors, so chocolate used
variable rods d., Satellitism
•• ! S , aureus produces V factor and
3, 10% KOH added to discharge causes releases Xfactor by hemolyzing
"fishy" odor blood
4. Tiny colonies at 48 hrs on BAP and
•!• Haemophilus will grow in the
chocolate
hemolytic zone surrounding staph
as satellite colonies on blood agar
5.. Catalase and oxidase negative; hippurate plate
and starch positive �· Need 5% horse or rabbit blood to
see hemolysis
BORDETELLA PERTUSSIS e, Infections
L Gram negative coccobacilli; causes : Meningitis ··· H. i.nfluenzae
••

I& 2-4 years


"whooping cough"
I& Preceded by nasopharyngeal
2,. Require special media colonization and bacteremia
a.. Classic= Bordet-Gengou (potato ! Epiglottitis
••

infusion with glycerol and 20% I& Ages up to 2 years; do NOT


SRBc 's) and penicillin collect throat culture (blood
b . Regan and Lowe (oxoid charcoal culture best)
agar; 10% horse blood, cephalexin); W May require intubation
longer shelf life •!• Pneumonia <5 years, >60 years
•:• Conjunctivitis ("pink eye")
3.. Old method for collection - cough plate; I& Very contagious
better to collect NP swab and plate :+ Chancroid - H ducreyi

directly; fluorescent antibody technique I& Painful genital ulcers or soft


more rapid chancres
W Gram stain of drainage shows
4 .. Colony
tiny gram negative coccobacilli
a., Incubate 72-96 hrs, 35'C
with a ''school of fish"
b , Pinpoint, "mercury droplet" colonies
anangement

3., Characteristics
a , Growth only on chocolate, NOT blood
agar plate (unless mixed with S
aureus)
b , Use X and V discs on Mueller-Hinton
agar to detect growth requirements
c.. Susceptibility zone inter·pretations
differ from conventional K- B zones
(use Haemophilus test media (HTM),
supplemented MuellercHinton agar
and incubate in 5-10% C02)
d. Perform beta lactamase to determine
sensitivity to ampicillin; cefuroxime ,
ceftriaxone, cefotaxime (meningitis)

HAEMOPHILUS
L Small, non--motile gram negative rod

2.. Requires gruwth factors


155
LEGION ELLA PNEUMOPHILA MYCOPLASMA AND UREAPLASMA
L Legionnaires disease - severe pneumonia; L Smallest free-living microorganisms (125-
Pontiac fever =milder form 250 nm)
2. 75% illness due to L . pneumophila 2. . Lack cell wall (bound by single triple
serogroups l and 6 layered membrane)
3. Sources - potable water, faulty air 3. Does not stain with Gram's stain; can use
conditioner vents, lakes and ponds Dienes stain
4 . Identification 4 . Center of colony grows into special media
E- a.. Specimen - sputum, bronchial (contains sterols) giving appearance of
washing, pleural fluid, lung aspirate inverted '·'fried egg"
or biopsy
b. Growth on BCYE (buffered charcoal 5 M. pneumomae
yeast extract) with or without a. Primary atypical pneumonia or
antibiotics but not on chocolate or "walking" pneumonia
blood b.. Causes positive cold agglutinin titer
c.. Direct exam - Giemsa, Dieterle and (> 1 :32); false positive RPR
Gram stain with basic fuschin counter c. Treat with erythromycin or
stain tetracycline
6.. Ureaplasma urealyticum
a.. Non-gonococcal, non-chlamydia!
urethritis, especially in males
b.. Produces urease
c. Treat with tetracycline or
spectinomycin
7. M. hominis
a May colonize GU tract; post partum
fever
b.. Tetracycline; resistant to
erythromycin (all other Mycoplasma
are sensitive)

ORGANISM DESCRIPTION DISEASE NOTES

Gardnerella vaginalis Tiny Colonies at 48 Hours on Blood and Bacterial Vaginosis "Clue" Cells; 10% KOH Added to
Chocolate; Catalase Neg; Oxidase Neg; Discharge = ''Fishy" Odor
Hippurate Pas; Starch Pas
�- --

Bordetella pettussis Gram Neg Coccobacillus; Pinpoint, Pertussis (Whooping Bordet-Gengou Media; NP Swab
"Mercury Droplet'' Colonies Cough) and Plate Directly
f--. - -

Haemophilus Small, Non-Motile Gram Neg Rods H influenzas -


Causes Require X and V Factors
• H ducreyi -
"School of Fish" Influenza, Meningitis,
and Epiglottitis
H. ducreyi - Causes
Genital Ulcers

Legionella pneumophilia Growth on BCYE Legionnaires Disease No Growth Routine Media


-

Mycoplasma!Ureaplasma Colony Appears as Inverted Fried Egg M, pneumoniae ·· Dienes Stain NOT Gram Stain
Causes Primary Atypical
Pneumonia (.+Cold
Agglutinin Titer)
156
Anaerobes

l Clues to anaerobic infection 3. Culture teclmiques


a .. Foul odor to specimen a . "Classic" principle of anaerobic
b Location i n close proximity to a culture
mucosal surface ! ]ar technique (Gas Pak jar)
••

c . Animal or human bite ·:· Catalyst - palladium pellets


d Gas in specimen •!• Envelope generates H2 and C02
e Previous therapy with when water is added
aminoglycosides ·:· Methylene blue or resazurin -
f Black discoloration of blood contain- indicators (blue and pink,
ing exudates respectively when oxidized; clear
g Presence of '·'sulfur granules" when reduced)
h Unique morphology on gram stain b. Other methods
i. Failure to grow organisms seen on ·:· Anaerobic bags - clear bag with
smear aerobically gas generating ampules; plates
J . Growth in anaerobic zone or bubbles can be read without opening bag
in fluid media !
•• Roll tube technique
I& PRAS (pre-reduced
2 Specimen collection and transport anaerobically sterilized
a .. Site containing a resident flora (oral, media) inoculated under
GI, GU) not appropriate for constant flow of 02 -fr·ee gas
anaerobic culture ! Anaerobic chamber
••

b . Best to aspirate with syringe and I& Plates put in chamber through
I
needle and place in a transport vial or a pass box that is reduced
tube under reduced conditions (swab I& Incubator in chamber; also
samples not as good) contains palladium catalyst
I

Vancomycin Kanamycin Colistin Indole Lipase Esculin Notes


(5 pg) (1 mg) (10 pg)
I
I
Bacteroides tragi/is R R ··
. .
R v - + Catalase +, black
- .. . ... . . . colonies on BBE
. ·. . May fluoresce brick-red,
Pigmented Gram R
-
s ..
. R t v may produce black
·
.
Negative Rods
piqment __
1---
.
---
t--
- - -
Bacteroides ureolyticus R s s Pits agar; urease +

f--· . . ..

I
·.
--

Fusobacterium R · + - Thin, fusiform rods,


s
.s . .

nucleatum ! ·. • speckled colonies


1-· �·
...

I
.
--

Fusobacterium + - Rods variable in length


R s s +
necrophorum -
.
... and width

\
Fusobacterium v - + Highly pleomorphic rods
R s s
mortiferum

I R resistant S = sensitive V = variable


I

1
=

I
l
L_
· . ·Differentiating Gram 'Pcs!tl\le. b . Incubate chocolate at 36'C under
· ·· ·
5-10% C02 and the anaerobe BAP at
Anaerobes '
36'C in a gas jar or anaerobe chamber
c.. If growth on both, the organism is
ORGANISM -· CHARACTERISTICS __ facultative; if growth only on
anaerobic blood, the organism is
Clostridium diffio/e Pseudomembranous Colitis; CCFA
an anaerobe
Agar ("Horse Stable" Odor};
Spore Former
f----- SPIRillACEAE
Clostridium perfringens Double Zone of Hemolysis;
1. Rigid , helically curved rods with one or
lecithinase +; Gas Gangrene; Spores
ar Seldom Observed more turns; corkscrew motility by polar
flagella; gr·am negative
Clostridium tetani Terminal Spores ("Racquet-5haped"J;
Tetanus 2 .. Spirillum minor - "rat bite" fever

'
1
Actinomyces israe/ii "Molar Tooth" Colony; Branchin a. Visualize by darkfield or stain with
Gram + Rods {"Lumpy jaw"}; Sulp ur Giemsa
Granules b . 2-3 spirals and bipolar polytrichous
tufts of flagella
P. anaerobius Sensitive to SPS
c . No growth on artificial media

Spirochetes
s
TREPONEMA PALLIDUM
gh L Stain with silver impregnation

2. Darkfield - slow motility and flexion

3. No growth on artificial media

4.. Sensitive to penicillin., tetracycline and


erythromycin

5. Detected through serological tests (see


Serology/Immunology for details)
EXAMINATION OF PRIMARY PLATE
l Pitting - Bacteroides ureolyticus (could be 6 Syphilis
Eikenella) a . Primary lesion
•) 2-10 weeks after infection,
2 Large colonies with double zone of chancre appears
hemolysis - Clostridium perfringens; set •:• Heals without treatment in J.-8
up egg yolk agar for Naegler test weeks; may perform darkfield or
direct FA on fluid
3 Bread crumb or speclded colonies; gram b Secondary lesion
negative slender fusiforms - •} Dissemination - skin rash
Fusobacterium nucleatum erosions on genitalia
c. Latent stage
4. Molar tooth colonies of gram positive •:• 2--20 yrs later
branching rods - Actinomyces •:• Affects skin, bone, joints, CNS
5.. BBE - dark colonies, > 1 mm ­
LEPTOSPIRA
Bacteroidesfragilis
L Spirals with hooked ends
6 KVLB - look for pigment and examine
under UV light 2. Animal pathogen passed to humans via
a.. Perform aerotolerance test on water contaminated with animal urine
colonies by subculturing each colony
(ex sewer workers, farmers, vets)
type to an anaerobic blood plate and
3. Positive clarkfield or direct FA
a chocolate plate

1
158 1
4. Can grow ill Fktcher 's semi-solid media 3, Dependent on host for ATP I

I
a.. Incuhate 6 wks at 30oC ill the dark
b . Perform clarkfield from several 4. Laboratory diagnosis
centimeters illto media a . Giemsa stain (purple inclusion bodies)
or iodine staill (glycogen synthesized

l
5. Weil's disease ··· severe form; CSF positive in large amounts by C . trachomatis
and surrounds elementary body;
stains brown with iodine; not
produced with other species) I
!
b .. Direct FA, tissue culture or

i
complement fixation

5 Growth in yolk sac of chick embryo or


I
tissue culture (McCoy cell)

6 .. C.. psittaci
a.. Psittacosis (panotfever') -
BORRELIA
occupational hazard for pet bird
l B . recurrentis handlers and poultry workers
a.. "Relapsillg fever" from ticks or lice b . Acute lower respiratory infection
b .. Looser coils ; best seen with Giemsa or
Wright's staill of blood smear 7 .. C . trachomatis
c. Mutates durillg disease; relapse due to a. Genital tract infections (sexually
illability to recognize new antigen transmitted disease)
d.. May exhibit cross reaction with ! Non-gonococcal urethritis and
••

Proteus OX K on febrile epididymitis in males


agglutinations with titer up to 1:80 !
•• Cervicitis and salpingitis (PID) in l
females
2 R burgdorferi •!• Can be passed to newborn as
a . Lyme disease conjunctivitis or pneumonia
b . Transmitted by Ixodes ticks (deer or b. Eye infections
mouse tick) !
•• Trachoma - leading cause of
c. Originally ill northeastern US blindness in underdevelped
(distribution spreading) countries
d .. Chronic migratory erythematous •!• Inclusion conjunctivitis
rash, fever, muscle and joillt pain; ll& Adults and newborns
later meningioencephalitis, ll& Can colonize nasopharynx
myocarditis and arthritis (leading to pneumonia) and
e Culture iu Kelly medium at 33oC - genital tract
clarkfield weeldy for 1 month ll@i' Appears 2-25 days after birth I

l
f. Serological diagnosis faster as a purulent eye discharge

I
Chlamydia Rickettsiae
L Obligate intracellular parasites 1. Small gram negative coccobacilli
2. Gram negative cell wall, with no 2. Obligate illtracellular parasites
peptidoglycan; possess ribosomes for
protem synthesis 3, Spread by arthropod vector

l
l:�_
4 Seen better with Giemsa 6 Weil-Felix test
a , Proteus OX-19, OX-2 and OX-K used
5.. Arthropod bite - causes fever, headache, as antigens to detect Rickettsial
lies) rash (Q fever - no rash and organism antibody
survives outside host) b ., 4-fold rise in titer or 1 : 160 titer

MostCommon ktck."ettsicie
ORGANISM VECTOR PROTEUS NOTES
OX 1 9 OX 2 OX K

R. akan House - - -

(Rickettsial Pox) Mites


�-
Coxiella burnett) Inhaled - - - Confirm with CF Test
(Q Fever)

R. prowazekii Louse + Variable ·-

(Typhus Fever)
f--
--

R. rickettsiae Tick + + - Characteristic Rash on Palms of Hand


(Rocky Mt Sp9tted Fever) and Soles of Feet

R typhi Rat Flea + + --

(Murine Typhus)

Fuf1gl:!s'-lik? each:itia
'
. ,• -- ;
- -
--
- '- \ ,-·- . -_ -- -- - - ' ' - __. - . • . - - \'
·
-

ORGANISM ATMOSPHERE CASEIN TYROSINE XANTHINE ACID FAST

Actinomyces israelii Anaerobic NA NA NA NA


-+-----
Nocardia a.steroides Aerobic - -· - Partially

Nocardia brasiliensis Aerobic + + - Partially

Acid Fast Bacilli

4, All stains based on presence of mycolic


acid (lipid-waxy) in cell wall

5 .. Any number seen on a smear is significant

6, Growth requirements
a Lowenstein-Jensen; 60% egg in
nutrient base; malachite green;
solidified into slants after inspissation
(heat to 85oC until protein
coagulates)
Mycobacteria b, Tween 80 (oleic acid) - aids in
1. Morphology - slim gr·am variable rods; dispersing colonies in liquid media
don't stain well due to high lipid content c., + C02 (especially in first 24 hrs)
in wall d Most grow at 36oC ; some require 30oC
e, Takes 3 - 6 weeks for gr·owth
2. Acid fast stain f Automated culture systems give rapid
a. Ziehl-Neilsen - "hot" stain indication of growth
b . Kinyoun - "cold" stain
7 .. Identification by nucleic acid probe
3. Auramine-Rhodamine - fluorescent stain
160

RF1MEM1 1ER!
SPECIMEN COLLECTION
l. Sputum (first morning; on 3 consecutive
mornings) , bronchial was�, gastrics ,
m·ines and tissue
''fg1ycb.Bo,Cterium •

2.. Collect aseptically and place in sterile,


tightly capped container
0 ct[.r6E:.tcurosis .
3. May be refrigerated overnight (neutralize
gastrics and urines if holding overnight)

SPECIMEN DECONTAMINATION
l. N-acetyl-1-cysteine (NALC) - mucolytic;
NaOH decontaminates; time dependent

2 .. Stain a�d report slides within 24 hrs of


processmg

3. Centrifuge decontaminated specimen for


20 min at 3000 rpm prior to making
smears and inoculating media (use
sediment)

!l}!l'Y�.f£6ift9terf:Uirr1eprae
; f�1���������t1rt��::1s,
······••··

•·. nerves -
(See Mycobacteria chart on following page .)
... u�·����(a 6Z:�gi�ssi'\'f ills�a8e th.at.
j�jgS;t���t@I�E �. • ... ··

� ·�n?��i���!'inH�rlij.Mmo. r<iotpads .
·

- _ -
- - _-'o -- ��- -"� -_,-_c� ,- ,,_·c.;:_:-�:0�-: , ·;- -
.. c.{)jff(jrentiating •cobdctetl&
. . . . ..
_;_ .,,, .. .._: .--. _. . _ . .,., _,. _ ,, ,
," -c'•: ',• _ -- ' ',<',_-0' :-.,,•-•,, ','_ ," \" - • ,•-' ),-,-, -, • c " ' .

I
-

1 Growth Niacm Nitrate Tween 80 Tellurite Growth on Notes


< 7 days Reductton Hydrolysis (3 days) MacConkey 1
I
M. tuberculosis
l f('i1.i(;£\!?'''' 1.,.•,;:\.• (];•: > 5 days Rough & buff, serpentine
1·-:,�/..<P::O:::: ·.:;)N:;;i y-::: ·-:;_:; �<:,<>\,_'\' : ;�;,:'_
..
cording on culture

M. bovis - - > 5 days - Rough & buff colony, serpentine


- : -
cording; susceptible to TCH

M. kansasii
! ;:'' '2\':";;•,\V)',.
i:'•·i(·• rc ·:,A.,.;: i'''
' .'/?•
;.y I
1
j Photochromogen !

M. martnum -
v
- ;:�t£f.li}7-:f:: -
- .!1 Photochromogen
�t�·�}i.:?i:�J _ (30" C optimum temperature)

M. scrotulaceum - - - - - - Scotochromogen

M. gordonae - - - 5-10 days - I -


I Scotochromogen

M. avwm
I ! Non-photochromogen

II
I - - -
Non-photochromogen

II
M. ulcerans
_
- - (32. C optimum temperature)

· �- - t .. ' • �,, _,

M. che/onei ::'_::.:.{t;'!�{�:;,(j::r.<;.�C,:;_
' v
- j__ v + Rapid grower

v = variable
Shaded areas = Key reactions differentiating similar genera

Photochromogen - produces pigment in light


Scotochromogen - produces pigment in dark
Non-photochromogen - produces no pigment
Rapid grower - growth in < 7 days 1
1

,_..

f-'
162

BODY "NORMAL" EXPECTED


SITES/SOURCE FLORA PATHOGEN NOTES
Throat, oropharynx Alpha Strep Strap Group A Pus pockets
-
Staph sp
Neisseria sp Corynebacterium diphtheriae Pseudomembranes when toxin produced
Gram + Rods --

Anaerobes Bordetella pertussis Whooping cough; confirm with DFA

Haemophilus influenzas Epiglottitis in children (No cuffure )

Same as above H influenzas "Pink eye"


except in Jesser
Neisseria gonorrhoea
J---·
Eye numbers Newborns

Ear
"
H influenzae
1--
Streptococcus pneumoniae

Lower Respiratory Tract None H. influenzas Early morning sputum specimen best; <1 o - 15
(sputum) squamous epithelial cells per LPF

S. aureus "

Streptococcus pneumoniae

l
"

Klebsiella pneumoniae "

l
1-·
··- --

Mycoplasma pneumoniae "

--

Mycobacterium tuberculosis "

--

l
Legionella "

Fungi

I
Transtrachial Aspirate None Anaerobes

I
Bronchial Washing Resident oral No anaerobic setwup; AFB and mycology setwup
flora

Gastric Specimens Helicobacter pylori Rapid urease test

Colon Profuse flora Shigella Selective and differential media on stools (MAC,
EMB, HE, XLD, selenite, or GN broth); subcul-
ture to selective media after 6 w 1 2 hours

Salmonella
"

--

Campylobacterjejuni Microaerophilic bag at 42"C


-

Yersinia enteroco/itica MAC after 48 hours at room temperature

Vibrio cho!erae TCBS


..

Toxigenic E coli Other tests better than culture

Clostridium difficile EIA best


163

BODY "NORMAL" EXPECTED


SITES/SOURCE FLORA PATHOGEN NOTES
Urinary Tract Normally sterile E coli Midstream catch with proper skin preparation;
(May be conta- >1 00,000 organisms per ml for infection
mlnated with (work up smaller number if pure culture and
fecal flora) white cells present)
I--·
Other gram neg rods

E. faecalis "

Staphylococcus sp "

Genital Tract Neisseria gonorrhoea Male - purulent discharge, do gram stain;


Female - gram stain not Sensitive or specific
enough, do culture tor GC (Thayer-Martin:
selective media. for GC), nucleic acid probes

Chlamydia trachomatis Nucleic acid probe

Group B Strep Significant in pregnant women

Herpes simplex HSV-·2

Trichomonas vagina/is Parasite, wet mount


Treponema pallidum Darkfield

Gardnerella vagina/is Implicated in vaginosis; look for "clue cells"

CSF Normally sterile H. influenzae Children under 5


--

Neisseria meningitidis Children through young adults


r--·
E.. coli Neonates

Group B Strep Neonates

Cryptococcus lmmunocompromised patients


--

Listeria lmmunocompromised patients

Deep Wounds/Abcesses Anaerobes and Aerobes; Bypass normal flora in collection; needle
depends on site aspirate better than swab

Superficial Wounds S. aureus


(pustules, dermatitis, --

rashes) Group A Strep

Pseudomonas aeruginosa
Enterobacteriacea

Blood Normally sterile In immunocompromised and prosthetic heart


device patients, any organism isolated from
more than 1 bottle is considered pathogenic
-
164

Virology b. If viral unknown "matches" clone, the


viral identity is confirmed
VIRAL STRUCTURE 2. Polymerase chain reaction (PCR)
L RNA or DNA - not both a. Method in which nucleic acid
sequences can be amplified in vitro
2.. Does NOT contain structural elements b . Carried out in cycles, each cycle
required for protein synthesis doubling the amount of desired
nucleic acid product
3.. Replicates in host cells

SPECIMEN COLL.ECTION AND HANDLING


L Pre- and post-convalescent sera - ship on
dry ice

2. . Specimen for viral culture - similar to


transport media for bacteria but contains
nutrients (fetal calf serum or albumin)
and antibiotics

LABORATORY METHODS
l ElA - presence of viral antibody or
antigen (ex . HbsAg and anti-HBsAb)

2 .. Viral culture

3. Electron microscopy

4. Molecular techniques (see below)


SPECIAL PROCEDURES
l. DNA probes
a . Molecular cloning of a specific DNA
sequence I
I
165
the

VIRUS DISEASE NOTES


Flavivirus Yellow Fever, Dengue; St. Louis Encephalitis Mosquito ·· Vector
-

Hantavirus Pulmonary Syndrome; Hemorrhagic Fever Rodent-Horne


-

Hepatitis A Virus (HA V? Hepatitis A Associated with Shellfish; One of Most Stable
Viruses Infecting Humans

Hepatitis C Virus (HCV} Hepatitis C Formerly Non· A. Non·.S Hepatitis

lnfluenzavirus Influenza
-

Morbillivirus Measles More Serious in Adults than Children

Mumps Virus Mumps


-- ·--

Parainfluenza Virus Parainfluenza

Poliovirus Poliomyelitis; Aseptic Meningitis Occurs Naturally Only in Humans

Respiratory Syncytial Serious Respiratory Infection in Young Children Giant Multinucleated Cells Due to Fusion of
Virus (ASV? Infected Cells

Rhabdovirus Rabies Negri Bodies in Brain Tissue at Infected


!- Animals; Rod or Bullet-Shaped;
Wildlife - Reservoir
-

Rhinovirus "Common" Cold


-- - -

Rotavirus Acute Infectious Infantile Diarrhea Can Cause Death in Infants

RubiVirus Rubella Vacclne Available; Contraindicated in


Pregnancy; Spread by Respiratory Secretions;
Serious Congenital Abnormalities

RETROVIRUS
Human AIDS EIA Techniques; Confirmed by Western Blot
Immunodeficiency Virus
(HIV !Ill}
-

Human T-Ce/l leukemia T-Ce/1 Leukemia; Tropica! Spastic Paraparesis EIA Techniques; Confirmed by Western Blot
Virus (1-{fL V I) (TSP)
-· �

Human Y.·Cell Leukemia Hairy·Ce/1 Leukemia EIA Techniques; Confirmed by Western Blot
Virus (HTL V II}
166

VIRUS
Adenovirus
DISEASE
Respiratory Infections
NOTES
f
I

Cytomegalovirus (CMV) Mental Retardation (most common viral cause); Most Common Congenital infection
Other Problems in Immunosuppressed
Depending on Site of Infection
--

Epstein-Barr Virus Infection Mononucleosis; Chronic Fatigue Heterophile Antibody


(EBV) Syndrome; Associated with Burkett's
Lymphoma
--

Hepatitis B Virus (HBV) Hepatitis B ELISA Techniques; Vaccine Available

Herpesvirus
H simplex ! Oral Infections
f1 simplex II Genital infections (STD)
1--- --

Human papillomavirus Genital Warts; Some Serotypes Associated with


(HPV) Cutaneous Warts Cervical Cancer
---
r---
Poxvirus Smallpox Supposedly Eradicated; Occasional Outbreaks
in Labs Where Virus Cultures Are Stored
-- ·- -

Varicella-Zoster Chicken Pox (children); Shingles (adults) Diagnosed by Clinical Picture

1
i..
11

MICROBIOLOGY SAMPI...E QUESTIONS


A " rice water stool" is characteristic of 7 Loeffler's medium is used as a primary
patients infected with isolation medium for
A , Clostridium botulinum A Bordetella pertussis
B Salmonella typhi B Corynebacterium diphtheriae
C. Shigella dysenteriae C Mycobacterium tuberculosis
D Vibrio choler ae D, Streptococcus pyogenes
2 A sample of material from bluish purulent 8 Autoclave sterilization of lab media requires
head lesions is submitted for analysis A which of the following pressure temperature
gram negative, motile, non-sporeforming and time parameters?
oxidase positive rod was isolated This A 15 lbs pressur e, ll5'C for 10 min
organism is most likely R 15 lbs pressure, 121 ' C for 1 5 min
A Proteus mirabilis C. 20 lbs pressur e, lOO'C for 1 5 min
B . Proteus vulgaris D. 20 lbs pressure, llO'C for 10 min
C Pseudomonas aeruginosa
9 Clinical diagnosis of rabies in infected
D Stenotrophomonas maltophilia
animals is dependent upon brain tissue
3 A gram stained smear from a genital soft obseNation of
chancre demonstrated small gram negative A. Metachromatic granules
rods arranged in tangled chains You would B. Multinucleated cells
suspect the cause of the chancroid to be C. Negri bodies
A Hemophilus ducreyi D Viral capsids
B . Herpes simplex II
1 0 Which of the following results is typical of
C .. Neisseria gonorrhoeae
Campvlobacter jejunn
D Treponema pallidum A Catalase negative
4 Organisms of this genus are gram negative, B . Non-motile
motile rods A few are chromogenic and C . Optimal growth at 42'C
produce a red non-water soluble pigment D . Oxidase negative
Some have been implicated in septicemia,
1 1 The use of penicillin/aminoglycoside to treat
pulmonary and urinary tract infections One
endocardttis due to Streptococci Group D
member of this genus is
represents a
A. Pseudomonas aeruginosa A Broad spectrum susceptibility
B Sarcina lutea R Multi-drug resistance
C. Serratia marcescens
C. "Shotgun" approach
D Staphylococcus aureus
D, Synergistic relationship
5 Which characteristic is most useful in
1 2 Characteristics indicating an appropriate
differentiating Citrobacter and Salmonella?
sputum collection would be microscopic
A H2S production
findings of
B. Indole production
A. > 25 epithelial cells, > 25 white cells
C Lysine decarboxylase
R > 25 epithelial cells, < 25 white cells
D. Urease production
C .. 10-25 epithelial cells, 10-25 white cells
6 A gram stain from a sputum specimen D < 1 0 epithelial cells , 10-25 white cells
demonstrates many gram positive cocci in
1 3 A clean catch urine specimen from a female
chains and pairs Numerous small alpha
with a suspected UTI showed gram positive
streptococci are obseNed on the primary
cocci that were catalase positive, coagulase
blood agar plate To determine if these
negative and Stap h latex negative. On the
organisms are Streptococcus pneumoniae
Microscan paneL growth in the novobiocin
which of the following tests should be well was noted The most likeiy organism is
performed?
A Staphylococcus aureus
A, Bacitracin susceptibility B.. Staphylococcus epidermidis
R Catalase C. Staphylococcus saprophyticus
C Esculin hydrolysis D. Enterococcus fecalis
D . Optochin susceptibility
168
14 The treatment of choice for methicillin 22 A gram negative rod. isolated from the urine
resistant Staphylococcus is of a female with recurrent UTI. was oxidase
A Cephalothin negative, urease positive showing A/A with
B. Nafcillin H2S on TSI and red/black on LIA is most likely 1
C.. Oxacillin A.. Escherichia coli
D. Vancomycin B. Klebsiella pneumoniae 01
C.. Proteus mir abilis
1 5 The most likely cause of subacute bacterial w
D .. Pseudomonas aeruginosa
endocarditis is b•
A. Staphylococcus aureus 23 An organism recovered from a diarrheal stool CC
B Staphylococcus epidermidis was K/A with no gas or H2S on TSI, lysine
2
C .. Streptococcus Group A negative, oxidase negative. urease negative
D. Streptococcus viridans and citrate negative The most likely
organism is
1 6 The organism known for its "stormy p
A. Aeromonas hydrophila
fermentation " and double zone of beta
B .. Escherichia coli 3
hemolysis under anaerobic conditions causes
C.. Proteus vulgaris
A Botulism
B. Gas gangrene
D. Shigella sonnei Sl
C.. Pseudomembraneous colitis 24 A cause of acute infectious infantile diarrhea c
D. Tetanus is c
A.. Hantavirus 4
1 7 The bacteremic Waterhouse··Friderichsen
B HIV
syndrome is associated with
C.. Rhabdovirus
A Corynebacterium jeikeium
D. Rotovirus r
B. Listeria monocytogenes
c
C Mycoplasma pneumoniae 25 Specimens for viral culture should be
D. Neisseria meningitidis transported in 5
A. Anaerobic containers
1 8 The specimen of choice in a case of
B. Bovine albumin (22%)
suspected epiglottitis is collected from the
C. Nutrient medium with antibiotics s
A. Blood t
D .. Sheep blood (5-10%)
B Spinal fluid
C.. Sputum E
D. Throat
1 9 A specimen from a female complaining of {
vaginitis emitted a "fishy" odor when mixed (
with 1 0% KOH A wet prep showed some 1
white cells and epithelial cells covered with (
small gram variable rods The most likely
organism is
A. Chlamydia trachomatis
B. Gardner ella vaginalis
C.. Neisseria gonorrhoea
D. Treponema pallidum
20 A photochromogenic mycobacterium
isolated at 30'C is most likely
A M. gordonae
B. M. marinum
C.. M ulcerans
D M. xenopi
21 A scotochromogenic mycobacterium
showing hydrolysis of Tween 80 in 7 days is
probably
A M. avium
B. M fortuitum
C. M. gordonae
D M. marinum
169

ANSWERS AND RATIONALE


1 D 9 c

Though options B and C may cause diarrhea, D emonstration of Negri bodies (cytoplasmic
only V: cholera causes the charact eristic "rice inclusion bodies) in brain tissue is the hallmark
water stool" . Option A may cause infant of rabies diagnosis.. Option A is not seen in
botulism which is characterized by 2-·3 days of viru ses.. Option B is associated with measles
constipation followed by flaccid paralysis. virus Option D is seen i n electron micrographs
of many viruses and bacteria..
2 c
10 c
Pseudomonas aeruginosa is the only oxidase
positive organism llsted. Campylobacter jejuni is oxidase positive,
motile, catalase positive and grows optimally at
3 A
42 c..
Opti on B is a virus not visible on a gram 11 D
stain. Option C are gram negative diplococci . .
Option D is a spirochet e that causes a hard T hese drugs in c ombination enhance
chanc re. bactericidal activity.
4 c 12 0

The only organism listed which produces a Greater than 10 epithelial cells indicates the
red pigment is Serratia . Options B and D are specimen is heavily contaminated with oral flora..
cocci ..
13 c
5 c
S . saprophyticus is resistant to novobiocin
The classic biochemical reaction which and can cause urinary tract infecti ons.
separates these two genera is lysine The other
14 D
bioc hemical reactions can be variable
6 D Vancomycin is the drug of c hoice for
methicillin resistant Staphylococci ..
Optochin susc eptibility separates Strepto­
15 D
coccus pneumoniae from the other Streptococci.
Option A is a characteristic of Streptococcus Streptococcus viridans is most commonly
pyogenes . Option B separates Staphylococcus associated with subacute bacterial endocarditis.
(catalase positive) h om Streptococcus (catalase
negative) . Group D Streptococcus are positive 16 B
with option C (esculin hydrolysis). Clostridium perfringens is the cause of gas
7 B gangrene and is noted for its stormy fermentation
and double zone of beta hemolysis.. Option A is
Microscopic morphology is best demonstrated caused by C. botulinum and is diagnosed by
on Loeffler's medium though cystine-tellurite demonstration of botulism toxin. Option C is
aga r is also used (C diphtheriae colonies caused by C . dif.ficile which grows on CCFA agar.
demonstrate a grey-to-black color on this Option D is caused by C. tetani and is identified
media) . Option A is seen on Bordet-Gengou
. by its "racquet' or " drumstick" shaped terminal
media.. Option C is associated with Lowenstein endospores..
Jensen media.. Option D grows well on blood
agar. 17 D

8 B Overwhelming DIC (due to large amounts of


endotoxin) with shock and destruction of adrenal
Autoclave st erilization requires 15 psi, at glands is caused by N meningitidis
121 "C for 15 min.
170
18 A

Options B and C are not related to the


diagnosis . Collecting a throat culture (option D)
could cause the airway to close
19 B

Gardnerella is associated with the


haracteristic "fishy" odor when vaginal
discharge is mixed with KOH. Option A is
diagnosed by EIA, DNA probes or FA. Option C
are gram negative cocci.. Option D is a spiro­
chete seen with clarkfield microscopy.
20. B

Option A is a scotochromagen. Option C


grows at 30'C but is a non-photochromagen ..
Option D is a scotochromagen and grows best at
3rc .
21 c

Option A is a non-photochromogen and does


not show hydrolysis of Tween 30. Option B is a
rapid grower and does not show hydrolysis of
Tween 80 . Option D is a photochromogen which
hydrolyzes Tween 80 in less than 5 days ..
22 c

Options A and B are H2S negative and


deaminase negative . Option D is KIK on TSI and
oxidase positive
23 D

Option A is oxidase positive Option B is


AlA on TSI and indole positive .. Option C is H2S
positive and urease positive
24 D

Option A causes hemmorrhagic fever. Option


B causes AIDS . Option C causes rabies .
25 c

Media for transporting specimens for viral


culture are similar to bacterial transport media
but must contain additional nutrients such as
albumin or fetal calf serum and antibiotics (to
prevent bacterial growth)

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