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BACTERIOLOGY

Gram + Aerobic Non-Spore Bacilli


Gram + Org
Virulence Factors
Mycobacterium Mycolic Acid protect
tuberculosis
Mycoside mycolic acid
+ CHO=glycolipid
(Kochs Bacillus)
Captain of All
Men of Death

Cord Factor 2 mycolic


acid + disaccharide;
inhibit neutrophil
migration; damage
mitochondria
Sulfatides mycosides
w/sulphate attached to
disaccharide; inhibits
phagosome
Wax D complicated
mycosides
(adjuvant=help
facilitate in inactivating
protective cellular
Immune system)

Associated Disease

Lab ID

1. 1 TB aerosol
droplet nuclei
a. Asymptomatic 1 TB

Specimen:
Sputum & Urine
3 Samples for 3
cons.days

casseous
granuloma=heal
w/fibrosis, calciferation,
scar
GHON/RANKE
complex = calcified
tubercle
b. Symptomatic 1 TB
large casseous
granuloma=liquefy and
extruded out

CSF,
Pericardial/Peritonial,
Pleural
Blood-decontaminated
(X contaminants) &
digested (x disulfide
bonds)
N-acetyl L-cysteine & 4%

digestion
4% NaOH
NaOH

2. 2 TB
Lungs Pulmonary TB
(most common site of
reactivation)
Heart Pericardial
Infection (fluids around
heart)
Lymph Node Scrofula
(most common
extrapulmonary
manifestation)
Kidney Sterile Purpura
(hematuria & pyuria, no
bacteria in gram
stain/culture)
Bones Polts Disease
Joints
CNS
Milliary TB tiny milletseed sized tubercles

decontamination

Culture
a. Liquid
Middlebrook 7H9 Medium
Middlebrook-Tween Alb
broth

b. Synthetic
Agar based
Middlebrook
7H10,7H11 AST
Selective 7-H11
(Mitchisons Medium)
Egg based
Lowenstein-Jensen
Medium Niacin test
Petragnanis Mediumspeciment w/contaminants
Carpens medium high
CHON, whole egg base;
malachite green, 8wks
monitoring; colonies tan,
no pigments, warty &
granular (cauliflower)

Stains
Acid-Fast: blue bacilli
against red bckgrnd
Fluorescent: yellow
green bacilli
1-Auramine O
2-Rhodamine
Virulence Test
Serpentine Cord
Formation
Neutral Dye Testsulfolipids bind red dye
soln

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Biochem Tests
Niacin + yellow clear
Nitrate Reduction
+pink to bright red
(tube)
+blue(paper strips)
Catalase + bubble
Tween 80 Hydrolysis pink to
red

Arylsulfatase pink to red


Tellurite Reduction black
ppt

Mycobacterium
leprae

>Strict INTRACELLULAR
parasite
>Hansens

Hansens
bacillus

disease/LEPROSY

>Multiplies w/in host


cells

1.
Lepromatous/Nodular
-severest form; patients no
cell-mediated immune
response
-skin, nerves, eyes &
testes
-skin=lumps & thickening
*Leonine facies
*Saddlenose deformity
*Internal testicular
damage
-peripheral nerves=thick,
loss of sensation

CANNOT be
CULTURED
Can be grown on foot
pads of armadillos

2.
Tuberculoid/Anesthetic
-cell-mediated immune
response
-milder, sometimes selflimiting disease
-localized unilateral skin &
nerve
-well defined
HYPOPIGMENTED
BLOTCHES w/ absent
sensation
-few orgs in skin lesions
+LEPROMIN TEST

Runyons Classification
Classification

Description

Organisms

Group I
PHOTOCHROMOGENS

Light: Pigmented
Dark: Non-pigmented

M.
M.
M.
M.

kansasii
asiaticum
marinum
simae

Group II
SCROTOCHROMOGENS

Light: Pigmented
Dark: Pigmented

M.
M.
M.
M.
M.

flavescens
gordonae
scrofolaceum
szulgai
thermoresistable (52)

Group III
NONPHOTOCHROMOGENS

Light: Non-pigmented
Dark: Non-pigmented

M. terrae-triviale, V
bacillus
M. haemophilium
M. avium-intracellulare
M. malmoense
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Group IV
RAPID GROWERS

Grows w/in 3-5days

M. fortuitum-chelonei
M. phlei
M. smegmatis

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Corynebactrium Diphteriae Exotoxin


diphteriae
(A-B toxin complex)
attacks mucus
Klebs Loefflers
membrane of respi tract
Fragment B-bind to cell
Bacillus
surface receptor
Fragment A-inhibits
Chinese
protein synthesis (cell
characters/
death & tissue necrosis)
palisades
Hemolysin
dermonecrotic act
K antigen-type
specificity of org

1. Respiratory
Diphteria
-local infection(throat)
-PSEUDOMEMBRANE
(thick greyish,
adherent exudates)
2. Cutaneous
Diphteria
-ulcer coated w/
PSEUDOMEMBRANE =
NON-HEALING
ULCER

O antigen-heat stable
Ag

Microscopic
-pleomorphic, palisades
(Chinese characters)
-BABES ERNST
GRANULES = clubbed
shaped swelling, beaded
& Barrel forms due to
metachromatic granules
Culture
Tinsdake Tellurite
grey-black colonies
Loefflers Serum Slant
POACHED EGG
Cysteine Tellurite
Agar(CTBA) Tellurite
H2S produced=black
zone & brown halo (GUN
METAL GRAY colonies)
Virulence Test for
Toxin Pro
In vivo-animal inoc
(Guinea pig) w/ toxin
In vitro-Eleks test
AST- Schick Test
Redness +
No reaction -

C. ulcerans

Nose & throats of horses


& humans

C. ovis

TONSILITIS

Ulcerative
lymphaginitis
Abscess & purulent
infe in sheep, horses,
cattle
ERYTHRASMA scaly
plagues (coral red in
fluorescence Woods
light)

C. pseudoTB

C.
minutissimum

Same as
C.diphteriae
Tellurite

Culture
Fetal serum coral
red to orange
fluorescence
BAP-no fluorescence

C. bovis

C. jeikeium

Resistant to most
ANTIBIOTICS

Listeria
monocytogenes

Multiply at ref temp 4C


TUMBLING MOTILITY at
RT

-hemolytic

Intestinal tract of mammals &


birds

Most common
Corynebact to CNS
SHUNT NEPHRITIS
Pneumonia
Endocarditis
Neonates, Elderly,
Immunocompromised
>Granulomatosis
infantiseptica
-transmitted to fetus
transplacentally

CAMP Test + (Grp. B


Strep)
Tumbling Motility wet
prep
Semi-solid medium-

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Soft cheeses & unwashed


vege
Raw/undercooked food of
animal origin

-early septicemic: 15days postpartum

umbrella like/inverted
xmas tree

-delay meningitic: 1020days following birth

Culture:
COLD ENRICHMENT
technique: 37C

>Intracellular pathogen
-cell-mediated & humoral
immunity develop
-only cell-mediated is
protective

BAP-Bhemolytic
Tryptose AgarBlueGreen
Phenyethylalcohol-inhibit
gram

Listeria
monocytogenes

Biochem Test:
Catalase +
H2S
Bile Esculine +
Salicilin

Erysipelothrix
rhusiopathiae

Occupational Infectfisherman, butchers,


vets, gardeners

Nonmotile

Inflammatory swelling of
hands & fingers

filamentous

Animal Inoc
(Pathogenic)
Rabbits eye-conjuctivitis
monocytosis
Catalase
H2S +
Gelatin Medium-Testtube
brush appearance

Erysipeloid (humans)
Erysipelas
(swine&turkeys)

Lactobacillus
acidophilus
Rothia

Normal flora GIT


Mouth

Endocarditis

Kurthia

Soil

Infection in
Immunocompromised px

Tomato Juice Agar

FUNGUS LIKE ORGANISMS


Gram Positive ++++, aerobic filaments, coccobacillus
Nocardia asteroides
Pulmonary Infection
grow at 46C
Casein hydrolysis --Tyrosine decomposition
--Gelatin Liquefaction --Ureas +++

N. brasiliensis
1 Mytecoma Pathogen
X grow at 46C
Casein hydrolysis +++
Tyrosine decomposition ++
+
Gelatin Liquefaction +++
Urease +++

Actinomyces Israeli and bovis


Specimen: Sputum, Pus, Scrapings
Gram stain: G+++ and filamentous (break into coccus and bacillus form)
Culture: Aerobically, Liquid and Solid
Liquid: Thioglycollate broth, freshly boiled and cooled; best: enriched w/ trypticase soy broth
Solid: Brain Hear infusion agar heaped rough lobar colony MOLAR TOOTH
CATALASE & INDOLE NEGATIVE ---

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A. israeli
Nitrate --Starch Hydrolysis
---

A. bovis
Nitrate +++
Starch Hydrolysis +
++

CLOSTRIDIUM
Obligate aerobes but some are aerotolerant like Cl. Histolyticum & Cl. Tertium
Motile w/ peritrichous flagellum except Cl. perfringens, Cl. ramosum, Cl. clostridioforme
All have SWOLLEN SPORANGIA except Cl. perfringens, Cl. bifermentans
All are ENCAPSULATED except Cl. perfringens
All are NAGLERS TEST NEGATIVE --- except Cl. perfringens, Cl. baratti, Cl. bifermentans, Cl.
sordelii
Toxic Clostridium: Cl. tetani; Cl. botulinum; Cl. difficile

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Gram + Org
Clostridium tetani
Tack-head bacillus
Lollipop bacilli
Tennis Rocket bacilli

Virulence Factors

Associated Disease

Lab ID

Tetanospasmin blocks

Tetanus (Lockjaw or
Trismus)
-spores infecting a wound

Microscopic
G+ w/ round terminal
spores; drumstick

release of inhibitory
neurotransmitter glycine &
-aminobutyric acid; Zc
dependent endopeptiase

Culture
Strict anaerobic
BAP- B-hemolytic
transparent
BURNT FLESH ODOR
Biochem DOESNT
ferment sugar/liquefy
gelatine
Animal Inoc
WASSERMANN TAKAKI
PHENOMENON rigid
spasm at hind legs of
mouse

Clostridium botulinum
Bacillus botulinus
Canned-good Bacillus

Clostridium difficile

Botulism Toxin attack


peripheral cholinergic
nerve terminals
-FLACCID PARALYSIS =
irreversible prevention of
acetycholine release from
neuromuscular junction

Toxin A- enterotoxin;
severe damage to
intestinal mucosa
Toxin B (cytotoxin)cytopathic for tissue
culture cell lines

Clostridium
perfringens
Clostridium welchii
Bacillus aerogenes
capsulatus

Toxins
1. Endotoxin toxin(lecithinase)
degrades lecithin = lysis
of var cells
2. Enterotoxin heat
labile protein in small
intestine
3. Degradative enzymes

1. Classic Botulinum
- food poisoning
2. Infant Botulism
(Floppy Baby
Syndrome)
-constipation, feeding
probs, lethargy & poor
muscle tone
3. Wound Botulism
- rare; wound
contamination
Pseudomembarnous
colitis (PMC)
Antibiotic-associated
diarrhea
Cl. difficileassociated
diarrhea(CDAD)
-lethal ds. In GIT
(overgrowth of orgs as
side effect of
antibiotics)
Cellulitis & Wound
infect
- necrotic skin; moist,
spongy crackling
consistency due to
pockets of gas
(CREPITUS)
Clostridial
Myonecrosis
-org inoc in muscle;

Culture
BAP & Cooked meat
medium slightly
hemolytic & slightly
brown under
transmitted light

Culture
BAP- yellowgreen
fluorescence horse
stable odor
Cycloserine-cefoxitine
egg yolk Fructose agar
yellow ground
glass

Microscopic: short plump,


box car-shaped g+ rod
Culture:
BAP-circular smooth w/
target/double zone
hemolysis
Chopped Meat Glucose
Medium-w/gas formation
Biochem Test
Milk Media- STORMY
FORMATION(excess gas
formation); ferments

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var of hydrolytic
enzymes; liquefy tissue;
promote infection spread

destroys adjacent
muscles; blackish
fluid which exudes
from skin
Acute Food
Poisoning
-spores from
improperly cooked
foods (enterotoxin)

Glucose, lactose,
malatose, fructose but
not xylose
Lecithinase Test-split
lecithin (egg yolk) to
insoluble disaccharides;
opaque Halo surrounding

Bacillus
Gram+++, aerobic, endospore, dust and soil, catalase +++

Gram + Org

Virulence Factors

Associated Disease

Lab ID

Bacillus anthracis

Antiphagocytic capsule
Lethal Factor
Protective Ag
Edema Factor

1. Cutaneous
anthrax
-org/spores germinate
a papulae that evolves
into a painless, black
severly swollen
MALIGNANT
PUSTULE which will
crust over

Culture
5% BAP: nonhemolytic; medusa

Anthrax Bacillus
Box-car shaped
Bamboo Fishing Rod

2. Pulmonary
Anthrax
-Wool Sorters Disease
-inhalation of spores
(lungs)
-most serious
3. Gastrointestinal
Form
-ingestion of spores
Bacillus cereus

head or caput medusa


lion head, comet tails,
Barristers wig

Polymyxin Lysozyme
EDTA Medium highly
sensitive
-BEATEN EGG WHITE
Solid medium
0.5ug/mL of
penicillin=STRING of
PEARLS
Animal Inoc
-confirmation ID (mice)
Ascoli Test ID from
animal carcass

Enterotoxin Food
Poisoning
1. Emetic rice
(vomiting, abdominal
cramps, nausea, heat
stable)
2. Diarrheal
vegetable & meat
(diarrhea, abd cramps,
nausea, heat labile)

Motility
Hemolysis
Litmus Milk
Methylene Blue
Growth at 45C
Penicillin
Sensitivity

B. anthracis
Non-motile
Non-hemolytic
Not reduced
Not reduced
No growth
Sensitive

B. cereus
Motile
B-hemolytic
Reduced in 2-3 days
Reduced in 24 hrs
Positive
Resistant
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B. subtilis
Hay Bacillus
B.
B.
B.
B.
B.

thuringensis
polymyxia
licheniforms
megaterium
stearothermophilus

Common lab contaminant


SLIMY BREAD
Spore strips- sterilization of dry heat and ETO
sterilizers
Biological control of mosquito larvae
Source of Polymyxin A
Industrial source of Bacitracin
BIG BREAST; largest spore former
Biological indicator for sterility testing

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Enterobacteriaceae
Gram negative ---; Facultative anaerobes, non-spore
Glucose fermenter w/ strong acid formation and often GAS
Cytochrome oxidase --- negative
Nitrate reduced to nitrite
Colon COLIFORM ORGANISMS
Heterogenous straight rods
Escherichiaciae
Citrobacter
Edwardsiella
Escherichia
Salmonella
Shigella

Klebsiella
Hafnia
Enterobacter
Klebsiella
Serratia

Protea
Proteus
Providencia

Yersinae Yersinia
Erwinae Erwinia
Antigenic Classification
1. O Ag LPS, differs from organisms depending on sugar and side chain substitution
2. K Ag capsule that covers O Ag
3. H Ag determinant; subunits of flagella
Associated
1. Diarrhea w/ or w/o systemic invasion
2. Various infections: UTI, Pneumonia, Bacteremia, Sepsis
Lab ID
1. Specimen: Feces, Blood, Spinal Fluid, Bile, Wound and Abscess, Urine and Throat Swab
2. Direct Smear: gram stain from liquid stools and material submit in preservative soln &
enrichment
3. Culture
A. Primary Isolation: Feces, Urine & Bile
B. Primary Media: BAP, MAC, XLD, SSA, BSA
BAP- non-inhibatory
MAC & XLD- slightly inhibitory (lightly inoculated)
SSA & BSA selective media (heavily inoculated)
Medium
CHO and other compo
Indicator
Results
1. MacConkey
Lactose
Neutral Red
(MAC)
Bile Salts & Crystal
Violet
-inhibit Gram +++ and
some fastidious gram-neg

2. Eosin Methylene
Blue Agar (EMB)
-selective medium for
E.coli

Lactose Levine EMB


Lactose & Sucrose
Holt Harris Teague
Formula
Aniline dye (Eosin &
Methylene Blue)
- inhibit Gram +++
and fastidious gramneg
-acid production (ppt)

Strong acid
fermenter
-green black w/
metallic sheen
Slow/weak
fermenter
- purple w/in 24-48hrs
Non-lactose
fermenters
-transparent
*Yersinia
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enterolitica
Levine transparent
Holt & Harris-purple
black

3. SalmonellaShigella Agar (SSA)


- highly selective to
inhibit growth of most
coliforms and permit
the growth of
salmonella & shigella
(heavy inoculums)

Bile Salts and


Sodium Citrate
-inhibit gram+++ and
many gram-neg
including coliform

Neutral Red

Salmonella
colorless w/ black
centers

Lactose
Sodium thiosulfate
-sulfur source

Shigella colorless
w/ no blackening

Ferric Citrate
-Sulfide Production
4. Hektoen Enteric
(HE Agar)
-selective &
differential
-increase yield of
Salmonella & Shigella
spp. From heavy #s
of normal flora

Bile Salts
-inhibits gram +++ &
retards growth of many
strains of coliform

Acid Fuchsin &


Thymol Blue

Sodium Thiosulfate
-Sulfur source

-selective &
differential
-detect SHIGELLAE in
feces after
enrichment in gramneg broth

Bile salts (low conc)


-less selective
Xylose, Lactose,
Sucrose
Sodium Thiosulfate
-Sulfur source
Ferric Ammonium
Citrate
-sulfide production

Proteus motile
dont swarm
Strong Lactose
Fermenter
-orange to salmon
pink
Salmonella
-blue-green, black
centers

Sucrose & Lactose

Shigella
-more green fading to
the periphery colony

Ferric Ammonium
Sulfate
-sulfide production

5. Xylose Lysine
Deoxycholate (XLD
Agar)

Salmonella arizonae
- lactose fermenter
- RED

Phenol Red

Proteus strains
inhibit
-transparent & more
glistening watery
E.coli, Klebsiella &
Enterobacter
- >1 CHO, bright
yellow
Proteus spp
-yellow or translucent
w/ black centers
Salmonella
-red colonies w/ black
centers (H2S)
Shigella,
Providencia
-translucent

-highly selective &


best for isolation of

Brilliant Green
-inhibit gram +++ and

Citrobacter
-yellow w/ black
center
S. typhi
-black surrounded by
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Salmonella typhi and


lactose +++
salmonella
-isolation of vibrio
cholera by pH to 9.2

7. Brilliant Green Agar


-highly selective for
Salmonellae other
than S. typhi

coliforms

a black zone &


metallic sheen

Dextrose

Other salmonellae
-green colonies

Ferrous SO4 H2S


indicator
Bismuth Sulfide
-CHO utilization
LACTOSE

Phenol Red

Salmonella
-whitish
snowflake
surrounded by
Brilliant Green

Brilliant Green inhibitor

S. typhi & Shigella


-inhibited, greenish
yellow
Enrichment medium
1. Selenite F Broth for isolation of Salmonella from specimens such as feces, urine or sewage
that has heavy concentrations of mixed bacteria
2. GN Broth designeed for recovery of Salmonella spp and Shigella when small in numbers in
fecal
Biochemical Tests
1. Lactose Fermentation
Lactose Fermenting
MAC & XLD inoculate a short
biochem test set
TSI
Christensen Urea Agar Slant
Methyl Red Broth
Simmons Citrate Agar
Decarboxylase Broth
ONPG (Slow Lactose Fermenters)

NLF
MAC & XLD subcultured to long set
of media
TSI
Christensen Urea Agar Slant
Simmons Citrate Agar
Phenylalanine Agar
SIM Agar
MR-VP Broth
Decarboxylase Broth
Bismuth Sulfite Agar (Typhoid)

2. Triple Sugar Iron Agar


- butt/slant medium; modification of KIA medium
Contents
a. CHO
TSI:

Glucose
Lactose
Sucrose

0.1%
1%
1%

KIA:

Glucose
Lactose

0.1%
1%

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b. Phenol Red pH indicator (Yellow=acid; Red=alk)


c. Sodium Thiosulfate H2S indicator (blackening)
POSSIBLE REACTIONS:
1. A/A = glucose & lactose and/or sucrose (SEEK: Serratia, Escherichia, Enterobacter,
Klebsiella)
2. K/A = glucose alone (SCiPPY: Shigella, Citrobacter, Providencia, Proteus, Yersinia)
3. K/K = no sugars (Pseudomonas)
4. CO2 = bubbles or breaks
5. H2S = blackening of butt
A/A, H2S+ (CAP: Citrobacter, Arizona, Proteus)
K/A, H2S+ (CASE: Citrobacter, Arizaona, Salmonella, Edwardsiella)
Biochemical Tests
3. IMVIC (Indole, Methyl Red, Voges-Proskauer, Citrate)
IMVIC Test
Indole Production

Principle
Oxidize tryptophan to
produce indole

Medium: peptone
broth

Methyl Red Test


Medium: Buffered
Peptone Glucose
Broth
Voges-Proskauer
Medium: Buffered
Peptone Glucose
Broth

Citrate Test
Medium: Simmons
Agar

Indicator
Kovacs reagent (pdimethylaminobenzaldehyde
)

Result
Red color rises to
top
+++:E.coli, P.
vulgaris

Produce enough acid


will overcome
neutralizing effect of
the buffer

Methy Red

Produce
acetylmethylcarbinol
(acetoin) from glucose

Barritts Reagent (5% naphthol + KOH)

Utilize Na+ citrate as


sole source of CHO
will grow changing
color to indicator

---: Klebsiella,
Enterobacter, P.
mirabilis
+++: E.coli
---: Klebsiella,
Enterobacter

Bromthymol Blue

+++ Red layer on


top(10mins):
Klebsiella
pneumonia,
Moraxella
--- No color, color
other than red:
E.coli, Acinetobacter
Green to Blue
+++: Klebsiella
pneumonia,
Moraxella,
Enterobacter
---: E.coli,
Acinetobacter

4.