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BACTERIOLOGY

COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELL

PROKARYOTE EUKARYOTE
Nuclear body

Cell division

Cell wall

Cytoplasmic membrane

Cell organelles

Site of energy production

Site of protein synthesis

BACTERIAL CYTOLOGY

CELL WALL

 Defines the shape of the bacteria

 Some components responsible for pathogenicity
*M protein
*Mycolic acid

* Main constituent: ____________________________________

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GRAM STAIN

REAGENTS GRAM POSITIVE GRAM NEGATIVE

Primary stain

Mordant

Decolorizer

Secondary stain

NOTE:

Gram positive cocci (aerobes): Micrococcus, Staphylococcus. Streptococcus

Gram positive cocci (anerobes): Peptococcus, Peptostreptococcus, Sarcina

Gram negative cocci (aerobes): Branhamella, Neisseria

Gram negative cocci (anaerobes): Veillonella

Gram positive bacilli (aerobes): Bacillus, Corynebacterium, Erysipelothrix, Lactobacillaus, Listeria,

Mycobacterium, Nocardia

Gram positive bacilli (anaerobes): Actinomyces, Clostridium, Propionobacterium

Gram negative bacilli (aerobe): Acinetobacter, Aeoromonas, Alcaligenes, Bordetella, Brucella,

Enterobacteriaacea, Francisella, Legionella, Pasteurella, Pseudomonas, Vibrio

Gram negative bacilli (anaerobes): Fusobacterium, Bacteroides

ACID FAST STAIN

ZIEHL-NELSEEN KINYOUN ACID-FAST NON-ACID FAST

ORGANISM ORGANISM
PRIMARY STAIN

MORDANT

DECOLORIZER

SECONDARY
STAIN
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CYTOPLASMIC MEMBRANE

 Selectively permeable membrane

 Site of energy production

MESOSOMES

 Point of attachment for chromosome

INCLUSIONS

1. Much granules

2. Babes-Ernst/metachromatic/volutin granules:

3. Bipolar bodies:

ENDOSPORES

 Resting cell, highly resistance to dessication, heat and chemical agents

 Composition : ____________________________
 Bacterial genera with spores:
1. ______________________________________
2. ______________________________________

CAPSULE

 Increase virulence by preventing Phagocytosis

 Antigenic; on the basis of serotyping by ________________________
 Demonstration
*Animal tissues and fluids
*Media containing milk or serum

* Colonies often slimy

* Stains: _________________________

_________________________

PILI

 Syn. ________________________
 Ordinary pili: adherence of bacteria to host cell
 Sex pili: bacterial conjugation

FLAGELLA

 Atrichous – no flagellum
 Monotrichous – flagellum on one pole:
 Amphitrichous – single flagellum on each pole
 Lophotrichous – tuft of flagella at one or both poles
 Perithrichous – flagella all over the organism:

*Periplasmic flagella _________________________

*Test for motility:

*Semisolid medium:

*Stains for flagella:

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BACTERIAL VIRULENCE FACTORS

1. Adherence pili

2. Antiphagocytic factors

3. Enzymes:

Ex. Coagulase, fibrinolysin, hyaluronidase

4. Toxins: Exotoxin and Endotoxin

COMPARISON EXOTOXIN ENDOTOXIN

SOURCE

RELEASE

COMPOSITION

HEAT STABILITY

IMMUNOLOGIC

PHARMACOLOGIC Cytotoxin:

Enterotoxin:

Neurotoxin:

TOXICITY

LETHAL DOSE

EX. DISEASES

TEST TO DETECT ENDOTOXIN IN BODY FLUIDS AND SURGICAL INSTRUMENTS:

_______________________________________________________________

_______________________________________________________________
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BACTERIAL GROWTH FACTORS

1. NUTRIENTS
a. Carbon :

*Lithotroph:

*Heterotroph:

b. Nitrogen:

c. Minerals: ex. Sulphur, magnesium

d. Salt

e. Others:

2. OXYGEN AND CARBON DIOXIDE AVAILABITY

a. Obligate/strict aerobe:

b. Obligate/strict anaerobe:

c. Facultative anaerobe:

d. Aerotolerant anaerobe:

e. Microaerophilic:

f. Capnophilic:

NOTE:

 Aerobes grow in ambient air, which contains 21% O2 and a small amount (0.03%) of CO2

 Anaerobes cannot grow in the presence of O2 and the atmosphere in anaerobe jars, bags or
chambers is composed of 5% to 10% H2, 5% to 10% CO2, 80% to 90% N2, and 0% O2.

 Microaerophiles grow under reduced O2 (5% to 10%) and increased CO2 (8% to 10%); this
environment can also be obtained in specially designated jars or bags.

 Capnophiles requires increased concentrations of CO2 (5% to 10%) and approximately 15% O2;
atmosphere can be achieved by a candle jar or CO2 incubator.

3. TEMPERATURE

a. Psychrophilic//cryophilic:

b. Mesophilic:

c. Thermophilic:
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4. PH
Optimum ___________________________________

a. Acidophile:

b. Alkaliphile:

GROWTH CYCLE

1. Lag phase
*Little or no multiplication:

2. Log/exponential phase

*Organisms grow at a maximum rate:

3. Stationary/plateau phase

*Growth ceases because nutrients are exhausted or toxic metabolic products have
accumulated.

4. Decline phase

*Viable count decreases

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CULTURE MEDIA

I. ACCORDING TO PHYSICAL STATE/CONSISTENCY

1. Liquid:

2. Semi-solid:

3. Solid:

a. Liquefiable:
b. Non-liquefiable:

II. ACCORDING TO COMPOSITION

1. Synthetic/chemically-defined:

2. Complex/non-synthetic:

3. Tissue:

III. ACCORDING TO DISPENSING/DISTRIBUTION

1. Plated 2. Tube

IV. ACCORDING TO DUNCTION AND USE

1. SIMPLE/BASAL/SUPPORTIVE/GENERAL ISOLATION/GENERAL PURPOSE MEDIA
o Support the growth of most nonfastidious bacteria
o Nutrient agar/broth
o Trypticase soy agar/broth

2. ENRICHED MEDIA
o Contain nutrient supplement for fastidious bacteria
o Blood agar plate
o Chocolate agar plate

3. ENRICHMENT MEDIA
o Enhance the growth of an organism
o Selenite broth
o Tetrathionate broth
o Alkaline peptone H2O ______________

4. SELECTIVE MEDIA
o Select for growth of a particular organism
o Contains inhibitors
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INHIBITORS FOR GRAM POSITIVE BACTERIA

INHIBITORS FOR GRAM NEGATIVE BACTERIA

EXAMPLES OF SELECTIVE CULTURE MEDIA

a. For MTB

Medium: __________________________
Inhibitor: __________________________

b. For C. diphtheria

Medium: __________________________
Inhibitor: __________________________

c. For gram positive bacteria

PEA (phenylethyl ROH): inhibits gram – bacteria

d. SELECTIVE FOR N. gonorrhoeae (contains antibiotics as inhibitors).

A. THAYER MARTIN

Vancomycin: ________________________________________
Colistin: ____________________________________________
Nystatin: ___________________________________________

B. MODIFIED THAYER MARTIN

Vancomycin + colistin + nystatin + _______________________:

C. MARTIN LEWIS

Vancomycin + colistin + nystatin + trimethroprim lactate + _______________:

D. NEW YORK CITY AGAR: ____________________________________________

Vancomycin + colistin + nystatin + trimethroprim lactate + _________________:

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e. Selective differential for staphylococcus sp.

Medium: __________________________
Inhibitor: __________________________
CHO: _____________________________
pH indicator: _______________________:
MF: _______________________________
Non MF: ___________________________

f. Selective differential for Vibrio sp.

Medium: __________________________
CHO: _____________________________
pH indicator: _______________________:
SF: _______________________________
Non SF: ___________________________

5. DIFFERENTIAL MEDIA
o Provides distinct colonial appearances of microorganisms to aid in their identification

ENTEROBACTERIACEAE

a. RAPID LACTOSE FERMENTERS: _________________________________________

b. LATE LACTOSE FERMENTERS: ___________________________________________

c. NONLACTOSE FERMENTERS: ___________________________________________

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EX. SELECTIVE DIFFERENTIAL MEDIA DOR GRAM NEGATIVE ENTERIC BACILLI

a. EOSIN-METHYLENE BLUE (EMB) AGAR

Sel/Diff for Gr- Enteric Bacilli

CHO: ____________________________

LF: ______________________________

NLF: _____________________________

Charac. Of colonies on EMB:

E. Coli: __________________________

Klebsiella: _________________________

Enterobacter: ______________________

b. MACCONKEY (MAC) AGAR

Sel/Diff for Gr- Enteric Bacilli

Inhibitor for Gr+: ___________________________

CHO: ______________________________

pH indicator: ________________________

LF: ______________________________

Non LF: _____________________________

c. HEKTOEN ENTERIC (HEA) AGAR

Sel/Diff for Gr- Enteric Bacilli

Inhibitor for Gr+: bile salt

CHO: lactose, sucrose and salicin

pH indicator: ________________________

LF, H2S negative:______________________________

LF, H@S positive: ______________________________

nonLF, H2S negative: ___________________________

nonLF, H2S positive: ____________________________

d. Salmonella Shigella (SS) Agar

Sel/Diff for Salmonella and Shigella

CHO: Lactose

pH indicator: _____________________________

H2S indicator: ____________________________

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Salmonella: _______________________________________________________________

Shigella: __________________________________________________________________

6. TRANSPORT MEDIA

7. MEDIUM FOR SUSCEPTIBILITY TESTING

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MEDIA FOR BIOCHEMICAL TESTING (TSI AND LIA)

TRIPLE SUGAR IRON AGAR

*Considered an initial step in the identification of Enterobacteriaceae

Composition:

Protein source

CHO: _________________________________

_________________________________

__________________________________

pH indicator: ___________________________

H2S indicator: __________________________

Sulfur source: __________________________

TSI REACTIONS TYPICAL ORGANISMS

A/AG H2S-

K/AG H2S+

K/A H2S-

K/K H2S-

LYSINE IRON AGAR

*Can be used to determine the ability of the organism to deaminate lysine, decarboxylate lysine
and produce H2S.

Composition:

Small amount of CHON

Glucose, lysine

pH indicator: ___________________

EX. LIA REACTION

K/K H2S+ ________________________

K/A H2S- ________________________

K/K H2S- _________________________

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LYSINE IRON AGAR

Can be used to determine the ability of the organism to deaminate lysine, decarboxylate lysine and
produce H2S.

Composition:

Small amount of CHON

Glucose, lysine
pH indicator ____________________________
H2S indicator ___________________________

Ex: LIA Reaction

K/K H2S+ _____________________________

K/A H2S- _____________________________

R/A H2S ______________________________

______________________________
______________________________

SUSCEPTIBILITY TESTING

1. TUBE OR MICROPLATE DILUTION

*Serial dilution of antibiotics is prepared, to each tube, a uniform amount of inoculum is added
*Minimum Inhibitory Concentration (MIC):
_________________________________________________________

*Minimum Bactericidal Concentration/Lethal:

_________________________________________________________

2. AGAR DILUTION
Varying concentration of antibiotics is incorporated to appropriate plating media

3. DISK DIFFUSION KIRBY BAUER

MUELLER-HINTON AGAR

Depth of agar: _________________________________________

pH of agar:____________________________________________

STEPS:
a. Pick 4-5 colonies into TSB. Incubate @ 37c for 2-5 hours.
b. Compare turbidity with 0.5 McFarland standard (___________________).
________________________________________________________
________________________________________________________
c. Inoculate to MHA.
d. Apply antibiotic disks
e. Invert plates and incubate 37c for 16-18 hrs.
f. Measure zone of growth inhibition
g. Interpret susceptibility from standard chart zone.
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POSSIBLE SOURCES OF ERROR

a. Use of mixed culture
b. Inoculum too light ___________________________________
c. Inoculum to heavy ___________________________________
d. Too much moisture on agar ____________________________
e. Very dry agar surface _________________________________
f. Improper storage of disk ____________________________________________
g. Reading and clerical error
h. Deterioration of turbidity standard or control strains.

4. E-TEST

5. AUTOMATED:

ANTIMICROBIAL AGENTS

TARGET SITES:

1. CELL WALL SYNTHESIS

*Beta-lactams: penicillins, cephalosporins, monobactams
*Glycopeptides: vancomycin, teicoplanin
*cycloserine
*Bacitracin: ___________________________________

2. PROTEIN SYNTHESIS

*Aminoglycosides: gentamicin, tobramycin, kanamycin

*Tetracyclines: _________________________
*Chlorampenicol: _______________________
*Erythromycin
*Fusidic acid

2. NUCLEI ACID SYNTHEIS

*Sulfonamides: ____________________________________________________
*Trimethroprim
*Quinolones
*Rifampicin

3. CELL MEMBRANE FUNCTION

*Polymyxins: colistin
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STERILIZATION:

I. PHYSICAL METHODS

1. HEAT
MOIST HEAT
a. Autoclaving:
*Most effective method
*__________________________________________
*Biologic indicator: _______________________________________

b. Fractional
Alternate heating (kill veg. cells), incubation (spores germinates), and heating
(kill remaining cells)

Tyndallization: ___________________________________________

Inspissation: _____________________________________________

DRY HEAT
a. Oven
*Temperature: __________________________
*Biologic indicator: __________________________________

b. Incineration: _______________________________________________
*Temp: ___________________________________________

2. Ionizing radiation
*plastic syringes, catheter or gloves, evacuated tubes
*short wavelength, high energy gamma rays

3. Filtration
*Antibiotic soln’, CHO soln’, and vaccines
*Liquid: pulling soln’ through cellulose acetate or cellulose nitrate with vaccum
*Air: HEPA filter

II. Chemical Methods

1. Ethylene oxide
2. Formaldehyde vapour and vapour phase H2O2:
3. Glutaraldehyde:
4. Peracetic acid

DISINFECTION:

I. Physical Methods
1. Boiling:

2. Pasteurization:
a. Batch/LTH: ____________________________________________
b. Flash/HTS: ____________________________________________

3. Non-ionizing radiation:
*Long wavelength low energy UV light
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II. Chemical Methods

ANTISEPTIC:

DISINFECTANT:

1. Alcohol:

2. Halogen:

3. Heavy metal

4. Quarternary ammonium compunds (QUATS)

*Inactivated by: _______________________

5. Phenol

PHENOL COEFFICIENT
*Expression of the bactericidal power of a particular substance as compared to pure phenol.

*PC > 1:

*PC <1:

*PC = 1:

BIOLOGICAL INDICATORS

1. Autoclave: ______________________________________________

2. Ionizing radiation: ________________________________________

3. Dry heat oven: ____________________________________________

4. Ethylene oxide (ETO): _______________________________________

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BIOLOGICAL SAFETY CABINETS (BSC)

CLASS I BSC
*Open-fronted, negative pressure, ventilated cabinets.
*Unsterilized room air enters and circulates within the cabinet, and exhaust air from the cabinet
is filtered by HEPA (_______________________________________) filter.

CLASS II BSC
*Sterilized both the air entering and circulating the cabinet and exhaust air.
*Used by most hospital microbiology laboratories.
*also known as: _________________________________________.

CLASS III BSC

*Provide the highest level of safety
*All air entering and leaving the cabinet is sterilized with HEPA filter.
*System is entirely close, and all infectious material are handled with rubber gloves that are
sealed to the cabinet.

COMPARISON OF BIOLOGIC SAFETY CABINETS

CABINETS APPLICATIONS
TYPE FACE AIRFLOW PATTERN RADIONUCLEOTIDE BIOSAFETY PRODUCT
VELOCITY S/TOXIC LEVEL(S) PROTECTION
CHEMICALS
Class I, *open 75 In at front; rear and No 2,3 No
front top through HEPA filter
Class II Type A 75 70% Recirculated No 2,3 Yes
through HEPA; exhaust
through HEPA
Type B1 100 30% Recirculated Yes 2,3 Yes
through HEPA; exhaust (low
through HEPA and levels/volatility
hard-ducted
Type B2 100 No recirculation; total Yes 2,3 yes
exhaust via HEPA and
hard-ducted
Type B3 100 Same as IIA, but yes 2,3 yes
plenums under
negative pressure to
room and exhaust air is
ducted.
Class III NA Supply air inlets Yes 3,4
through 2 HEPA filters.
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CLINICAL SPECIMENS

BLOOD CULTURE
*Antiseptic: ROH-iodine-ROH
*Anticoagulant: _______________________________:
*Neutralizes bactericidal effect of human serum
*prevents Phagocytosis

THROAT AND NASOPHARYNGEAL CULTURES

*Most abundant normal flora: _________________________________________
*Most common pathogen: ____________________________________________
*Cultures must include anaerobic conditions for beta Streptococcus (some are Nonhemolytic
unless conditions are anaerobic).
*Culture on Todd-Hewitt broth for fluorescence microscopy of beta Streptococcus
*Nasopharyngeal swab
*H. influenzae
*B. pertussis
*N. menigitidis

MAJOR LABORATORY RESULTS FOR THE DIFFERENTIAL DIAGNOSIS OF MENINGITIS

BACTERIAL TUBERCULAR VIRAL FUNGAL

*Elevated WBC count *Elevated WBC count *Elevated WBC count *Elevated WBC count
*Neutrophils present *Lymphocytes and *Lymphocytes present *Lymphocytes and
*Marked protein monocytes present *Moderate protein monocytes present
elevation *Moderate to marked elevation *Moderate to marked
*Markedly decreased protein elevation protein elevation
glucose level *Decreased glucose Normal glucose level *Decreased glucose
*Lactate level Normal lactate level level
level>35mg/dL *Lactate level *Lactate
*Positive Gram Stain >25mg/dL level>25mg/dL
and bacterial culture Positive India Ink with
Pellicle formation C. neoformans
Positive limulus lysate
test result with gram- Positive immunologic
negative organisms test for C. neoformans.

SPUTUM CULTURES
*Deep cough and examine immediately
*Specimens with too many squamous epit. Cells and /or few PMNs arte not suitable for culture.
*BARTLETT’S CLASSIFICATION
__________________________________
__________________________________

URINE CULTURES
*Midstream cleancatch urine
*Catheterized urine
*Suprapubic urine

CSF
*Examine immediately or hold in incubator for no longer than 1 hr.
*Centrifuge, use sediment for:
*Smears, Gram stain, India ink
*Culture- H. influenza, N. meningitides, S, pneumonia

GENITAL CULTURES
*Specimens: cervical (female), urethral (male), rectal and throat
*STD include infections caused by T. pallidum, N. gonorrhoeae, C. trachomatis, G. vaginalis, T.
vaginalis, C. albicans and HSV.
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FAMILY MICROCOCCACEAE

Genera: Staphylococcus
Micrococcus
Planococcus
Stomatococcus

LAB
1. Gram stain
Staphylococcus _________________________________________

Micrococcus _________________________________________

2. Growth on BAP
Staphylococcus _________________________________________

TYPES OF HEMOLYSIS

Alpha (incomplete hemolysis):_____________________________

Beta (complete hemolysis):________________________________

Gamma (non haemolytic): ________________________________

APLHA PRIME:

S. aureus on BAP: _______________________________________

3. Growth on Loeffler’s Serum Salt (LSS): ______________________________________________

S. aureus: ______________________________

S. citreus: ______________________________

S. albus: _______________________________

4. Growth on Mannitol Salt Agar (MSA)

Sel/Diff for Staphylococcus sp.

Inhibitor: _______________________________
CHO: __________________________________
pH indicator: ____________________________
S. aureus: ______________________________
S. epidermidis: __________________________
S. saprophyticus: ________________________

5. Catalase test
Differentiate Staphylococcus from Streptococcus

Rgt.: ___________________________________
Positive result: ___________________________
Staph.: _________________________________
Strep.: _________________________________
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6. Tests to differentiate Staphylococcus from Micrococcus

TESTS STAPHYLOCOCCUS MICROCOCCUS
Aerobic growth
Anaerobic growth
Lysostapin
Bacitracin
Modified oxidase
Glucose utilization

Modified oxidase:
Rgt: __________________________________________________________
Positive result: ___________________________

CHO Oxidation Fermentation (OF) Test

CHO: ___________________

pH indicator: _______________________________________

OPEN TUBE CLOSED TUBE

STAPHYLOCOCCUS
MICROCOCCUS

7. Coagulase:
Most important pathogenic determinant of: ________________________________
*Slide Coagulase

*Detects: _____________________________________

*Rgt: ________________________________________

*Positive: _____________________________________

*Tube Coagulase

*Detects: _____________________________________

*Rgt: _________________________________________

*Positive: _____________________________________

DIFFERENTIATION AMONG COAGULASE-POSITIVE STAPHYLOCOCCI

ORGANISM TUBE COAGULASE VOGES-PROSKAUER PYR

S. aureus subsp. + + -
aureus
S. intermedius V - +
S. hyicus V - -
S. scleiferi subsp. + + +
coagulans

8. DNASE test
Medium: ________________________________________________________
Positive result: ___________________________________________________
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9. Novobiocin

S. epidermidis
Associated with bacterial endocarditis following the insertion of artificial heart valves
NOVOBIOCIN ______________________________________

S. saprophyticus
Important cause of UTI in young women
NOVOBIOCIN ______________________________________

PATHOGENIC DETERMINANTS OF S. AUREUS

 Protein A: _________________________________________
 Coagulase- conversion of fibrinogen to fibrin; may coat neutrophils with fibrin formed to protect
the organisms from phagocytosis.
 Staphylokinase (fibrinolysin)- dissolves fibrin clots and may enable the infection to spread once
clot is dissolved.
 Lipase- hydrolyzes lipids in plasma and skin; associated with initiation of skin infections such as
boils.
 Hyaluronidase- hydrolyzes hyaluronic acid in connective tissue, spreading factor= ___________
 Dnase- degrades DNA.
 Exfoliatins- hydrolyze tissue through cleavage of stratum granulosum; associated with
staphylococcal scalded skin syndrome.
 Leukocidins- lysis of neutrophils and macrophages; inhibit phagocytosis.
 Hemolysins- lyse erythrocytes.
 Enterotoxins- food poisoning and toxic shock syndrome= _______________________________
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FAMILY STREPTOCOCCACEAE

Streptococci

*Gram-positive cocci occurring in pairs or in chains

*Nonmotile
*Non-spore forming
*Facultative anaerobes
*Catalase negative

CLASSIFICATION

LANCEFIELD CLASSIFICATION

*By antigenic character of a group-specific cell wall polysaccharide

*Almost 20 Lancefield groups are identified

A ___________________ Infections include pharyngitis,

tonsillitis, or “strep throat”
Scarlet fever, impetigo,
cellulitis, wound infections and
erysipelas
May be associated with acute
rheumatic fever (ARF)
B ____________________ Associated with neonatal
infections, including
pneumonia, meningitis and
bacteremia
C S. equisimilis Causes severe pharyngitis
S. zooepedimicus followed by bacteremia
S. equi Opportunistic pathogen that
S. dysagalactiae may also be associated with
pneumonia, cellulitis, and
abscess
D Enterococci UTI and wound infections
E faecalis
E faecium
E durans
E avium

Non-Entrecocci
S. bovis
S. equinus

BROWN’S CLASSIFICATION

Classified according to the pattern of hemolysis

ALPHA (Incomplete hemolysis)

BETA (Complete hemolysis)

GAMMA (No hemolysis)

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LAB

1. Gram Stain __________________________________________

S. pneumonia __________________________________

2. Growth on BAP __________________________________________________

3. Catalase ____________________________

4. Bile esculin hydrolysis

*determine ability to grow in 40% bile and esculin hydrolysis

*positive result: esculetin reacts with ferric chloride to form brown-black

TESTS TO DIFFERENTIATE GROUP D ENTEROCOCCUS FROM GRP D NON-ENTEROCOCCUS

GROWTH IN 6.5% NaCl PYR

D Enterococcus

D Non-Enterococcus

5.PYRase ________________________________
Test for _____________________________
Substrate _____________________________________
Positive result ______________________________
Positive for: _________________________________

6.CAMP Reaction
Positive result ______________________________________
Positive for ________________________________________

7.Hippurate hydrolysis
Determines hydrolysis of sodium hippurate to benzoic acid and glycine
*Positive for Group B strep (S. agalactiae)

8.Bacitracin and sulfamethoxazole-trimethroprim susceptibility tests

BACITRACIN SXT NOTES

TAXO ______
A (S. pyogenes)

B (S. agalactiae)

D Enterococcus

D Non-Entercoccus
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Streptococcus pneumoniae: causes: ____________________________________

Not Lancefield grouped, alpha hemolysis on BAP

Viridans Streptococci
Not Lancefield grouped, alpha hemolysis on BAP
S. mutans (associated with dental carries)
S. uberis
S. mitis
S. sanguis
S. salivarius
S. constellatus
S. intermedius

TESTS TO DIFFERENTIATE S. PNEUMONIAE FROM VIRIDANS STREP.

S. PNEUMONIAE VIRIDANS
Mouse virulence

Inulin fermentation

Bile solubility

Optochin

TAXO _________

Chem. Name: __________________

Neufeld Quellung

NUTRITIONALLY VARIANT STREPTOCOCCI (ABIOTROPHIA SPP.)

*Streptoccocci that need cysteine or pyridoxal (vitamin B6)
*Satelliting-streptococci, thiol-requiring streptococci
*S. adjacens
*S. defectivus
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GRAM-NEGATIVE COCCI
Neisseria
Important characteristics:
*Gram-negative diplococcic resembling coffee beans or kidney beans except N. elongate (rod-
shaped)
*Obligate aerobes
*Capnophilic-requires 5-10% CO2
*Nonmotile
*Catalase positive except N. elongate
*Oxidase positive

***Neisseria organisms establish disease through attachment in the mucous membranes of the
host through pili.

ORGANISM DISEASE
N. gonorrhoeae A leading cause of STD. Genital infections include
purulent urethritis in males and cervicitis in females.
These infections also may be asymptomatic. Other
localized infections include pharyngitis, anorectal
infcetions and conjunctivitis (eg. Opththalmia
neonatorium of newborns acquired during birth from the
infected mother). Disseminated infections result when
the organism spreads from a local infection to cause
pelvic inflammatory disease or disseminated gonococcal
infection that includes bacteremis, arthritis and
metastatic infections at other body sites.
N. meningitidis Life-threatening, acute, purulent meningitis. Meningitis
may be accompanied by appearance of petechiae (rash)
that is associated with meningococcal bacteremia
(meningococcemia). Bacteremia leads to
thrombocytopenia, disseminated intravascular
coagulation, and shock. Disseminated disease is often
fatal. Less common infections include conjunctivitis,
pneumonia and sinusitis.
Waterhouse-Friderichsen syndrome (hemmorhage in
adrenal gland)
Other Neisseria spp. Rarely involved in human infections. When infections
occur, includes bacteremia, endocarditis and meningitis.
Moraxella catarrhalis Most infections are localized to sites associated with the
previous name: Branhamella catarrhalis, respiratory tract and include sinusitis and pneumonia.
Neisseria ctarrhalis) Lower respiratory tract infections often target elderly
patients and those with chronic obstructive pulmonary
disease. Rarely cause disseminated infections such as
bacteremia and meningitis.

GLUCOSE MALTOSE SUCROSE LACTOSE

M. catarrhalis - - - -
N. gonorrhoeae + - - -
N. meningitides + + - -
N. subflava + + +/- -
N. lactamica + +- +

CULTIVATION
*N. meningitides, M. catarrhalis and saprophytic Neisseria spp. grow well on 5% sheep blood
and chocolate agars; N. gonorrhoeae is more fastidious and requires an enriched chocolate agar for
growth on primary culture.
*Modified Thayer-Martin, Martin Lewis and New York City Agar
*NYC medium: genital mycoplasma (M. hominis and U. urealyticum) will also grow on this agar.
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GRAM-NEGATIVE BACILLI

Enterobacteriaceae

*Gram-negative rods
*Motile with perithrichous flagella except klebsiella and Shigella (nonmotile)
*Facultative anaerobes
*Often with gas production
*Catalase positive:
*Reduce nitrate to nitrite except for Erwinia and Pantoea agglomerans

Antigenic Structure

K Antigen
*Capsular antigen
*Heat-labile
*Some salmonellae have capsular (K) antigens, referred to as Vi:

O Antigen
*Somatic antigen
*Heat-stable
*Antibodies to O antigens are predominantly IgM.

H Antigen
*Located in the flagella
*Denatured or removed by heat or alcohol
*H antigens agglutinate with anti-H antibodies, mainly IgG

LAB

1. Growth on media: EMB/ MAC/ HEA/ SSA

2. TSI
*Sugars: 10 parts lactose, 10 parts sucrose, 1 part glucose
*pH indicator: phenol red
*H2S indicator: sodium thiosulfate is reduced to SO2 and H2S, and H2S reacts with ferrous
sulphate to produce black ferric sulphide.
3. LIA
*detects lysine deamination and decarboxylation
*pH indicator: bromcresol purple
*H2S indicator: ferric ammonium citrate
4. Indole
*detects: _________________________
*Medium: ________________________
*Indicator: ________________________
*Positive result: ____________________
5. MRVP test
*Medium: _________________________
*Methyl Red: _______________________
*organism that produce enough acid will overcome the neutralizing effect of the buffer
*Positive result: _____________________________________
*Voges Proskauer
*Detects: ___________________________________
*Reagents: __________________________________
6. Citrate Utilization
*determine of an organism can utilize citrate as sole source of carbon.
*Medium: _______________________________________
*pH indicator: ____________________________________
*Positive result: ___________________________________
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7Malonate utilization
*determine if an organism can utilize malonate as sole source of carbon
*positive: BLUE
*Negative: Green or Yellow
8. Urease test
*Medium: _______________________________________
*pH indicator: ____________________________________
*Positive: ________________________________________
*Rapid urease producer’s _________________________________________________________
*Slow urease producer’s __________________________________________________________
9. ONPG
*Rapid test to detect: ______________________________
*Positive: ________________________________________
10. Decarboxylase
*Enzyme that removes the carboxyl group (COOH) from an amino acid
*Uses decarboxylase basal medium with indicator (bromcresol purple) + sugar (glucose) + amino
acid (lysine, ornithine, and arginine) + control tube with no amino acid.
*Moller’s medium better
*Positive: purple to yellow to purple
11. Phenylalanine deaminase
*Enzyme that removes amino group (NH2)
*Phenylalanine agar, overnight culture + 10% FeCl3
*Positive: green slant and fluid
12. Gelatin liquefaction
*Determine if an organism can breakdown gelatin into: ___________________________
*Positive: Liquefaction.

Escherichia
*UTI – 90%
*Sepsis
*Meningitis – (also Group B Strep) causes meningitis in infants
*Diarrheal disease

1. Enterotoxigenic E. coli (ETEC)

*Produces heat-labile (LT) and/or heat stable (ST) enterotoxins; genes of both toxin reside on
plasmid.
*LTs are closely related in structure and function to cholera toxin; STs result in net intestinal
fluid secretion by stimulating guanylate cyclase.
*”Montezuma”s revenge” or “turista”, traveler’s and childhood diarrhea characterized by
profuse watery stool.

2. Enteroinvasive E. coli (EIEC)

*Invades the intestinal epithelium causing Shigella-like-infection
*Dysentery (necrosis, ulceration and inflammation of large bowel); usually in young children
living in areas of poor sanitation.
*Stool with rbc, neutrophils and mucus.

3. Enteropathogenic E. coli (EPEC)

*Non-invasive, produce no toxin
*Nosocomial, seen in newborn and infants
*Watery diarrhea with mucus but no blood

4. Enterohemorrhagic E. coli (EHEC)/Verotoxic E. coli (VTEC)

*Produce verotoxin – named for its cytotoxic effect on Vero cell, a line of African grren monkey
kidney cells, a cytotoxin that resembles that of S. dysenteriae.

Note: E coli 0157:H7 on SMAC ______________________________________________

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5. Enteroaggregative E.coli (EAEC)

*Probably involves binding by pili, ST-like, and hemolysin-like toxin; actual pathogenic
mechanism not known
*watery diarrhea

Klebsiella

IMVIC: ______________________ TSI: ________________________

*Exhibit mucoid growth, large polysaccharide capsule
*K. pneumonia – Friedlander’s bacillus, encapsulated and appears as mucoid colonies that tend
to string
*K. ozaenae – purulent sinus infection
*K. rhinoscleromatis – granuloma of the nose and orophrarynx

Enterobacter

IMVIC: ______________________ TSI: _______________________

*Opportunistic infections: UTI, RT and wound infections
*Most predominant isolate is E. cloacae
*Produces yellow pigmentation that intensifies at 25c ________________________________

Note:

LDC ODC ADH

K. pneumoniae + - -
K. oxytoca + - -
E. aerogenes + + -
E.Cloacae - + +
P. agglomerans - - -

Serratia: ________________________________________
*S. marcescens and S. rubidea produce a red pigment: __________________________________
*Common opportunistic pathogen in hospitalized patients.

Salmonella
*Sources of infection: water, milk and dairy products, shellfish (from contaminated water), dried
or frozen eggs, meat and meat products and household pets.
*Cause enteritis, systemic infection and enteric fever (typhoid fever)
*Cause enteric fever:
*S. paratyphi A (serogroup A)
*S. paratyphi B (serogroup B)
*S. cholerasuis (serogroup C)
*S. typhi (serogroup D) – most important cause
*Bacteremia-S. cholerasuis
*Entrocolitis/gastroenteritis – most common manifestations of salmonella infection – S.
typhimurium (most common)
*Widal’s test – tube dilution agglutination test
*At least two serum specimens, obtained at intervals of 7-10 days, are needed to prove
a rise in antibody titer.
*Serial dilutions of unknown serum are tested against antigens from representative salmonellae
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Shigella: ______________________________________
*Natural habitat of shigellae is limited to the intestinal tracts of humans and other primates,
where they produce bacillary dysentery.

PRESENT GROUP TYPE CATALASE ONPG MANNITOL

DESIGNATION
S. dysenteriae

S. flexneri

S. boydii

S. sonnei

Citrobacter
*Causes UTI and sepsis
*Organisms resemble Salmonella but are ONPG and LDC negative

Proteus: ______________________________________
*Produce infections in humans only when they leave the intestinal tract
*UTI, bacteremia, pneumonia, nosocomial infections
*P. mirabilis and many strains of P. vulgaris exhibit swarming motility.
*Certain strains share specific polysaccharides with some rickettsia and are agglutinated by sera
from patients with rickettsial diseases:
*a. ___________________________________
*b. ___________________________________

Providencia
*Normal intestinal flora
*UTI and occasionally other infection and are often resistant to antimicrobial therapy

Edwardsiella
*Isolated from the environment and many cold-blooded and warm blooded animals including
reptiles, freshwater and aquarium fish, frogs and turtles.
*E. tarda – most human species
*to differentiate from E. coli
*IMVIC: ___________________________________

Yersenia
Yersenia pestis: ( )
*Plague-infection of wild rodents transmitted from one rodent to another and occasionally by
the bite of fleas.
*Common vector: Xenopsylla cheopsis – rat flea
*Produced pandemics of Black Death with millions of fatalities
*Broth cultures exhibit a characteristic “stalactite pattern” in which clumps of cells adhere to
one side of the tube.
*Inclusion ______________________________________
*Stains ________________________________________
Yersenia enterocollitica
*Enetrocolitis – char by fever, diarrhea and abdominal pain
*Bacteremia
*Cultured on ___________________________________
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IMVIC REACTIONS

Escherichia
I M V C
E.coli + + - -
Klebsiella
I M V C
K. pneumoniae - - + +
K. oxytoca + V + +
K. ozaenae - + - V
Enterobacter
I M V C
E.aerogenes - - + +
E.cloacae - - + +
E.agglomerans V V V V
Serratia
I M V C
S.marcescens - V + +
S.liquefaciens - + + +
Hafnia
I M V C
H.alvei - V V -
Salmonella
I M V C
Most serotypes - + - +
Shigella
I M V C
ABC V + - -
D - + - -
Citrobacter
I M V C
C.freundii V + - V
C.diversus/koseri + + - +
Proteus
I M V C
P.mirabilis - + V V
P.vulgaris + + - V
P.penneri - + - -
Providencia
I M V C
P.rettgeri + + - +
P.stuartii + + - +
Morganella
I M V C
M.morganii + + - -
subsp. morganii
Edwardsiella
I M V C
E.tarda + + - -
Yersenia
I M V C
Y.enterocolitica V + - -
Y.frederiksenii + + - V
Y.intermedia + + - -
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OTHER GRAM-NEGATIVE BACILLI

Pseudomonas aeroginosa
*Obligate aerobe
*Motile and rod-shaped
*Sweet or grape-like or corn-taco like odor
*Grows well at _______, its growth at _______ will differentiate it from other Pseudomonas sp.
*Oxidase positive
*Grape-like or “corn-tortilla-like” odor resulting from production of 2-aminoacetophenone
*Does not ferment carbohydrate: TSI ______________
*Produces blue-green pigment:
*___________________________________
*___________________________________
*Produces infection of wound and burns, giving rise to blue-green pus
*Meningitis
*Pneumonia
*Sepsis
*Mild otitis externa/media - _________________________
*Skin lesion - _____________________________________
*Resistant to a number of disinfectants and has been responsible for serious nosocomial
infections.

Burkholderia mallei
*Cuaes ___________________________: a disease of horses and similar animals transmissible
to humans.
*Horses-pulmonary involvement
*Human-fatal, begin as ulcer of skin and mucous membrane followed by lymphangitis and
sepsis.

Burkholdria pseudomallei
*Causes ___________________________, an endemic glander’s like disease of animals and
humans.

Vibrio
*Faculatative anaerobe
*Monotrichous
*Oxidase_____________________.
*Halophilic except: _________________ ________________

Vibrio cholera
*Characteristic rice water stool
*Cultured on APW and TCBS
*String test
*Reagent_______________________________.

Serogroups
*Inaba-
*Ogawa-
*Hikojima-
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Biotypes (Biovars)
CLASSICAL EL TOR
Red cell hemolysis
VP
Polymyxin B
Aggln with chicken RBCs

Vibrio alginolyticus
*Clinical significance: wound and ear infections associated with marine environment
*Halophilic
*Sucrose fermentation positive.

Vibrio parahemolyticus
*Clinical significance: gastroenteritis, usually associated with contaminated seafood.
*Halophilic
*Sucrose fermentation negative

Vibrio mimicus
*Clinical significance: gastroenteritis and ear infections associated with marine environment
*Nonhalophilic
*Sucrose fermentation negative

Vibrio vulnificus
*Clinical significance: septicaemia and wound infections involving marine environment.

Haemophilus
*Non-motile, non-spore forming
*Facultative anaerobes
*Most sp are oxidase and catalase positive
*Preferred incubation: ____________________________________.

SPECIES REQUIREMENT FOR BETA HEMOLYSIS D-ALA

X FACTOR V FACTOR
H.influenzae

H.parainfluenzae

H.hemolyticus

H.aegypticus

H.aphrophilus

H.paraphrophilus

H.ducreyi
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H. influenzae
*Formerly known as _____________________________
*Six serotypes (a, b, c, d, e and f); most frequently encountered serotype in infection _________
*Encapsulated strains are pathogenic
*Main cause of meningitis in children <5 years old
*Associated with respiratory conditions including epiglottitis

H. aegypticus
*Formerly known as ____________________________
*Closely resembles H. influenza biotype III
*Causes ________________________________

H. ducreyi-
*Infective agent of ______________________, venereal disease characterized by painful ulcers
in the genetalia.
*Direct examination ____________________________.

Campylobacter
*C. jejuni, C. coli (associated with gastritis and diarrhea)
*Microaerophilic and capnophilic, motile, gram negative rods that are associated with gastritis
and diarrhea.
*Optimum temperature for growth________________

*Motility_____________________
*Culture medium_______________________________

Helicobacter
*Associated with ____________________________
*The natural habitat is the human stomach, where the organism is found in the mucus secreting
cells.
*Current evidence may suggest a role between H. pylori and peptic and duodenal ulcers.
*Histology staining and culture or biopsies obtained from the stomach or duodenum are
recommended for the identification.

Bordetella
*B. pertussis – agent of _______________________________

*Three stages of pertussis (whooping cough)

*Catarrhal: general flulike symptoms
*Paroxysmal: repetitive coughing episodes
*Convalescent: recovery phase

*Ideal specimen____________________________
*Culture medium
*______________________________
*______________________________

*Colonies________________________

*B. parapertussis – pertussis-like syndrome

*B. bronchoseptica – inhabits respiratory tract of canines (kennel’s cough) infrequently
responsible for chronic respitatory tract infcetions in humans.
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UREASE MOTILITY NITRATE OXIDASE

B.pertussis

B.parapertussis

B.bronchiseptica

Brucella
*Agents of Brucellosis, undulant fever, Malta fever
*Small, nonmotile, aerobic, gram-negative coccobacilli or short rods
*Organisms are normal flora of the urinary tract and gastrointestinal tracts of sheep/goat (B.
melitensis), cattles (B. abortus), pigs (B. suis), and dogs (B. canis).
*Humans acquire infections through the ingestion of contaminated animal products, including
meats and, milk; farmers can directly acquire infections through direct animal contact.

CO2 H2S GROWTH IN THE PRESENCE OF

THIONINE BASIC FUCHSIN
B.abortus

B.melitensis

B.suis

B.canis

CULTURE FOR BRUCELLA: _________________________________

Legionella
*L. pneumophilia (Legionnaire’s disease, Pontiac fever)
*L. micdadei (Pittsburgh pneumonia)
*L. bonzemanni (Wigas’s agent of pneumonia)
*Naturally found in both natural and artificial water sources; the bacteria have been found in
ponds, creeks and streams, wet soil, water-cooling towers for air-conditioning and heating systems,
shower heads, and plumbing systems
*CULTURE MEDIUM ______________________________________

Pasteurella
*P. multocida (agent of pasteurellosis)
*The organism is carried in the oral cavity and respiratory and gastrointestinal tracts of cats and
dogs.
*Agent of shipping fever in cattles, a hemorrhagic septicaemia.
*Humans may acquire the infection following contact with domestic animals that harbour the
bacterium; most frequently the route of infection is from the bite or scratch of an infected animal,
usually a cat.
*Wound infections are the most common type of Pasteurella infection.
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Francisella
*Faintly staining, gram-negative coccobacilli that are non-motile and obligate aerobic
*Tularemia is a disease of the rodents, primarily the rabbits
Individuals who handle the infected animal, acquire this zoonosis through direct contact with
blood or through animal bite and scratch.
*Bacterenium can also be inhaled or acquired indirectly through insect vectors primarily ticks
*CULTURE MEDIUM ___________________________________________

Gardnerella
*G. vaginalis (associated with bacterial vaginosis)
*Gram-variable to gram-negative bacillus, previously known as Haemophilus vaginalis and
Corynebacterium vaginalis
*Bacterial vaginosis (BV) is characterized by foul-smelling, grayish, vaginal discharge
*Amsel and Nugent scoring systems are used to diagnosi BV

*Cytology/Pap’s _____________________________________________

*Whiff or Sniff test __________________________________________

*Organism can be isolated on __________________________________

Streptobacillus moniliformis
*Agent of Rat-like fever

Capnocytophaga
*Capnophilic, fusiform or filamentous bacilli
*caharacteristic motility: _________________________________

Calymmatobacterium granulomatis
*Agent of: _____________________________________________
*Encapsulated, pleomorphic gram-negative bacillus usually observed in vacuoles in large
mononuclear cells
*Groups of organisms are seen with mononuclear cells; this pathognomonic entity is known as
Donovan body.
*Closely related to Kliebsiella.
*Based on DNA-DNA hybridization, it has been recently proposed that the name of this
organism be changed to Kliebsiella granulomatis.

Eikenella coorodens
*fastidious, capnophilic rod; part of the gingival and bowel flora
*Corroding bacterium
*Characteristics in agar ________________________________
*Produces bleachlike odor

Chromobacterium violaceum
*Found in soil and water, has the ability to produce violacein a purple pigment

Cardiobacterium hominis
*Normally found in the upper respiratory tract and is isolated as a rare cause of endocarditis
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