Microbiology Notes 2

MICROBIOLOGY
INTRODUCTION
Taxonomy
 Orderly classification and grouping of organisms into o Subspecies or Serotype
categories  Example: Escherichia coli
 Levels of classification: o Domain: Bacteria
o Domain o Kingdom: Bacteria or Monera
o Kingdom o Division/Phylum: Proteobacteria
o Division/Phylum o Class: Gammaproteobacteria
o Class o Order: Enterobacterales
o Order o Family Enterobacteriaceae
o Family o Tribe: Escherichia
o Tribe o Genus: Escherichia
o Genus o Species: coli
o Species
Eukaryote vs. Prokaryote

Characteristic Eukaryote Prokaryote
Cell division Mitosis Binary fission
Cell organelle Present Absent
Cell wall No cell wall on animal cells; Has a cell wall made up of peptidoglycan layer
except for Mycoplasma and Ureaplasma
Contains cellulose and chitin for plants
and fungi
Cytoplasmic membrane Fluid phospholipid bilayer with protein Fluid phospholipid bilayer without protein and
and sterol sterol except for Ureaplasma it contains a sterol
Nuclear body Nucleus is bound to a membrane; Nucleoid is not bound to any membrane;
Contains DNA and Histones Has a single chromosome only
Site of energy production Mitochondria Cytoplasmic membrane
Site of protein synthesis Rough endoplasmic reticulum Free ribosome
BACTERIAL STRUCTURE
A. Cell wall
 Responsible for the shape of the cell
 Prevents rupture of cell when the when the water pressure inside is greater than the outside of the cell
 Point of anchor of the flagella
 Also known as “peptidoglycan/murein”
 Also made up of teichoic acid/lipoteichoic acid
 Chemicals that can destroy cell wall:
o Penicillin – inhibits transpeptidation (process of replicating peptidoglycan)
o 70% alcohol – contact time should be at least 1 minute
o HCl
 Abnormal cell wall:
o Protoplast = Gram positive bacteria; produced by lysozyme in hypotonic solution
o Spheroplast = Gram negative bacteria; produced by penicillin in hypertonic solution
GRAM POSITIVE GRAM NEGATIVE

Peptidoglycan Peptidoglycan
Teichoic acid Lipoprotein
Contains Exotoxin Phospholipid
Lipopolysaccharide
Contains Endotoxin
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Prepared by: Jessa D. Felix, RMT, MLS (ASCPi)
MICROBIOLOGY
B. Plasma membrane
 A lipoprotein that surrounds the cytoplasm
 Functions:
o Generate ATP
o Site of respiration
o Regulates osmotic pressure
o Transports solutes
C. Inclusions
 Reserve food deposits inside the cytoplasm of prokaryotic cells
Common Bacterial Inclusions
Metachromatic granules C. diphtheriae
Polysaccharide granules P. aeruginosa
Lipids Bacillus, Mycobacterium, Spirillum, Azotobacter
Sulfur granules Thiobacillus, Nocardia, Actinomyces
Carboxysomes Cyanobacterium, Thiobacillus
Gas vacuoles Cyanobacteria, Halobacteria
Magnetosome Magnetospirillum
D. Slime layer – unorganized material
E. Cell appendages:
 Pili
o Somatic pili/ordinary pili: for adhesion (virulent factor)
o Fertilizing/Sex pili: for genetic transferfrom donor to recipient via conjugation
o NOTE: N. gonorrheae is non-motile but w/ pili
 Glycocalyx
o outward complex of polysaccharides
o attachment of bacteria to the cell
o resists phagocytosis/desiccation
 Flagellum
o organ for locomotion
o Contains a protein known as flagellin
o Motility is seen in 25°C
o 37°C is inhibitory for Listeria and Yersinia
o can be stained using Leifson, Gray, Fisher and Conn
o Flagellar arrangements:
▪ Atrichous – no flagella
▪ Monotrichous – Flagellum in one pole (e.g., V. cholerae)
▪ Amphitrichous – single flagellum in each pole (e.g., Aquaspirillum serpens)
▪ Lophotrichous – tuft of flagella at one or both sides
▪ Peritrichous – flagella all over the organism
▪ Periplasmic flagella/axial filament/Endoflagella (e.g., Spirochetes)
F. Endospore
 Resist extreme environment conditions
 Made up of calcium dipicolinate (dipicolinic acid + calcium)
 Seen in Clostridia and Bacillus
 Location of spores:
o Terminal Spore – C. tetani
o Subterminal Spore – C. botulinum
o Central Spore – B. anthracis
 Sporogenesis/Sporulation: process of spore production
 Germination: the end of the spore’s dormant stage
G. Capsule
 Organized material attached to cell wall
 Resist phagocytosis
 Organisms with capsule: H. influenzae, N. meningitidis, S. pneumoniae
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MICROBIOLOGY
QUICK SUMMARY!
MOTILE NON-MOTILE
Alcaligenes Bacillus anthracis
Bacillus cereus Bordetella
Bacillus subtilis Brucella
Campylobacter Clostridium perfringens
Clostridium botulinum Corynebacterium diphtheriae
Clostridium tetani Erysipelothrix
Escherichia coli Haemophilus
Listeria Mycobacterium
Proteus Pasteurella
Providencia Staphylococcus
Pseudomonas Streptococcus
Salmonella Shigella
Vibrio Klebsiella pneumoniae
Neisseria
W/ ENDOSPORE
Bacillus
Clostridium
W/ CAPSULE
Bacillus anthracis
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis
Klebsiella pneumoniae
Streptococcus agalactiae
CLASSIFICATION OF BACTERIA
I. Morphology
A. Cocci – spherical or ovoid in shape
▪ Diplococci – pairs of cocci (e.g., Neisseria)
▪ Streptococci – chains of cocci (e.g., S. pyogenes)
▪ Staphylococci – a cluster or group of cocci (e.g., S. aureus)
▪ Tetrads – tuft of four cocci arranged within the same plane (e.g., Micrococcus luteus)
▪ Sarcina – tuft of cocci arranged in cube (e.g., Sarcina lutea)
B. Bacilli – rod shaped bacterium
▪ Diplobacilli – bacilli arranged side-by-side with each other (e.g., C. burnetti, K. rhinoscleromatis)
▪ Streptobacilli – bacilli arranged in chains (e.g., S. moniliformis)
▪ Coccobacilli – circular shaped bacilli (e.g., H. influenzae, G. vaginalis)
▪ Palisade – bacilli bent at the point of division (e.g., C. diphtheriae)
C. Spiral
▪ Vibrio – comma shaped organisms
▪ Spirochete – helical in shape with flexible bodies that move with the use of axial filaments (e.g.,
T. pallidum, B. burgdorferi, L. interrogans)
▪ Spirillum – rigid spiral structure and does not contain any axial filament for locomotion (e.g., C.
jejuni, H. pylori, S. minor)
D. Filamentous – very thin with long filamentous bodies that may form a mycelium when grouped together
(e.g., Candidatus liberibacter)
E. Pleomorphic – do not have any definite/characteristic shape (e.g., M. pneumoniae)
II. Staining Characteristics
A. Gram staining
Reagent Function
Crystal violet Primary stain, stains all bacteria blue to purple
Mordant, enhances reaction between cell wall and
Gram’s Iodine
primary stain
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MICROBIOLOGY
Gram positive bacteria retain the primary stain because

of the peptidoglycan and techoic acid cross-links. Gram
Ethyl alcohol or acetone
negative bacteria lose the primary stain because of the
large amount of lipopolysaccharide in the cell wall
Counterstain, no effect on gram positive bacteria, stains
Safranin
gram negative bacteria pink to red
Gram Positive cocci (aerobe) Micrococcus, Staphylococcus, Streptococcus

Gram Positive cocci (anaerobe) Peptococcus, Peptostreptococcus, Sarcina
Gram Negative cocci (aerobe) Branhamella, Neiserria
Gram Negative cocci (anaerobe) Veilonella
Gram Positive bacilli (aerobe) Bacillus, Corynebacterium, Erysipelothrix, Lactobacillus,
Listeria, Mycobacterium, Nocardia
Gram Positive bacilli (anaerobe) Actinomyces, Clostridium, Propionobacterim
Gram Negative bacilli (aerobe) Acinetobacter, Aeromonas, Alcaligenes, Bordetella,
Brucella, Enterobacteriaceae, Fransicella, Legionella,
Pasteurella, Pseudomonas, Vibrio
Gram Negative bacilli (anaerobe) Fusobacterium, Bacteroides
QUICK SUMMARY!
All cocci are gram positive EXCEPT:
Neisseria
Veilonella
Moraxella/Branhamella
All bacilli are gram negative EXCEPT:
Bacillus
Mycobacteria
Actinomyces
Clostridium
Corynebacterium
Propionebacterium
Erysipelothrix rhusiopathiae
Listeria monocytogenes
Lactobacillus
Nocardia
B. Acid Fast staining
Ziehl-Neelsen Pappenheim Baumgarten
Kinyuon Method
Method Method Method
3 grams of Carbol 4 grams of Carbol Carbol Fuschin Carbol
Primary Stain
fuschin + 5% phenol fuschin + 9% phenol Fuschin
Mordant Heat Tergitol
Dil. Alcohol
Decolorizer 3% Acid alcohol Rosolic Acid
Fuschin
Secondary Methylene Methylene
Methylene Blue
Stain Blue Blue
Acid Fast Organisms:
✓ Mycobacteria
✓ Nocardia
✓ Coccidians
✓ NOTE: mycolic acid/hydroxymethoxy acid – the substance unique found in the cell wall
of acid fast organisms
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MICROBIOLOGY
C. Special Stains
Capsule Welch Stain
Cell Wall Dyar Stain
DNA Feulgen
Endospore Schaeffer-Fulton
Flagella Leifson Stain
Metachromatic Granule Albert Stain, Burke’s Modification of Gram Stin
III. Nutrition and Growth Requirements

A. According to Oxygen Requirement
Obligate Aerobe Requires 15-21% oxygen and 1% CO2

Bordetella, Brucella, Mycobacteria, Pseudomonas
Obligate Anaerobe Requires 0% O2, 5-10% CO2, 80-90% N2, 5-10% H2
Bacteroides, Clostridium
Microaerophile Requires 5% O2, 10% CO2, 85% N2
Campylobacter, T. pallidum
Capnophilic Requires 15% O2, 5-10% CO2
Neisseria, S. pneumoniae, HACEK group
Facultative anaerobe
Aerotolerant anaerobe grow in the presence of oxygen but no metabolic process

unless placed in anaerobic environment
Propionibacterium and Lactobacillus
B. According to Temperature
Psycrophile/Cryophile 0-20°C L. monocytogenes, Y. enterocolitica
Mesophile 20-45°C Most pathogenic bacteria
Thermophile 50-125°C B. stearothermophilus, Sulfolobus, Pyrococcus
Extremophile Organisms that live in unusual conditions (B.

infernus)
C. According to pH Requirement
Acidophile 0-5.5 Sulfolobus, Picrophilus, Acontium
Neutrophile 5.5-8.0 Most pathogenic bacteria
Alkalophile 8.5-11.5 B. alcalophilus, Natronabacterium
D. Carbon Source
▪ Autotroph – use CO2 as sole source of carbon
▪ Heterotroph – use reduced, preformed, organic molecule from bacteria
E. Energy Source
▪ Phototroph – uses light
▪ Chemotroph – uses energy by oxidation of organic or inorganic compounds
F. Electron Source
▪ Lithotroph – use inorganic molecules
▪ Organotroph – uses organic molecules
G. Barophlic – grow rapidly in the presence of high pressure (600-1100 atm. pressure)
H. Fastidious – requires additional nutrient such as vitamins, purines, pyrimidines, and hemoglobin
I. Saprophyte – obtain nutrients from dead organic substances
J. Parasitic – obtain organic nutrients from living tissue
LABORATORY SAFETY
Biologic Safety Cabinet
 Device that encloses workspace to protect workers from aerosol of infectious disease agents
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MICROBIOLOGY
 HEPA filter removes particle larger than 0.3 um in diameter

 QC: there should be an area for 3 feet from cabinet during operation of air-circulating system to ensure
infectious material is directed through HEPA filters.
 NOTE: certification is required whenever BSC is moved more than 18 inches
Classification Based on Effective Level of Biologic Containment

Class I − Allows air to pass into cabinet and around the area and material within
− Sterilizes only the air to be exhausted

− Have negative pressure and external ventilation
− Operation is through an open front
Class II − Vertical laminar flow
− Sterilize air that flows over infectious material and air to be exhausted
− Air flows in sheets creating barriers to particles from outside cabinet and directs flow
of contaminated air into filters
− Design: various sash opening through which operators gains access to work surface
− Class IIb: self-contained and 70% of air is recirculated; most commonly used in
hospital clinical microbiology laboratory
− Class IIa: exhaust air is discharged outside of building
Class III − Completely enclosed with negative pressure
− Afford most protection to worker

− Rubber gloves are attached and sealed to the cabinet
− Air flow: incoming air → filter → exhaust air → filter
STERILIZATION AND DISINFECTION

Sterilization – removal or destruction of all forms including spores
A. Incineration – materials are burned to ashes; safest method

B. Moist Heat
▪ used for biohazardous trash and heat stable objects
▪ Autoclaving: 121°C for 15 minutes at 15 psi
o fastest and simplest method of sterilization
o Principle: hot steam under pressure
o Can kill spores BUT not prions
o indicators: B. stearothermophilus or Clostridium
▪ Tyndallization/Fractional or Intermittent Sterilization: 100°C for 30 minutes (3 consecutive
days)
▪ Inspissation: 70-80°C for 2 hours; thickening through evaporation
C. Dry heat
▪ Flaming – direct heating
▪ Oven – 160-170°C for 1.5 to 2 hours; for glassware, oil, petroleum, and powders
▪ Incineration – burning materials into ashes; 300-400°C or 870-980°C (for hazardous materials)
▪ Cremation – to control the spread of communicable disease
D. Filtration – method of choice for sterilization of antibiotic solutions, radioisotopes, vaccines, and
carbohydrates which are all heat sensitive
E. Radiation – creates free hydrogen and hydroxyl radicals and peroxidases → intracellular damage →
sterilize disposables
F. Desiccation – removes water from microbes; significant in food microbiology
G. Lyophilization – long term preservation of microbial cultures
H. Chemical Sterilant
▪ Ethylene dioxide – used to sterilize heat sensitive objects
▪ H2O2 – sterilize HEPA filters in BSCs
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MICROBIOLOGY
▪ Periacetic acid – used to sterilize medical equipment

Disinfection – destroys pathogenic organisms but not necessarily all microorganisms
A. Antiseptic – topically applied to skin to prevent sepsis formation
B. Disinfectant – for inanimate objects (Lysol, chlorine, sodium hypochlorite)
C. Bactericidal – kills bacteria; strong acid
D. Bacteriostatic – inhibit the growth of microorganism
SPECIMEN COLLECTION
General Guidelines During Specimen Collection
✓ Collect during acute phase of an illness or within 2-3 days of viral infection
✓ Standard cleaning: 70% alcohol for 1 minute followed by an iodophor or 2% iodine
✓ Specimens should be collected before antibiotics are administered
✓ Aspirated specimen must be placed into a sterile tube or transport vial
✓ Labels attached on the body of the container:
o Patient identification, specific anatomic site (especially for wound specimen)
o Do not settle for a mislabeled specimen or request form over the phone
Specimen Rejection
✓ DO NOT reject bone marrow, spinal fluid, or surgical specimen
✓ Inadequate specimens require decision of physician, and must be documented
✓ NOT appropriate for culture:
o 2 hours delay time from collection
o specimen in formalin
o dried up specimen
✓ Microscopic criteria for sputum rejection: <25 WBC/HPF and >10 Epithelial cells/LPF
Specimen Transport
✓ Allowable delay time:

o Generally, 30 minutes of collection
o 10 minutes for anaerobic bacteria
o 15 minutes for CSF specimen (after 1 hour, S. pneumoniae may no longer be detected)
✓ Container: triple packaged for shipping, leak-proof, w/ biohazard label
✓ Transport media must be maintained at certain temperature and pH
o Temperature sensitive: N. meningitidis, Haemophilus
o pH-sensitive: Shigella spp.
✓ Add charcoal to transport media for N. gonorrheae and B. pertussis
Specimen Preservation
✓ Boric acid for urine culture (for 24 hours)

✓ Refrigeration: stool
✓ Anticoagulants for blood, bone marrow, and synovial fluid: 0.025% SPS
o ADVANTAGE: prevents phagocytosis and complement activity
o DISADVANTAGE: higher concentration may be toxic to Neisseria and anaerobic bacteria; also
neutralizes aminoglycoside (can be neutralized by 1.2% gelatin)
✓ Heparin: for mycobacteria and viral culture (inhibits the growth of gram + bacteria and yeast)
Prioritization
LEVEL DESCRIPTION EXAMPLES
1 Critical/Invasive Amniotic fluid, blood, CSF, brain, heart valves, Pericardial fluid
2 Unpreserved Bone, feces, sputum, tissue, body fluids
3 Quantitation required Catheter tip, urine tissue
4 Preserved Urine, feces, swabs
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MICROBIOLOGY
Specimen Storage
▪ Refrigerator: urine, stool, viral specimen, sputum, swabs
▪ Incubator: CSF
▪ Room temperature: fungal specimen
▪ Freezing: longer storage of tissues, serological specimens
Panic Values (Communicated to the physician immediately)

1. Positive blood cultures
2. Positive CSF gram stain or culture
3. Group A strep in a surgical wound
4. Gram stain suggestive of gas gangrene
5. Blood smear positive for malaria
6. Positive for cryptococcal antigen
7. Positive AFS
8. Detection of a significant pathogen (e.g., Legionella, Brucella, Vancomycin resistant S. aureus, or other
antibiotic-resistant bacteria)
MICROBIAL CULTURE
Classification of Culture Media
A. According to Consistency e. Slant/butt
a. Liquid medium D. According to use
b. Semi-solid medium a. Simple media
c. Solid medium b. Enrichment media
B. According to Composition c. Enriched media
a. Synthetic medium d. Differential media
b. Non-synthetic medium e. Selective media
c. Tissue culture medium f. Special media
C. According to how the medium is dispensed
a. Plated
b. Tubed
c. Slanted
d. Butt
LIST OF COMMON CULTURE MEDIA
Medium Component/Comment Primary Purpose
Bile Esculin Agar Nutrient agar based with ferric citrate. Hydrolysis of Differential isolation and
(BEA) esculin by Group D Streptococci imparts a brown presumptive identification of Group
color to the medium; sodium desoxycholate inhibits D Streptococci and Enterococci
many bacteria
Bile esculin azide Contains azide to inhibit Gram-negative bacteria, Selective and differential for
agar with Vancomycin to select for resistant Gram-positive cultivation of Vancomycin-resistant
vancomycin bacteria, and bile esculin to differentiate Enterococci enterococci from clinical and
from other Vancomycin-resistant bacteria that may surveillance specimens
grow
Blood Agar Trypticase soy agar, Brucella agar, or beef heart Cultivation of non-fastidious
infusion with 5% sheep blood microorganisms, determination of
hemolytic reactions
Bordet-Gengou agar Potato-glycerol-based medium enriched with 15- Isolation of Bordetella pertussis and
20% defibrinated blood; contaminants inhibited by Bordetella parapertussis
methicillin (final concentration of 2.5 um/mL)
Brain Heart Infusion Dextrose, pork brain and heart dehydrated infusions Cultivation of fastidious organisms
agar or broth
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MICROBIOLOGY
Buffered Charcoal Yeast exract, agar, charcoal, salts supplemented with Enrichment for Legionella spp.,
Yeast Extract agar L-cystein HCl, ferric pyrophosphate, ACES buffer, and Francisella and Nocardia
(BCYE) a-ketoglutarate
Buffered Charcoal BCYE supplemented with polymyxin B, vancomycin, Enrichment and selection for
Yeast Extract agar and ansamycin, to inhibit Gram-negative bacteria, Legionella spp.
with antibiotics Gram-positive bacteria, and yeast respectively
Burkholderia Bile salts, gentamycin, ticarcillin, polymixin B, For recovery of B. cepacia from
cepacia selective Peptone, and yeast extract cystic fibrosis patients
agar
Campy-blood agar Contains Vancomycin (10mg/L), trimethoprim (5 Selective for Campylobacter spp.
mg/L), polymixin B (2500 U/L), amphotericin B (2
mg/L), and cephalothin (15 mg/L) in Brucella agar
base with sheep blood
Campylobacter Thioglycollate broth supplemented with antibiotics Campylobacter spp. incubated at 4oC
thioglycollate broth for cold enrichment
CDC anaerobe 5% Tryptic soy broth, 5% sheep blood and added Improvement growth of obligate,
sheep blood agar nutrients slow-growing anaerobe
Cefoperazone, Blood-supplemented enrichment medium containing Selective medium for isolation of

Vancomycin, cefoperazone, vancomycin, and amphotericin to Campylobacter spp.
Amphotericin (CVA) inhibit growth of most Gram-negative bacteria,
medium Gram-positive bacteria, and yeast respectively
Cefsulodin-Irgasan- Peptone base with yeast extract, mannitol, and bile Selective for Yersinia spp.; may also
Novobiosin (CIN) salts; supplememented with cefsulodin, irgasan, and be useful in the isolation of
agar novobiocin; neutral red and crystal violet indicators Aeromonas
Chocolate agar Peptone base, enriched with solution of 2% Cultivation of fastidious

hemoglobin or isoVitalex (BBL) microorganisms such as
Haemophilus spp., Brucella spp., and
pathogenic Neisseria spp.
Chromogenic media Organism-specific nutrient base, selective Chromogenic media are designed to
supplements and chromogenic substrate optimize growth and differentiate a
specific type of organism.
Chromoagars are routinely used in
the identification of yeasts,
methicillin resistant Staphylococcus
aureus (MRSA), and a variety of
other organisms
Columbia colistin- Columbia based agar with 10mg colistin per liter, Selective isolation of Gram-positive
nalidixic acid (CNA) 15mg nalidixic acid per liter, and 5% sheep blood cocci
agar
Cysteine-tellurite Infusion agar base with 5% sheep blood; reduction of Isolation of C. diptheriae
blood agar potassium tellurite by Corynebacterium diphtheriae
produces black colonies
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MICROBIOLOGY
Eosin Methylene Peptone base containing lactose; eosin Y and Isolation and differentiation of
Blue (EMB) agar methylene blue as indicator lactose-fermenting and non-lactose-
(Levine) fermenting enteric bacilli
Gram-negative Peptone base broth with glucose and mannitol; Selective enrichment liquid medium
broth sodium citrate and sodium desoxycholate act as for enteric pathogens
inhibitory agent
Hektoen Enteruc Peptone base agar with bile salts, lactose, sucrose, Differential, selective medium for
(HE) agar salicin, and ferric ammonium citrate; indicators the isolation and differentiation of
include bromthymol blue and acid fuschin Salmonella and Shigella spp. from
other Gram-negative enteric bacilli
Loeffler’s medium Animal tissue (heart muscle), dextrose, egg and beef Isolation growth of Corynebacterium
serum, and sodium chloride
MacConkey agar Peptone base with lactose; Gram-positive organisms Isolation and differentiation of
inhibited by crystal violet and bile salts; neutral red lactose fermenting and non-lacotse
as indicator fermenting enteric bacilli
MacConkey sorbitiol A modification of MacConkey agar in which lactose For the selection and differentiation
agar has been replaced by sorbitol as the primary of E. coli O157:H7 in stool specimens
carbohydrate
Mannitol salt agar Peptone base, mnnitol, and phenol red indicator; Selective differentiation of
salt concentrationof 7.5% inhibits most bacteria Staphylococci
New York City agar Peptone agar base with cornstarch, supplemented Selective for Neisseria gonorrhoeae,
with yeast dialysate, 3% hemoglobin, and horse also supports the growth of
plasma; antibiotic supplement includes vancomycin Ureaplasma urealyticum and some
(2ug/mL), colisitin (5.5 ug/mL), amphotericin B (1.2 Mycoplasma
ug/mL), and trimethoprim (3 ug/mL)
Phenylethyl Alcohol Nutrient agar base. Phenylmethanol inhibits growth Selective isolation of aerobic Gram-
(PEA) agar of Gram-negative organism positive cocci and bacilli and
anaerobic Gram-positive cocci and
negative bacilli
Regan Lowe Charcoal agar supplemented with horse blood, Enrichment and selective medium
cephalexin and amphotericin B for isolation of Bordetella pertussis
Salmonella-Shigella Peptone base with lactose, ferric citrate, and sodium Selective for Salmonella and some
(SS) agar citrate, neutral red as indicator; inhibition of Shigella spp.
coliforms by brilliant green and bile salts
Schlaeder agar Peptone and soy protein base agar with yeast Nonselective medium for the
extract, dextrose, and buffers; addition of hemin, L- recovery of anaerobes and aerobes
cystein, and 5% blood enriches for anaerobes
Selective for Campylobacter and

Helicobacter spp.
Selenite broth Peptone base broth; sodium selenite toxic for most Enrichment of isolation of
Enterobacteriaceae Salmonella spp.
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MICROBIOLOGY
Skirrow agar Peptone and soy protein base agar with lysed horse Selective for Campylobacter
blood; vancomycin inhibits Gram-positive organsims;
polymixin B and trimethoprim inhibits most Gram-
negative organsims
Streptococcal Contains crystal violet, colisitin, and trimethoprim Selective for Streptococcus pyogenes
selective agar (SSA) sulfamethoxazole in 5% sheep blood agar base and Streptococcus agalactiae
Tetrathionate broth Peptone base broth; iodin and potassium iodide, bile Selective for Salmonella and Shigella
salts, and sodium thiosulfate inhibit Gram-positive spp. except Salmonella typhi
organisms and Enterobacteriaceae
Thayer-Martin agar Blood agar based enriched with hemoglobin; Selective for N. gonorrheae and N.
(modified Thayer- contaminating organisms are inhibited by colistin, meningitidis;
Martin agar) nystatin, vancomycin, and trimethoprim lactate
Supports the growth of Francisella
and Brucella spp.
Thioglycollate broth Pancreatic digest of casein, soy broth, and glucose Supports the growth of anaerobes,
enrich growth of most microorganisms; includes aerobes, microaerophiles, and
reducing agents thioglycolate, cystine, and sodium fastidious organisms
sulfite; agar is semi-solid medium with a low
concentration of agar reducing oxygen diffusion in
the medium
Thiosulfate citrate- Peptone base agar with yeast extract, bile salts, Selective and differential for Vibrio
bile salts (TCBS) agar citrate, sucrose, ferric citrate, and sodium spp.
thiosulfate; bromthymol blue acts as an indicator
Todd-Hewitt broth Todd-Hewitt, an enrichment broth for Streptococci, Selection and enrichment for
supplemented with supplemented with nalidixic acid and gentamicin or Streptococcus agalactiaee in female
antibiotics (LIM) colistin for greater selectivity; thioglycollate and agar genital specimens
reduce redox potential
Trypticase Soy broth All-purpose enrichment broth that can support the Enrichment broth used for
(TSB) growth of many fastidious and non-fastidious subculturin various bacteria from
bacteria primary agar plates
Xylene lysine Yeast extract with lysine, xylose, lactose, sucrose, Isolation and differerentiation of
desoxycholate (XLD) and ferric ammonium citrate; sodium desoxycholate Salmonella and Shigella spp. from
agar inhibits Gram-positive organisms; phenol red as other Gram-negative enteric bacilli
indicator
ANTIMICROBIAL SUSCEPTIBILITY TESTING
ANTIBIOTICS
 Chemical substances produced by organisms with the capacity to inhibit or kill other microorganisms
o Narrow-spectrum:
▪ Bacitracin, Clindamycin, Diapsone, Erythromycin, Gentamicin, Isoniazid, Penicillin, Polymyxin B,
Vancomycin
o Broad-spectrum:
▪ Ampicillin, Cephalosporin, Chloramphenicol, Ciprofloxacin, Rifampicin, Sulfonamides,
Trimethoprim
o Classes according to manufacture
▪ Natural → produced by bacteria or fungi (e.g., penicillin)
▪ Semisynthetic → chemically modified (e.g., methicillin)
▪ Synthetic → chemically produced
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MICROBIOLOGY
o Therapeutic index:
▪ Ratio of toxic dose to the therapeutic dose
▪ The higher the TI = more effective
Genetic Elements Contributing to Resistance

▪ PBP → Penicillin-binding protein; cross-links peptidoglycan
▪ Transposon → DNA elements encoding for transposition and excision function → antibiotic resistance
▪ Integrans → Genetic elements capable of integrating genes by integrin encoded site-specific recombinase
Classes of Antibiotics
▪ Cell wall inhibitors
o Most selective antibiotics with high TI
o Inhibits transpeptidation enzymes
o B-lactam drugs: penicillin, cephalosporin → targets gram positive and gram negative
o Glycopeptides: vancomycin and teicoplanin
▪ Cell membrane inhibitors
o Polymyxin
▪ Interacts with the phospholipid and increases permeability → last resort to P. aeruginosa
▪ Targets gram-negative bacteria; very poor against gram-positive
o Colistin
▪ Protein synthesis inhibitors
o Aminoglycosides
▪ Binds to 30s
▪ Target: gram-positive and gram-negative
▪ Contraindication: NOT for Anaerobes
▪ Tobramycin, Amikacin, Streptomycin, Kanamycin
o Tetracycline
▪ Binds to 30s
▪ Target: gram-positive and gram-negative, as well as intracellular organism
o Glycylglycines
▪ Binds to 30s
▪ A tetracycline derivative
▪ Target: gram-positive and gram-negative, as well as tetracycline-resistant organisms
o Macrolides
▪ Binds to 50s
▪ Target: usually gram-positive bacteria
▪ Erythromycin, clindamycin, azithromycin
o Oxazolidinones
▪ Binds to 50s
▪ Target: gram-positive
▪ Linezolid
o Chloramphenicol
▪ Binds to 50s
▪ NOTE: toxic to bone marrow
o Ketolides
▪ Binds to 50s
▪ Target: gram-positive cocci, macrolide-resistant organisms, and fastidious gram-negative
organisms
▪ DNA and RNA synthesis inhibitors
o Fluoroquinolones
▪ Nalidixic acid derivatives
▪ Inhibits DNA gyrase and topoisomerase IV
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MICROBIOLOGY
▪ Ciprofloxacim, Ofloxacin
o Metronidazole
▪ Also effective for protozoans
o Rifampin
▪ Inhibits RNA synthesis by binding DNA dependent RNA polymerase
▪ Inhibitors of metabolic process
o Sulfonamides
▪ Inhibits bacterial folic acid pathway
▪ Binds to dihydropteroate synthase
o Trimethoprim
▪ Inhibits folic acid pathway; binds to dihydrofolate reductase
o Nitrofurantoin
▪ Uncertain mechanism
▪ NOTE: may cause pulmonary fibrosis
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Primary goal
▪ To determine whether bacterial isolate is capable of expressing resistance (acquired) to antimicrobial agents
selected for treatment
▪ Factors to consider before performing AST:
o Clinical significance of bacterial isolate
o Predictability of susceptibility to therapeutic drug of choice
o Availability of reliable standardized methods for testing isolate
▪ NOTE: AST is necessary when there is known intrinsic resistance of identified bacteria against test antibiotics
Commonly required Staphylococci, S. pneumoniae, Viridans streptococci, Enterococci,
Enterobacteriaceae, P. aeruginosa, Acinetobacter
Occasionally required H. influenzae, N. gonorrheae, M. catarrhalis, Anaerobic bacteria
Rarely required Beta-hemolytic streptococci, N. meningitides, L. monocytogenes
Considerations for Standardization

▪ Inoculum preparation
o Pure inoculum: obtained by selecting 4-5 colonies of same morphology → inoculate into broth medium
→ incubate for 3 to 5 hours (for most organism) or 16 to 24 hours
o Standard inoculum size:
→ Compare the turbidity of suspension with the standard against dark background
→ Standard: 0.5 McFarland Standard = 99.5 mL of 1% H2SO4 + 0.5 mL 1.175 BaSO4
▪ Growth medium
o Base: MHA (beef infusion, nucleic acid, vitamins, casein hydrolysate)
→ + 5% sheep’s blood → for streptococci and fastidious organisms
▪ Guidelines and Streaking
o Turn 60 degrees between each streaking
o Allow the surface of the medium to dry for 3 to 5 minutes (no longer than 15 minutes)
o 24 mm: the distance from the center of once disc to another center
o 10-15 mm: the distance from edge of plate to the disc
o Incubation: 35’C for 16-18 hours (must be inverted to avoid contamination of moisture)
▪ Reading of Plates
o Lawn of growth must be confluent
o Use caliper to measure the diameter of ZOI
o Examine 2-4 inches above black or nonreflecting surface and measure at the back side of plate
o For media containing blood → remove the lid before examination
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MICROBIOLOGY
o Ignore the swarming of Proteus and discontinuous poor growth of tiny colonies near the end of zone
size
o DO NOT ignore colonies within clear zone → sign of contamination or mix culture → RETEST
▪ Interpretation
o Susceptible
→ ZOI noted
→ The organism responds to therapy using recommended dosage
o Intermediate
→ ZOI falls into a range of susceptibility in which MIC approaches or exceeds level of antimicrobial
agent → clinical response is less likely to be less than susceptible
o Resistant
→ Microorganisms is not inhibited by the usual dosage of antimicrobial agent tested
→ NOT appropriate for treatment
o Nonsusceptible
→ NOT susceptible and NOT resistant too
▪ Factors of ZOI
o Inoculum
→ Use pure fresh culture
→ Too light = false susceptibility
→ Too heavy = false resistance
o Thickness of agar
→ 4 mm is ideal
→ Too thick = false resistance
→ Too thin = false susceptibility
o Growth rate of test organism
→ Most are 16-18 hours (for N. gonorrheae 20-24 hours)
→ Lower temperature → larger zones of inhibition
→ Higher temperatures (>35’C) → false detection of MRSA
→ Prolonged incubation = false resistance
→ Less than 24 hours of incubation = undetection of MRSA
o pH
→ 7.2 to 7.4 = ideal pH
→ Decreased pH = decreased activity of aminoglycosides, erythromycin, and clindamycin, BUT
increased activity of tetracycline
o Number of discs per plate
→ 12 discs per 150 mm plate is ideal
→ >12 discs = overlapping zones
o Cations (Ca and Mg)
→ Elevated concentrations leadst o diminished activity of aminoglycosides against P. aeruginosa
and decreased activity of tetracyclines against bacteria
Conventional Techniques
▪ Broth dilution
o Challenge the organism of interest with antimicrobial agents in a liquid environment
o Concentration range is determined by pharmacological properties of drug
o Dilution: series of doubling dilution of antibiotic but fixed amount of organism
o Medium: usually MH broth
▪ MH broth w/2% NaCl → detection of oxacillin-resistant Staphylococci
▪ MH broth w/ 2-5% lysed horse blood → streptococci
▪ Agar dilution
o Antimicrobial and organism are brought together on a molten and cooled medium
o Standard inoculum: 1 x 104 CFU/mL
o Medium: series of plates with varying concentration of antibiotics are prepared
▪ MHA = aerobes
▪ MHA + 5% sheep’s blood = fastidious bacteria
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MICROBIOLOGY
▪ MHA + 2% NaCl = MRSA

o Advantage: examination of one or more bacterial isolates per plate
o The reference method for anaerobes and N. gonorrheae (has tendency to lyse in broth media → false
susceptibility)
▪ Kirby-Bauer Test (Disk Diffusion)
o Limited to aerobic and facultative anaerobic bacteria
o Qualitative method
o Maximum of 12 antimicrobial agents on a 150 mm-MHA plate
o Disc storage:
▪ -20’C or below for long term storage
▪ 2-8’C for at least 1 week
o Standard medium: MHA, 4 mm depth, pH of 7.2-7.4
o Inoculum size: 1.5x108
Criteria for evaluation of antibiotics

▪ MIC → lowest concentration of antimicrobial agent which inhibits bacterial growth
▪ MLC → lowest concentration of antimicrobial agent that kills bacteria when subcultured in a fresh medium
▪ MBC → lowest concentration of antibiotic needed to kill bacterial growth
▪ Breakpoint → cutoff point; concentration of antimicrobial agent that coincides with susceptible or intermediate
MIC breakpoint for a drug
Alternative Approaches to AST

▪ E-test
o Dilution test based on diffusion of continuous concentration gradient of antimicrobial agent from strip
into agar medium
o Positive: ellipse of growth of inhibition → read at the point on scale where ellipse intersect the strip
▪ MBC test
o A 0.01 mL aliquot of clear MIC tube or well is subcultured agar medium
o Standard inoculum size: 5x105 CFU/mL
o Final dilution: 1:1000 spread per plate
o Volume spread per plate: 0.1 mL
o Dilution factor: 1:10,000
o Interpretation: 99.9% killing accomplished if 5 or fewer colonies grown on subculture of each well
o Paradoxic effect: decreased bactericidal activity at higher drug concentration
o Tolerance: MBC to MIC ratio of ≥32
▪ Automated
o Principle: based on turbidimetric detection; fluorometry
o Examples: VITEK 1 and 2, BD Phoenixes, Microscan WalkAway SI, Crystal ID, TREK Sensitire, API20
Supplemental Methods for Detection of Antimicrobial Resistance

▪ Oxacillin agar screen
o Detects staphylococcal resistance to penicillinase-resistant to penicillin
▪ Vancomycin agar screen
o Detection of enterococcal resistance to vancomycin
▪ Aminoglycoside screen
o Detection of acquired enterococcal high level resistance to aminoglycosides that would compromise
synergy with antibiotics targeting cell wall
▪ Oxacillin disk screen
o Detects S. pneumoniae resistant to penicillin
▪ D test
o Differentiate constitutive and inducible resistance of S. aureus to clindamycin
o Constitutive resistance: rRNA methylase is always produced and organism is resistant to both
Erythromycin and Clindamycin
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MICROBIOLOGY
o Inducible resistance: methylase is produced only in the presence of inducing agent; isolates are resistant
to erythromycin BUT susceptible to clindamycin
o (+) result: blunting of clindamycin zone giving a “D” pattern
o Interpretation: clindamycin resistance has been induced
CATALASE POSITIVE – GRAM-POSITIVE COCCI

LABORATORY TESTS:
▪ Catalase test
 Separates gram positive cocci into two groups
 Reagent: 3% H2O2
 Principle: the enzyme catalase breaks down H2O2 to O2 and H2O
 (+) result: bubble formation/effervescence
NEGATIVE
POSITIVE
Micrococcaceaea Streptococcaceae
(Staphylococcus, Micrococcus, (Streptococcus)
Planococcus, Stomatococcus)
Pseudocatalase positive: Aerococcus, Enterococcus, Rothia;

*Colonies from BAP
Micrococcus vs. Staphylococcus
Staphylococci Micrococci
Oxidase test NEGATIVE POSITIVE
Furoxone Tween 80 Oil Red O agar NEGATIVE POSITIVE
Acid production from Glycerol w/

POSITIVE NEGATIVE
erythromycin
Oxygen requirement FACULTATIVE ANAEROBE STRICTLY AEROBE
Glucose utilization FERMENTER OXIDIZER
Bacitracin/Taxo A disk (0.02 – 0.03 units) RESISTANT SUSCEPTIBLE
Furazolidone (100 ug) Susceptibility test SUSCEPTIBLE RESISTANT
Lysostaphin Sensitivity test SUSCEPTIBLE RESISTANT

▪ Coagulase test
 To differentiate Staphylococcus aureus from other Staphylococcus species
 Reagent: Rabbit EDTA plasma
 (+) organisms: S. aureus, S. intermedius
SLIDE METHOD TUBE METHOD

Detects cell bound coagulase Detects free coagulase
(+) result: clot formation within 30 (+) result: clot formation after 1-4 hours at 35’C
seconds
NOTE: extend incubation for 24 hours if negative
Screening method Confirmatory method
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MICROBIOLOGY
False (+): citrate-utilizing organisms → DO NOT use citrated plasma
False (-): production of staphylokinase that lyses fibrin clot → check at regular intervals
(produced after 6 hours)
▪ Mannitol Fermentation Test

 To differentiate pathogenic Staphylococci from non-pathologic Staphylococci
 Principle: S. aureus ferments mannitol and produce acid
 Reagents: MSA (1% mannitol + 7.5% NaCl)
 pH indicator: phenol red
 (+) result → colonies surrounded by yellow halos
▪ VP (Voges-Proskauer) test
 To differentiate S. aureus from S. intermedius
 Positive: S. aureus
 Negative: S. intermedius
▪ DNAse test:
 Reagent: DNA medium and methyl green dye
 (+) result: clearing of dye → S. aureus
 Reagents:
▪ 0.1% toluidine blue
• (+) result: pink color
• (-) result: blue color
▪ HCl precipitation
• (+) result: pink w/o precipitation
▪ PYRase
 Positive: S. lugdunensis, S. intermedius, S. schleiferi
 Negative: S. aureus
▪ Serological test
 Latex agglutination test for detection of clumping factor, protein A, and PBPs (penicillin-binding
protein)
▪ Molecular test
 Specimen of choice: anterior nares swab
 Gold standard method = nucleic acid probes or PCR amplification
▪ Other tests
 Tellurite Glycine Agar → (+) jet black colonies
 Polymyxin Sensitivity test → (+) resistance: S. aureus
 Phage typing: susceptibility to lysis by standard panels of bacteriophages
 Pulse field gel electrophoresis
▪ Culture
 BAP
 MSA
 PEA → for heavily contaminated specimens
 CAP, BHI, Thioglycollate
 Vogel-Johnson, Chapman, Tellurite Glycine → yields jet black colonies
VIRULENCE FACTORS OF Staphylococcus aureus
▪ Catalase
 Decomposes H2O2 to H2O and O2
▪ Coagulase
 Enzyme that coagulates fibrinogen in plasma
 Evades phagocytosis by formation of fibrin around abscess
 Cell-bound/clumping factor → bound to cell wall
 Unbound/free coagulase → extracellular
▪ Hyaluronidase
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MICROBIOLOGY
 Also known as Duran-Reynal factor

 Spreading factor
 Breaks down hyaluronic acid → allowing the spread of bacteria in deep tissues
▪ Staphylokinase
 Fibrinolytic activity
▪ Lipase
 Fat-splitting enzyme
 Produced by all Staphylococcus species
 Targets sebaceous glands → causing furuncles, carbuncles, and boils
▪ DNAse and Phosphatase
 Lowers viscosity, giving the pathogen more mobility
▪ Beta-lactamase
 Breaks down beta-lactam drugs → resistance
▪ Enterotoxins
 Heat stable at 100’C for 30 minutes
 Resistant to gastric acid and jejunal enzymes
 Neurotoxins: stimulates the vagus nerve (causing vomiting)
 A,B,D: responsible for causing food poisoning; diarrhea
 B: causes pseudomembranous enterocolitis
 F: superantigen; associated with toxic shock syndrome
▪ Leukocidin
 Also known as Panton Valentine Leukocidin (Cytolytic toxin)
 Necrotizing skin and soft tissue infection
 Pore-forming exotoxin that attacks and kills WBCs → evading phagocytosis
▪ Hemolysin
 Alpha: destroys RBCs, platelets, and macrophages → causes severe tissue damage
 Beta: destroys sphingomyelin (sphingomyelinase C) and RBC;
 Gamma: less toxic; injury to RBC in culture and edematous lesion
 Delta: destroys RBC and is associated with PVL
▪ Exfoliatin A & B
 Responsible for causing Ritter disease or SSS
 Causes sloughing of the stratum granulosum (epidermolytic toxin)
▪ TSST-1
 Also known as enterotoxin F
 Superantigen
▪ Protein A
 Immunologically active substance found in the cell wall
 Antiphagocytic property by competing with neutrophils for the Fc portion
DISEASES ASSOCIATED w/ Staphylococcus aureus
▪ Food poisoning
 The no. 1 cause of food poisoning in the USA
 Enterotoxin A, B, D
▪ Scalded Skin Syndrome
 Also known as Ritter disease
 Extensive exfoliative dermatitis in newborns and healthy children
▪ Toxic Shock Syndrome
 Potentially fatal; multisystem shock
 Often recovered from female patients
 Associated with the use of highly adsorbent tampons
 Virulence factor responsible: TSST-1
▪ Cutaneous infection
 Impetigo:
▪ superficial; crusty lesions surrounded by red border
▪ common in children
 Folliculitis:
▪ Damage to the hair follicle or sebaceous glands
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MICROBIOLOGY
 Furuncles
▪ Also known as boils (pus-filled bumps)
▪ Large, raised; extended folliculitis
 Carbuncles
▪ Clusters of several boils
▪ Deeper tissues; systemic
▪ Others
 Bacteremia/sepsis (MRSA)
 UTI
 Acute bacterial endocarditis
 Osteomyelitis
ANTIBIOTIC RESISTANCE
▪ MRSA
 Hospital acquired antimicrobial resistance especially after receiving broad spectrum antibiotics
 Penicillin resistance: mecA gene coding for an altered penicillin binding protein (PBP2a) → cell wall is
incapable of binding penicillin
 Chromogenic test: (+) result → mauve colored colonies within 24 hours
 Rapid tests: ID-MRSA; BD Gene Ohm Assay
 Borderline-Oxacillin-Resistant Isolates → isolates that have MIC right above the breakpoint for
oxacillin susceptibility
▪ Heteroresistant
 Two subpopulations in a culture, one that is susceptible and one that is resistant to antibiotics
 Some strains have mecA gene but doesn’t express altered PBP
 Resistant population grows more slowly than susceptible colonies
▪ Erythromycin resistance
 Erm gene → methylation of 23s RNA → resistance to erythromycin
 Msr A gene → efflux mechanism → erythromycin resistance but susceptible to clindamycin
CONS (Coagulase-negative Staphylococcus)
▪ Staphylococcus epidermidis
 # 1 normal flora of the skin (major inhabitant of the skin)
 Most common isolate seen in medical instruments and blood culture
 Most common cause of UTI among catheterized patients
 Implicated in causing nosocomial infections
 Virulence factor: Poly-gamma-DL-glutamic acid (provides adherence to devices and biofilm → capable
of producing slime)
▪ Staphylococcus saprophyticus
 # 1 cause of UTI among sexually-active young females
 Capable of adherence to epithelial cells of urogenital tract
NOVOBIOCIN SUSCEPTIBILITY TEST (5ug)
S. epidermidis SUSCEPTIBLE
S. saprophyticus RESISTANT
▪ Staphylococcus lugdunensis
 Associated with catheter-related bacteremia and endocarditis
 Can mimic S. aureus infection
 Possess mecA gene coding for oxacillin resistance
 More aggressive than other CONs
 Slide coagulase test (+) but tube coagulase negative
 PYR test (+)
OTHER CATALASE-POSITIVE GRAM (+) COCCI
▪ Planococcus – associated with marine life
▪ Stomatococcus
 Implicated in causing endocarditis following cardiac catheterization
 Weakly catalase (+); produces white or transparent sticky colonies
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MICROBIOLOGY
CATALASE – NEGATIVE GRAM-POSITIVE COCCI

STREPTOCOCCI:
1. Normal flora of the human body but causes life-threatening conditions when it gains access to sterile sites
2. Capsulated bacteria → has a layer of C-Carbohydrate → w/c evades phagocytosis
3. Biochemical test:
✓ Catalase (-)
✓ Oxidase (-)
✓ Non-motile
✓ Ferments carbohydrate
4. All are facultative anaerobes or capnophiles EXCEPT for Peptostreptococci (strict anaerobe)
CLASSIFICATION OF STREPTOCOCCI:
A. Bergey’s Classification
 Based on temperature requirement
o 37°C only
− Pyogenic group
− Produces pus and mostly Beta-hemolytic
− Groups A,C, and G
o 37°C and 45°C
− Viridans group
− Alpha hemolytic or non-hemolytic
o 10°C and 37°C
− Lactococci group
− Non-hemolytic
− Group N
− Found in dairy products (e.g. S. lactis)
B. Smith and Brown Classification
 Based on hemolytic pattern
o Alpha
− Partial hemolysis around colonies; green hemolysis
− S. pneumoniae
o Beta
− Complete hemolysis
− S. pyogenes, S. agalactiae, S. dysagalactiae,
o Gamma
− No hemolysis
− E. faecalis
o α, β, γ
− Enterococci, Viridans
o α, β
− S. bovis
o Alpha prime (α’)
− Alpha around the colonies + beta outside the alpha hemolysis (bull’s eye hemolysis)
C. Lancefield
 Based on antigen serogrouping of the C-Carbohydrate from Streptococci cell wall
 NOTE: C-Carbohydrate can be extracted by diluting w/ acid and heating for 10 minutes.
Group A S. pyogenes
Group B S. agalactiae
Group C S. dysagalactiae, S. zooepidemicus, S. equi, S. equisimilis
Group D S. bovis, Enterococci,
Group F S. anginosus
DIAGNOSTIC ALGORITHM FOR IDENTIFICATION OF GRAM-POSITIVE COCCI
CATALASE
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MICROBIOLOGY
POSITIVE NEGATIVE
Staphylococci Streptococci
Micrococci
Planococcus
Stomatococcus
Hemolysis
ALPHA BETA GAMMA
DIFFERENTIAL TEST FOR BETA-HEMOLYTIC STREPTOCOCCI:
Species Lancefield Group PYR Test VP Test Hippurate Hydrolysis CAMP Test
S. pyogenes Group A + - - -
S. anginosus Group A - + - -
S. agalactiae Group B - - + +
DIFFERENTIAL TEST FOR ALPHA-HEMOLYTIC STREPTOCOCCI:
OPTOCHIN SUSCEPTIBILITY TEST
S. pneumoniae RESISTANT
Viridans group SUSCEPTIBLE
ADDITIONAL TESTS:
▪ Gram stain
 Appear in chains when grown in broth cultures
 S. pneumoniae often appears in pairs; lancet-shaped
▪ Culture
 BAP, PEA, CAN, CAP, Carrot broth
 BAP w/ SXT: more selective for Group A
 Growth factors:
✓ Cyanocobalamin → for E. faecalis
✓ Thiol compounds (cysteine, vitamin B6) → for Abiothrophia, and Granulicatella
 Culture of S. pneumoniae:
✓ Considered as fastidious
✓ Incubate at CO2
✓ Young colonies: dome shaped, mucoid, glistening and moist
✓ Old colonies: coin shaped with raised rim
▪ Serological tests
 ASO titer: used to indicate infection with S. pyogenes (4-fold rise is significant)
 Latex agglutination test
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MICROBIOLOGY
 ELISA → for detection of hyaluronidase, streptokinase, DNAse

▪ Bacitracin and SXT test
 For beta-hemolytic streptococci species only
S. pyogenes S. agalactiae S. pneumoniae Non-A/Non-B
BACITRACIN S R S R/S
SXT R R S S
▪ CAMP test (Christie-Atkins-Munch-Petersen)

 Principle: there is hemolytic interaction seen on blood agar between β-hemolysin by most strains of S.
aureus and the CAMP factor
 CAMP factor: an extracellular, thermostable antigen that enhances β-hemolysis
 (+) result: arrowhead β-hemolysis near the growth of S. aureus
▪ Hippurate hydrolysis test
 Purpose: for detection Group B beta-hemolytic streptococci
 Reagents: sodium Hippurate + ninhydrin (color indicator)
 (+) result: purple color after the addition of ninhydrin reagent
 (+) organisms: group B streptococci, group D
▪ Bile Esculin test
 Principle: Group D streptococci hydrolyzes esculin → esculitin and grows in 4% or 40% bile salts
 Procedure: bile esculin slant is inoculated → incubation for 24-48 hours in ambient incubator
 (+) result: blackening
 (+) organism: Group D streptococci
▪ 6.5% NaCl tolerance test
 Purpose: to differentiate Enterococci Group D from Non-Enterococci Group D
 Principle: Enterococci will grow in the presence of 6.5% NaCl
 (+) result: turbidity of broth w/ 6.5% salt after incubation
 Other positive organisms: Group B streptococci → can be differentiated by bile esculin hydrolysis
▪ LAP test (Leucine Aminopeptidase Test)
 Used for presumptive identification of Streptococci
 Principle: LAP disk detects leucine aminopeptidase enzyme
 Reagent: disk is impregnated with LAP
 (+) organism: E. faecalis
▪ PYR
 Principle: Group A and Group D possess PYRase enzyme that hydrolyzes PYR
 (+) result: bright cherry red color
 Other (+) organisms: Enterococcus, Aerococcus, Gemella
▪ Bile Solubility Test
 Evaluates the ability of the organism to lyse bile salts in the presence of amidase
 (+) result: clearing of broth after 3 hours of incubation
 (+) organism: S. pneumoniae
▪ Optochin disk test (5 ug)
 Also known as Taxo P
 Used as presumptive test for identification S. pneumoniae
 Reagent: ethylhydroxycuprein HCl
 (+) result: susceptibility (>16 mm zone of inhibition)
OPTOCHIN SUSCEPTIBILITY TEST

S. pneumoniae SUSCEPTIBLE
Viridans group RESISTANT
▪ Francis test
 Immunological test (skin test) that detects antibodies against S. pneumoniae
▪ Mouse virulence
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MICROBIOLOGY
 In vivo testing by injecting the organism to the mouse

 (+) result: death of the animal
 For S. pneumoniae
MOUSE INOCULATION TEST

S. pneumoniae DEAD
Viridans group ALIVE
▪ Staphylococcal Streak test

 Principle: streak of Staphylococcus provides nutrients needed by NVS (nutritionally variant
streptococci)
 (+) result: very small colonies can be seen growing in adjacent to the streak (satellitism phenomenon)
▪ Penicillin Susceptibility test
 To differentiate Enterococci group from Viridans group
 Resistant: Enterococci
 Susceptible: Viridans group
GROUP A STREPTOCOCCI (S. pyogenes)
▪ General Characteristics:
 NOT a normal flora; can be acquired through contaminated droplets by coughing or sneezing
 Resistant to drying
 Culture media: BHI, TSA with 5% sheep’s blood, CAP
 Young colonies appear as dome-shaped, glistening, wet and mucoid
 Old colonies appear as coin-shaped with raised rim
VIRULENCE FACTORS:
▪ M protein
 Principal virulence factor
 Antiphagocytic properties
 Adheres to mucosal cells
▪ Protein F
 Mediates epithelial cell attachment
▪ Lipoteichoic acid
 Adheres to respiratory epithelium
▪ Streptolysin O
 Oxygen labile
 Highly antigenic
 Causes lysis to WBCs, platelets, tissue cells
 Responsible for subsurface hemolysis → incubated anaerobically
▪ Streptolysin S
 Oxygen stable
 Nonantigenic
 May cause lysis to WBCs
 Responsible for surface hemolysis → incubated aerobically
▪ DNAse
 Types A,B,C,D
 Lowers the viscosity of exudates → facilitating more mobility
▪ Streptokinase
 Fibrinolytic; responsible for the spread of infection due to mobility of bacteria from clotted area
▪ Hyaluronidase
 Spreading factor
▪ Pyrogenic toxins
 Also known as Erythrogenic toxin (A,B,C)
 Considered as superantigens
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MICROBIOLOGY
 Exotoxin B: degrades protein and mediates rash seen in scarlet fever
DISEASES CAUSED BY S. pyogenes:
▪ Pharyngitis/Tonsillitis
 MOT: droplets or close contact (causes outbreaks in pre-school)
 Diagnosis: throat culture or direct antigen detection
▪ Scarlet fever/Scarlatina
 Characterized by diffuse erythema appearing initially on neck and upper chest 1-2 days following strep
throat infection
 MOT: inhalation of infectious respiratory droplets
 S/S: red rash appears on the upper chest and spreads to the trunk and extremities; strawberry colored
tongue
▪ Dick’s test
− For detection of erythrogenic toxin
− (+) result: erythema or redness of test site
▪ Schultz Charlton test
− Diagnostic test
− For detection of anti-erythrogenic toxin
− (+) result: blanching phenomenon
▪ Skin infection
 Cellulitis: diffuse spreading infection of subcutaneous skin tissue leading to erythema and edema
 Erysipelas: acute infection and inflammation of the dermal layer → painful reddish patches
 Necrotizing fasciitis: galloping gangrene or flesh-eating bacteria
▪ Rheumatic fever
 Post-streptococcal sequelae infection
 Characterized by fever, inflammation of heart, joints, and blood vessels
▪ Post-streptococcal Glomerulonephritis (Bright’s disease)
 Post-streptococcal sequelae infection
 Inflammatory disease of the renal glomeruli due to deposition of immune complexes
▪ TSS
 Once the organism gains access to the circulation (due to cellulitis, wound infection, etc.) → entire
organ system shutdown
GROUP B STREPTOCOCCI (S. agalactiae)
▪ Normal flora of the female genital tract and lower GIT

▪ Can be fatal to newborn patients
▪ Diseases:
o Pneumonia, meningitis, neonatal sepsis, postpartum infection, osteomyelitis, UTI, endocarditis
▪ MOT:
o unwashed hands of the mother or health care personnel handling the infant
o passage of baby through the birth canal
▪ Virulence factor: capsule with sialic acid
▪ Culture media: Todd Hewitt (suppresses vaginal flora), Carrot broth (produces orange/red pigment), Granada
agar
ENTEROCOCCI
▪ Normal flora of the GIT; Opportunistic pathogen

▪ Associated with nosocomial infection (UTI, endocarditis, wound infection)
▪ Can grow in extreme conditions (alkaline pH, 45°C, salt solutions)
▪ Virulence factors: serine proteinase, gelatinase, cytolysin
▪ Most common isolate: E. faecalis
▪ Other species: E. faecium, E. avium, E. gallinarum, E. raffinosus
▪ NOTE: E. faecalis requires cyanocobalamin as growth factor
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MICROBIOLOGY
GROUP C and G
▪ Normal flora of the URT, vagina, skin

▪ Virulence factor: M protein, streptokinase
Streptococcus pneumoniae
▪ Diplococcus/Pneumococcus
▪ Normal flora of the URT among preschool children
▪ Virulence factors: capsule (made up of polysaccharide)
▪ Disease:
o Lobar pneumonia
− Characterized by bloody, rust-tinged sputum
− MOT: bacteria residing in the URT may infect immunocompromised host (disturbance of
the normal defense barriers)
o Meningitis: as complication of pneumonia and otitis media
o Otitis media: most common among children (<3 years old)
o Others: bacteremia, endocarditis, peritonitis
VIRIDANS GROUP
▪ Normal flora of the oropharyngeal area and throat, URT, female genital tract, GIT
▪ Most common cause of SBE (subacute bacterial endocarditis)
▪ Virulence factors: capsule, cytolysin, dextran, and adhesins
▪ Infection: SBE, gingivitis, dental carries, meningitis, osteomyelitis, empyema
▪ Causes gastrointestinal carcinoma (bovis group)
▪ Most common isolate: S. mutans
▪ Biochemistry: bile insoluble, optochin resistant, no growth in 6.5% NaCl, LAP (+), PYR (-)
Abiotrophia and Granulicatella
▪ Nutritionally variant streptococci

▪ Pyridoxal dependent (vitamin B6), thiol dependent, symbiotic streptococci
▪ Normal flora of the oral and GIT
▪ Forms satellite colonies around pyridoxal-producers (E. coli, Klebsiella, Enterobacter, Yeasts, S. aureus)
▪ Related infection: endocarditis, otitis media, breast implants, chronic sinusitis
Other Genus
▪ Aerococcus
 Species: A. viridans (bile esculin and PYR positive), A. urinae (bile esculin and PYR negative)
▪ Gemella: easily decolorized thereby appearing as gram (-) cocci
▪ Lactococcus: Group N; non-fermentative
▪ Leuconostoc:
 cross-react with Group D antiserum
 (+): PYR, LAP, bile esculin, and 6.5% NaCl
▪ Pediococcus: bile esculin hydrolysis positive
GRAM NEGATIVE COCCI

LABORATORY TESTS
▪ Specimen collection
 Dacron swab is recommended
 Transport media must have charcoal
 SPS should not exceed 0.025%
 N. gonorrheae: pus and secretions from urethra, cervix, prostate, rectal mucosa, throat, joint
 N. meningitides: CSF, blood, nasopharyngeal swab, petechial skin lesion
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MICROBIOLOGY
▪ Gram stain
 Gram-negative intracellular diplococci
 Avirulent form → extracellular
 IMPORTANT NOTES:
✓ Oropharyngeal specimen should not be gram stained because commensal Neisseria may be
present
✓ Cytocentrifugation is best for body fluids because of its concentrating ability
✓ One should consider the presence of Chlamydia if there are more than 5 PMNs per field w/o
bacteria
DIAGNOSTIC ALGORITHM FOR IDENTIFICATION OF Neisseria gonorrheae
GRAM STAIN
MALE PATIENT FEMALE PATIENT
Specimen: urethral Specimen: vaginal

discharge discharge
Gram-negative Gram-negative
diplococci intracellular diplococci intracellular
DIAGNOSTIC Confirm by culture
NOTE: the rectum and endocervical secretions contain normal flora that may resemble Neisseria among females;
gram stain is only presumptive evidence of gonorrhea → should be confirmed with culture
▪ Culture media
Thayer Matin Chocolate agar w/ IsoVitalex VCN (vancomycin, colistin, nystatin)
MTM Chocolate agar w/ IsoVitalex VCNT (+ trimethoprim lactate)
Martin Lewis Chocolate agar w/ IsoVitalex VCAnT (+ anisomysin, trimethoprim)
New York Medium Lysed horse blood + yeast dialysate VCAmT (+ amphotericin, trimethoprim)
GC-LECT Chocolate agar w/ IsoVitalex VCAmTL (+ lincomycin)
OTHER CULTURE MEDIA: Transgrow, Cary-Blair, Amies, JEMBEC
▪ Culture
 For sterile specimen → BAP and CAP
 Direct inoculation → swab rolled onto media in “Z pattern” and cross-streaked
 Incubation: capnophilic environment (candle jar, JEMBEC) for 72 hours
▪ Oxidase test
 For presumptive identification of Neisseria and Moraxella
 Principle: the enzyme cytochrome oxidase catalyzes the reaction → produce dark blue end product
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MICROBIOLOGY
 (+) result: purple color w/in 10 seconds

▪ Superoxol
 Principle: catalase breaks down H2O2 to H2O and O2
 Reagent: 20% to 30% H2O2
 (+) result: vigorous bubbling
 Positive: N. gonorrheae, Moraxella
▪ GGA test (gamma-glutamyl aminopeptidase test)
 Positive: N. meningitides
▪ B-galactosidase test
 Purpose: to differentiate N. lactamica from other Neisseria species
 Principle: N. lactamica possess B-galactosidase that allows it to ferment lactose
▪ DNAse test
 To differentiate Moraxella from Neisseria species
 Positive: Moraxella
 Negative: Neisseria
▪ Penicillin (10 unit)
 Purpose: to differentiate M. catarrhalis from other Moraxella species
 Principle: M. catarrhalis retains the typical coccal morphology after overnight incubation on blood agar
with 10-unit penicillin; other species form long filaments
▪ CHO Utilization test
 Standard method for identification of Neisseria species
 Medium: CTA medium + 1% CHO + phenol red
 Detects acid production from glucose, maltose, lactose, fructose, and sucrose
 (+) result: yellow color within 24-72 hours of incubation
ORGANISM GLUCOSE MALTOSE FRUCTOSE SUCROSE LACTOSE
N. gonorrheae + - - - -
N. meningitides + + - - -
N. lactamica + + - - +
N. sicca + + + + -
N. flavescens + + + - -
M. catarrhalis - - - - -
▪ Butyrate disk test

 Rapid test for detection of butyrate esterase produced by M. catarrhalis
 Reaction: bromochlorindonyl butyrate → indoxyl → turns indigo color in the presence O2 within 5
minutes
 (+) result: indigo color
▪ Immunologic
 Fluorescent antibody test → highly specific and sensitive
 Coagglutination test
▪ Molecular
 Specimen: endocervical and urethral swab, urine
▪ Direct detection
 Nucleic acid probe → rapid, direct detection of gonococcal rRNA in genital and conjunctival specimen
NEISSERIACEAE
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MICROBIOLOGY
✓ Obligate aerobe, non-motile, non-hemolytic

✓ Gram-negative diplococci with coffee or kidney bean-shaped in appearance EXCEPT N. elongate and N. weaveri
✓ Normal flora of the mucous membranes
✓ Oxidase (+), catalase (+) EXCEPT N. elongate
✓ Capnophilic (requires 2%-8% CO2)
✓ Requires moist temperature (sensitive to drying and extremes) → direct inoculation of samples at bedside is
recommended
N. gonorrheae
▪ NOT a normal flora; infects urogenital tract, anorectal area, oropharynx and conjunctiva
▪ MOT: sexual contact, infected mother to newborn during birth
▪ # 1 cause of sexually transmitted disease
▪ Glucose fermenter
▪ Virulence factors: common pili (major), IgA protease, lipooligosaccharide endotoxin
▪ AHU strains: auxotypes that requires arginine, hypoxanthine, and uracil
▪ Penicillin susceptible
▪ Diseases:
o Gonorrhea:
− Short incubation period (2 to 7 days)
− Patients may be asymptomatic (AHU strains)
− Symptomatic patients: purulent discharge, lower abdominal pain, dysuria, vaginal bleeding
o Fitz-Hugh-Curtis syndrome
− Purulent urethritis and cervicitis → perihepatitis
o Pharyngitis
− Symptomatic oropharyngeal infection
o Conjunctivitis
− Ophthalmia neonatorum
− Gonococcal eye infection during vaginal delivery through the infected birth canal
N. meningitides
▪ Normal flora of the URT; colonize mucous membrane

▪ Serotypes (based on capsule): A,B,C,Y,W
▪ Virulence factors:
o Pili, polysaccharide capsule, lipopolysaccharide endotoxin, IgA protease
▪ MOT: oral secretion and respiratory droplets
▪ Risk factor: C5-C8 deficiency
▪ Diseases:
o Waterhouse-Friderichsen syndrome:
▪ Endotoxin → released after bacterial lysis → activation of coagulation cascade → fibrin
deposition in blood vessels (petechiae) → hemorrhage in adrenals → shock and death
o Meningococcal meningitis
o Meningococcemia
M. catarrhalis
▪ Normal flora of the oropharynx; opportunistic pathogen

▪ 3rd most common cause of otitis media
▪ Specimen: eye or ear
▪ Encapsulated organism with pili and non-motile
▪ B-lactamase producer, asaccharolytic
▪ Positive: oxidase, catalase, DNAse, butyrate esterase, tributyrin hydrolysis
▪ Culture:
o Hockey puck appearance colonies → intact when pushed across the plate with loop at 22’C
o Wagon wheel appearance → elevated center, thinner wavelike periphery
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MICROBIOLOGY
o Doesn’t grow in gonococcal media because it is inhibited by colistin
Other Neisseria species
N. cinerea Colonies are morphologically similar to T3 of N. gonorrheae

Susceptibility test to differentiate
N. flavescens Yellow pigmented Neisseria; asaccharolytic
N. lactamica NF of the nasopharynx of infants and children; rare on adults
ONPG (+)
N. mucosa Colonies are large; adherent to agar and mucoid
NF of the nasopharynx of children or young adults
Isolated in dolphin airways
N. sicca Colonies are dry, wrinkled, adherent (breadcrumb-like)
N. elongata Rod-shaped; weakly positive
Catalase (-)
N. weaver Normal microbiota in dogs; infects humans after dog bites
Non-fermenter, PAD (+)
ENTEROBACTERIACEAE
GENERAL CHARACTERISTICS
▪ Gram-negative, Non-spore-forming
▪ Facultative anaerobic bacilli
▪ Most members are present in the intestinal tract as commensal flora EXCEPT Shigella, Salmonella, Yersinia
▪ All members are motile EXCEPT Shigella, Klebsiella, Yersinia
▪ All are non-encapsulated EXCEPT Klebsiella and Enterobacter
▪ All members are glucose fermenters and can reduce nitrate to nitrite
▪ All are nonhemoloytic EXCEPT E. coli (beta-hemolytic)
▪ Oxidase (-) EXCEPT P. shigelloides
▪ Glucose fermenters
▪ Nitrate reduction (+)
ANTIMICROBIAL RESISTANCE
▪ ESBL producers: E. coli, K. pneumoniae, Shigella

▪ Generally antibiotic resistant: Citrobacter, Enterobacter, Serratia
LABORATORY DIAGNOSIS
▪ Specimen:
o Stool, rectal swab, blood urine
o NOTE: enterics from sterile sites are highly significant
▪ Microscopy: gram-negative rods or coccobacilli
▪ Culture:
o Transport media: Amies, Cary-Blair, Stuart
o Optimal growth temperature:
 1-5°C → Yersinia and Serratia
 45-50°C → E. coli
 37°C → the rest of the members
MAC For enteric gram-negative rods
MAC w/ Sorbitol For E. coli O157:H7
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MICROBIOLOGY
XLD For Salmonella and Shigella
HEA For Salmonella and Shigella
EMB For differentiation of LF vs. NLF
BSA Selective for Salmonella
ANTIGENIC DETERMINANTS FOR SEROLOGICAL ID
▪ Somatic O antigen
 Heat stable
 Found on the cell wall
▪ Flagellar H antigen
 Heat labile
 Represents the flagella
▪ Capsular antigen
 Heat labile
 Requires heating → may mask the O antigen
▪ V and W antigen
 Present in Y. enterocolitica
BIOCHEMICAL TESTS:
▪ ONPG (Orthonitrophenyl β-galactopyranoside)

 Determines whether an organism is a late-lactose (LLF) or a true non-lactose fermenter (NLF)
 Salmonella arizonae is the only ONPG-positive Salmonella serotype
 Principle: B-galactosidase acts on ONPG, cleaves it into galactose and orthonitrophenol
 Positive test result: yellow color within 20 minutes
▪ TSIA (Triple Sugar Iron Agar)
 Used to determine whether a gram-negative rod utilizes glucose, lactose or sucrose fermentatively and
forms hydrogen sulfide
 pH Indicator: Phenol Red
 H2S Indicator: Ferrous sulfate and sodium thiosulfate
 Uninoculated TSI is at pH 7.4 (red color)
Interpretation of TSIA reaction:
K/K No fermentation
K/A Non-lactose, non-sucrose fermenter;

Glucose fermenter
A/A Lactose/Sucrose fermenter
Black precipitate Hydrogen sulfide production
Formation of Gas production

bubbles/splitting of
the media in the butt
Summary of TSIA reaction:

E. coli
A/A
Enterobacter
GAS (+)
Klebsiella
H2S (-)
K/A M. morganii
GAS (+) C. koseri
H2S (-)
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MICROBIOLOGY
Edwardsiella
K/A C. freundii
GAS (+) Salmonella
H2S (+) Proteus
Plesiomonas
Providencia
K/A
Shigella
GAS (-)
Serratia
H2S (-)
Yersinia
▪ KIA (Kligler’s Iron Agar)

 Similar to TSIA but no sucrose
 To differentiate lactose fermenters from sucrose fermenters
 Sucrose negative = no growth (Serratia, Citrobacter)
▪ SIM (Sulfide Indole Motility)
 Determines the ability of an organism to split tryptophan to form the compound indole
 Positive sulfide: Black color formation
 Positive indole: Pink to wine colored ring after the addition of Kovac’s reagent
 Positive motility: Movement away from the stab line/hazy appearance throughout the medium
▪ MR test (Methyl Red test)
 Principle: acid products are formed when glucose is metabolized by mixed acid fermentation pathway
 Mixed acid: Lactic acid, Acetic acid, Succinic acid
 pH indicator: Methyl red (pH 4.4)
 Positive result: red color after addition of methyl red
 (+) E. coli
▪ VP test (Voges-Proskauer)
 Principle: Acetoin is produced from alternate pathway for glucose metabolism
 Reagents: 40% KOH and alpha-naphthol
 Positive result: red color after addition of VP reagent
 (+) Klebsiella, Enterobacter, Serratia, Hafnia
▪ Citrate utilization test
 Determine the ability of an organism to utilize sodium citrate (due to citrate permease) as its only
carbon source and inorganic ammonium salts as its only nitrogen source
 Positive Reaction: from green → blue color slant
 pH indicator: bromthymol blue
 Citrate (+) = Klebsiella, Enterobacter, Citrobacter, Salmonella
 Citrate (-) = E. coli, E. tarda
▪ IMViC Reaction (Indole, MR, VP, Citrate)
E. coli
++-- E. tarda
M. morganii
Klebsiella
--++ Enterobacter
Serratia
-+-+ Salmonella
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MICROBIOLOGY
▪ LIA (Lysine Iron Agar)

 Determine whether a gram-negative rod decarboxylase or deaminates lysine and forms hydrogen sulfide
 pH indicator: Bromcresol purple
 Important in differentiating Salmonella (+) from Citrobacter (-)
Interpretation of LIA reaction:
K/K (-) lysine deamination
(+) lysine decarboxylation
K/A (-) lysine deamination

(-) lysine decarboxylation
R/A (+) lysine deamination

(-) lysine decarboxylation
▪ Urease test
 Principle: Urea → (urease) → ammonia + CO2 → ammonia increases the pH → pH indicator changes the
color of the medium
 Christensen’s Urea agar
 pH indicator: phenol red
 Rapid urease producers: (+) Proteus and Morganella
▪ PAD test (Phenylalanine Deaminase)
 Principle: Phenylalanine + 10% Ferric chloride oxidative deamination phenyl pyruvic acid
 (+) result: green color after addition of ferric chloride
 (+) PPM group
▪ Nitrate reduction test
 Principle: Nitrate + sulfanilic acid + a-naphthylamine nitrite (red, water-soluble azo dye)
 Positive result: Red color
 (+) E. coli
 NOTE: Zinc dust is used to confirm negative reaction
▪ Moeller’s Decarboxylation test
 Principle: If organism has enzyme to decarboxylate amino acids (lysine, ornithine, arginine), the pH
increases → pH indicator changes in color
 pH indicator: bromcresol purple
 Positive result: yellow → purple
 Important in differentiating Klebsiella (-) from Enterobacter (+)
▪ Acetate utilization test
 Principle: To determine the ability of an organism to utilize sodium as sole source of carbon releasing
alkaline substance
 pH indicator: bromthymol blue
 Positive result: Blue
 (+) E. coli
▪ Malonate test
 Principle: To determine the ability of an organism to utilize malonate as sole source of carbon
 pH indicator: Bromthymol blue
 Positive result: blue color (@pH 7.6)
 (+) Enterobacter, Salmonella 2, 3a, and 3b
▪ Salmonella and Shigella Typing
 Serotyping of Salmonella spp. is based on the heat-stable somatic O antigen and heat labile flagellar “H”
antigen
 Shigella is based on somatic O antigen
 Serogroups of Shigella:
▪ Serogroup A – S. dysenteriae
▪ Serogroup B – S. flexneri
▪ Serogroup C – S. boydii
▪ Serogroup D – S. sonnei
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MICROBIOLOGY
DISEASES
UTI E. Coli (most common)

P. mirabilis (may cause renal stone)
K. pneumoniae
Enterobacter
Diarrhea Shigella, Salmonella, E. coli, Yersinia
Pneumonia K. pneumoniae (foul atrophic rhinitis; currant jelly-like sputum)
Plague Y. pestis
Neonatal meningitis E. coli (due to K1 antigen)
Mesenteric adenitis Y. pseudotuberculosis, Y. enterocolitica
HUS EHEC, Shigella
Enteric fever S. typhi
MEMBERS OF ENTEROBACTERIACEAE
Escherichia coli
▪ Colon bacillus
▪ Normal bowel flora of humans and may also inhabit female genital tract
▪ Primary marker of fecal contamination in water purification
▪ Culture:
o Flat dry, pink colonies on MAC
o B-hemolytic on BAP
o Green metallic sheen on EMB
▪ IMVIC Reaction: ++--
▪ TSIA Reaction: A/A, (+) gas, (-) H2S
▪ Virulence factor: Endotoxin, pili, K1 antigen
STRAINS OF E. coli
EPEC (Enteropathogenic E. coli) • Causes diarrhea in infants
• Pathogenesis: adhesive property → attach to intestinal epithelial
cell → loss of microvilli → cell damage → infantile diarrhea
• MOT: contaminated formula and food w/ fecal material
• Stool: watery diarrhea w/ mucus (no blood)
ETEC (Enterotoxigenic E. coli) • Traveler’s diarrhea/Montezuma’s revenge
• Pathogenesis: enterotoxins → colonization of the small intestine →
traveler’s diarrhea
• Stool: Profuse, watery (NO blood)
EIEC (Enteroinvasive E. coli) • Dysentery-like
• Common among children in areas of poor sanitation
• Pathogenesis: Invasin → penetrates and multiplies within intestinal
epithelial cells → Dysentery-like diarrhea
• Stool: Bloody diarrhea w/ mucus and WBCs
• (+) Sereny test: ability to produce keratoconjunctivitis in guinea pig
EHEC (Enterohemorrhagic E. coli) • Also known as Shiga-toxin producing- (STEC) or Verotoxin
producing- (VTEC)
• E. coli O157:H7 strain is the most common isolate of the group
(most pathogenic)
• Pathogenesis: Cytotoxin (verotoxin or shiga-like toxin) → destroy
vascular endothelial cell → bloody diarrhea → hemorrhagic colitis,
HUS, TTP, Hamburger disease
• Biochemical test: sorbitol fermentation (-), MUG (-), Intimin (+)
EAEC (Enteroadherent E. coli) • Pathogenesis: Enteroadherent → fimbriae adhere to the surface of
intestinal mucosa
• EAEC adheres to Hep2 cells forming clumps of bacteria (stacked
bricked appearance)
• Stool: watery diarrhea
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MICROBIOLOGY
Klebsiella
▪ Usually found in the GIT of humans and animals
▪ Isolated species:
 K. pneumoniae subs. pneumoniae
 K. pneumoniae subs. ozanae
 K. pneumoniae subs. rhinoscleromatis
 K. oxytoca
 K. ornithinolytica
▪ K. pneumoniae subs. pneumoniae
 Also known as Friedlander’s bacillus
 Causative agent of community acquired pneumonia
 Most common isolate
 Pink mucoid colonies in MAC
 Virulence factor: Polysaccharide capsule
 Currant Jelly-like sputum: hallmark of infection
 Differential test
▪ String test
▪ Neufeld Quellung test
 TSIA: A/A + GAS – H2S
 IMViC: - - + +
Citrobacter
▪ Species: Citrobacter freundii, C. koseri (C. diversus)
▪ Citrobacter is a common urinary pathogen seen in hospitalized patients
▪ Found in soil, water, intestinal tract of animals, and in human clinical samples.
▪ Biochemically and serologically, it resembles Salmonella
▪ Cultures: Late lactose fermenter colonies
▪ IMViC reaction:
o C. freundii: - - + +
o C. diversus: + + - +
▪ TSIA reaction:
o C. freundii: A/A, GAS + , H2S +
o C. diversus: K/A, GAS +, H2S +
▪ C. koseri
o associated with cases of neonatal meningitis and brain abscess (nursery outbreaks)
▪ C. freundii
o Causes extraintestinal diseases (e.g., endocarditis in IV drug users)
o Produces cephalosporinase → ESBL resistance
Proteus
▪ Isolated from urine, wound and ear infection
▪ May cause Acute glomerulonephritis
▪ Rapid urease produces
▪ Human pathogens: P. mirabilis and P. vulgaris
▪ Culture: Swarming phenomenon with “burnt chocolate” like or “burnt gunpowder” like odor
▪ Biochemical reaction:
o IMViC reaction:
▪ P. mirabilis: - + v v
▪ P. vulgaris: + + - v
o TSIA reaction:
▪ P. mirabilis: K/A, + Gas, + H2S
▪ P. vulgaris: K/A, +/- Gas, + H2S
Providencia
▪ P. rettgeri
o Pathogens of UTI, diarrhea among travelers
▪ P. stuartii
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MICROBIOLOGY
o Isolated from nosocomial outbreaks in burnt units

▪ P. alcalifaciens
o Commonly found in feces of children with diarrhea
▪ Biochemistry:
o IMViC reaction: + + - +
o TSIA reaction: K/A, GAS (-) , H2S (-)
Morganella
▪ Gram-negative rod commonly found in the environment and in the intestinal tracts of humans, mammals, and
reptiles as normal flora
▪ Despite its wide distribution, it is an uncommon cause of community-acquired infection and is most often
encountered in postoperative and other nosocomial settings
▪ M. morganii
o IMViC: + + - -
o TSIA Reaction: K/A, GAS (+) , H2S (-)
Plesiomonas shigelloides
▪ NOT part of the normal flora
▪ The only Oxidase positive member of the Enterobacteriaceae
▪ Culture: white to pink colonies in IBGBS agar (Inositol Brilliant Green Bile Salt agar)
▪ Apron-like colonies on CIN agar
▪ Cross reacts with Shigella
▪ Mode of acquisition: ingestion of undercooked oyster, clamps and shrimps
Shigella
▪ Inert bacillus
▪ An intracellular organism
▪ Reservoir: Humans only
▪ MOT: 4F (flies, fingers, food, fecal-oral)
▪ Specimen of choice: rectal swab
▪ Culture: clear, fragile NLF, colorless colonies
▪ Virulence factor: Shiga toxin
▪ Serology: ID based on O antigen, K antigen (heat labile)
▪ Cross-reactions: E. coli (Alkalescens dispar strain)
▪ Susceptible to disinfectants, high concentration of acids and bile, sensitive to pH
▪ Species:
o A – S. dysenteriae (most severe)
o B – S. flexneri
o C – S. boydii
o D – S. sonnei (most common in the US)
▪ Biochemical tests:
o Non-motile
o IMVIC: - - + -
o Lactose negative
o Indole negative
▪ Dysentery (Shigellosis)
 Characterized by acute inflammatory colitis and bloody diarrhea
 Highly communicable; found primarily in crowded or substandard conditions (e.g., jails, prisons, day care
centers)
 Sources: human carriers
 Symptoms: fever, chills, abdominal cramp and painful bowel movement
 Diarrhea: bloody w/ mucus and WBCs (hallmark of infection)
Salmonella
▪ Most serious pathogenic enterobacteria in humans
▪ Acquired through ingestion of contaminated food or of human carriers
▪ Inhabits the GIT of animals and gall bladder of humans (carriers)
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MICROBIOLOGY
▪ Species: S. enterica and S. bongori

▪ Subspecies of S. enterica:
o I (enterica)
o II (salamae)
o IIIa (arizonae)
o IIIb (diarizonae)
o IV (houtenae)
o V (indica)
▪ Subspecies of S. bongori:
o S. typhi
o S. paratyphi
o S. pullorum
▪ Virulence factor: Fimbriae and enterotoxin
▪ Antigenic factors: Somatic O, Flagellar H and Vi Antigen
▪ Biochemical reaction:
o All are motile EXCEPT: S. pollorum and S. gallinarum
o All are H2S producers EXCEPT: S. paratyphi A
o All are LDC positive EXCEPT: S. paratyphi A
o All are gas producers EXCEPT: S. gallinarum and S. typhi
o All are K/A, gas +, H2S + EXCEPT: S. typhi (K/A, gas -, H2S +)
o All are IMVIC - + - + EXCEPT: S. typhi (- + - -)
▪ Salmonella infection: Gastroenteritis, Enteric Fever, Bacteremia
o Specimen of choice:
 1st week → blood
 2nd week → urine
 3rd week → stool
o Gastroenteritis:
 Most common form of food poisoning
 Mostly due to S. enterica subsp. enterica
 “Peanut-butter Outbreak” → caused by Serotype typhimurium
 Causes:
→ Utensils and chopping boards
→ Inadequate refrigeration
→ Poultry and egg
 Symptoms: nausea, vomiting, fever and chills, watery diarrhea and abdominal pain
o Enteric Fever
 Also known as “typhoid fever” caused by Serotype typhi (resistant to gastric acid)
 Sources: human carriers, contaminated food and water
 Cause of outbreaks: improper disposal of sewage, poor sanitation and lack of modern water
system
 Best Specimen: BONE MARROW
 Clinical Characteristics:
→ Malaise, anorexia, myalgia and continuous frontal dull headache
→ Fever with “rose spots”
→ Necrotizing cholecystitis
o Bacteremia
 Prolonged fever and intermittent bacteremia
 Caused by typhimurium, paratyphi, cholerasuis
Yersinia pestis
▪ Also known as “Plague bacillus”
▪ Class A bioterrorism agent
▪ Facultative intracellular
▪ Transmitted by bite of an infected vector: Xenopsylla cheopis (fleas)
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MICROBIOLOGY
▪ Causative agent of bubonic plague (black death)

▪ Microscopy: short, plump, rod with bipolar staining or “safety pin” appearance
▪ Broth culture: stalactite pattern of growth
▪ Optimal growth temperature: 25-35°C
▪ Plague infection:
o MOT: close contact with a person infected and acquired thru respiratory droplets
o Bubonic fever
o Pulmonary infection
Yersinia enterocolitica
▪ Causative agent of enterocolitis or waterborne gastroenteritis
▪ Most commonly isolated species
▪ Motile at 22ºc but not at 35ºc
▪ Requires cold enrichment
▪ Culture: Bull’s eye colonies in CIN agar
▪ MOT: consumption of infected or undercooked pork, pork intestines, dairy products such as chocolate milk and
handling pets
Yersinia pseudotuberculosis
▪ Pathogen of rodents
▪ Reservoir: Farm and domestic animals
▪ Acquired thru close contact with infected animals or their fecal materials
▪ Motile at room temperature
▪ Urease (+), Rhamnose fermenter
Edwardsiella tarda
▪ Rarely cause human infection
▪ Isolated from cold blooded and warm-blooded animals
▪ IMViC: + + - -
▪ TSIA: K/A, + Gas, + H2S
Enterobacter
▪ Gelatin liquefaction test (+) EXCEPT E. aerogenes
▪ Malonate test (+) EXCEPT E. cloacae
▪ H2S (+)
▪ Sucrose (+)
▪ IMVIC: - - + +
▪ E. sakazakii causes meningitis and bacteremia (from powdered infant formula)
▪ yellow pigmented colonies; capsulated
Serratia
▪ Opportunistic pathogens associated with nosocomial outbreaks
▪ Slow or late lactose fermenter
▪ Species: S. marcescens, S. odorifera, S. rubideae and S. plymuthica
▪ S. marcescens:
o Most clinically significant species
o Bacteremic outbreaks
o Contaminant in antiseptic solutions
o IMViC: - - + +
o TSIA: K/A, + Gas, - H2S
Hafnia alvei
▪ Late lactose fermenter
▪ Delayed citrate reaction
▪ Not known to cause gastroenteritis but isolated from stool cultures
Erwinia
▪ Plant pathogen
▪ Grows poorly at 37’C and fails to grow on MAC and EMB
MYCOBACTERIA
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MICROBIOLOGY
▪ Non-spore formers, non-encapsulated, strictly aerobic organisms

▪ Catalase (+)
▪ Cell wall:  lipid content
▪ Resist decolorization
▪ Produce Much’s granules
▪ pH requirement: 6.5-6.8
▪ Generation time: >12 hours
▪ 2 Groups:
o MTC (causes tuberculosis)
o NTM (causes pulmonary infection, other than TB)
MYCOBACTERIUM TUBERCULOSIS COMPLEX (MTC)

M. tuberculosis Virulence factor: cord factor and sulfatides
Culture: cauliflower colonies; has the longest replication time among Mycobacteria (20-22 hours)
Biochemical: Catalase (+), Niacin (+) and Nitrate reduction (+)
Other tests: Inhibition by Nitroimidazopyran
M. bovis TB in cattle, dogs, cats, swine, parrots and humans

MOT: ingestion of contaminated milk
NOTE: attenuated strain is used for production of BCG vaccines
Biochemical: Niacin (-) and Nitrate reduction (-)
M. africanum TB prevalent in Africa

Spacer Oligotyping is required for diagnosis
M. canettii Smooth strain of M. tuberculosis

Isolated from AIDS patients with mesenteric tuberculosis (First human isolate from cervical lymph
node)
Biochemical: Niacin (+) and Nitrate reduction (+)
Rapid grower
Mycobacterium tuberculosis
▪ Also known as Koch bacillus
▪ Most virulent
▪ Microscopy: slender, beaded rods (X, V, Y and L forms)
▪ Culture: Slow growers, buff in color, raised and dry “Cauliflower appearance”
▪ Virulence factor → cord factor: Glycolipid molecule on the cell wall, responsible for the arrangement of M.
tuberculosis to form chains
▪ Clinical infections: MDR-TB (Multidrug-resistant TB)
o Pulmonary tuberculosis ✓ Develops when a patient on multidrug
o Pott’s disease (Tuberculosis Spondylitis) therapy fails to complete the course of
o Miliary tuberculosis medication
✓ Caused by spontaneous mutation as a
▪ Extrapulmonary form of TB
result of inappropriate use and lack of
▪ Seeding of organism through various organs compliance
Mycobacterium bovis
▪ TB in cattle, swine and domestic animals
▪ Attenuated strains are used as vaccines for TB for newborns
▪ Slow growers with white nonpigmented colonies
Mycobacterium canettii
▪ Known to be the smooth strain of M. tuberculosis-colonies
▪ reduces nitrate to nitrite
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MICROBIOLOGY
▪ Grows rapidly than M. tuberculosis (6 days on solid media)

▪ First human isolate was from a cervical lymph node
NONTUBERCULOUS MYCOBACTERIA
▪ AKA anonymous, atypical, unclassified and MOTT (Mycobacteria other than tuberculosis)
▪ Widely distributed: Soil, water, even in healthy individuals
▪ Causes chronic pulmonary disease that may resemble TB
▪ Immune status of host is a contributing factor, AIDS contributes to the incidence of infection
▪ Can be classified by using the Runyon’s Classification
Mycobacterium avium complex (MAC)
▪ Most common NTM causing tuberculosis
▪ Pulmonary infection is similar to MTB but usually self-limiting
▪ Saprophyte: present in soil, water and house dust
▪ Portal of entry: respiratory tract and GIT (most common site of colonization and dissemination among AIDS
patients)
▪ Specimen: Sputum, blood, and bone marrow aspirate
▪ Biochemical test:
o (+) Heat-stable catalase
o (+) Thiophene-2-carboxylic hydrazide test (T2H test)
▪ M. avium → disease of poultry and swine
▪ M. avium subsp. paratuberculosis → Agent of Johne’s disease
NOTE: Johne's disease is a contagious, chronic and sometimes fatal infection that primarily affects the small intestine of ruminants
Mycobacterium kansasii
▪ Also known as “Yellow Bacillus”
▪ Second to M. avium complex as a cause of NTM lung disease
▪ NOT contagious from person to person
▪ Microscopy: appears as long rods with crossbanding resembling a shepherd’s crook
▪ Biochemical:
o (+) Tween 80 hydrolysis
o Strong nitrate reducer
Mycobacterium marinum
▪ Causes disease in fish → isolated from aquariums

▪ Causes swimming pool granuloma in humans → tender red subcutaneous nodules in traumatized skin that made
contact with inadequately chlorinated freshwater or salt water
▪ MOT: direct inoculation
▪ Culture: rough and wrinkled deep blue colonies (photochromogen)
▪ Prefers low temperature (28-32)
▪ Biochemical:
o (+) Urease
o (+) PYRase
Mycobacterium ulcerans
▪ 3rd most common mycobacterium species after MTB and M leprae
▪ A rare causative agent of Buruli ulcer → painless nodule under the skin after previous trauma
▪ Culture: non-pigmented smooth to rough colonies (6-12 weeks incubation)
▪ Biochemical: Heat stable catalase (+)
Mycobacterium gordonae
▪ Also known as “tap water bacillus”
▪ Can be grown in Ogawa medium: potassium, sodium glutamate, homogenized whole egg and malachite green
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MICROBIOLOGY
▪ Rarely cause infection to humans

▪ Biochemical:
o (+) Heat stable catalase
Mycobacterium xenopi
▪ Recovered from hot and cold-water taps, especially those water storage tanks
▪ First isolated from an African toad
▪ Potential pathogen → may cause pulmonary infection in adults
▪ Culture: young colonies: exhibits stick line projections resembling a “Bird’s nest appearance”
▪ Grows well at 42’C
▪ Biochemical:
o (+) PYRase
o (+) Heat stable catalase
o (+) Arylsulfatase
Mycobacterium terrae complex

▪ normal saprophytes and rarely cause infections to humans
▪ Biochemical: Tween 80 hydrolysis (+) and Heat stable catalase (+)
▪ M. trivial – rough and dry colonies
▪ M. terrae – Smooth colonies
▪ M. nonchromogenicum – smooth to rough and white to buff colonies
RUNYOUN’S CLASSIFICATION OF NTM
CLASS SPECIES COLOR PRODUCED GROWTH RATE

“MASK” NOT pigmented unless
exposed to light
GROUP I M. marinum 10-21 days
M. asiaticum Colonies:
M. kansasii cream/buff/orange/yellow
“XGFSS”
M. xenopi Pigmented in BOTH dark

GROUP II M. gordonae 10-21 days
and light
M. flavescens
M. szulgai
M. scrofulaceum
“GATHU”
M. gastri NON-PIGMENTED in BOTH

GROUP III M. avium complex 10-21 days
dark and light
M. terrae complex
M. haemophilum
M. ulcerans
“FSCP”
RAPID GROWERS M. fortuitum 3-7 days

M. smegmatis
M. chelonei
M. phlei
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MICROBIOLOGY
NTM: RAPID GROWERS

▪ Potential pathogens
▪ Saprophytic
▪ Enters the host thru skin lesions
▪ Culture: Colonies appear on solid media in 7 days or less
▪ Common isolates:
o M. fortuitum → most common rapid-growing mycobacteria; causes localized cutaneous infection
o M. chelonei → causes disseminated cutaneous infection among immunocompromised patients;
resistant to multiple antibiotics
o M. smegmatis → causes pulmonary, skin, soft tissue, and bone infection
✓ Incubation: 35°C in the dark with 5-10% CO2
✓ 30°C – 32°C : M. marinum and M. ulcerans
✓ 42°C: M. xenopi
✓ Must be examined weekly
✓ Malachite green → must be added to inhibit the growth of NTM
Culture Media
1. Solid-based media
a. Middlebrook – 2% glycerol which enhances growth of MAC IMPORTANT NOTE:
b. Middlebrook 7H10 – With carbanecillin for inhibition of
combination of solid-based media and
pseudomonads, polymyxin B, trimethoprim lactate and
a liquid-based medium is
amphotericin B recommended for primary isolation
c. Middlebrook 7H11 – 0.1% enzymatic hydrolysate of casein
which improves recovery of isoniazid-resistant MTB
2. Egg-based media → infused w/ fresh whole eggs, potato flour, glycerol, milk, potato and malachite green
a. Lowenstein Jensen Agar
b. L-J Gruft (w/ penicillin and nalidixic acid)
c. L-J Mycobactosel (w/ cycloheximide, lincomycin, and nalidixic acid)
d. L-J w/ Pyruvic acid
e. L-J w/ Glycerol
f. Petragnani medium
g. Wallenstein medium
h. American Thoracic Society Medium
3. Liquid/broth media
a. BACTEC 12B and BACTEC 13B
b. Middlebrook 7H9
Specimen
1. Sputum
 Specimen of choice
 Early morning collection (3 consecutive days; 1 day → DOH recommendation)
 5-10 mL
 Criteria for culture: <10 epithelial cells with >25 PMNs
2. Gastric lavage
 Recovers mycobacteria swallowed during the night
 Requirements: 3 specimens within 3 days after overnight fast
 20-25 mL of gastric secretion
 Process within 4 hours of collection; neutralized with Na2CO3
3. Urine
 First morning midstream clean catch (3 consecutive days)
 15 mL
 Indwelling catheters
4. Bronchial washing
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MICROBIOLOGY
 specimen of choice for NTM; brushing is more diagnostic than washing or biopsy
5. Fecal specimen
 for detection of MAC; no preservatives; freeze if delayed
6. Body fluids
 CSF: 2-5 mL
 Abdominal and Chest fluids: 10-15 mL
 EDTA/Heparin – used to liquefy clotted specimens
7. Blood
 For individuals with AIDS (BACTEC 13A)
Decontamination and Digestion

▪ Purpose: to kill contaminating organisms and dissolve mucous substances and enable the bacteria to use the
nutrients present in the medium
▪ Factors to consider: exposure time & concentration
 Specimen for decontamination – sputum, urine, tissue, abdominal fluid, exudates
 Decontamination and digestion – sputum, gastric washing, BAL, bronchial washing, tracheal aspirate
 No need for decontamination – CSF, synovial fluid, deep organ biopsy
▪ Reagents:
o NALC-NaOH (2-4%) – decontamination and digestant (ratio is Purpose of NALC: __________________
1:1)
o Oxalic acid (5%) – for sputum containing Pseudomonas Purpose of NaOH: _________________
o Zephiran-trisodium PO4 – decontamination and digestant w/

buffer
o Cetyl-Pyridium Chloride (1%) – prolongs the shelf-life of sputum
STAINING
▪ General rule:
✓ 2 out of 3 sample must be positive to confirm diagnosis
✓ If none or only the first specimen is positive, additional sample must be added
✓ 300 fields are required to examine before ruling out a negative diagnosis
✓ 5,000-10,000 organisms/mL –needed for positive culture
▪ AFB Staining method:
o Ziehl-Neelsen
o Kinyoun method
o Auramine-Rhodamine – more sensitive (bright yellow-orange against dark background)
GRADING AND REPORTING OF AFB
Ziehl-Neelsen/Kinyoun Method Fluorochrome Method Quantitative Report

0 0 No AFB seen
1-2/300 fields 1-2/70 fields Doubtful; Recollect
1-9/100 fields 1-2/70 fields 1+
1-9/10 fields 2-18/50 fields 2+
1-9/field 4-36/field 3+
>9/field >36/field 4+
SUMMARY OF BIOCHEMICAL TESTS
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MICROBIOLOGY
TESTS PRINCIPLE ORGANISM
30% H2O2 @68°C M. avium complex

M. ulcerans
Positive: >44 mm column of bubbles M. gordonae
Heat-Stable Catalase
M. xenopi
M. terrae
MTB (-)
Niacin test Detects deficiency of niacin- MTB

connecting enzyme that converts M. bovis (-)
free niacin to niacin ribonucleotide M. canettii
Reagents: cyanogen bromide +

aniline
Positive: Yellow color
Nitrate reduction Nitrates → (nitroreductase) → MTB

nitrite M. kansasii
M. szulgai
Reagents: sulfanilamide + n- M. fortuitum
naphthylenediamine M. smegmatis
dihydrochloride + zinc M. canettii
Positive: Red color
Breaks down phenolphthalein M. fortuitum

disulfate into free phenolphthalein M. chelonae
Reagents: potassium M. xenopi
Arylsulfatase test
phenolphthalein sulfate + sodium M. triviale
bicarbonate M. marinum
Positive: Pink color M. szulgai
Tween 80 Hydrolysis Separate nonphotochromogens M. gordonae

from scotochromogens M. terrae
Detects the ability of lipase to
hydrolyze Tween 80 into oleic acid
and polyoxyethylated sorbitol
pH Indicator: neutral red
Positive: Pink
T2H inhibition Also known as Thiophene-2- M. bovis = no growth

carboxylic hydrazide test MTB = growth (+)
Used to differentiate M. bovis and
MTB
Ability of MAC to reduce tellurite in MAC

3-4 days NTM Rapid growers
Tellurite reduction test
Reagent: Potassium tellurite
Positive: Black metallic
PYRase Pyrazinamide → PYRase → M. kansasii

Pyrazinoic acid + ammonia M. marinum
Reagent: ferrous ammonium sulfate M. xenopi
Positive: red pigment
Iron uptake Used to differentiate M. smegmatis M. smegmatis (+)

and M. chelonae
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MICROBIOLOGY
Positive: rusty brown colonies M. chelonae (-)
Urease 4 mL Urea @ 37’C → pink color M. scrofulaceum (+)

Used to differentiate M. M. gordonae (-)
scrofulaceum and M. gordonae M. marinum
NAT Most rapid

Requires only one colony
NON-CULTIVATABLE NTM
Mycobacterium leprae (Hansen’s bacillus)

▪ Invades peripheral nerve and skin cells
▪ Mostly found on SCHWANN Cells
▪ Microscopy: Cigar pocket or pocket fence appearance
▪ Culture: Only grow biological living tissue media
▪ Clinical Infection:
o Hansen’s disease/Leprosy
 MOT:
• Person to person thru inhalation of nasal secretions
• Contact with infected skin
• Breast feeding
• Vertical transfer
 2 FORMS:
• Tuberculoid/Localized leprosy
− Benign form; Nonprogressive disease
− Associated with delayed type hypersensitivity
− S/S: Skin lesion, damaged nerves
− Positive for Lepromin test
• Lepromatous/Disseminated Leprosy
− Malignant form and slowly progressive
− S/S: loss or deformities of facial features and fingers, toes and anatomical
structure
− Negative for Lepromin test
 Laboratory Diagnosis:
• Specimen: nasal/mucosal smear and skin nips
• Serologic testing: ELISA, DNA amplification
• Definitive test: Inoculation of specimen to footpads of armadillo
• Specimen: Biopsy material
• Skin test: Fernandez and Mitsuda test
• Biochemical: Heat stable catalase test (+)
GRAM POSITIVE BACILLI
AEROBIC GRAM-POSITIVE BACILLI

BACILLUS
▪ Sporeforming
▪ Aerobic or facultative anaerobic organism
▪ Motile EXCEPT: B. anthracis
▪ Can survive in varying temperature and extreme environment
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MICROBIOLOGY
▪ Catalase (+), glucose fermenters, starch hydrolyzers

▪ Microscopy: large, box-car appearance, gram-positive rods
Bacillus anthracis
▪ Also known as Anthrax bacillus
▪ NOT a normal flora
▪ Bioterrorism agent
▪ Non-motile and halophilic organism
▪ Microscopy:
o Gram-positive large encapsulated and square-ended rod
o Centrally-located spore
o Resembles bamboo fishing pole
▪ Culture (BAP)
o Non-hemolytic
o Flat irregular with swirling projections
o Medusa head appearance or “beaten egg white appearance
▪ Penicillin Susceptibility Test:
o B. anthracis → susceptible → forms “string of pearls”
o D-glutamic acid capsule
o Exotoxin → edema factor, protective antigen, lethal factor
▪ PA + EF = EDEMA
▪ PA + LF = DEATH
▪ Clinical Infection
o Cutaneous anthrax
o Pulmonary anthrax (Woolsorter’s disease)
o GI Anthrax
Laboratory Diagnosis
▪ Specimen:
o Malignant sputules
o Sputum
o Blood
Bacillus cereus
▪ Also known as Fried Rice Bacillus
▪ Causes food poisoning (from ingestion of contaminated rice)
▪ Can be part of the normal fecal biota
▪ Most commonly encountered Bacillus in opportunistic infection
▪ Motile and resistant to penicillin
▪ Culture on BAP:
o Large, feathery, spreading and frosted glass colonies
o Beta hemolysis
▪ Virulence factor: enterotoxins
▪ Clinical Infections:
o Emetic type
▪ Associated with ingestion of improperly stored fried rice and reheated rice
▪ Short incubation
▪ S/S: abdominal cramps and profuse vomiting
▪ Production of heat stable enterotoxin
o Diarrheal type
▪ Ingestion of contaminated meat and pultry and vegetable products
▪ S/S: abdominal pain, watery dirrhea without fever
▪ Produces heat labile enterotoxin
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MICROBIOLOGY
Bacillus subtilis
▪ Also known as Hay Bacillus
▪ Halophilic organism
▪ Primary source of bacitracin
▪ Culture: large, flat, dull with ground glass appearance, maybe beta hemolytic and may be pigmented
CORYNEBACTERIUM
▪ Aerobic non-spore forming gram-positive bacilli

▪ Normal flora of the skin and mucous membranes of animals
▪ Non motile, non-spore forming and non-encapsulated
▪ Closely related to Mycobacterium and Nocardia
▪ Club-shaped under the microscope
▪ Catalase and oxidase positive
▪ Nitrate reducer
Corynebacterium diphtheriae
▪ Also known as Kleb-Loeffler’s bacillus
▪ Pleomorphic rods
▪ Inhabits the nasopharynx
▪ Enriched medium is recommended (w/ cystine, serum, and K tellurite)
▪ Heat labile
▪ Virulence factor: Diptheria toxin
▪ Clinical infection:
o Diphtheria
▪ Respiratory → pharyngitis and tissue necrosis (pseudomembranous formation)
▪ Cutaneous (Velat sore/Barcoo rot) → Slow healing ulcer and membrane formation
▪ Laboratory Diagnosis:
o Gram stain
▪ Highly pleomorphic slightly curved rods with rounded ends
▪ “Chinese letter” formation appearance
▪ May appear as club shaped swelling and beaded form
o Culture
▪ BAP → small, granular with irregular edge and may have small zone of hemolysis
▪ CTBA (Cystine Tellurite Blood Agar)
→ preferred for identification of diptheria (selective and differential for tellurite reduction)
→ contains sheep’s blood agar, bovine serum, cystine and potassium tellurite
→ (+) Black brown colonies with brown halos
→ 3 colonial types:
o Mitis → small black colonies with gray periphery, bleach like odor
o Intermedius → small, flat, dry grayish black colonies
o Gravis → large dark gray, daisy-like colonies
o Schick test
▪ Immunological test → injection of toxin
▪ (+) redness in 2-4 days
▪ No reaction = protective; immunity present
Other Coryneform bacteria
Nonlipophilic Lipophilic
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MICROBIOLOGY
C. ulcerans C. jeikeium
C. pseudotuberculosis C. urealyticum
C. xerosis
C. striatum
C. amycolatum
C. auris
C. pseudodiphtheriticum
C. glucuronolyticum
LISTERIA
Listeria monocytogenes
▪ Important cause of wide spectrum of disease in humans and animals

▪ Can survive in wide range of environments NOTE:
▪ Important foodborne pathogens ✓ tumbling motility can be demonstrated
▪ Short gram-positive, non-spore forming rod only at temperatures of 22°C -28°C but
NOT at 37°C
▪ Catalase positive
✓ This motility differentiates it from
▪ Has a tumbling end-over-end motility diphtheroids
Culture and Growth Requirements
▪ Grows well in 5% sheep’s blood agar → exhibits small zone of hemolysis

▪ Catalase (+) and esculin hydrolysis (+)
▪ Produces acid but not gas
▪ Inverted Christmas tree or inverted umbrella pattern on tube media
▪ Primary Virulence: Listeriolysin O
Clinical Infection
▪ 2 Forms of Listeriosis:
o Granulomatosis infantiseptica
▪ Neonatal sepsis
▪ Pustular lesion
▪ Granulomas in multiple organs
o Listeria meningoencephalitis in adults
ERYSIPELOTHRIX
Erysipelothrix rhusiopathiae
▪ Produce small transparent glistening colonies in BAP

▪ May look like gram negative on staining (decolorize easily)
▪ Appear singly, in short chains or long branching filamentous
▪ Catalase, oxidase and indole negative
▪ H2S positive
▪ Clinical Infection:
o Erysipeloid
o Occurs in fingers by direct inoculation into cuts or abrasions “Seal finger, Whale finger”
ANAEROBIC GRAM-POSITIVE BACILLI
GENERAL INDICATIONS
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MICROBIOLOGY
▪ Foul odor upon opening of an Anaerobic Jar

▪ Presence of sulfur granules
▪ Brick red fluorescence
▪ Growth of anaerobic culture plates
▪ Double zone of hemolysis
CLOSTRIDIUM
▪ Exogenous anaerobic infections

▪ MOT: ingestion and via open wounds
▪ Obligate anaerobic, Gram-positive spore forming bacilli
▪ Catalase positive
▪ Contributing virulence: Collagenase, Hyaluronidase, Lecithinase or Phospholipase (Cell destruction)
Clostridium perfringens
▪ Also known as Gas Gangrene Bacillus

▪ Formerly known as Clostridium welchii
▪ Most commonly isolated Clostridia specie in blood culture
o Deoxyribonuclease → lowers viscosity of exudates → mobility of bacteria
o a-toxin → lecithinase, phospholipase → lysis of WBC, RBC, platelets → necrosis of tissue
o enterotoxin
▪ Microscopy:
o Box-car shaped bacilli
o Central to subterminal spore
▪ Culture:
o BAP:
▪ Dome-shaped, gray to white colonies
▪ Double zone of hemolysis
o Litmus milk medium
▪ Stormy fermentation of milk
▪ Biochemical testing:
o (+) Lecithinase
o (+) Nagler test
o (+) Reverese CAMP test
▪ Clinical infections:
o Gas Gangrene (Myonecrosis)
− Life threatening destruction of muscle and other tissues
− It is caused by a-toxin and lecithinase
− Acquired through wound contamination (frost-bite, surgery, trauma)
− Accompanied by bullae, pain, swelling and serous discharges
− Treatment: Hyperbaric oxygen therapy
o Food poisoning (Enteritis necrotans)
− Ingestion of enterotoxin
− 2 types:
• Type A food poisoning → mild and self-limiting
• Type C food poisoning → more serious but are rarely encountered in humans
Clostridium tetani
▪ Soil and environmental inhabitants

▪ Microscopy:
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MICROBIOLOGY
o Terminal spore and swollen sporangia

o Drumstick or tennis racket appearance
▪ Biochemical testing
o Motile, geletinase and indole +; Lecithenase and lipase –
▪ Virulence factor: Tetanospasmin
▪ Clinical infection:
o Tetanus
− Characterized by trismus and risus sardonicus
− Symptoms:
• Mascular rigidity
• Difficulty swollowing
• Rigidity of the abdomen
− Tetanus neonatorum:
• Contaminated instruments used for newborns
Clostridium botulinum
▪ Also known as Canned goods bacillus
▪ Potential agent of bioterrorism
▪ Characterized by the presence of subterminal spore
▪ Virulence factor
o Botulism toxin
▪ Clinical significance
o Foodborne botulism
o Infant botulism
Clostridium difficile
▪ The most common cause of antibiotic associated diarrhea and pseudomembranous colitis
▪ Virulence factor
o Toxin A and Toxin B
▪ Microscopy
o Endospores maybe oval and subterminal
▪ Culture
o Yellow ground glass colonies on CCFA
GUIDELINES FOR SPECIMEN COLLECTION AND TRANSPORT

✓ Specimen
▪ Blood, CSF and abscess
✓ The specimen must be taken from the actual site of infection
✓ Food and fecal specimen for C. perfringens isolation should be stored at 4°C
✓ Should be transported under anaerobic conditions
NON-SPORE FORMING ANAEROBIC BACILLI
ORGANISM CHARACTERISTIC
Bacteroides fragilis Intra-abdomenal abscesses
Microscopy: pleomorphic with capsule
Actinomyces israelii Causative agent of Actinomycosis
Microbiota of the oral cavity
Propionibacterium acnes Causes contamination in phlebotomy procedures
Lactobacillus acidophilus Normal flora of the mouth, GIT and vaginal canal
Protects host from urogenital infection
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MICROBIOLOGY
NONFERMENTATIVE BACILLI
OXIDASE POSITIVE, GROWS ON MAC

▪ Opportunistic pathogens that thrives in the environment such as in medical devices, soil, and water
▪ NOT normally found in the normal flora of the human body
▪ Isolated from nebulizers, saline catheters, and other hospital devices
▪ Resistant to chlorhexidine and quaternary ammonium compounds (QATS)
BIOCHEMICAL CHARACTERISTICS
▪ All are motile EXCEPT: B. mallei (glanders disease)
▪ Non-lactose fermenters
▪ TSIA: K/K H2S(-)
▪ Oxidase (+)
▪ Gram stain
 Appearance: medium-sized, straight rods
 Bipolar staining observed in P. pseudomallei
▪ NAT Testing (Nucleic acid)
 Rapid screening for evaluation of rapid outbreak or epidemiologic studies
 PCR for 16s rRNA, heat shock protein and exotoxin A
▪ Culture
 5% SBA
 CAP
 Broth-blood culture
 Thioglycollate
 Brain-heart infusion
 Selective media:
 B. cepacia
• Pseudomonas cepacian agar
• Oxidative-Fermentation-base Polymyxin Bacitracin Lactose (OFPBL)
 B. pseudomallei
• Ashdown medium
▪ Biochemical Tests
 Hugh and Leifson Test
 Purpose: to determine if an organism is assacharolytic, fermentative, or oxidative
 Reagents: 1% CHO + bromthymol blue (indicator) yellow w/ acid production
 Setup:
• Tube 1: covered with oil
• Tube 2: not covered with oil
 Results:
• Assacharolytic: green in both tubes
• Fermentative: yellow in both tubes
• Oxidative: Yellow tube 1; green tube 2
 Oxidase test
POSITIVE NEGATIVE
Pseudomonas species Acinetobacter
Alcaligenes Stenotrophomonas
Burkholderia
Chryseobacterium
▪ Pigment production
 P. aeruginosa (may not be exhibited by mucoid strains from patients with cystic fibrosis)
 Pyoverdin (yellow-green/yellow-brown)
 Pyocyanin (blue)
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MICROBIOLOGY
 Pyorubin (red)
 Pyomelanin (brown)
 B. cepacia: yellow to yellow-green
 Chryseobacterium: yellow
 Stenotrophomonas: brown pigment on BHIA w/ tyrosine; lavender green-light purple in MAC
▪ Mannitol fermentation
 Positive: P. aeruginosa
 Negative: P. putida
▪ Nitrate reduction
 Principle: nitrate → (nitrate reductase) → nitrite
 Positive: P. aeruginosa
 Negative: P. putida, Acinetobacter
▪ Growth at 42’C
 Positive: P. aeruginosa, P. stutzeri
 Negative: P. putida, Stenotrophomonas
▪ Motility
 Motile: Pseudomonas, Alcaligenes, Stenotrophomonas, Burkholderia
 Non-motile: Acinetobacter, Chryseobacterium
▪ Growth on MAC
 NO growth: Chryseobacterium, M. lacunata
 NLF: Pseudomonas
▪ Cetrimide agar
 Principle: to determine the ability of organism to grow in the presence of cetrimide
 Cetrimide: a toxic substance that inhibits the growth of bacteria by release of nitrogen and phosphorous
(P. aeruginosa is resistant)
 Enhances pigment production
 Also allows the growth of P. fluorescens
▪ AST (Antimicrobial Susceptibility)
 P. aeruginosa: susceptible to Aminoglycosides and polymyxin
 NOTE: AST is necessary for every clinical isolate
▪ Fluorescence
 Positive: P. aeruginosa, P. fluorescens, P. putida, P. veronii, P. motenlii
Pseudomonas aeruginosa
▪ Most commonly isolated non-fermentative bacilli
▪ Causative agent of “blue pus” → due to pyoverdin pigment
▪ Causative agent of swimmer’s ears
▪ Leading cause of nosocomial respiratory tract infection
▪ It can exist in hot tubs and swimming pools
▪ Mode of acquisition: ingestion of contaminated food or water; contaminated medical device; penetration of
open wounds
▪ Colonies: flat spreading pigmented metallic colonies, beta-hemolytic with grape or tortilla like odor (due to
alginate) on BAP
▪ Resistant: resistant to QUATS and aminoglycosides, survive in chlorinated water, hot tubs, contact lens,
disinfectant, whirlpools
o Exotoxin A – kills host cells by protein synthesis inhibition
o Pili for attachment to host cells
o Proteolytic enzymes: lecithinase, elastase, protease
o Alginate: polysaccharide polymer that inhibits phagocytosis
o Pyocyanin: Damage cells by producing reactive oxygen species
o Hemolysin
o Capsule
o Catalase
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MICROBIOLOGY
▪ Spectrum of Disease:
o Cystic fibrosis
o Nosocomial infections
o Ecthyma gangrenosum
▪ Organism invade and destroy the walls of subcutaneous blood vessels
▪ Formation of cutaneous papules that become black and necrotic
o Swimmer’s ear (acute otitis externa) → a painful condition resulting from inflammation, irritation, and
infection
o Malignant otitis externa
▪ Seen in patients with diabetes
▪ Severe infection of external ear canal that may progress to nerves and skull
o Others:
▪ Hot tub or Jacuzzi syndrome (necrotizing skin rash)
▪ Nail bed infection
▪ 3rd most common cause of gram-negative dysentery
▪ Transfusion-associated septicemia (growth at 4’C)
▪ Biochemical Tests
 Catalase and Oxidase (+)
 Obligate aerobes, non-spore forming, motile (w/ monotrichous flagella)
 Can grow in a wide range of temperature (4°C and 42°C)
 Gluconate production (+)
 Arginine dihydrolase (+)
 Citrate utilization (+)
 Can be distinguished from other Pseudomonas species by its ability to grow at 42°C
 Acetamide Utilization Test (+)
▪ Based on utilization of acetamide as sole source of carbon
▪ Positive result: change in color (green → blue)
▪ Culture Media:
o BAP (bluish green to brown colonies)
o CAP
o MAC (yellow colonies; NLF)
o Seller’s medium
o Cetrimide agar – enhance pigment production (pyocyanin and pyoverdin)
o Irgasan
o C390
o TSIA = K/A reaction
▪ Other Pseudomonas species:
o P. stutzeri
o P. fluorescens
o P. aeruginosa, P. fluorescens, P. putida, P. veronii, P. monteilli, P. mossellii
Burkholderia pseudomallei
▪ The only pathogenic Burkholderia species
▪ Causative agent of Melloidosis
 Glander’s-like disease
 Vietnamese time bomb
 Ranges from asymptomatic to severe
 MOT: inhalation or direct inoculation
 Non-motile
 Bipolar staining
 Colonies: wrinkled dry colonies with earthy odor
 Ashdown medium w/ colistin
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MICROBIOLOGY
 Diagnosis: Serological tests

Burkholderia cepacia
▪ Causes low-grade nosocomial infection due to ability to resist antibiotics and survive in hospital environment
▪ NOTE: should be suspected whenever a non-fermentative organism decarboxylate lysine
▪ If isolated in a patient with cystic fibrosis, confirm it with phenotypic and genotypic methods
▪ Culture: yellow to yellow-green colonies
▪ Biochemical reactions:
o Oxidase (+)
o LDC (+)
o ONPG (+)
Burkholderia mallei
▪ Causative agent of Glander’s disease/Farcy disease
▪ Causes severe infection in horses and donkeys; rarely associated with humans
▪ Bioterrorism agent
▪ MOT: via inhalation or direct inoculation through skin or mucous, close animal contact
▪ Non-motile, coccobacillus, nonpigmented
OXIDASE NEGATIVE, GROWS ON MAC
Acinetobacter
▪ A member of the family Moraxellaceae
▪ Isolated from hospital equipments
▪ 3rd most frequently isolated gram-negative bacilli in clinical specimens
▪ Related infections: UTI, pneumonia, endocarditis and meningitis
▪ Isolated from catheters, humidifiers, and ventilators
▪ Optimum growth at 30°C – 35°C at pH 5.5-6.5
A. baumanii
 Water organism and preferentially colonizes aquatic environments
 This organism is often cultured from hospitalized patients' sputum or respiratory secretions, wounds,
and urine
Laboratory Diagnosis:
✓ Gram stain: plump coccobacilli that tend to resist alcohol
decolorization → may appear gram (+)
✓ Purplish colonies on MAC
✓ Gummy colonies on BAP
✓ Catalase (+), Oxidase (+)
✓ Non-motile
✓ Saccharolytic group: A. baumanii
✓ Assaccharolytic group: A. lwoffi
✓ Beta-hemolytic strains: A. haemolyticus
Stenotrophomonas maltophilia
▪ 4th most commonly isolated gram-negative bacilli in clinical specimen
▪ Clinical significance:
o High mortality rates in debilitated patients
o Endocarditis, bacteremia, wound infection
▪ Strictly aerobic, grows at 42°C
▪ Catalase (+), Esculin (+), Gelatin hydrolysis (+)
▪ DNAse (+), LDC (+), ONPG (+)
▪ Culture:
o Lavender-green to light purple colonies
o Odor: Ammoniacal
o Brown pigment in BHIA with tyrosine
▪ AST: multiple resistance to antibiotics
▪ Therapy of choice: trimethoprim-sulfamethoxazole
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MICROBIOLOGY
NON-FERMENTATIVE, DOESN’T GROW ON MAC

Alcaligenes faecalis
▪ Obligate aerobe
▪ Motile (peritrichous flagella)
▪ Isolated in soil, water, moist hospital environment, medical devices, and solutions
▪ Fruity odor colonies resembling apples or strawberries
▪ 6.5% NaCl (+)
▪ Oxidase (+)
Chryseobacterium
▪ Non-motile
▪ Pigmented colonies (yellow)
▪ Fails to grow on MAC
▪ Oxidase (+)
▪ Esculin hydrolysis (+), ONPG (+), DNAse (+)
▪ AST Profile: resistance to penicillin and polymyxin
▪ Species:
o C. indologenes: most common isolate; indole (+) after xylene extraction
o C. meningosepticum: nosocomial; causes meningitis and pneumonia
Moraxella lacunata
▪ Also known as Morax-Axemfeld bacillus
▪ Causative agent of blephanoconjunctivitis or Angular conjunctivitis
▪ Normal flora of mucous membrane, nose, throat, URT, conjunctiva
▪ Produces small colonies that pit the agar
▪ Catalase (+), Oxidase (+)
▪ Non-motile
▪ Strict aerobe
OTHERS
▪ Shewanella
o S. putrefasciens
o The only non-fermentative gram-negative bacilli that produces H2S
▪ Chromobacterium violaceum
o Opportunistic pathogen in immunocompromised patients with neutrophil defect
o Fermentative
o Oxidase (+), motile, grows at 42’C
o Produces violacein pigment
▪ Oligella
o Colonizes distal urethra
o Non-saccharolytic
GRAM NEGATIVE NON-ENTERIC GIT PATHOGEN

▪ Gram stain: Curved or comma-shaped
▪ Culture:
o Requires 0.5% NaCl EXCEPT: V. cholerae, V. mimicus
o TCBS (Thiosulfate Citrate Bile salt Sucrose)
 Sucrose fermenters:
→ yellow colonies on TCBS
→ V. cholerae, V. alginolyticus, V. metschnikovii
 Non-sucrose fermenters:
→ green colonies on TCBS
→ V. mimicus, V. vulnificus, V. parahemolyticus
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MICROBIOLOGY
o APW (Alkaline Peptone Water Broth)

 1% NaCl; pH 8.5
▪ String Test
 0.5% sodium desoxycholate
 Differentiates Vibrio from Aeromonas
 Positive result: lysis of Vibrio → releases DNA which can be pulled up into a string
▪ Vibriostatic (Susceptibility Test)
 Use of 150 ug of Vibriostatic 0/129
 Susceptible: Vibrio and V. cholerae 01
 Resistant: Aeromonas and non-01 V. cholerae
▪ Serology
 V. cholerae non-01 phenotypically resembles V. cholerae but fail to agglutinate 01 antisera
 V. parahemolyticus can be serotyped by O and K antigen
VIBRIO
▪ Not part of the normal flora
▪ Found in brackish, marine, or saltwater
▪ Isolated from algae, plankton, fish, and shellfish
▪ Temperature sensitive (>20°C)
▪ Some are halophilic EXCEPT V. cholerae and V. mimicus
▪ Facultative anaerobic
▪ All species are NLF on MAC except V. vulnificus
▪ Mode of acquisition: consumption of raw or undercooked seafood
▪ Infectious disease: cholera, wound infections, septicemia, and necrotizing fasciitis
▪ Biochemical testing:
o All are oxidase (+) and nitrate reducers EXCEPT V. metchnikovii
o All are glucose fermenters
V. cholerae
▪ Causative agent of cholera
▪ Pathogenesis:
 Spread mainly through water
 Acquired by ingestion of improperly cooked seafood
 Hallmark: rice watery stool
▪ Motility: Rapid darting or shooting star
▪ Virulence factor: Choleragen 2
▪ Taxonomy:
o V. cholerae can be divided into V. cholerae 01 and V. cholerae non-01
o V. cholerae serotypes:
 Ogawa
 Inaba
 Hikojima
o V. cholerae biotypes:
 El tor
 Classical
V. parahemolyticus
▪ Second most important specie of the genus
▪ Causes gastroenteritis
▪ Etiologic agent of “summer diarrhea” in Japan
▪ Mode of acquisition: eating of contaminated seafood like oyster, scallops, crabs, lobsters and shrimps and
sardines
▪ Virulence: heat-stable hemolysin
▪ Pathogenecity: Kanagawa phenomenon
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MICROBIOLOGY
▪ Selective medium: Wagatsuma agar

▪ LDC (+)
V. vulnificus
▪ Second to V. cholerae in terms of producing serious type of vibrio associated infection
▪ Can cause potentially fatal necrotizing fasciitis
▪ Infections: primary septicemia and wound infection
▪ Mode of infections: eating raw oysters and fish and open wound infection to contaminated seas water
▪ Healthy individuals may produce symptoms after 16 hours of ingestion
▪ Symptoms: vomiting, diarrhea and abdominal pain
▪ Halophilic
▪ Lactose positive Vibrio specie
▪ LDC (+)
V. alginolyticus
▪ Least pathogenic vibrio for humans
▪ Strict halophile organism (1-10% NaCl)
▪ An occupational hazard organism
▪ Infections: Eye, ear and wound infections
AEROMONAS
▪ Associated with disease among warm- and cold-blooded animals BIOCHEMISTRY:
including fish, reptiles and amphibians ✓ Facultative anaerobe
▪ Found in fresh water or chlorinated water, and can be isolated from ✓ Oxidase (+), Catalase (+), DNAse (+)
meat products ✓ Beta-hemolytic
▪ NOT part of the normal flora ✓ Motile
▪ Causative agent of “red-leg disease” ✓ Glucose and Lactose fermenter
✓ Inositol fermentation (+)
▪ Disease of frogs
✓ Bulls-eye colonies on CIN
▪ Causative agent of cellulitis, wound infections, and nebulous ✓ Growth at 4°C to 42°C
syndrome
▪ 2 Groups:
o Mesophilic:
 A. hydrophilia complex (water-loving; causes gastroenteritis and cellulitis)
 A. veronii complex
 A. caviae complex (most common isolate)
o Psychrophilic
 A. salmonicida (fish pathogen)
• Non-motile
• Growth at 22-25’C
AEROMONAS vs. VIBRIO
Vibriostatic Gelatin
Oxidase 6% NaCl Fermentation
O129 Liquefaction
Aeromonas + R - Glucose +
Glucose
Vibrio +/- S + +
Mannitol
CAMPYLOBACTER
▪ Most recognized antecedent cause of Guillain-Barre LABORATORY DIAGNOSIS:
syndrome Growth at 25°C C. fetus subsp. fetus
▪ Mode of acquisition: Ingestion of contaminated water, Growth at 42°C C. jejuni
poultry and dairy products C. coli
▪ Animal pathogen of swine and cattle (sterility and Hippurate hydrolysis C. jejuni
Motility Darting motility
abortion)
▪ Microscopy: Long spirals or seagull winged shaped
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MICROBIOLOGY
▪ Culture: Runny spreading colonial growth; “tailing effect along streak line”
C. jejuni
▪ Most common cause of bacterial gastroenteritis worldwide (similar to Shigella)
▪ Acquired from eating contaminated chicken and turkey
▪ Pathogenesis: Invades the epithelium of the small intestines → inflammation
▪ Secretes toxin similar to cholera toxin
▪ Optimum growth at 37°C and 42°C
▪ Fastidious organism
▪ Unable to grow in 3.5% NaCl
▪ Hippurate hydrolysis (+)
▪ Culture:
o Specimen: Feces, rectal swab (less preferred), and blood
o Transport media: Cary Blair
o Selective media: CAMPY-BAP, Butzler agar, Skirrow, CCDA (Charcoal Cefoperazone Desoxycholate)
o Brucella agar or TSB: for motility
o Incubation time: 2 weeks
o Incubation condition: increased CO2
HELICOBACTER
H. pylori
▪ Microaerophilic organisms
▪ Most common cause of type B gastritis, peptic ulcer, and gastric carcinoma
▪ Primary habitat: human gastric mucosa LABORATORY DIAGNOSIS:
▪ Motility allows the organism to escape acidity of the ✓ Specimen: Tissue biopsy, urine (ammonia
stomach test), feces
✓ Gram stain: 0.1% basic fuchsin as
▪ Routes of transmission: oral-oral route, fecal-oral route
counterstain to enhance the morphology
▪ Strong Urease producer ✓ Other stains: Warthin-Starry, Silver stain,
▪ Oxidase (+) and Catalase (+) Giemsa
▪ Susceptible to Metronidazole ✓ Incubation: 5 days, capnophilic, requires
hemin for enhancing the growth
✓ Urea Breath test: excellent specificity and
sensitivity
CAMPYLOBACTER vs. HELICOBACTER

Hippurate Nitrate
Urease Cephalothin Nalidixic acid Growth at 42°C
Hydrolysis Reduction
C. jejuni - + R S + +
H. pylori + - S R v -
SMALL PLEOMORPHIC GRAM-NEGATIVE BACILLI

HAEMOPHILUS
▪ Normally inhabit the URT of humans EXCEPT H. ducreyi
▪ Fastidious, Non-motile, non-spore forming, capnophilic and facultative anaerobic bacteria
▪ Susceptible to drying and extreme temperature
▪ Microscopy:
o Gram negative, small, pleomorphic coccobacilli or rods
▪ Culture:
o BAP – moist, smooth, convex colonies on agar plate
▪ Biochemical Testing
o Catalase positive and Oxidase positive
▪ Growth Factors
o X (hemin) and V (NAD)
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MICROBIOLOGY
Haemophilus influenzae
▪ Also known as Pfeiffer’s bacillus
▪ Main cause of meningitis in children
▪ MOT: person to person by contaminated respiratory droplets
▪ Culture:
o Translucent, convex, tan mucoid colonies with “mousy or bleach-like odor”
▪ Virulence Factors:
o Polysaccharide capsule
o The only member of the genus that produces IgA protease (unique to H. influenzae)
o B-lactamase
▪ Typeable strains
o Based on capsular characteristics
o Type b – causes meningitis in unvaccinated children
▪ Non-typeable strains
o Normal inhabitants; causes otitis media
Haemophilus ducreyi
▪ NOT part of the human flora
▪ Infects mucosal epithelium, genital and non-genital skin and regional lymph nodes
▪ Causative agent of chancroid or “soft chancre”
▪ Requires X factor
▪ Culture: Transparent, small, flat, smooth tan or yellow colonies on CAP
Haemophilus aegypticus
▪ Also known as “Koch-Weeks bacillus”
▪ Etiologic agent of “Pink-Eyed Conjunctivitis” or Brazilian purpuric fever
▪ Genetically related to H. influenzae
▪ Biochemistry:
o Glucose (+), Urease (+), Oxidase (+), Catalase (+)
o Indole (-) and Xylose (-)
▪ Specimen
o CSF, Sputum, genital/ulcer lesions, vaginal swabs, swabs from conjunctiva, bronchial washing and blood
o CSF samples should be centrifuged
o H. haemophilus are very sensitive to drying → direct bedside plating is recommended
▪ For H. ducreyi:
o Clean ulcer with sterile gauze moistened with saline; pre-moisten cotton swab with phosphate buffered
saline
▪ Gram staining:
o Microscopy: small gram negative pale pink cocobacilli to long filaments
o School of fish or fingerprint appearance (H. ducreyi)
▪ Culture
o Chocolate agar plate (X and V factor)
o In Blood agar medium all V factor producing bacteria will grow as satellites around colonies producing
NAD
o NAD-producing bacteria: S. aureus, S. pneumoniae, and Neisseria
o All clinically important Haemophilus require V factor EXCEPT for H. ducreyi
o Horse Blood-Bacitracin → for isolation of H. influenzae from respiratory secretions of patients with
cystic fibrosis
o CAP with 1% IsoVitalex → for isolation of H. aegypticus
o GC agar base with 2% bovine hemoglobin + 5% fetal calf serum + vancomycin → for H. ducreyi
▪ Differential test
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MICROBIOLOGY
DISTINGUISING REQUIRES X REQUIRES V HEMOLYSIS ON

SPECIES PORPHYRIN
CHARACTERISTIC FACTOR FACTOR BAP
H. influenzae Mousy/bleach-like + + 0 0
odor
H. aegypticus Pink eye + + 0 0
H. haemolyticus Beta-hemolytic + + 0 +
H. ducreyi School of fish + 0 0 0
appearance
H. parahemolyticus Mannose fermenter 0 + + +
H. parainfluenzae Lactose and 0 + + 0
mannose fermenter
Important notes:
✓ Porphyrin and X factor requirement are opposite
✓ Species with “para” only requires V factor and porphyrin
✓ Species with “haemolyticus” are only hemolytic on blood agar
▪ Porphyrin test
o Differentiates heme-producing species of Haemophilus to establish X-factor requirements
o Principle: ALA dehydrase converts d-ALA → porphyrins or porphobilinogen
o Reagent: Kovac’s reagent → end product: porphobilinogen (red), porphyrins (reddish orange)
HACEK GROUP
▪ Small, non-motile, non-spore forming, gram negative coccobacilli that will not grow on MacConkey agar
▪ Part of the normal oral flora; Opportunistic
▪ They are dysgonic pathogens on BAP and CAP (slow growers)
▪ Causes slow, progressive bacterial endocarditis
Haemophilus aprophilus
▪ Most prevalent species under HACEK group
▪ Causes endocarditis
▪ Isolated from dental plaque and gingival scrapings
▪ Lactose fermenter and Porphyrin (+)
▪ Culture: raised, convex, granular and yellowish
Aggregatibacter actinomycetemcomitans
▪ Recognized as the causative agent of periodontitis (main habitat: mouth)
▪ Isolated from lungs, blood, abcesses of the mouth, brain and sinuses
▪ Virulence: collagenase and leukotoxin
▪ The only Catalase (+) of the HACEK group
▪ Culture: “star-shaped” with 4-6 points in the center of the colonies
Cardiobacterium hominis
▪ Infects the aortic valve
▪ Culture on BAP produces pitting of the agar
▪ Oxidase (+)
Eikenella corrodens
▪ Corroding bacilli
▪ The least common isolate of the HACEK group
▪ Normal flora of the URT and mouth
▪ Causes cellulitis among drug abusers
▪ Causes infection from bites or clenched fist
▪ Seen after trauma to head and neck and human bite
▪ Culture:
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MICROBIOLOGY
o yellow colonies, pits and corrode the agar with sharp bleach odor
▪ Lysine and Ornithine Decarboxylase (+)
Kingella kingae
▪ Have the tendency to resist decolorization
▪ Isolated from degenerative bone and joint infection in children below 3 years old
▪ May grow on TMA and resemble N. gonorrheae
▪ Oxidase (+), Catalase (-)
Biochemical Testing
▪ All HACEK are catalase negative EXCEPT Aggregatibacter actinomycetomas
▪ All HACEK are oxidase positive EXCEPT A. actoinomycetomas and A. aphrophilus
▪ All HACEK are glucose fermenters EXCEPT Eikenella corrodens
▪ All HACEK are non-lactose fermenters EXCEPT H. aphrophilus
BORDETELLA
▪ Obligate aerobic gram-negative fastidious coccobacilli
▪ Non-carbohydrate fermenter (asaccharolytic)
▪ Non-motile EXCEPT B. bronchiseptica
▪ Bipolar staining
▪ Catalase (+), Oxidase (+), Citrate (+)
▪ Species: Pertussis, Parapertussis, bronchiseptica and avium
Bordetella pertussis
▪ Etiologic agent of whooping cough
▪ It only causes disease in human
▪ Does not survive well outside the host (obligate intracellular)
▪ Culture: appears as mercury drops
▪ Virulence factor: pertussis toxin
▪ Specimen: nasopharyngeal swab
Whooping cough
▪ Highly contagious infection of the URT
▪ Acquired through aerosol route
▪ 7-14 days incubation
▪ 3 stages:
o Catarrhal
o Paroxysmal
o Convalescent
▪ Specimen:
o Nasopharyngeal swab (Calcium or Dacron swab)
▪ Culture media:
o Regan Lowe Agar (Preferred) → charcoal+agar+10% horse blood + Cephalexin)
o Bordet-Gengou Potato Infusion agar → charcoal+10% horse blood+40mg/L cephalexin w/ methicillin
o Modified Jones-Kendrick Charcoal → yeast extract + cephalexin
Bordetella bronchiseptica
▪ Motile
▪ Causes respiratory disease in animals
▪ (+) Urease
Differential Tests
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MICROBIOLOGY
NITRATE
SPECIES OXIDASE UREASE MOTILITY BAP
REDUCTION
B. pertussis - + - - -
B. parapertussis - - + - +
B. bronchiseptica + + + + +
BRUCELLA
▪ Bang’s bacillus
▪ An important human and animal pathogen
▪ MOT: close contact with animals
▪ B. melitensis – most common isolate
▪ Known as a category B bioterrorism agent
▪ Class 3 biohazard organism
▪ Strict aerobe and intracellular parasites
▪ Localized in tissues rich in erythritol → sugar present in placental tissue → induce spontaneous abortion
▪ Causes spontaneous abortion among animals (B. abortus)
▪ Often recovered from blood and bone marrow
▪ Causes Undulant fever in humans
Undulant Fever (Malta Fever)

▪ Normal temperature in the morning, high temperature in the afternoon and evening
▪ MOT: unpasteurized poultry products, inhalation, direct contact/inoculation
▪ Specimen: blood, bone marrow, tissue
▪ Must be handled in a BSL 3 cabinet (aerosol)
▪ Gram stain: carbol fuchsin must be substituted to safranin
▪ Culture: BAP, Castaneda agar – biphasic medium (for blood and bone marrow)
▪ Serology: febrile agglutinins (>160 titer)
BARTONELLA
▪ Doesn’t grow on primary culture media
▪ Causative agent of cat scratch disease (CSD)
▪ Biochemically inert
▪ Diagnosis: DNA amplification
CAPNOCYTOPHAGA
▪ Normal flora of the oropharynx
▪ Causes periodontitis, bacteremia, DIC
▪ Motility: gliding
▪ Culture: requires 5% CO2, slow-growers on BAP and CAP
FRANCISELLA
Francisella tularensis
▪ A category A biological agent (bioterrorism agent)
▪ Small, obligate aerobic, coccobacillus and extremely invasive
▪ Microscopy: with faint bipolar staining; capsulated
▪ Culture: round, smooth gray to white and slightly mucoid colonies
▪ Growth factor: cysteine and thiosulfate
▪ Clinical infection: Tularemia
o A zoonotic disease which can be acquired through ingestion, inhalation, arthropod bite or handling of
infected tissue or carcasses
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MICROBIOLOGY
o Specimen: lymph node biopsy (BEST), scrapings, ulcers, sputum
o Gram stain: acridine orange
o Culture:
▪ CAP, MTM, BCYE, TSB
▪ Doesn’t grow on MAC
▪ Cysteine blood agar – media of choice
o Growth factors: X factor and cysteine
o Biochemical:
▪ Oxidase (-)
▪ Non-motile
▪ Biochemically inert
LEGIONELLA
▪ Obligate aerobic
▪ Growth requirements: iron and L-cysteine buffered to pH 6.9
▪ Mode of acquisition: exclusively from environmental sources through inhalation
▪ Can survive in extreme ranges of environment; adhere to fomites
▪ Can tolerate chlorine
▪ Motile
Legionella pneumophilia
▪ Habitat: both natural and man-made environments (hospital)
▪ Isolated from air condition ducts
▪ Has the ability to invade the bronchoalveolar macrophages (intracellular pathogen)
▪ Colony: grayish white or blue green glistening colonies
▪ Clinical infection: Legionnaire’s disease:
o Febrile and pneumonic illness
o Clinical manifestations:
▪ Pneumonia
▪ Pontiac fever
▪ Wound abscesses
▪ Laboratory diagnosis:
o Specimen: sputum and BAL; urine (for antigen detection)
o Gram stain: small to filamentous
o Culture:
▪ Plate onto BCYE w/ l-cysteine (most important media)
▪ Colonies: iridescent with sticky consistency
▪ Selective media: w/ polymyxin B, anisomycin, and vancomycin
PASTEURELLA
Pasteurella multocida
▪ Most frequently isolated species
▪ Commensal in the URT of mammals
▪ Facultative anaerobe, non-motile
o Endotoxins
o Capsule
▪ It grows only in BAP and susceptible to penicillin
▪ Has a characteristic mushroom smell of the colonies
o Oxidase, ornithine decarboxylase and indole positive
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MICROBIOLOGY
o Urease and ONPG negative
Pasteurella bettyae
▪ Isolated from amniotic fluid, placenta, blood and urogenital specimen
▪ Glucose and fructose fermenter
▪ Non-motile
o catalase and indole positive
o oxidase positive
▪ May grow on Mac
SPIROCHETES
▪ Family Spirochitales
▪ Helically curved unicellular bacteria
▪ Motile w/ periplasmic flagella
▪ Corkscrew motility
▪ Microaerophilic
▪ Appear as gram negative because they don’t stain very well
▪ Culture in vivo
TREPONEMA
▪ Treponema (Greek word = turning thread; twisting motion)

▪ Infects only humans
▪ Cannot be cultivated in vitro
▪ Diagnosis: serology
▪ Rapidly killed by 42’C but can survive for 3 days in ref temperature
▪ Best observed by dark-field microscopy
T. pallidum subsp. pallidum

▪ Causative agent of syphilis, a sexually-transmitted infection
▪ Has tropism to aerterioles
▪ Susceptible to disinfectants and drying
Syphilis
▪ Also known as French disease, Italian disease, Great Pox, Great imitator
▪ MOT: Sexual contact, congenital, skin contact with active lesion, blood transfusion, needles
▪ S/S: chancre, fever, sore throat, headache, rash, gummas
▪ Specimen: Skin lesions (cleaned w/ saline)
▪ Microscopy: direct examination of exudates
▪ Stains used:
o Levaditi’s impregnation stain
o Fontana Tribondeau stain
Serologic Test
NONTREPONEMAL TESTS TREPONEMAL TESTS

VDRL TPI
RPR FTA-ABS
USR (unheated serum reagin) TPHA
TRUST (Toluidine Red Unheated Serum Test) MHA-TP
✓ Screening tests ✓ Confirmatory tests
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MICROBIOLOGY
✓ Monitor the efficacy of treatment ✓ Uses either live or dead T. pallidum to demonstrate
the presence of antibodies to the organism
Other Spirochetes
▪ T. pallidum subsp. pertenue

o Yaws, frambesia tropica, parangi, paru, babu, bouba
o MOT: direct contact thru breaks in the skin
▪ T. pallidum subsp. endemicum
o Endemic non-venereal syphilis among children (bejel)
o MOT: direct contact with active lesions and contaminated fingers or utensils
▪ T. pallidum subsp. carateum
o Pinta, carate, mal de pinta, azul
o Skin infections acquired through direct contact
BORRELIA
Borrelia recurrentis
▪ Louseborne (Pediculus humanus)
▪ Causative agent of Epidemic relapsing fever or European relapsing fever
▪ Humans are the only reservoir host
Borrelia hermsii
▪ Tickborne (Ornithodoroes/soft ticks)
▪ Causes relapsing fever (Endemic/American)
Relapsing fever
▪ Acute infection marked by recurrent febrile episode → 2 to 10 relapses
▪ Relapses are due to the ability of the organism to alter antigenicity
Borrelia burgdorferi
▪ Causative agent of Lyme disease
▪ Tickborne (Ixodes ticks/hard ticks)
▪ NOTE: spirochete can be found in all stages of the ticks
▪ Ticks natural host: deer and rodents (white footed mouse)
Lyme disease
▪ Acute, recurrent inflammatory infection involving joints
▪ Cardinal sign: erythema migrans (bull’s eye rash)
o 1st stage:
▪ Appearance of erythema migrans
▪ red, ring-shaped lesion with central clearing at the site of the bite
nd
o 2 stage:
▪ Weeks to months after infection
▪ Dissemination of organisms
▪ Meningitis, nerve palsy, (Bannwarth syndrome)
o 3rd stage:
▪ Chronic arthritis that may continue for years
▪ Neuron demyelination
▪ Alzheimer’s
▪ Microscopy: darkfield microscopy
▪ Relapsing fever: peripheral blood is the specimen of choice (Wright-Giemsa)
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MICROBIOLOGY
▪ Lyme disease: Biopsy, Blood, CSF → Warthin-Starry, Acridine orange, and Giemsa
▪ Culture:
o Keller’s medium or Chick-embryo
o Slow grower (2 weeks)
o Microaerophilic
▪ Serology
o Relapsing fever: Proteus OX K antigen up to 1:80 titer
o Lyme disease: Serology is the standard method; IgM and IgG detection
▪ Molecular
o PCR
LEPTOSPIRA
Leptospira interrrogans
▪ Microscopy: tightly-coiled organisms, question-mark-like shape organisms
▪ Stain: silver stain
▪ Motility: spinning
▪ Obligate aerobic
▪ Can be grown in artificial media
▪ In vivo culture: hamster and guinea pigs
▪ Inhabits the lumen of renal tubules → shed into urine
▪ Causative agent of Leptospirosis
▪ MOT: direct contact with urine or bodies of water
▪ Pathogenesis: rapid invasion of bloodstream → spread throughout CNS and kidneys
Leptospirosis
▪ 2 syndromes:
o Anicteric leptospirosis: septicemic stage; high fever, aseptic meningitis
o Icteric/Weil Syndrome: Liver, kidney, and vascular dysfunction → death in 10% cases
Laboratory Diagnosis:
▪ Specimen: blood, CSF, tissues (bacteremic phase), urine (2 weeks of illness)
▪ Microscopy: motile spirochetes
▪ Culture:
o Fletcher media
o EMJH agar
o Few drops of heparinized or oxalated blood → directly inoculated onto agar → incubation for 4-6 weeks
→ observe for growth
▪ Serodiagnosis
o ELISA, RIA (fourfold rise in the titer of antibodies is diagnostic)
o Reference method (Gold Standard): Microscopic agglutination method
▪ Molecular tests: Leptospiral DNA in infected patient, PCR
CELL WALL-DEFICIENT BACTERIA

MYCOPLASMA
▪ Smallest bacteria (0.3 um-0.8 um)
▪ Free-living organism
▪ Capable of growing on artificial media
▪ Normal flora found in nasopharynx , URT, GUT
▪ Parasites of genital tract
▪ Microscopy: pleomorphic, no cell wall
▪ Culture:
o Capable of growing on artificial media
Page 65 of 69
MICROBIOLOGY
o Slow grower
o Fastidious
o Facultative anaerobe
o Requires sterol
M. pneumoniae
▪ Eaton agent
▪ Etiologic agent of primary atypical pneumonia (Walking pneumonia)
▪ NOT normal commensal
▪ Pleuropneumonia-like organism with gliding motility
▪ MOT: respiratory droplets
▪ Culture: extremely fastidious
GENITAL MYCOPLASMA
▪ Recovered from genital tract of healthy adult or nose and throat of infants
▪ Causes invasive disease in immunosuppressed patients, prostatitis, bacterial vaginosis
▪ Causes non-gonococcal urethritis
M. hominis
▪ Causes postabortal and postpartum fever
▪ Grown on M agar containing arginine and phenol red
▪ Colonies: fried-egg appearance with red holes
Ureaplasma urealyticum
▪ T-strain of mycoplasma
▪ Isolated from genital specimens on U agar containing urea and phenol red
▪ Subculture media: A7/A8 medium
▪ Colonies: small and golden brown
▪ Specimen:
o M. pneumoniae – throat swab, serum, BAL, sputum
o Genital mycoplasma – urethra, vagina, endocervical swab, blood, urine, prostatic secretion, semen
▪ Stain: Dienes or acridine orange stain for M. hominis
▪ Culture:
o PPLO broth agar
o Modified NYC medium
o Biphasic SP-4
▪ pH requirements:
o 5.5 to 6.5 for Ureaplasma
o 6.0 to 8.0 for Mycoplasma
▪ Growth factors:
o Glucose – M. pneumoniae
o Urea – U. urealyticum
o Arginine – M. hominis
▪ Colonial Characteristics:
o Mycoplasma: fried-egg appearance
o Ureaplasma: dark brownish clumps in A7/A8 medium
▪ Definitive ID for M. pneumoniae
o Principle: M. pneumoniae is known to hemadsorb
o Procedure: add 0.5% guinea pig RBC in phosphate buffered saline to suspicious colonies
o Result: after 20-30 minutes at RT, colonies are observed to adhere to RBC
▪ Serodiagnosis:
Page 66 of 69
MICROBIOLOGY
o ELISA – most widely used

o BEST approach: EIA + DFA
▪ Molecular:
o PRC – most sensitive but may detect M. pneumoniae in the absence of infection
Manganous Chloride Urea Test

▪ For rapid ID for U. urealyticum
▪ Principle: U. urealyticum utilizes manganous chloride in the presence of urea
▪ Positive result: dark brown precipitate of manganese oxide around colonies
▪ Reaction is observed under dissecting microscope
OBLIGATE INTRACELLULAR BACTERIA
CHLAMYDIACEAE
▪ Nonmotile, obligate intracellular parasite
▪ Tropism to columnar epithelial cells
▪ Resemble gram-negative cell wall
▪ 2 morphological forms:
o Reticular body – replicative; non-infectious; metabolically active; intracellular
o Elementary body – vegetative; infectious; extracellular
C. trachomatis
▪ Major STD pathogen
▪ Causes pelvic inflammatory disease and ocular trachoma
▪ Infertility and ectopic pregnancy
▪ Serovars:
o Endemic trachoma (A, B, C)
o LGV (L)
o Others (D, K, I, J)
▪ Trachoma
o Chronic inflammation of conjunctiva
o Leads to blindness; distortion of the eyelids
o MOT: inanimate objects
▪ Lymphogranuloma venereum
o STD; multisystem involvement
o Small painless ulcer or papule → nodules
o Intradermal skin test: Frei test
▪ Laboratory diagnosis
o Specimen: urethra, cervical secretion, conjunctiva discharge, nasopharynx, rectal swab (Dacron)
o Culture: cell culture (reference method; time consuming)
o Culture media: McCoy, Hela 229, BGMK (Buffalo Green Monkey Kidney cells)
o Incubation: 2-3 days
o Serodiagnosis:
▪ EIA, MIF, antibody detection, antigen detection
▪ PCR: may be performed on urine
OTHER SPECIES
C. psittaci
▪ Causative agent of ornithosis (Parrot fever)
▪ Endemic pathogen of birds
▪ Causes outbreaks among turkey-processing workers
▪ MOT: inhalation of infected aerosols from dried bird excreta or handling of infected birds
▪ Serological tests:
o Complement fixation test – widely used; a >1:32 titer is presumptive identification of the bacteria
Page 67 of 69
MICROBIOLOGY
o Direct MIF test – sensitive method

o PCR
C. pneumoniae
▪ TWAR strain
▪ Human pathogen; 3rd most common cause of infectious respiratory disease
▪ MOT: aerosol droplets
▪ Culture: HEP-2 cell line culture
▪ Serology: MIF test
Differential Properties of Chlamydia Species

C. trachomatis C. psittaci C. pneumoniae
Host range Humans Birds Humans
Inclusion bodies Halbertstadler-Prowazek bodies Levinthal-Cole Lillie bodies Round-dense
Stains used Lugol’s iodine Machiavelo and Giemsa Giemsa

stain
Glycogen inclusion Present Absent Absent
Susceptibility to S R R
Sulfonamides
No. of serovars 20 10 1
RICKETTSIACEAE
▪ The simplest bacterial form
▪ Gram-negative, pleomorphic, small bacilli
▪ Nonmotile
▪ Considered transitional organism (between viruses and bacteria)
▪ Arthropod-transmitted infection
▪ ALL requires a living cell for growth EXCEPT B. quintana
▪ Fastidious, small pleomorphic gram-negative bacilli
▪ Reside in cytosol of host cell
▪ Phospholipase A2 contributes to intracellular activity
▪ Direct method
o Immunohistology – sensitive and specific
o Specimen: skin biopsy (for RMSF)
o Giemsa or Wright stain for morulae detection during febrile stage of Ehrlichiosis and Anaplasmosis
▪ Culture
o Yolk sacs of embryonated eggs and tissue culture
o Incubation conditions:
▪ Coxiella: acidic; to activate metabolic enzymes
▪ Rickettsia, Ehrlichia, and Anaplasma: antibiotic-free centrifugation- enhanced shell vial cell
culture
▪ B. henselae and B. quintana: lysis of intraerythrocytic bacteria → centrifugation and incubation
at 35’C in humid atmosphere on Columbia agar or CAP for >1 month
▪ B. bacilliformis: supplement with 5% defibrinated blood or hemin-supplemented media after 18
days
▪ Serodiagnosis
o The only test performed for diagnosis of Rickettsial diseases
Page 68 of 69
MICROBIOLOGY
▪ IFA – gold standard test (>1:64 titer)

▪ Weil-Felix test – presumptive test (agglutination of certain strains of P. Vulgaris by serum from
patients)
▪ Microimmunofluorescent Dot Test – excellent sensitivity (antibody detection)
ORGANISM DISEASE VECTOR/MODE OF TRANSMISSION

SPOTTED FEVER
Rickettsia conorii Mediterranean spotted Ticks (Rhipicephalus sanguineus)
fever
Rickettsia rickettsii Rocky mountain spotted Wood and Dog Ticks
fever
Rickettsia akari Rickettsial pox Mouse mite
TYPHUS GROUP
Rickettsia prowazekii Epidemic typhus/ Brill- Louse and Squirrel flea
Zinsser disease
Rickettsia typhi Endemic murine typhus Rat fleas
SCRUB TYPHUS GROUP
Orientia tsutsugamushi Scrub typhus Chigger bite
EHRLICHIA
Ehrlichia chaffeensis Human monocytic Lone star tick
Ehrlichiosis
Ehrlichia ewingii Ehrlichiosis ewingii Tick bite
Anaplasma Human Granulocytotropic Deer tick
phagocytophila Anaplasmosis
OTHER RICKETTSIAL FEVER
Rickettsia felis Flea-borne spotted fever Flea bite or feces
Coxiella burnetti Q fever Inhalation of aerosol from infected animals
Bartonella Quintana Trench fever Feces of louse
Page 69 of 69

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Microbiology Notes 2

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MICROBIOLOGY

Eukaryote vs. Prokaryote

Cell division Mitosis Binary fission

Cell organelle Present Absent

Contains DNA and Histones Has a single chromosome only

Site of energy production Mitochondria Cytoplasmic membrane

Site of protein synthesis Rough endoplasmic reticulum Free ribosome

GRAM POSITIVE GRAM NEGATIVE

Gram positive bacteria retain the primary stain because

Gram Positive cocci (aerobe) Micrococcus, Staphylococcus, Streptococcus

III. Nutrition and Growth Requirements

Obligate Aerobe Requires 15-21% oxygen and 1% CO2

Aerotolerant anaerobe grow in the presence of oxygen but no metabolic process

Mesophile 20-45°C Most pathogenic bacteria

Thermophile 50-125°C B. stearothermophilus, Sulfolobus, Pyrococcus

Extremophile Organisms that live in unusual conditions (B.

Neutrophile 5.5-8.0 Most pathogenic bacteria

Alkalophile 8.5-11.5 B. alcalophilus, Natronabacterium

 HEPA filter removes particle larger than 0.3 um in diameter

Classification Based on Effective Level of Biologic Containment

− Sterilizes only the air to be exhausted

Class II − Vertical laminar flow

Class III − Completely enclosed with negative pressure

− Afford most protection to worker

STERILIZATION AND DISINFECTION

A. Incineration – materials are burned to ashes; safest method

▪ Periacetic acid – used to sterilize medical equipment

✓ Allowable delay time:

✓ Boric acid for urine culture (for 24 hours)

LEVEL DESCRIPTION EXAMPLES

2 Unpreserved Bone, feces, sputum, tissue, body fluids

3 Quantitation required Catheter tip, urine tissue

4 Preserved Urine, feces, swabs

Panic Values (Communicated to the physician immediately)

Medium Component/Comment Primary Purpose

Cefoperazone, Blood-supplemented enrichment medium containing Selective medium for isolation of

Chocolate agar Peptone base, enriched with solution of 2% Cultivation of fastidious

Selective for Campylobacter and

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Genetic Elements Contributing to Resistance

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Considerations for Standardization

▪ MHA + 2% NaCl = MRSA

Criteria for evaluation of antibiotics

Alternative Approaches to AST

Supplemental Methods for Detection of Antimicrobial Resistance

CATALASE POSITIVE – GRAM-POSITIVE COCCI

Pseudocatalase positive: Aerococcus, Enterococcus, Rothia;

Micrococcus vs. Staphylococcus

Oxidase test NEGATIVE POSITIVE

Furoxone Tween 80 Oil Red O agar NEGATIVE POSITIVE

Acid production from Glycerol w/

Glucose utilization FERMENTER OXIDIZER

Bacitracin/Taxo A disk (0.02 – 0.03 units) RESISTANT SUSCEPTIBLE

Furazolidone (100 ug) Susceptibility test SUSCEPTIBLE RESISTANT

Lysostaphin Sensitivity test SUSCEPTIBLE RESISTANT

SLIDE METHOD TUBE METHOD

Screening method Confirmatory method

False (+): citrate-utilizing organisms → DO NOT use citrated plasma

▪ Mannitol Fermentation Test

 Also known as Duran-Reynal factor

CATALASE – NEGATIVE GRAM-POSITIVE COCCI

DIAGNOSTIC ALGORITHM FOR IDENTIFICATION OF GRAM-POSITIVE COCCI

ALPHA BETA GAMMA