BACTERIOLOGY - CSA-B In-House Review 2021 2.

Gram-negative Cell Wall

 Its outer membrane is composed of proteins,
Bacteria – prokaryotic organisms which lack true nucleus. phospholipids, and lipopolysaccharides (LPS).
Karyo  nut in a shell, Pro before formation of nucleus.  The inner membrane is composed of a thin
peptidoglycan layer, which is the reason for its high
Bacterial Cell Structures susceptibility to mechanical breakage.
A. Cytoplasmic Structures  It has porins that contribute to the permeability of the
 Contains a single, closed, circular, double stranded cell wall.
DNA associated with virulence.  It contains a periplasmic space which is involved in
 Plasmid - an extra chromosomal circular DNA. peptidoglycan synthesis.
Associated with conjugation. A bacterial cell may or  It does not contain teichoic acid.
may not contain a plasmid  The outer membrane has the following functions:
 Donor cell  there is a copying of the a. The strong negative charge of the outer
characteristic which is passed on the membrane is an important factor in evading
recipient cell. Projection is sex pili (fertility phagocytosis.
pili). Passes antibiotic resistance and have b. It allows hydrophilic compounds to enter the
toxin production. cell through the porins.
 Recipient cell  carries the new c. It acts as a barrier to toxic substances that
characteristics prevents movement inside the cell.
 Ribosomes - are found free and are the sites of  The LPS have 3 regions:
protein biosynthesis which gives cytoplasm granular a. Lipid A - major constituent; an endotoxin
structure. Size is 70s (svedberg unit). Two subunits: b. Core polysaccharide
Smaller (30s) and larger subunit (50s) = total is 70s c. Antigenic O-specific polysaccharide
 Endospores - which are small, dormant, asexual  The LPS have the following important functions:
spores developed in response to harsh conditions a. They are vital in evading the host defenses.
(Becomes vegetative when the harsh condition is b. They contribute to the negative charge of the
removed.) Aid in the survival of bacteria against harsh bacterial surface, which stabilizes the
conditions. They are produced in the vegetative cells membrane structure.
of gram-positive bacteria. Composed of dipicolinic c. LPS is also considered as an endotoxin.
acid and calcium ions. Spores are used in identifying
the bacteria 3. Acid-fast Cell Wall
 _____________ - are organisms with spores  It has a Gram-positive cell wall structure.
 Aside from the peptidoglycan layer, it contains a waxy
B. Cell Wall Structures layer of glycolipids and fatty acids (mycolic acid) that
 Cell membrane surrounds the cytoplasm; composed are bound to the exterior of the cell wall.
of a phospholipid bilayer but unlike eukaryotes, it  Mycolic acid has a strong hydrophobic structure that
lacks sterols. affects the permeability of the acid-fast cell wall. e.g.,
 Site of respiration and photosynthesis. Mycobacterium and Nocardia
 Mesosomes  provides respiration.
 Cell wall is a rigid structure which maintains the shape 4. Absence of a Cell Wall
of the cell.  Prokaryotes that do not have a cell wall contain
 Its synthesis and structure have been the primary sterols in their cell membrane. e.g., Mycoplasma and
target of antimicrobial agents. Ureaplasma
 It has the following functions:
 It prevents bacterial cells from rupturing when
osmotic pressure inside the cell is greater than Bacterial Classification Based on Cell Wall
the pressure outside the cell Characteristics
 It serves as a point of anchorage for flagella Gram Positive Organisms Gram Negative Organism
 It determines the staining characteristics of a Thick peptidoglycan layer
Thin peptidoglycan layer
species. (murein)
 Cell membrane surrounds the cytoplasm; composed Contains lipopolysaccharide:
of a phospholipid bilayer but unlike eukaryotes, it 1. Core
lacks sterols. Contains teichoic and polysaccharide
 Cell wall is a rigid structure which maintains the shape lipoteichoic acid 2. Antigenic O specific
polysaccharide
of the cell.
3. Lipid A / Endotoxin
 Its synthesis and structure have been the primary
LOW lipid content HIGH lipid content
target of antimicrobial agents. NO endotoxin (except
 It has the following functions: ENDOTOXIN
Listeria monocytogenes)
a. It prevents bacterial cells from rupturing NO periplasmic space PERIPLASMIC SPACE
when the osmotic pressure inside the cell is NO porin channel PORIN CHANNEL
greater than the pressure outside the cell. Sensitive to lysosome and Resistant to lysosome and
b. It serves as a point of anchorage for flagella. penicillin attacks penicillin attacks
c. It determines the staining characteristics of a
species. Gram Positive Organisms: “SSEC-LBC-MAN”
Staphylococcus
Types of Cell Wall
Streptococcus
1. Gram-positive Cell Wall
Enterococcus
 It is composed of a very thick protective peptidoglycan
Clostridium
(murein) layer.
Listeria monocytogenes
 It consists of glycan chains of alternating N-acetyl-D-
Bacillus
glucosamine (NAG) and N-acetyl-D-muramic (NAM)
Corynebacterium
acid.
Mycobacterium
 It contains a negatively charged teichoic acid and
Actinomyces
contributes to the charge of the cell wall.
Nocardia
 It is the prime target of antimicrobial agents like
penicillin which prevents the synthesis of
peptidoglycan.
NOTE:  Endotoxin - produced by gram negative organisms
 Some organisms contain mycolic acid (hydroxy- and are released only when bacteria are destroyed.
methoxy acid)
 Some organisms lack cell walls and are seen in Motile Bacteria Non-motile Bacteria
various shapes (pleomorphic). - Mycoplasma, Alcaligenes Bacillus anthracis
Ureaplasma Bacillus cereus Bordetella
 Spirochetes are indistinct when viewed under light Bacillus subtilis Clostridium perfringens
microscope. (Borrelia, Treponema, Leptospira)
Campylobacter Corynebacterium diphtheriae
C. Surface Polymers Clostridium tetani Erysipelothrix
 Capsule may be composed of polysaccharide or Escherichia coli Haemophilus
polypeptide. Virulence factor which helps bacteria Helicobacter Klebsiella pneumoniae
evade phagocytosis, resist phagocytosis and Listeria Mycobacterium tuberculosis
desiccation. Alcaligenes
 Flagella - exterior protein filaments (flagellin) that Bacillus cereus
rotates and cause bacteria to be motile. It is important Bacillus subtilis
to survivability and pathogenicity of bacteria.
Bacteria with Capsule
Classification according to flagella Bacillus anthracis
a. Atrichous  no flagellum Klebsiella pneumoniae
b. Monotrichous  single flagellum at one end Streptococcus pneumoniae
c. Amphitrichous  single flagellum at 2 ends
d. Lophotrichous  tuft of flagella at one end or both ends Bacteria with Spores
e. Peritrichous  flagella on all sides of the bacterium Bacillus
Clostridium
 Pili – are non-motile, long, hollow (way of copied plasmid)
protein tubes which can either serve for adherence or
Bacteria with Inclusion bodies or granules
conjugation.
Corynebacterium Babes-Ernst bodies
 These are hair-like, proteinaceous structures that
extend from one cell membrane to the external Mycobacterium Much's granules
environment (2um in length) Nocardia and Actinomycetes Sulfur granules
 These aid in the attachment of bacteria to Pasteurella and Bordetella Bipolar bodies
surfaces
 A virulence factor or organ of attachment is the Bacterial Growth
COMMON or SOMATIC PILUS  Refers to the multiplication of bacteria which is
 An essential part of the genetic possible through binary fission.
transfer/conjugation process is the SEX PILUS  Generation time (DT or Doubling time) – refers to
the amount of time it takes for a bacterial cell to divide
Encapsulated Organisms Flagellated Organisms
“SHNES-KSS-PBB” “SHE-CCAVS” into two cells.
S. agalactiae Salmonella typhi  Fast-growing bacteria (e.g., E. coli  20 min.) and
Haemophilus influenza (type B) Helicobacter pylori slow growing bacteria (e.g., Mycobacterium  does
Neisseria meningitidis E. coli not divide after 24 hours)
E. coli Campylobacter
Strep pneumoniae Caulobacter  Growth Curve – there are four phases of bacterial
K. pneumoniae Aeromonas growth
Strep pyogenes Vibrio 1. LAG phase – no cellular division but
Salmonella typhi Selenomonas bacteria prepare to divide.
Pseudomonas aeruginosa  0 means no bacteria; don’t start
B. anthracis with zero.
Bordetella pertussis
2. LOG phase – bacterial population increases
exponentially
Organisms with Pili:  Gliding motility:
 Logarithmic phase
“PENS-BYC” Capnocytophaga,
Pseudomonas Myxobacteria,  Susceptible to antibiotics
E. coli cyanobacteria 3. STATIONARY phase – nutrients are limited;
Neisseria gonorrhea  Darting motility: number of bacteria remains constant / no net
Salmonella Campylobacteria growth
Bordetella (seagull appearance)  Plateau phase
Yersinia  Tumbling motility:  Some bacteria die (because they
Campylobacter Listeria are using the nutrients and
monocytogenes becomes limited) and some divide =
 Shooting star motility: no net growth
Vibrio  pH is altered  not conducive for
the growth of bacteria
Microbial Toxins  Spores are produced
 Biochemically active substances released by bacteria 4. DEATH phase – number of non-viable
to have a particular effect on its host cell bacteria exceeds number of viable cells.
 Exotoxin - produced by gram positive organisms  complete cessation of bacteria
(EXCEPT: L. monocytogenes)
 release during bacterial death Bacterial Metabolism
a. Neurotoxins – Botulinum toxin,  Include various biochemical reactions that breakdown
Tetanospasmin organic compounds as energy sources
b. Enterotoxin: Diarrhea – Vibrio, E. coli,  Phenotypic identification is possible through the
Campylobacter, Shigella dysenteriae determination of:
c. Enterotoxin: Food Poisoning: B. cereus, S. a. Substrate utilized
aureus b. End products being produced
d. Pyrogenic  cause fever – S. pyogenes, S. c. Change in pH
aureus
  Two mechanisms for bacterial metabolism:  Dry heat: Makes use of higher temperature for longer
a. FERMENTATION – anaerobic environment hours.
 Organic compound is the final electron a. Oven
acceptor  used to sterilize glasswares, petrolatum, powders
Note: All enterics are capable of fermenting  160-180°C, 1.5-3 hours
glucose because most of them are  indicator for dry heat oven: Bacillus subtilis var.
fermenters of other sugars. niger
b. OXIDATION – aerobic environment b. Incineration
 Oxygen is the final electron acceptor  used to sterilize infectious wastes
OF test (OXIDATION-FERMENTATION  870-980°C
TEST) – to differentiate enterics  not environment-friendly
 without cap – oxidizer c. Flaming – used for loops and needles
 with cap – fermenter
 Filtration: Used to sterilize thermolabile / heat-sensitive
Mechanisms of Gene Transfer substances
 Genetic material may be transferred from one 1. HEPA filter (High Efficiency Particulate Air)
bacterium to another by:  Used in biosafety cabinet
 Removes microorganisms/objects which are
a. TRANSDUCTION larger than 0.3 um
 Transfer of bacterial genes by a bacteriophage  Can filter viruses
 Inject genetic material 2. Membrane filter
 Biosynthesis  more pieces of bacteriophage  For experiments and research involving
 Temperate bacteriophages – are those which undergo injection
lysogeny (lysogenic conversion)  composed of plastic polymers or cellulose
 Bacteriophage stays in the bacterial cell ester containing pore
 pore sizes may include 0.45 um or 0.80 um
b. TRANSFORMATION  used to sterilize vaccines, antibiotic, and
 Uptake and incorporation of naked DNA into a parenteral solutions
bacterial cell.  Enough to filter bacteria
 2 cultures with 2 strains of bacteria in which there will 3. Micropore filter
be a transfer of naked DNA.  pore size of 0.22 um – 100% sterility
 Bacteria capable of transformation are called  used for filtering Pseudomonas-like
competent bacteria  transfer naturally or readily. organisms
 S. pneumoniae, N. gonorrhoeae, H. influenzae
 Ionizing Radiation
c. CONJUGATION  Used to sterilize plastic syringes, catheters, and
 Transfer from a donor bacterial cell to recipient gloves before use.
bacterial cell  Can achieve sterilization
 Close contact is required for this mechanism  Utilizes gamma rays: short wavelength and high
 Donor bacterial cell forms a hollow appendage sex energy rays.
pilus for transfer to the recipient cell.  Non-ionizing  can only achieve disinfection

Laboratory Safety: Microbial Control A.2 Chemical Methods of Sterilization

 Sterilization – the process of destroying all microbial  Ethylene oxide (EtO)
life including spores.  most commonly used chemical agent for sterilization
 Disinfection – the process of destroying pathogens  indicator for ethylene oxide: Bacillus subtilis var.
but not all microorganisms and their spores. globiggi
 Killing of microorganisms by these methods is
affected by several factors.  Vapor Formaldehyde / Hydrogen peroxide – used to
sterilize HEPA filter
A. Physical Methods of Sterilization
 Moist heat: “STEAM UNDER PRESSURE” (MOIST si  Glutaraldehyde – sporicidal (3-10 hours) used to sterilize
ATI) medical equipment [COLD STERILIZATION]
 used to sterilize biohazardous wastes and heat-
stable objects.  Peracetic acid – effective in the presence of organic
1. Autoclave material and is also used for medical equipment [COLD
 Irreversible denaturation of enzymes and STERILIZATION]
structural proteins
 moist heat in the form of saturated steam under B1. Physical Methods of Disinfection
15 psi (pounds per square inch)  Boiling
 121°C – 15 min. (media, liquids, instruments)  kills vegetative bacteria at 100°C for 15 minutes
 132°C – 30-60 min. (biohazardous wastes)
 Indicator for autoclaving: Bacillus  Pasteurization
stearrothermophilus  kills food pathogens
 at 63°C for 30 minutes (Batch method)
2. Tyndallization  at 72°C for 15 seconds (Flash Method or HTST =
 flowing steam using Arnold sterilizer High Temperature Short Time)
Fractional  100°C – 3 consecutive days  at 140°C for 3 seconds (Ultrahigh temperature)
sterilization
This process
3. Inspissation  Non-ionizing Radiation
is not
continuously  Thickening through evaporation  uses UV rays: longer wavelength and low energy
done  used to sterilize media with increased rays.
protein content
 75-80°C for 2 hours for 3
consecutive days
B2. Chemical Methods of Disinfection  transmitted by aerosol
 transmitted by: Aerosol
 Alcohols or unknown
and inhalation
 volatile substances used as antiseptic for the skin transmission
Examples:
(non-sporicidal = to be filtered) Example:
 M. tuberculosis
 most effective at 70% concentration  Ebola virus
 Why? – Water takes part in protein denaturation
BSL-1 = BSC-1
 Halogens BSL-2 & 3 = BSC-2
1. Chloride BSL-4 = BSC-3
 in the form of sodium hypochlorite
 suggested dilution for disinfection is 1:10 Specimen Collection and Handling
 substitute for bleach as disinfectant for blood spills: A. Collection General Guidelines
diluted vinegar  Specimens must be collected in sterile containers
EXCEPT stool specimens which may be collected with
2. Iodine clean, leak-proof containers.
 iodine + neutral polymer (detergent) = iodophore  Swabs are used for URT, eyes, external ear and genital
 iodine + alcohol = tincture tract specimens but not for others since they are easily
 NOTE: Disinfection prior to blood collection is contaminated.
commonly done by applying alcohol then povidone- a. Cotton
iodine. b. Calcium alginate
c. Dacron (for Neisseria)
3. Heavy Metals  Lesions, wounds, and discharges should be collected
 1% Silver nitrite – used as a prophylaxis for the eyes through needle aspiration; label must contain the exact
in infants born of mothers infected by N. gonorrhoeae anatomic site.
 Crede’s prophylaxis  What is the specimen of choice for corneal
ulcer?
4. Quaternary ammonium compounds Corneal scraping
 QUATS is used to disinfect bench tops - Instill local anesthetic, scrape with sterile
 Easily inactivated by organic compounds spatula, and inoculate directly to the
 P. aeruginosa is RESISTANT to this disinfectant agar.

5. Phenolic B. Clinical Specimens

 cleans and disinfects at the same time 1. Blood – most common anticoagulant used: SPS
 At concentrations between 2-5% these products are (sodium polyanethol sulfonate – inhibits Neisseria, G.
widely used for cleaning benchtops vaginalis, P. anaerobius
 Bacterial inhibition by SPS is counteracted by:
 Biosafety Cabinets – serve to protect the worker from 1% gelatin
aerosolized transmission of organisms while processing  Routine blood culture is held for: 5-7 days
specimens.  Brucellosis: 3-4 weeks; Leptospirosis: 8 weeks

3 Types of Biosafety Cabinets 2. CSF – collected using 3 tubes (#1: Chemistry; #2:
BSC Class I BSC Class II BSC Class III Microbiology; #3: Hematology)
 Filters both  Examine immediately or hold either at room temp
exhausted and / 37 0C
recirculated air  Centrifuge: use the sediments for smears and
 Most commonly cultures
used
 Pathogens: N. meningitidis, H. influenzae
Filters Filters supplied
IIa. Exhausts air
exhausted air and exhausted
into the room Cause of meningitis:
only air
5 years old H. influenzae
Product Entirely sealed;
IIb. Exhaust air 29 years old and above Streptococcus pneumoniae
contamination is accessible only
outside the building Elderly Listeria monocytogenes
possible thru glove ports.
5-29 years old Neisseria meningitidis
 Biological Safety Level – presented by the CDC
consisting of 4 guidelines or levels were established for 3. Throat and Nasopharyngeal swab
protection.  Specimen of choice to detect carrier state of N.
meningitidis and detect B. pertussis
BSL – 1 BSL – 2  Most abundant normal flora: Viridans group
 Most common pathogen: S. pyogenes (Strep
 organisms which pose  organisms which pose
minimum threat on lab moderate on lab throat)
personnel and personnel and 4. Sputum – Usually contaminated with normal flora
 Specimen of choice: Early morning specimen
environment environment.
 Rinse or gargle before collection; dentures must
 transmitted by: mucus
be removed first
membrane ingestion
 Bartlett’s Classification: To determine if the
and percutaneous
Examples: specimen in question is good.
transmission
 B. subtilis  More than 25 epithelial cells/LPF and
Examples:
 M. gordonae (Tap less than 10 PMN cells/LPF  saliva
 S. aureus
water Bacillus)  More than 10 PMN cells/LPF and less
 HIV
than 25 epithelial cells/LPF  sputum
 Enterics
 Three separate first morning specimens for
 Toxoplasma spp.
mycobacteria or fungal infections
BSL – 3 BSL – 4
 Fungal infections mimic fungal infections 
 organisms which pose  organisms which
systemic fungal infections like Valley fever or
high risk on lab expose extreme risk on
those with cough symptoms. Sometimes this is
personnel and lab personnel and
mistaken as tuberculosis.
environment. environment.
 5. Urine – Specimen of choice: Midstream clean catch 3. Serves as a guide to the ongoing workup of the
 Catheterized - for patients incapable of specimen.
producing urine but prone to contamination.
 Suprapubic - best for urine sample intended for C. Ways of Identifying and Classifying Bacteria
anaerobic culture because there is no  Morphology / Appearance – morphological
contamination. characterization of microorganisms includes the
observance of their shape, arrangement, motility, and
6. Stool – Specimen of choice for detection of staining characteristics.
gastrointestinal pathogens According to shape:
 Specimens should not be taken from the toilet 1. Cocci
and should not be contaminated by urine 2. Bacilli
because urine already contains bacteria. 3. Spirochetes
4. Pleomorphic
7. Genital Tract Specimens – to detect microorganisms
causing cervicitis According to arrangement:
 You can collect using swabs. 1. Singly
2. In pairs
C. Specimen Transport 3. In chains
 Primary aim: To maintain the specimen as near as 4. In clusters
possible to its original state 5. Pallisading
 To keep the specimen safe to handle
 Specimen must be transported within 30 minutes to 2 According to motility
hours to the laboratory. 1. Motile
 If impossible to transport within the allotted time, or if 2. Non-motile
processing is delayed, specimens must be stored and
preserved. According to staining characteristics
 Staining – artificial way of coloring microorganisms by
 TRANSPORT MEDIA: a.k.a. HOLDING MEDIA – used to using stains and dyes.
maintain the specimen without supporting the growth of
any microorganism present. Some staining techniques are as follows:
1. Cary-Blaire a. Simple staining
2. Stuart’s or Amie’s – commonly used holding media b. Differential staining
 Charcoal may be used to absorb excessive c. Negative staining  look for capsule (special)
fatty acids d. Special staining
3. JEMBEC system – John E. Martin Biological
Environmental Chamber System Special Staining
 Contains a selective media and a CO2 Capsular stains Muir, Anthony’s, Tyler, Hiss
supplying tablet. Leifson, Fisher and Conn,
 Transport system for N. gonorrhoeae Flagellar stains
Gray
Schaeffer and Fulton, Wirtz
D. Specimen Storage and Preservation Spore stains
& Conklin, Dorner’s
Polar bodies stains 
Specimen Storage and Preservation Wayson  after staining
seen in Yesinia spp.
Anticoagulants are used Nuclei acid stains  DNA Feulgen
- SPS concentration must Warthin starry, Levaditi,
not exceed 0.025% Spirochetes stains fontanna tribondeau, silver
- Heparin may be used for nitrate
Blood viral or Methylene blue, Albert’s,
Mycobacterium isolation Metachromatic granules Neisser, Ljubinsky, Ponder,
- Citrate, EDTA should not stains Lindergran, Burke’s
be used for Technique
microbiological studies
Stored at 36°C – 6 hours GRAM STAINING
CSF EXCEPT for viral isolation –  used to differentiate microorganisms as gram-
Refrigerator temperature positive or gram-negative
Throat and Stored at Refrigerator  originally developed by Hans Christian Gram
Nasopharyngeal swabs temperature  Modifications are later on made such as the Hucker’s
Boric acid is used as (1% ammonium oxalate)
Urine preservative for maintaining  PRINCIPLE: Microorganisms have cell walls which
urine colony count vary based on the thickness of the peptidoglycan
Refrigeration layer and the presence of teichoic acid.
Stool
Formalin and PVA
Genital Tract specimens Stored at room temperature GRAM STAINING:
GRAM POSITIVE GRAM NEGATIVE
V-I-A-S
 Aerobes – Refrigerator Temperature (USSS: Urine, Primary Stain:
Purple Purple
Stool, Sputum, Swab) Crystal Violet
 Anaerobes – Room Temperature (Sterile specimens: Mordant: Gram’s
Purple Purple
CSF, Genital tract specimen, body fluids) Iodine
Decolorizer:
Purple Colorless
Study and Identification of Organisms Acetone Alcohol
A. Gross Examination Secondary Stain:
Purple Red
 take note of the volume of the specimen, Safranin Red
consistency, presence of blood or mucus

B. Direct Microscopic Examination

1. Useful in checking the quality of the specimen collected.
2. Early indication of what causes the infection or disease.
RULES IN GRAM STAIN: 2. Solid phase – contains solidifying agent called agarose
1. Gram stain is not applied to the following organisms: (2-3%)
a. Mycoplasma and Ureaplasma
b. Rickettsia and Chlamydia  Agar is the term used to refer agarose-containing
c. Spirochetes media
2. Cells in a direct smear must appear pink.  Colony formation serves as the indication of
3. All cocci are gram positive EXCEPT: NVMAK bacterial growth in an agar.
4. All bacilli are gram negative EXCEPT: MACCBELL 3. Semi-solid phase – also contains a solidifying agent
but of lower concentration 0.5-1%
Gram negative cocci: Gram positive cocci: 4. Biphasic media – media containing both a liquid and
Neisseria Mycobacterium solid phase
Veilonella Actinomyces  E.g., Castaneda (Brucella) & HBT (Human Blood
Moraxella/Bramhanella Clostridium Tween Agar)  G. vaginalis = Bacterial vaginosis,
Acinetobacter Corynebacterium Clue cells
Klebsiella Bacillus
Erysipelothrix
Functions of Growth Media – media used in the laboratory
Listeria
for transport and cultivation
Lactobacillus
ACID FAST STAINING  provides the nutritional requirements
 used to differentiate microorganisms as Acid fast or
Non-Acid fast 1. Enrichment media
 PRINCIPLE: Some microorganisms contain mycolic  used to enhance the growth of certain organisms
acid (a.k.a. hydroxyl-methoxy acid) which envelopes by the presence of specific nutrients required
the bacteria and retains the primary stain. 2. Supportive media
 used to support the growth of most organisms as it
Ziehl-Neelsen Kinyoun Method contains basic requirements for bacterial growth
 Routinely  Modified 3. Selective media
applied for method  used to inhibit the growth of all organisms
sputum applied for EXCEPT the one being sought after.
specimens stool and  Inhibition is possible because of the presence of
tissue some factors:
specimens  BALDAA: Bile salts, Alcohol, Dyes,
Antibiotics, Acids
Carbol fuchsin Carbol fuchsin
Primary stain: 4. Differential media
3g + 5% phenol 4g + 9% phenol
 used to differentiate organisms by certain metabolic
Steaming (5
Mordant: No. 7 Tergitol or colonial characteristic observed by employing
minutes)
certain factors in the medium
Decolorizer: Acid Alcohol Sulfuric acid
Secondary
Methylene blue/Malachite green  Esculin –
Stain:
hydrolysis of
which imparts a Identification and
 AFO (acid fast organisms) appear red against a blue Bile Esculin black or brown differentiation of
background (Mycobacterium, Nocardia, etc.) Agar coloration streptococci and
 N-AFO (non-acid fast organisms) appear blue or green  Sodium enterococci
(depending on the secondary stain used) desoxycholate
 Ferric citrate
 What is the difference between Ziehl-Neelsen method For the growth of
used in sputum specimens and the modified method for most organisms
stool in the determination of cocidians? (Decolorizer: Blood Agar  5% sheep blood and differentiation
Sulfuric acid) by hemolytic
patterns
 Potato-glycerol
FLUOROCHROME STAINING
based Isolation of
Used when Bordet-
 15-20% Bordetella
microorganisms Gengou agar
defibrinated pertussis
Acridine are suspected but Bright orange against blood
orange cannot be a dark background Buffered  Yeast extract
detected by light Charcoal Yeast  Charcoal Isolation of
microscopy Extract (BCYE)  Salts Legionella spp.
Used to enhance Bright yellow or agar  Cysteine
Auramine-
detection of bright orange against (CVA)  Antibiotics to
Rhodamine Selective for
Mycobacterium a dark background. Cefoperazone inhibit gram
Campylobacter
Calcofluor Strains of fungal Bright apple green or Vancomycin pos, gram neg
spp.
white cell wall (chitin) bluish white Amphotericin and yeasts

 2% hemoglobin Cultivation of
Colonial Characteristics
 Isovitalex fastidious
 Bacterial cultivation is a method by which bacteria are Chocolate agar  X factor = organisms such
transferred from an in vivo environment (human hosts) to Hematin/Hemi as Haemophilus
an in vitro environment (laboratory) in which they are n and Neisseria
artificially allowed to grow and isolated.  V factor = NAD
 Mannitol
(CIN) Selective for
Phases of Growth Media  Antibiotics
Cefsulodin Yersina
1. Liquid phase – does not contain solidifying agent  Indicators:
Irgasan enterotolitica
 nutrients for bacterial growth are dissolved in  Crystal violet
NOvobiosin (Red bull’s eye)
water  Neutral red
 e.g., Broth = BHI, TSB, ALP  Colistin and
Selective isolation
 Turbidity or cloudiness – indication of bacterial (CN) nalidixic to
of
growth in liquid media Colistin inhibit gram
Gram positive
Nalidixic negative
cocci
organisms
  5% sheep blood Except B.  Alkaline Peptone Water: Vibrio spp.
anthracis  Cetrimide Agar: Pseudomonas aeruginosa
 5% sheep blood  Barbour-Stoenner-Kelly: Borrelia burgdorferi
 Potassium
Cystine
Tellurite- Isolation of C. MEDIA for PATHOGENIC NEISSERIA spp. ISOLATION:
Tellurite blood
reduction of diphtheriae a. Thayer-Martin: VCN = Vancomycin, Colistin,
agar
which produces Nystatin
black colonies
b. Modified Thayer-Martin: TVCN = Trimethoprim,
Isolation and
Vancomycin, Colistin, Nystatin
differentiation of
 Lactose & c. Martin-Lewis: TVCAn = Trimethoprim, Vancomycin,
Lactose-
(EMB) Eosin sucrose Colistin, Anisomycin
fermenting and
Methylene  Indicators: d. New York City: TVCAm = Trimethoprim,
non-lactose
Blue  Eosin Vancomycin, Colistin, Amphotericin B
fermenting
 Methylene
enterics (gram
blue
negative)  Cell Cultures – are composed of layers of viable host
cells growing on a surface of a solid medium
 Glucose &
Selective
MaNnitol Chlamydia McCoys cells
enrichment
Gram Negative  sodium Rickettsia Chick embryo cells
medium for
broth desoxycholate
Salmonella, M. leprae Armadillos, Foot pads of mice
and sodium
Shigella spp. HSV 1 & 2 Rabbit kidney
citrate
Influenza,
Parainfluenza,
 Lactose, Measles, Mumps, Monkey kidney
Isolation and Rubella,
sucrose, and
differentiation of
Hektoen- salicin Enteroviruses
Salmonella,
Enteric Agar  Indicators: Adenoviruses,
Shigella from
 Bromthymol Varicella, Human embryonic kidney
other enterics
blue Rhinoviruses
 Acid fuchsin Human diploid fibroblast or
Isolation and CMV
Human embryonic fibroblast
differentiation
Adenoviruses,
between lactose A549 – Human lung carcinoma
and non-lactose Varicella zoster
 Bile salts and RSV, Adenoviruses, HELA cells – Human cervical
fermenting
MacConkey Crystal violet Rhinoviruses adenocarcinoma (Henrietta Lacks)
enterics
Agar  Lactose
NOTE: Lactose is Hep2 cells – Human Laryngeal
 Indicator: RSV, Adenoviruses
replaced by Carcinoma
 Neutral red
sorbitol for
isolation of E. coli Evaluation of Colony Morphology
O157:H7 (EHEC)  Colony size – pinpoint, small, medium, large
 Colony pigmentation – inherent characteristic of a
 Mannitol
specific organism
Mannitol Salt  Sodium chloride Selective isolation
 Green or Metallic sheen – P. aeruginosa
Agar (7.5%) of S. aureus
 Indicator:  Greenish metallic sheen on EMB – E. Coli
 Phenol red  Brick red – Serretia marcescens
Selective isolation  Purple – C. violaceum
Phenyl Ethanol  Phenylethyl  Brown-black – Prevotella melalinogenica
of gram-positive
Agar (PEA) alcohol  Colony shape
organisms
 Bile salts, a. Edge/margins – smooth, filamentous, rough
Salmonella- Brilliant green, Selective for or rhizoid, irregular
Shigella Agar sodium citrate Salmonella,  “Medusa heads” are filamentous
(SSA)  Indicator: Shigella appearance observed in B.
 Neutral red anthracis
 Vancomycin,  Swarming – hazy blanket of growth
Selective for
Skirrow Agar Trimethoprim, on surfaces that extends beyond
Campylobacter
Polymyxin the streak lines observed in
Proteus spp.
 Bile salts,
b. Elevation – flat, raised, convex or dome,
Citrate, Sodium
Thiosulfate Selective and umbilicate and umbonate
Thiosulfate
Citrate Bile Differential for  S. pneumoniae – umbilicate
 Sucrose
Salt (TCBS) Vibrio spp.  S. pyogenes – flat colonies
 Indicator:
 Bromthymol  S. aureus – convex
Blue  Hemolytic Patterns
a. Beta-hemolytic – complete hemolysis
Isolation and b. Alpha-hemolytic – partial hemolysis or
 Lysine
differentiation of
Xylose Lysine  Xylose, incomplete hemolysis (until biliverdin only)
Salmonella,
Desoxycholate Lactose, c. Gamma hemolytic – non-hemolytic
Shigella from
(XLD) Agar sucrose d. Alpha-prime or Wide zone – intact RBCs
other gram
 Indicator: surrounded by wide zone of complete
negative enterics
 Phenol red hemolysis (prolonged refrigeration)

Other Significant Media:  Odor – allow the odor to dissipate. NEVER INHALE
 Selenite broth: Salmonella DIRECTLY.
 Tetrathionate broth: Salmonella, Shigella  Old socks – S. aureus
 Todd-Hewitt broth: Transport media – Streptococcus  Grape-like or fruity – P. aeruginosa
agalactiae  Putrid – P. mirabilis
 Tinsdale agar: Corynebacterium
  Mousy, musty basement or mouse nest – 6. Salt concentration = Halophilic
Haemophilus a. 6.5% = Enterococci
 Freshly plowed field – Nocardia b. 7.5% = S. aureus
 Horse stable/barn yard – Clostridium c. 8-10% = Vibrio  All Vibrios are salt loving, EXCEPT:
difficile V. cholerae, V. mimicus

Environmental Requirements for Bacterial Growth

1. Oxygen Requirement
a. Aerobic – organisms require the presence of oxygen
for growth.
 requires 21% O2, 0.03% CO2
b. Strictly Anaerobic – organisms that grow in the
absence of oxygen such as Clostridium and
Bacteroides
c. Facultative Anaerobes – organisms that grow either
in the absence or presence of O2
 They are most clinically significant bacteria
 These organisms do not require oxygen but
grow better in its presence
 Enterobactiaceae
 Note: Obligate aerobes and facultative anaerobes
contain protective enzymes (superoxidase dismutase
and catalase) that counter the toxic effect of oxygen.
d. Microaerophiles – organisms which require low
levels of oxygen
 85% nitrogen, 10% CO2, 5% O2 (Campy
gas)
e. Aerotolerant – organisms that do not grow well but
can survive in the presence of air. e.g., Lactobacillus,
Propionibacterium acnes

2. Carbon Dioxide requirement

a. Capnophilic – organism requires the presence of
CO2 (5-10%) for growth
 Most aerobic and facultative aerobic bacteria
need 0.3% CO2
 Neisseria, Haemophilus, Capylobacter
Helicobacter, Moraxella

3. Temperature Requirement
a. 35-37°C = temperature where most organisms grow
b. 42°C = temperature where Pseudomonas and
Campylobacter can grow
c. 0-20°C = temperature where Yersinia and listeria
monocytogenes can grow
d. 5-125°C = temperature where B.
stearothermophilus and subtilis var niger can grow
 thermophiles

Psychrophiles/Cryophiles
 Grow well at 0°C to a maximum of 20°C
 Listeria and Yersinia enterocolitica
Mesophiles
 Grow between 20°C to 45°C
 These are the most commonly encountered
pathogenic bacteria in the clinical laboratory
Thermophiles/Hyperthermophiles
 Grow between 50°C to 125°C
 Bacillus stearothermophilus, Sulfolobus, Pyrococcus,
Pyrodictium, and Thermus aquaticus
Extremophiles
 These are prokaryotes that are able to survive in an
unusual condition like the absence of oxygen,
increased temperatures and living below the earth’s
surface
 Bacillus infernus

4. pH Requirement
a. Acidophilic – requires acid medium at a pH range of
0 and 5.5
b. Neutrophilic – neutral pH for growth (5.5-8.0)
c. Basophilic – requires alkaline (8.5-11.5) medium for
growth = Vibrio

5. Moisture = Humodophilic  require water

 METABOLIC & BIOCHEMICAL CHARACTERISTICS

ENZYMATIC TESTS
Positive Result
 Bubble formation
 Effervescence Staphylococcus
Neisseria
Catalase is an enzyme which hydrolyses hydrogen 3% = Staph vs. Strep Micrococcus
Catalase Test
peroxide. Listeria
15% = Anaerobes
Corynebacterium
30% = Superoxol test Mycobacterium
(N. gonorrhoeae)

 S. aureus –
free coag
Coagulase is an enzyme that alters fibrinogen Positive result:
 S. lugdunensis
causing it to form clots. It is diagnostic test for S.  Clot formation or
– bound
aureus (+) clumping
 S. schleiferi -
Coagulase test bound
A. Slide Test – determines bound coagulase or clumping factor
 directly reacts with fibrinogen
B. Tube Test – determines free coagulase of
 reacts with CRF (Coagulase-Reacting Factor) to form a complex that is thrombin-like
Human, Rabbit, or Pig plasma may be used.

a. Dye method  Green color /Clear zone S. aureus

 Methyl green around the colony M. catarrhalis
DNAse Test  Toluidine blue  Pink zone S. marcescens
b. HCl method  Clear zone
(SMS)

 cytochrome oxidase acts in the substrate Neisseria

Tetramethyl-p-phenylenediamide-dihydrochloride  Dark purple Aeromonas
Oxidase Test
to Indophenol Plesiomonas
(NAP)
Microdase Test
- modification of  also detects presence of oxidase (DMSO)
oxidase test  primarily differentiates Micrococcus from Micrococcus
Staphylococcus  Blue to purple
(Dimethylsulfoxide)
DMSO
Indole is a product of tryptophan degradation Indicator:
catalyzed by tryptophanase p-dimethyl-
E. coli
 PAI = Pyruvic acid, ammonia, indole aminobenzaldehyde
Indole Test (acetoin)
A. Ehrlich’s method – xylene is added for extraction
 more sensitive than Kovac’s
B. Kovac’s method – no xylene is needed  red ring at the tube
Positive Result
Urease hydrolyzes urea to form ammonia, CO2, and  Pink/Red Proteus
Urease Test
water. H. pylori
Indicator: Phenol red
Positive Result
 Red
PYR (pyrolidonyl-a-naphthylamide) is acted by
PYR Test enzyme pyrrolidonyl amino peptidase / arylamidase S. pyogenes
to form beta- naphthylamide Indicator:
p-dimethyl-am-
cinnamaldehyde
Positive Result
Hippurate is hydrolyzed by hippuricase to form  Deep purple color S. agalactiae
Hippurate
glycine. Only S. agalactiae can produce hippuricase. L. monocytogenes
Hydrolysis Indicator:
Glycine is oxidized. C. jejuni
Ninhydrin
CARBOHYDRATE UTILIZATION
Differentiation
Oxidation  Bromcresol purple;
between
 carbohydrate utilization which requires oxygen. Andrade’s acid fuchsin;
Oxidation- Enterobacteriaceae
Fermentation Bromthymol blue; phenol
Fermentation and Pseudomonas
 carbohydrate utilization in the presence or red.
(O-F Test) spp. As well as
absence of oxygen.  Yellow is the positive
Staphylococcus
Mineral oil is used as barrier to oxygen. color.
from Micrococcus
A/A Citrobacter
 contains glucose and lactose or sucrose with a Yellow/yellow E. coli
1:10 Enterobacter
Triple Sugar Iron  Sodium thiosulfate and ferrous sulfate to Klebsiella
(TSI) observe for H2S production. K/A Proteus
 Gas formation is also observed using this agar. Red/yellow Providencia
 Displacement and bubble formation Morganella
Shigella
 Salmonella
K/K Pseudomonas
Red/red Alcaligenes
Achromobacter
Acinetobacter
Lactose fermenters:
 β-galactose permease,
directly uses lactose as
 For the differentiation of LLF and NLF source of energy
Serratia
 ONPG is hydrolyzed by β-galactosidase and Late lactose fermenter:
ONPG H. alvei
produces galactose and o-nitrophenol.  β-galactosidase only, acts
Y. enterocolitica
on lactose to use it
Positive Result:
 Yellow color

Positive Result
 Red
 Further degradation of pyruvic acid via the mixed
Methyl Red Indicator:
acid pathway
 Methyl red

Positive Result
 For further degradation of pyruvic acid via the
 Red
butene diol pathway
Voges-Proskauer Indicators:
 Pyruvic acid  Acetoin Diacetyl + KOH and α-
 KOH
naphthol = 2,3-butenediol
 α-naphthol

AMINO ACID UTILIZATION

Amino acids may be
decarboxylized to produce
amines. Positive Result
decarboxylized to:  Purple
Decarboxylation Lysine  cadaverine
Ornithine  putrescine Indicator:
Arginine  ornithine  Bromcresol blue
Requires an anaerobic
environment.
Positive Result
 Phenylalanine is used to  Green Proteus
Deamination (PAD) Test determine the presence of Providencia
deaminase. Indicator: Morganella
 10% Ferric chloride
 Determine whether
bacterium is capable of
Deaminase:
decarboxylation or
 Red is positive.
deamination.
Decarboxylase:
Lysine Iron Agar  Contains lysine, glucose,
 Purple is positive
peptone, ferric ammonium
 Yellow = glucose fermenter
citrate
 Black = H2S production
 Sodium thiosulfate for H2S
production.
MISCELLANEOUS TESTS
Positive Result
 Blue
 Determine citrate utilization
Citrobacter
as a sole source of carbon. Indicator:
Hafnia
 Simmon’s Citrate agar is  Bromthymol blue
Enterobacter
Citrate utilization used for this test.
Klebsiella
 Christensen’s citrate medium Positive Result
Serratia
can be used as the  Pink
Providencia
alternative medium.
Indicator:
Phenol red
 Sulfanilic acid and NNDAP  If no color is developed
(n-n-dimethyl-a- initially, zinc dust is added. C. diphtheriae
Nitrate Reduction Test
naphthylamide) indicates (+) = no color change M. tuberculosis
reduction (-) = red color
 Determination of motility,
Sulfide Indole Motility (SIM) It is a semi-solid medium for motility of bacteria.
H2S and indole production
 Dissolution of colonies in the
Bile solubility presence of sodium  S. pneumoniae identification
desoxycholate
GRAM POSITIVE COCCI o Gamma – less toxic than alpha and beta,
 organisms observed to be gram positive cocci under produce by all Staph strains and produce
the microscope injury in culture
 include: o Delta - Destroys red blood cell and white
a. Staphylococcus blood cells
b. Micrococcus  Enzymes:
c. Streptococcus - Coagulase – fibrin formation around the bacteria
d. Enterococcus, Pneumococcus, Viridans as protection from phagocytosis
 These four can further be classified using the - β-lactamase / Penicillinase – destroys the beta-
catalase test. lactam unit of an antibiotic making it inactive.
 Catalase positive: Staphylococcus and - Protein A – interferes phagocytosis (anti-
Micrococcus  OF test to differentiate phagocytic)
 Catalase negative: Streptococcus and  Found on the cell wall of Staph aureus
Enterococcus, Pneumococcus, Viridans - Staphylokinase – promotes fibrinolysis (aka
fibrinolysin)
A. Staphylococcus spp. - Hyaluronidase – breaks down proteoglycans in
 catalase positive, non-motile, non-spore formers, connective tissues
facultative anaerobes  a.k.a. Spreading factor or the Duran-
 Resistant to Bacitracin, a.k.a. Taxo A Reynal factor
 they are arranged in a “grape-like” clusters - Lipase – degrades fats and oils
 able to grow in 7.5% NaCl - Proteinase – destroys tissue proteins
 normal flora of the skin and the nasopharynx
 Staphylococci are generally differentiated using the  Diseases caused:
Coagulase test.  Skin infections
 Folliculitis, furuncles (boils), carbuncles
COAGULASE POSITIVE COAGULASE NEGATIVE (systemic), bullous impetigo
“CoPS” “CoNS”
S. aureus  most S. epidermidis  most  Food Poisoning
common and pathogenic common and pathogenic  Ingestion of pre-formed toxins
S. delphini S. saprophyticus  most  common in products such as mayonnaise,
S. intermedius common and pathogenic cream fillings, and dairy products
S. lutrae S. haemolyticus  symptoms are manifested 2-8 hours of
S. hyicus S. schleiferi contaminated food
S. lugdunensis
 Toxic Shock Syndrome
Clinically significant species:  characterized by fever, diarrhea, vomiting, and
1. S. aureus rashes that later on leads to hypotension and
 General Characteristics: shock.
 exhibits golden/creamy yellow pigmentation in BAP
 Beta-hemolytic  SSSS Staphylococcal Scalded Skin Syndrome
 Mannitol fermenter = yellow halo around colonies on  a.k.a. as the Ritter’s Disease
MSA (Mannitol Salt Agar)  characterized by cutaneous erythema followed by
 DNAse positive profuse peeling of the epidermis.
 more common in children and neonates
 Virulence Factors:
 Enterotoxins:  Other infections: Pneumonia, Osteomyelitis, Septic
 Heat-stable toxins – Arthritis, and Endocarditis
 E.g., Enterotoxins A, B, C1, C2, D, E, and G to J
 Enterotoxins A, B, and D = responsible for food 2. S. epidermidis
poisoning  Exhibits gray-white colonies
 The infected food handler is the source of  Gamma-hemolytic, normal flora of the skin
contamination  Biofilm formation - capable of producing slime which
 Enterotoxin B = associated with enhances attachment to surfaces.
pseudomembranous enterocolitis and is often  Hospital-acquired infections with predisposing
found in contaminated milk products factors which include catheters, IV, prosthetic devices.
 TSST-1 = Toxic Shock Syndrome Toxin-1
 Interacts with T-cells causing an over-reactive 3. S. saprophyticus
immune response which leads to shock  exhibits white, glossy colonies (which may also
 Menstruation associated toxic shock syndrome appear yellow or orange)
 Enterotoxin F  gamma-hemolytic
 Associated with tampon use.  Novobiocin resistant
 Associated with UTI in young menstruating women
 Exfoliative toxin / Exfoliatin: Epidermolytic toxin
 Causes the sloughing off of skin’s epidermal layer MRSA (Methicillin-Resistant S. aureus)
 responsible for Staphylococcal Scalded Skin  strains of S. aureus which are resistant to antibiotics
Syndrome such as that of methicillin, nafcillin and oxacillin.
 Causes sloughing off of the skin  It can be acquired:
 Produce burn like effect on the patient  after a prolonged stay in the hospital (ICU
 Also known as the Ritter’s disease and burn patients)
 close contact with individuals who are
 Cytolytic toxins carriers of this organism
 Types of hemolysins: alpha, beta (hot-cold) lysin),  After-effects of a broad spectrum of antibiotic
gamma, Panton-Valentine Leukocidin (delta) treatments
o Alpha – major hemolysin  Exposure to nasal secretions
o Beta – destroys sphingomyelin around  MeC A gene is the one which codes for penicillin
nerves binding protein (PBP) which gives the strain its
methicillin resistant feature.
  It is controlled by the proper isolation of the organism,  Pyrogenic or Erythrogenic Exotoxins – causes a
rapid identification of the bacteria, hand hygiene, red spreading rash (Scarlet fever  Exotoxin B)
treatment of sources and most importantly a strict  Streptolysin – responsible for hemolysis
compliance to infection control programs
 It may also be resistant to semi-synthetic penicillins Streptolysin O Streptolysin S
 Three types of MRSA (Oxygen labile) (Oxygen stable)
1. Hospital -acquired (HA) MRAA Hemolysis on BAP in an Hemolysis on BAP in an
2. Community-acquired (CA) MRSA anaerobic environment aerobic environment
3. Healthcare-associated community-onset  exhibits subsurface  exhibits surface
(HACO) MRSA hemolysis hemolysis
 (+) Chromogenic test – Changes in color of MRSA  Highly immunogenic –
colonies within 24-48 hours using CHROM agar  Non-immunogenic
ASO test
against colorless colonies of non-MRSA.
 MRS may be detected by:  Diseases caused:
a. Nitrocefin disk test (colored colony in  Streptococcal pharyngitis (Strep Throat)
chromogenic agar)  inflammation of the tonsils and pharynx
b. PCR – detection of mecA gene and PBP
 gold standard  Skin Infections
 Impetigo (non-bullous)
B. Micrococcus spp.  Erysipelas /St. Anthony’s Fire – a spreading
 catalase positive, coagulase negative, strict aerobe skin lesion, intensely erythematous with
 arranged in tetrads under Gram stain demarcated irregular edge.
 susceptible to Bacitracin  Cellulitis – develops following deeper invasions
 produce opaque, non-hemolytic colonies with various  Necrotizing fasciitis
pigments (white, tan, yellow, orange, or pink)  a flesh-eating disease
 rapid inflammation and invasion in the fascia
Differentiation of Staphylococci and Micrococci
Oxygen Facultative  Scarlet fever aka Scarlatina
Strict aerobe
Requirement anaerobe  fever with scarlet red rash (from trunk to
Microdase - + extremities)
Glucose  Dick’s test = skin test for scarlet fever
Fermenter Oxidizer
Utilization  Blanche phenomenon – fading of rashes with
Bacitracin (0.04 the administration of anti-erythrogenic toxin
Resistant Susceptible
U)  Diagnostic test – Schultz Charlton Test
Furazolidone
Susceptible Susceptible
(100 ug)  Streptococcal Shock Syndrome
Lysostaphin  Post-streptococcal infections
Resistant Resistant  Rheumatic fever – caused by cross-
(200 ug)
reactivity between streptococcal antigens
C. Streptococcus spp. and heart tissue This is a complication of
 catalase negative, non-motile, with colonies arranged pharyngitis not wound infection.
in chains or in pairs  Acute glomerulonephritis syndrome –
 capnophilic antigen-antibody complexes are deposited in
 Streptococcus spp. may be classified based on the the glomeruli, complement system is
following: activated causing the destruction of
 Hemolytic pattern: complexes but also damages glomerulus.
 Alpha-hemolysis – S. pneumoniae, Viridans group
 Beta-hemolysis – S. pyogenes, S. agalactiae & 2. S. agalactiae
Group C strep  General Characteristics:
 Gamma-hemolysis – Group D non-enterococci and  Catalase negative
Enterococci  Bacitracin resistant
 Alpha’ hemolysis – caused by prolonged  Hippurate hydrolysis positive  only S. agalactiae
refrigeration contain hippurase enzyme
 Lancefield Classification by Rebecca Lancefield  CAMP test positive
 Streptococcus spp. are classified based on their
antigenic C carbohydrate (polysaccharide) found in  Virulence Factors:
the cell wall.  Polysaccharide capsule (sialic acid) – anti-phagocytic
 This classification is then designate by letters:  CAMP factor – diffusible extracellular protein which
a. Group A – S. pyogenes enhances RBC lysis by S. aureus
b. Group B – S. agalactiae  S. agalactiae exhibits arrow-head pattern of
c. Group C – S. dysagalactiae, S. equi hemolysis (enhanced hemolysis)
d. Group D – Non-enterococci, S. bovis
e. Other Groups:  Diseases caused:
- Enterococci  Newborn diseases
- Pneumococci Early Onset Late Onset
- Viridans group At least 7 days old to 3
Less than 7 days after birth
months after birth
Clinically significant species: Associated to obstetric Not associated with obstetric
1. S. pyogenes / Group A Strep complications complications
 General Characteristics: Manifested as neonatal Manifested as neonatal
 M protein – antiphagocytic and enhances pneumonia and sepsis meningitis
attachment Organism is found in the Organism is rarely found in
 Protein F – Fibronectin – functions to mediate vagina of the mother the vagina of the mother
attachment to host epithelial cells  It is recommended that all pregnant women be
 Hyaluronic acid capsule – prevents opsonization screened in Group B Strep at 35-37 weeks of
 Weakly immunogenic, prevents opsonized gestation.
phagocytosis and masks antigen
  Samples (vaginal or rectal swabs) are 3. Viridans Group
inoculated in selective broth, Todd-Hewitt  Bacitracin resistant
(transport media).  Optochin resistant
 Post-partum endometriosis  SXT susceptible
 Endocarditis  Normal flora of the mouth
 Can also cause UTI
 Diseases caused:
3. S. dysagalactiae, S. equi / Group C  Dental carries and gingivitis – S. mutans
 Beta-hemolytic and are subdivided as large or small  Subacute bacterial endocarditis – S. sanguis
colonies
 Usually classified as animal pathogens – when STREPTOTOCCUS-LIKE ORGAN
causing infections to man, resembles Strep pyogenes Known as nutritionally
infection. Abiotrophia variant strep requiring
Vitamin B6
4. Non-Enterococci & S. bovis / Group D Pseudocatalase reaction,
 Bile esculin positive Aerococcus
grows in 6.5% NaCl
 No growth in 6.5% NaCl Lactococcus Infecting
 May cause endocarditis and UTI Pediococcus immunocompromised
Leoconostoc patients
Non-Enterococci Enterococci
Bile esculin GRAM NEGATIVE COCCI
+ +
hydrolysis  includes Neisseria, Veilonella, Moraxella
Growth in 6.5%
- +
NaCl
A. Neisseria
PYR test - +
 The species in this genus are obligate aerobes, non-
Penicillin
Sensitive Resistant motile, and non-hemolytic.
susceptibility
 The species are fastidious, capnophilic, and grow
D. Enterococcus, Pneumococcus, Viridans optimally in a moist temperature.
1. Enterococcus spp. (E. faecalis, E. faecium)  Most species are carbohydrate fermenters.
 bile esculin positive  The species grow best in media with blood and
 grows in 6.5% NaCl cholesterol.
 PYR positive  They are sensitive to heat and drying, thus requiring a
 Normal flora of the GUT direct inoculation of specimens “at the bedside.”
 Causes nosocomial infections such as UTI  GS: gram negative diplococci that are coffee- or
kidney bean-shaped
2. S. pneumoniae  Catalase positive EXCEPT N. elongate
 General Characteristics:  Oxidase positive EXCEPT N. flavescens–yellow
 Young culture: dome-shaped and mucoid (contains pigmented colonies
capsule) colonies  Culture: colonies appear small, grayish-white,
 Old culture: coin with a raised rim appearance opaque, convex, and glistening.
(undergoes autolysis)  Their natural habitats are the mucous membranes of
 Exhibits Quellung / Neufeld in which capsule swells the respiratory and urogenital tracts
in the presence of anti-capsular serum  susceptible to: drying, cold chemicals, calcium
 GS: gram positive cocci arranged in pairs (diplococci) alginate, cotton swabs
= oval/lancet shape/bullet shape
 Bacitracin susceptible  Species may be classified according to carbohydrate
 Optochin susceptible utilization:
 SXT sensitive Gluco Malto Lacto Sucro Fruct
 Bile soluble – with the presence of bile salts, colonies se se se se ose
are lysed
N. gonorrhoeae + - - - -
 Normal flora of the upper respiratory tract (when lower N. meningitidis + + - - -
RT is infected, it will cause pneumonia) N. lactamica + + + - -
N. elongate - - - - -
 Virulence Factors: N. flavescens - - - - -
 Polysaccharide capsules – strains without capsule are N. cinera - - - - -
non-pathogenic. N. sicca and N. + + - + +
mucosa
 Diseases caused: Note: Only N. lactamica is able to utilize sugar lactose.
 Lobar pneumonia – most common bacterial
pneumonia characterized by chills, dyspnea, and Clinically significant species:
cough 1. N. gonorrhoeae
 Meningitis in individuals > 29 years old – usually  General Characteristics:
follows S. pneumoniae infections (pneumonia and  kidney-shaped
otitis media)  Grows in CAP but not in BAP, requires 3-5%
 Otitis media in children CO2 (selective media may also be used)
 Utilizes glucose only
 Vaccines  Superoxol positive –rapid testing using 30%
 PCV7 = Pneumococcal Conjugated Vaccine H2O2
 purified polysaccharide of 7 serotypes conjugated with  (positive result = bubbling)
a diphtheria protein  Virulence Factors:
 used for children  Pili –anti-phagocytic and enhances attachment
 PS23 – composed of 23 pure capsular polysaccharide  Diseases Caused:
 used for adults  N. gonorrhoeae causes sexually transmitted
disease
 Specimen used: swabs / exudates of cervix,
urethra, rectum, throat
  Diseases Caused: Gonorrhea - Waterhouse Friderichsen Syndrome–
 came from the Greek words gonos, which means refers to the bleeding of the adrenal
“seed,” and rhoia, which means “flux.” glands
 It is an acute pyogenic infection of non-ciliated,  Adrenal gland function is affected
columnar, and transitional epithelium. resulting to low blood pressure and
 It may be asymptomatic among females. tachycardia which may lead to
 sites of infection: endocervix, conjunctiva, shock and death.
pharyngeal surface, anorectal area, urethra  Treatment: PENICILLIN G and
A. In men –SYMPTOMATIC = “Flow of seed” CEFTRIAXONE, RIFAMPIN for
– pus formation close contact
 characterized by pain in urinating and - Notes to Remember:
purulent urethral discharge.  Petechial skin lesions may develop
 complications may lead to inflammation during bacteremic spread due to the
of prostate and epididymis release of endotoxin after a
B. In women - ASYMPTOMATIC bacterial cell lysis.
 Complications may lead to inflammation  Penicillin is the drug of choice for
of uterus, fallopian tube, and ovaries the treatment of meningococcal
together known as Pelvic Inflammatory meningitis.
Disease (PID)
 further complications of PID may lead to: B. Moraxella
- sterility 1. M. catarrhalis
- ectopic pregnancy  A commensal of the respiratory tract (but may cause
- Fitz-Hugh-Curtis Syndrome otitis media)
o perihepatitis characterized  “Hockey-pucky” colonies; remains intact when pushed
by pain in the upper right across plate with a loop
quadrant  Catalase negative
 Diseases Caused: Ophthalmia neonatorum  Oxidase positive
 transmitted by an infected mother to her baby  DNase positive
during delivery  Assachrolytic–neither fermenter nor oxidizer
 cornea of the newborn is infected which may  Butyrate disc positive = detects butyrate esterase (rub
lead to blindness colonies on disc = (+) blue color)
 prophylaxis: Crede’s prophylaxis using 1%
silver nitrate and Erythromycin eye drops GRAM POSITIVE BACILLI

Clinically significant species: SPORE FORMERS –includes Bacillus and Clostridium spp.
2. N. meningitidis A. Bacillus – AEROBIC, Catalase positive
 General Characteristics:  General Characteristics:
 Intracellular or extracellular gram negative  The species of this genus are spore-forming,
diplococci aerobic or facultatively anaerobic, rod-shaped
 Utilizes glucose and maltose only bacteria, and can be isolated from the soil.
 A commensal of the nasopharynx  The species only form endospores aerobically.
 It is the causative agent of meningococcemia or  The species are motile with peritrichous flagella,
spotted fever. except for B. anthracis and B. mycoides.
 It is the leading cause of fatal bacterial  Microscopy: large, boxcar-shaped, Gram-
meningitis. positive rods with clear, unstained central spores
 Microscopy: encapsulated strains can have a or “empty spaces”
halo around the organism
 Culture: 1. B. anthracis
a. Encapsulated strains are mucoid in  General Characteristics:
appearance.  Colony: Medusa head/ lion face colony, cut
b. Colonies appear bluish gray on BAP. glass appearance
c. Colonies appear small, tan, and mucoid on  Exhibits gamma hemolytic pattern
CAP.  Exhibits beaten egg white consistency when
 Virulence Factors: colonies are lifted using inoculating loop
 Capsule–antiphagocytic (positive for Neufeld)  Microscopic: Square ends, bamboo rods/ box
 Endotoxins–which damages blood vessels car appearance
causing the formation of petechiae  Exhibit inverted pine/fir tree in gelatin agar tube
 IgA protease –cleaves the IgA into two  String of pearl test –inoculate isolate on (0.05
 Diseases Caused: u/mL) penicillin-containing agar.
 Infants (6 months to 2 years) and army recruits  incubate at 37 C for 3-6 hours
are prone to these diseases  Virulence Factors:
 Specimen used: CSF, blood, joint fluid, swabs  Capsule –unique since it is made of a protein
–carriers glutamic acid
 Transmitted through respiratory droplets in the  Spores –which releases the exotoxin: TRIPLE
form of meningitis EXOTOXIN:
A. Meningitis a. Edema factor (EF) –causes massive edema
- inflammation of the meninges c. Protective antigen (PA) –allows the entry of
characterized by fever, stiff neck and the 2 other factors
lethargy d. Lethal Factor (LF) –inactivates protein kinase
- Usually affects individuals who are 5-29  EF + PA = edema
years old  LF + PA = death
B. Meningococcemia  Disease caused:
- intravascular multiplication of the A. Cutaneous Anthrax
organism - Most commonly transmitted, least severe
- characterized by fever, chills, joint and - Characterized by “malignant which
muscle pain and petechial rash eventually forms a black necrotic central
- DIC may eventually develop called black eschar
 B. Pulmonary Anthrax  Exhibits double / target hemolysis on BAP
- Also known as Woolsorter’s disease, Rag  REVERSE CAMP Positive= reverse/enhanced
picker’s disease, Hide porter’s disease hemolysis (arrow-head pattern)
- Transmitted by the inhalation of spores  Known organism: S. agalactiae
C. Gastrointestinal Anthrax  Microscopy: “Boxcar-shaped” bacilli with oral,
- Rarest form of anthrax, most severe subterminal spores
- Transmitted by the ingestion of spores  Culture: BAP –colonies are dome-shaped and
***Ascoli test –serological test for the diagnosis grayish-white with double zones of hemolysis (alpha
of anthrax and beta zones)
 Biochemical test: Very fermentative
2. B. cereus  (+) lecithinase–detected using egg yolk agar
 General Characteristics: (EYA)
 Beta-hemolytic, motile, non-encapsulated  (+) Reverse CAMP test –formation of an
 Virulence Factors: “arrowhead-shaped” zone of hemolysis towards
 Spores –which releases the enterotoxin the test organism
 diarrheal enterotoxin –diarrhea (ingestion of  Virulence factors: capsule, alpha toxin
meat, vegetables, sauce) (Lecithinase), beta toxin
 emetic enterotoxin –vomiting, shorter incubation  Diseases Caused:
period (fried rice) a. Food poisoning –necrotizing enteritis
 Diseases Caused: - it is caused by the ingestion of β-enterotoxin
 Food poisoning –commonly associated to fried in contaminated food.
rice (Fried rice bacillus) - improperly stored food allows the
a. Diarrheal type germination of the spores and growth of
- It is associated with the ingestion of vegetative bacteria.
contaminated meat, poultry, and - symptoms: bloody diarrhea and abdominal
vegetables. pain
- Incubation period: 8-16 hours (long b. Cellulitis/ Wound infections –associated with
incubation) CREPITUS which is a spongy moist consistency
- symptoms: abdominal pain and watery of skin due to gas production.
diarrhea without fever. c. Gas gangrene / Myonecrosis–involves muscle
- (+) production of heat-labile degradation
b. Emetic type - it is a life-threatening destruction of muscle
- It is associated with the ingestion of and other tissues.
improperly stored cooked rice. - it is accompanied by bullae (fluid-filled
- It is caused by B. cereus type 1. blisters), pain, swelling, serous discharge,
- Incubation period: 1-6 hours (short discoloration, and tissue necrosis.
incubation)
- (+) production of heat-stable enterotoxin 2. C. difficile – “difficult to treat
 Eye infections  it is the most common cause of antibiotic-associated
diarrhea and pseudomembranous colitis (blood
3. Other Bacillus species diarrhea with necrosis of colonic mucosa).
A. B. subtilis –also known as the Hay Bacillus–causes  it is an “infection control dilemma” among hospitalized
eye infection in drug addicts especially with heroine patients.
- It is the most commonly encountered laboratory  virulence factor: Toxin A and Toxin B
contaminant.  Microscopy: chains up to 6 cells that are aligned
- It is the source of the bacitracin antibiotic. It can from end to end with oval, subterminal endospores
cause an eye infection among prohibited drug  Normal flora of the colon
users.  Spores are subterminal
- Culture: BAP–colonies are large, flat, and dull  In CCFA (Cycloserine cefoxitine fructose agar) –
with a ground with a ground glass appearance horse stable / barnyard odor
B. B. stearothermophilus  produces TOXIN A: enterotoxin and TOXIN B:
C. B. subtilis varniger Cytotoxin
D. B. subtilis varglubiji  causes Antibiotic-associated Pseudomembranous
colitis (Clindamycin, Ampicillin, Cephalosporin)
Hemolysis Motility Capsule Penicillin  Treatment includes discontinuing initial antibiotic
B. therapy and administering metronidazole or
Gamma - + S
anthracis vancomycin orally.
B.
Beta + - R
cereus 3. C. tetani – “drumstick bacillus”, “tennis racket
Growth at Salicin Gelatin hydrolysis bacillus”, “lollipop bacillus”, “tack head bacillus”
45°C fermentation and PEA  it is a soil and environmental inhabitant
B.  the endospores are found in the soil, dust, and feces
- - -
anthracis of many farm animals.
B.  virulence factor: Tetanospasmin(neurotoxin)
+ + +
cereus  Culture: BAP –colonies exhibit a slow, anaerobic,
heavy, smooth, and swarming growth and have a
B. Clostridium – ANAEROBIC, Catalase negative
matte surface with a narrow zone of β-hemolysis.
 The species of this genus are obligate anaerobes,
 Characterized by a terminal swollen spore
catalase-negative, Gram-positive, spore-forming
 Commonly found on soil or animal feces, iron-loving
bacilli.
bacterium
 The toxins produced by the pieces are acquired
 Produces the exotoxin: TETANOSPASMIN–heat
through ingestion or open wounds that have been
labile toxin
contaminated with soil.
 Inhibits the inhibitory neurotransmitters (GABA and
1. C. perfringens – also known as the Welch bacillus, Gas
Glycine) leading to spastic paralysis:
gangrene bacillus by William Welch
a. lockjaw / trismus–muscle spasms of the jaw
 Only clostridium species that is: non motile,
b. risus sardonicus–muscle spasms of the face
encapsulated, lactose and sucrose +
 c. opisthotonus–severe muscle spasm at the 1. C. diphtheriae – also known as the Kleb Loeffler’s
back bacillus
 TETANOSPASMIN  it is facultative anaerobe but grows best under aerobic
 it is an endopeptidase conditions.
 it causes tension or cramping and twisting in  it is not part of the indigenous microbiota of the
skeletal muscles that surround the wound, respiratory tract, and only inhabits the human
and tightness of the jaw muscles. nasopharynx in a carrier state.
 TETANUS  it is acquired through inhalations of contaminated
 characterized by trismus or lockjaw and risus respiratory droplets or direct contact with infected
sardonicus or distorted grin. cutaneous lesions (hand-to-mouth transmission).
 it occurs when the organism (spore) enters  rarely enters the bloodstream.
an open wound and spreads a potent toxin  is readily killed by heat and by most of the usual
that mediates generalized muscle spasms. disinfectants.
 Symptoms: muscular rigidity (jaws, neck,  is a glucose and maltose fermenter.
and lumbar region), difficulty in swallowing,  virulence factor: Diphtheria toxin
rigidity of the abdomen, chest, back, and  preferred medium: enriched medium with serum,
limbs cysteine, and potassium tellurite
 incubation period: 3-21 days –the long  microscopy:
incubation is related to the distance from the a. Its cells have rounded ends and “club-shaped
injury to the central nervous system swellings.”
 DPT vaccine is given during childhood and booster b. Its highly pleomorphic cells are arranged in pairs
dose is given every 10 years (tetanus toxoid-formalin and create X, V, Y, and L formations that closely
inactivated) resembles Chines letters.
 best specimens: nasopharyngeal and throat swabs
5. C. botulinum – “canned good bacillus”  Contains Babes-Ernst granules giving a beaded
 it is a potential bioterrorism agent. appearance when stained with Methylene blue
 it is characterized by the presence of subterminal  Nitrate positive, Urease negative
spores.  CTBA –black colonies are formed from the
 virulence factor: Botulism toxin –a neurotoxin that is reduction of potassium tellurite
considered as one of the most potent natural toxins  CTBA = Cysteine Tellurite Blood Agar
known to man.  Tinsdale medium – brown-black colonies with
 Culture: BAP –β-hemolytic colonies brown black halo
 Produces the exotoxin:  PAI / Loeffler’s medium –enhances pleomorphism of
 BOTULINUM TOXIN which is neurotoxic the organism and granule formation
- Inhibits the release of acetylcholinesterase  Produces Diphtheria toxinwhich is composed of 2
leading to flaccid paralysis. fragments:
- it requires a small amount to produce a. Fragment A = cytotoxicity= blocking of protein
paralysis and death synthesis
 Causes Botulism: b. Fragment B = entrance of fragment A into the
- characterized by double or blurred vision, cells
impaired speech, difficulty in swallowing,  Produces Diphtheria toxin
weakness, and paralysis. - it is heat-labile
a. Adult botulism–ingestion of preformed - it is produced by strains with a lysogenic β-phage
toxin from home-made / canned products/ that carries the TOX gene.
smoked fish - it causes tissue necrosis and exudates
 Characterized by diplopia, formations (pseudomembrane lining) over the
difficulty in swallowing and may tonsils, larynx, and pharynx
lead to respiratory paralysis. - it favors an alkaline pH (7.8-8.0), an aerobic
b. Infant botulism–ingestion of the organism environment, and sufficient amount of iron in the
(spore) from fresh honey medium for production
 characterized by the floppy baby - it is released when the iron in the medium is
syndrome consumed.
 Elek’s test–determine toxin production
Lactose  Unknown isolates are streaked with
Motility Lecithinase Lipase and Glucose controls on an agar and a filter paper
Sucrose
(containing anti-diphtheria toxin) is
C.
- + - + + placed at a right angle to the streaked
perfringens
lines. Incubate at 35 C and observe
C. difficile + - - - +
after 24-48 hours.
C. tetani + - - - -
C.  Positive: Arch of Identity
+ - + - -  Causes Diphtheria
botulinum
- Transmitted via droplets
NON-SPORE FORMERS - Characterized by low-grade fever, malaise, and
A. Actinomyces (Aerobic) mild sore throat
1. Nocardia - Grayish-White pseudomembrane is formed.
 Beaded gram positive bacilli - Schick’s test –a serological test for diphtheria
 Weakly acid fast organism / partially acid fast a. Respiratory diphtheria
 Causes Pulmonary infections (N. asteroides) and  an acute, infectious disease that is
Cutaneous infections (N. brasiliensis) characterized by the production of a
 Colonies are chalky/powdery (bread crumbs) in systemic toxin and a false membrane
appearance lining (pseudomembranous formation) of
the throat mucous membrane that may
B. Corynebacterium eventually lead to respiratory obstruction.
 club-shaped or coryneform appearance  signs and symptoms: low grade fever,
 Pleomorphic organism which may exhibit palisade, thick mucopurulent nasal discharge, and
Chinese letters, picket-fence morphology cough
  control measure: Immunization (DPT  Has long filaments, non-motile, catalase negative,
vaccine) H2S production
 diphtheria antitoxin is administered to  exhibits a test tube brush appearance in gelatin
neutralize any unabsorbed exotoxin in the culture at 22°C
patient’s tissues.  Causes erysipeloid which is a localized skin infection
b. Cutaneous / Skin diphtheria (Veldt sore / characterized by a painful swollen lesion in the hands
Barcoo rot) and fingers.
 Characterized by slow-healing ulcers and
membrane formations. E. Lactobacillus acidophilus
 toxin is absorbed systematically but is less  also known as the Doderlain Bacillus
severe.  Non-pathogenic, produces large amount of lactic acid
 Normal flora of the mouth, GIT, and vaginal canal
2. Other Corynebacterium  Can be cultivated using tomato juice
a. C. jeikium – JK bacillus (Johnson & Kaye)
- causes prosthetic valve endocarditis F. Gardnerella vaginalis
b. C. ulcerans – diphtheria like illness and causes  Associated to bacterial vaginosis characterized by
mastitis in cattles malodorous discharge and increased pH
c. C. urealyticum – associated with UTI; rapid urease  Clue cells are diagnostic of BV. Epithelial cells with
positive gram positive bacilli clustered on the edges.
 Human blood tween agar is the media of choice for
C. Listeria monocytogenes G. vaginalis
 is aerobic or facultatively anaerobic and non-spore-
forming ACID-FAST BACILLI
 motile with peritrichous flagella and exhibits a
characteristic “tumbling motility” 1. Mycobacteria
 can grow in a high salt medium with up to 10% NaCl  Gram-positive,
 causes miscarriage or stillbirth in humans  acid-fast bacilli,
 MOT: ingestion of contaminated food such as meat,  strictly aerobic,
chicken, dairy products, and vegetable  non-motile,
 Microscopy: coccobacillary in form and are arranged  non-encapsulated,
singly or in short chains that resemble streptococci  slender and slightly curved or straight rods.
 Culture: BAP –colonies are small, translucent,  Often have beaded appearance due to the presence
grayish-blue, and are surrounded by a narrow zone of of Much granules (which are non-acid fast, gram-
β-hemolysis positive granules.
 Able to survive cold enrichment  May have palisade (slipping) or snapping formation.
 Motile, bile esculin positive, Hippurate hydrolysis  Stain with difficulty, but once stained, they are
positive resistant to decolorization.
 CAMP positive = blocked hemolysis–shovel-like,
substitute: S. equi  CLASSIFICATION
 Exhibits tumbling motility in wet preparations A. Potentially pathogenic species
 In motility media – exhibits umbrella-like or inverted  Mycobacterium tuberculosis – Koch’s Bacillus
Christmas tree when incubated at 20 C  Mycobacterium leprae – Hansen’s Bacillus
 Virulence Factor: Listeriolysin O  Mycobacterium bovis – Bovine Tubercle Bacilli
 Diseases caused:  Mycobacterium lepraemurium
 Listeriosis B. Saprophytic species
 a serious infection that affects neonates,  Mycobacterium phlei –Timothy Bacillus
pregnant women, and immunocompromised  Mycobateriumsmegmatis – formerly
hosts. Mycobacterium lacticula
 processed meat products should be  Mycobacterium butyricum
thoroughly cooked or heated before the C. Opportunistic or Atypical Mycobacteria (Runyon’s
consumption as a primary preventive Classification) – based on pigmentation of the
measure. organism
 immunocompromised individuals and
pregnant women are predisposed to  RUNYON’S CLASSIFICATION OF MYCOBACTERIA
listeriosis after ingesting contaminated dairy Group I – PHOTOCHROMOGENS
products like cheese.  Produce bright yellow to orange-red pigmented
 an infected pregnant woman may pass the colonies when exposed to light.
organisms onto the fetus  Require 14-21 days incubation
 Listeriosis – acquired through ingestion of
contaminated food coleslaw, cheese, chicken Group II – SCOTOCHROMOGENS
 Spontaneous abortion and still birth in pregnant  Produce reddish orange pigmented colonies when
women at 3rd trimester incubated in the dark
 Meningitis in elderly and immunocompromised  Require 10-14 days incubation
patients
Group III – NON-PHOTOCHROMOGENS
D. Erysipelothrix rhusiopathiae  Usually non-pigmented, if ever produced, it is weak
 not part of the indigenous human microbiota and light-independent.
 the only catalase-negative, non-spore-forming, Gram-  Require 14-21 days incubation
positive rod-shaped bacterium that produces
hydrogen sulfide. Group IV – RAPID GROWERS
 microscopy: thin, pleomorphic rods with the  Produce colorless and creamy-yellow colonies
tendency to form long filaments that are arranged in  Grows within 2-7 days incubation
single, short chains, or in a V-shaped formation
 Gelatin stab culture: has a pattern of a “pipe
cleaner” or a “test tube brush” at 22 C.
 MOT: direct contact with infected excreta, blood and
flesh of animals through skin breaks
Group I – PHOTOCHROMOGENS
A. Mycobacterium kansasii
 Yellow Bacillus
 causes pulmonary disease like TB but milder
B. Mycobacterium marinum/balnei
 causes swimming pool granuloma (lesions usually in
lower extremities
C. Mycobacterium simae M. Chelonei –MB 7H11
 causes rare cases of pulmonary disease
D. Mycobacterium asiaticum

M. Chelonei iIncubated in the

M. Kansasii on MB 7H11

Group II – SCOTOCHROMOGENS
A. Mycobacterium scrofulaceum
 causes lymphadenitis in children
B. Mycobacterium zsulgai
 causes pulmonary, bone and joint diseases
C. Mycobacterium gordonae
 Tap Water Bacillus
 causes joint and bone diseases also
D. Mycobacterium flaviscens
 considered as saprophytic
E. Mycobacterium xenopi

Group III – NON-PHOTOCHROMOGENS

A. Mycobacterium haemophilum
B. Mycobacterium avium
C. Mycobacterium intracellulare–Battey Bacillus
D. Mycobacterium gastri–J Bacillus
E. Mycobactrium terrae–Raddish Bacillus
F. Mycobacterium triviale–V Bacillus
G. Mycobacterium shimoidei

Smooth colony of M. avium Rough colony of M. avium

A. Mycobacterium tuberculosis
 Also known as Koch’s Bacillus or Human Tubercle
Bacillus
 Considered as the “captain of all men of death”
 Identified by Robert Koch in 1882
 Slender and sometimes curved rods
 Measures 0.2 to 0.6 um in diameter and 1 to 4 um in
M. Avium –MB 7H11 length
 Non-motile, non-spore former

 BACTERIAL COMPONENTS
 Mycolicacid– long chain fatty acid responsible for the
acid-fastness of the organism
 Cord Factor– (6-6 dimycolol-alpha-trehalose);
responsible for the organisms to grow in filaments
forming a cord-like arrangement
M. Avium –  Wax D Protein–a complex peptidoglycolipid which act
LJ Medium as the antigenic factor of the organism
 Much Granules–non-acid fast but gram-positive
granules inside the organisms which form a bead-like
Group IV – RAPID GROWERS appearance
A. Mycobacterium fortuitum  Sulfatides–glycolipids responsible for neutral-red
B. Mycobacterium chelonei reaction; associated with virulence
C. Mycobacterium ulcerans–causes buruli ulcers of the skin
D. Mycobacterium phlei  CULTURAL CHARACTERISTICS
E. Mycobacterium smegmatis  Able to grow in simple synthetic medium, but for
F. Mycobacterium vaccae primary isolation, a more complex medium containing
either egg potato basedor serum agar is required.
 Very slow grower organism requiring an increase
CO2tension (5-10%) at 37°C (it takes 10-20 days
before it can be visualized.
 The first growth appears small (1-3 mm) friable and
granular colony (rough) which then becomes typical
 colonies like CAULIFLOWER at the center after  These boxes should be kept in the cooler conditions
several weeks. and then transported to the laboratory.
 The growth is luxuriant –enhanced by glycerol.
 PROCEDURE IN PROCESSING OF SPUTUM
 PATHOGENESIS SPECIMEN (WHO Guidelines)
 TUBERCULOSIS –disease produced by this  In a small glass, sealable bottle, mix equal volumes of
organism which begin in the middle or lower lung to sputum and 4% sodium hydroxide (digestant).
the lymphaticsthen spread to the other organs via the  Shake well and incubate at room temperature (25-
blood. 30°C) for 15-20 minutes with regular shaking every 5
 TB of the skin –Lupus vulgaris minutes.
 TB of the spine –Pott’s disease  Centrifuge at high speed (>13,000 g) for 8-10 mins.
 TB of the cervical lymph nodes of the neck –  Discard the supernatant.
Scrofula  Neutralize the sediment by adding drop by drop, 2
 Clinical Manifestations: mol/litre HCl containing 20 ml of phenol red solution
 Chronic coughing per litre until the mixture remains pink.
 Night sweats  Inoculate the neutralized deposit on to at least 3 tubes
 Hemoptysis of Lowenstein-Jensen (L-J) medium-one with glycerol
 Shortness of breath and another with sodium pyruvate.
 Chest pain  Incubate the L-J tubes for 2-3 days at 35-37°C in a
 Fatigue horizontal position with the caps loosened half a turn.
 Afternoon fever Thereafter incubate at 37°C for 6 weeks and inspect
 Rapid weight loss for growth at weekly intervals. Initial incubation of the
culture tubes in the presence of 5-10 percent
 SPECIMEN CO2improves the growth of M. tuberculosis.
 Specimen:  Note the growth of bacteria on the surface during
 Sputum – early morning by deep expectoration these weekly inspections, and if present stain by
minimum of 3 specimens at different time intervals. Ziehl-Neelsen method.
(Note: 24-hour pooled collection yields more  If the isolate has the typical colonial appearance and
positive culture but increased contamination rate) the Z-N stained smear from a colony is also typical
 Urine –early morning is preferred than 24-hour report the growth as Mycobacterium spp.
collection  Send the isolate to a reference laboratory for further
 CSF –require centrifugation for concentration of characterization and susceptibility testing.
microorganisms  The growth of typical human strains is "rough,
 Tissues, draining sinuses and gastric tough and buff" and can sometimes be seen after
washings 2-3 weeks of incubation but seldom earlier.
 Diagnosis of pulmonary TB by sputum microscopy is  Contaminated specimens are exposed to digestion
simple, easy, inexpensive, rapid, technically not very and decontamination as well as concentration
demanding and more reliable than X rays. The techniques because:
purpose of the sputum microscopy is two fold:  of the presence of Non -Mycobacterial
1. diagnosis of the patients with infectious contaminating bacteria that might kill the tubercle
tuberculosis bacilli
2. monitoring the progress. For diagnosis, 3 sputum  of the presence of mucus traps, thus, mucus
examinations are performed (spot, morning, spot) should be liquefied to facilitate concentration by
and for follow up 2 sputum examinations (morning, centrifugation.
spot) are performed.
 Digesting Decontaminating Agents
 COLLECTION OF SPUTUM SAMPLE 1. NALC (N-acetyl-L-Cysteine)–digesting or mucolytic
 Select a good wide-mouthed sputum container, which agent that breaks the disulfide bond that may trap the
is disposable, made of clear thin plastic, unbreakable organism
and leak proof material. 2. 4% NaOH (Petroff’sMethod)–traditional method
 Give the patient a sputum container with the which act as decontaminating agent
laboratory serial No. written on it. Show the patient 3. DTT (Dithiothreitol)with 2% NaOH–also called
sputulysin
how to open and close the container and explain the
 effective mucolyticaction
importance of not rubbing off the number written on
4. 13% Na3PO4+ Benzalkonium Chloride (Zephiran)
the side of the container. 5. 1% Cetylpyridium chloride + 2% NaCl–used for
 Instruct the patient to inhale deeply 2-3 times, cough mailed specimen
up deeply from the chest and spit in the sputum 6. 4% H2SO4–suitable for urine decontamination
container by bringing it closer to mouth. 7. 5% Oxalic acid–suitable for specimen contaminated
 Make sure the sputum sample is of good quality. A with Pseudomonas aeruginosa
good sputum sample is thick, purulent and sufficient in
amount (2-3 ml).  LABORATORY DIAGNOSIS
 Give the patient another container with laboratory A. Microscopic Method
serial number written on it for an early morning  Acid-Fast Staining
specimen. Explain to the patient to rinse his/her  Auramine-phenol staining
mouth with plain water before bringing up the sputum. B. Culture
 Egg-based
 STORAGE AND TRANSPORTATION  Agar-based
 If the specimen is collected in the field and cannot be  Liquid-based
immediately processed, it should be transported to the C. Biochemical Tests
laboratory within 3-4 days of collection. D. Skin Test
 The specimen should be collected in the containers  Tuberculin Test
meant for the purpose, lid tightly secured, properly
labeled and kept away from the sun and heat.
 These can be placed in a special box which can
withstand leakage of contents, shocks and other
conditions incident to ordinary handling practices.
 Acid-Fast Staining (Ziehl-Neelsen Method)  ArylsulfataseTest
No. of fields  Iron Uptake Test
Examination Result Grading to be  Urease Test
examined  Growth on 5% NaCl
More than  Growth on Mac Conkey Agar
10 AFB per
oil Positive 3+ 20 A. Niacin Test
immersion  All Mycobacterium species produce niacin but not all
fields
of them produce an enzyme that will convert niacin to
1-10 AFB
ribonucleotide.
per oil
Positive 2+ 50
immersion
fields Principle:
10-99 AFB Cyanogen Bromide + Free Niacin  yellow color
per 100 oil (toxic) (ribonucleotide)
Positive 1+ 100
immersion
fields
1-9 AFB per Record Result:
100 oil exact Positive:
Scanty 100
immersion number Mycobacterium
fields seen tubercolosis
No AFB per Negative:
100 oil Mycobacterium bovis
Negative 0 100
immersion
fields

 How to Prevent False Positives

 Only use new, unscratched slides.
B. Tween 80 Hydrolysis Test
 Always use filtered carbolfuchsin.
Principle:
 Do not allow the carbol fuchsin to dry during staining.
 Certain species of Mycobacteria are capable of
 Do not allow the carbol fuchsin to boil during staining.
hydrolyzing Tween80, a derivative of sorbitan
 Decolorize adequately with sulphuric acid.
monoleate with the production of oleic acid.
 Make sure there are no food particles or fibers in the
sputum sample. Neutral red indicator
Tween 80 olieic acid (pink)
 Never allow the oil immersion applicator to touch a
slide.
Result: (+) Mycobacterium tuberculosis
 Auramine-phenol Staining
C. Nitrate Reduction Test
 Also Fluorescent Dye Staining which uses
 The presence of nitrosoreductase in Mycobacterium
fluorochrome dye which causes the bacilli to glow
tuberculosis produces a colored end product from a
when exposed to UV light making them more visible
substrate that combines with nitrites.
 Primary stain: auramine dye
 Decolorizer: Acid alcohol Nitroso-reductase
Nitrate Nitrite
 Counterstain: KMnO4 or Rhodamine
Result: (+) Mycobacterium tuberculosis
 CULTURE
 Incubation period for the isolation takes 2-4 weeks or
D. Heat Stable Catalase Test
even up to 6 weeks; the culture should have an
Principle:
increase protein containing inhibitor (e.g.malachite
 Most species of Mycobacteria are able to split
green)
hydrogen peroxide into water and oxygen
 Cultures are examined weekly for growth. Negative
growth should be discarded after 8 weeks of Heat 68°C for 20 min
H2O2 H2O + O2
incubation
Result: (-) MTB complex (M. tuberculosis, M. bovis, M. leprae)
Agar based:
(+) MOTT
1. Dubos oleic acid albumin
2. Middlebrook7H-10
E. Arylsulfatase Test
 most preferred for susceptibility testing
ArylsulfataseTest - test for rapid growers
3. Middlebrook7H-11
Principle:
4. Mitchison’s selective 7H-11 arylsulfatase
Tripotassium phenolphthalein free
phenolphthalein (pink)
Egg based: (contains potato flour, eggs, glycerol, with
malachite green)
Result: (+) Mycobacterium fortuitum
1. Petragnani
(-) Mycobacterium chelonei
2. Lowenstein Jensen
3. Dorset egg medium
F. Iron Uptake Test
4. American Thoracic Society Medium
Principle:
 This test is used mainly to differentiate M. fortuitum
Liquid culture media:
and other rapid growers from Mycobacterium
1. 1.Bactec12B medium
chelonei.
2. Middlebrook 7H-9 broth – for aseptically collected
 M fortuitum and other rapid growers –have the ability
specimens from normally sterile sites.
to convert ferric ammonium citrate to an iron oxide.
(reddish brown or rust color on the colonies)
 BIOCHEMICAL TESTS
 Niacin Test
 Tween80 Hydrolysis Test
 Nitrate Reduction Test
 Heat Stable CatalaseTest
 c. < 5 mm or without induration  negative and
should be retested with 250 IU.
 Positive tuberculin test indicates:
1. person had TB infection in the past
2. person is in close contact with TB cases
3. person had BCG vaccination

 Treatment/Prevention
 Treatment: (PRISE)
G. NaCl Tolerance Test  Isoniazid (INH), Rifampicin–liver
Principle: toxicityEthambutol–decrease visual acuity
 Some species of Mycobacteria including the rapid  Pyrazinamide–increase uric acid
growers, except for Mycobacterium chelonei can grow  Streptomycin –hearing damage
at 28°C on an egg-based medium containing 5%  Prevention:
NaCl.  BCG Vaccination (Bacillus of Calmetteand Guerin)
Result: - live attenuated strain of M. bovis
M. chelonei (negative) – left  INH Prophylaxis for close contact with TB cases
M. fortuitum (positive) – right
B. Mycobacterium leprae
 Also called Hansen’s Bacillus which causes
Hansen’s disease (leprosy)
 Obligate intracellular parasite that multiply slowly in
mononuclear phagocytes and has strong predilection
on nerves.
 Other Biochemical Tests  Morphology:
 Urease Test – positive (+) M. scrofulaceum, M. bovis,  Closely resemble tubercle bacillus
M. gastri  Long (up to 8 um) slender rods, usually straight
 Mac ConkeyAgar –no growth for Mycobacterium but sometimes slightly curved
tuberculosis  Non-motile, non-spore-former
 Positive: M. fortuitum, M. chelonei for 5 days  Acid-fast, predominantly in mononuclear or
 Negative: other Group 4 (Rapid Growers) epithelial structures known as LEPRA cells
 Tuberculin Test  Found in parallel or palisade arrangement which
- Skin test for tuberculosis appear like packets of cigar
- A diagnostic test which identifies tuberculosis  Contains PHENOLASE from skin nodules unique
infections recent or past, with or without disease. to the organism.
- It is based on the fact that persons infected with
tubercle bacilli develop hypersensitivity to the  Leprosy
proteins of the organism.  a chronic disease affecting the skin, mucous
- Types of Tuberculin Preparations: membranes and peripheral nervous system.
 Old Tuberculin (OT)–the original test reagent  Mode of Transmission:
prepared from boiled 6-week old broth 1. Humans are known to be the natural host
cultures from which organisms were filtered 2. Contact in particles with lepromatous leprosy in
and concentrated by steaming; the active nasal secretions and ulcer exudates
component of the filtrate is heat-stable 3. Major portal of entry is respiratory tract
protein. 4. Biting insects and breast milk may also be
 Purified protein Derivatives (PPD)–a potential sources
partially purified preparation of OT using
ammonium sulfate fractionation.  Forms of Leprosy
 Method of Administration: Standard dose: 0.1 I. Intermediate Type
ml PPD or 5 TU (tuberculin units)  characterized by hypopigmentation of the skin which
 Mantoux Test–subcutaneous injection may mimic Tinea versicolor fungal infection
(forearm)
 Vollmer-patch Test–a piece of cloth or II. Tuberculoid Type
plaster is soaked in OT or PPD and placed  benign form of the disease
over the skin (over shoulder blade)  non-progressive to slow progressive (18 years
 Moro-percutaneous Test–OT or PPD with average duration)
the addition of lanolin ointment into the skin  characterized by few hypopigmented macules
 Von Pirquet Test–scratching the tuberculin in  few bacilli in the lesions affecting primarily the skin
the skin and nerves
 Heaf Multiple Tine Test –aka: Multiple  positive Lepromin test (skin test)
Puncture Technique; the use of multiple
puncture needles to introduce the tuberculin III. Borderline Tuberculoid
preparation into the forearm.  more skin lesions produced and more thickenings
 Chamber Test–the use of small, concave  positive lepromin skin test
button-like capsule made of aluminum filled
with cream containing different concentrations IV. Borderline Lepromatous
of tuberculin. The capsules are taped into the  presence of skin nodules, papules and macules all
forearm for 48 hours. over the body
 stained smear is strongly positive
Tuberculin Test  nodules ca be found on testes, nose, eyes and bones
 Result: (+) redness and induration (wheal and flare)
within 48-72 hours after injection. V. Lepromatous or Nodular Type
 For Mantoux Test:  most severe and extensive form
a. > 10 mm  infection with Mycobacterium  with little or no immunity developed and disease is
tuberculosis more generalized
b. > 5 mm but < 10 mm  doubtful since it maybe  with multiple skin lesions with numerous bacilli
due to MOTT (leproma)
  face becomes thickened (LIONARE’S FACE) Colonial Characteristics and Appearance
 Saddle nose deformity maybe present Organism Media Appearance
 AFB (+), lepromin skin test (-) MAC LLF, NLF after 24 hrs
Citrobacter spp. HEA Colorless
 Laboratory Diagnosis XLD Red, yellow or colorless
 Specimen: scrapings from skin lesions and nasal MAC NLF
smears Edwardsiella
HEA Colorless
 Microscopic examination: spp.
XLD Red, yellow or colorless
 AFB staining (Baumgarten’s Method) – uses MAC LF; maybe mucoid
pyridine/phenolase unique to M. leprae Enterobacter
HEA Yellow
 Bacillary Index (BI) – refers to the number of spp.
XLD Yellow
fragmented organisms as it denotes destruction of MAC LF; flat, dry pink
the organism due to treatment Escherichia coli HEA Yellow
 Culture – organisms not readily grown in artificial XLD Yellow
media MAC NLF
 Footpads of mice and armadillos – animals with Haffnia alvei HEA Colorless
low body temp XLD Red or yellow
MAC LF; mucoid
 Skin Test Klebsiella spp. HEA Yellow
 Lepromin Skin Test XLD Yellow
- Intradermal inoculation of material prepared from MAC NLF
leprous nodules to demonstrate hypersensitivity Morganella spp. HEA Colorless
usually developed by Leprous persons XLD Red or colorless
- not a specific test since it cross react with NLF; may swarm; foul
MAC
tuberculosis and BCG vaccination. smell
Proteus spp. HEA
Colorless
 Reactions to LeprominTest XLD
Yellow or colorless
- Fernandez Reaction –induration appears 24-48
MAC NLF
hours after ID injection Providencia spp. HEA Colorless
- Mitsuda Reaction –late reaction appearing 3-4 XLD Yellow or colorless
weeks from the inoculation of lepromin materials. MAC NLF
Salmonella spp. HEA Green
 Treatment/Prevention XLD Red with black center
 For Lepromatous Leprosy –combined regimen of LLF; maybe pigmented
MAC
dapsone/DDS (4,4-aminodiphenyl sulfone), rifampicin (prodigiosin)
Serratia spp. HEA
and clofacimine Colorless
XLD
 For Tuberculoid Type –combined dapsone and Yellow or colorless
rifampicin MAC NLF
Shigella spp. HEA Green
 PREVENTION: XLD Colorless
 BCG vaccination NLF; maybe colorless to
MAC
Chemoprophylaxis with DDS peach
 Yersinia spp. HEA
Salmon
XLD
Yellow or colorless
GRAM NEGATIVE BACILLI
 Tests for Enterobacteriaceae
ENTEROBACTERIACEAE
A. Indole:
 Gram-negative, non-spore-forming
 Detects tryptophanase enzyme (tryptone broth)
 Motile with peritrichous flagella, except Klebsiella and
 Kovach’s/Ehrlich’s reagent tryptophanase
Shigella
 Glucose fermenter tryptophanase
Tryptophan Indole (red ring at surface)
 Some are lactose fermenters:
o RLF – lactose permease & B-galactosidase
o LLF – B-galactosidase
o NLF – no enzyme present
 Oxidase negative Brilliant red ring
 Catalase-positive except S. dysenteriae (sometimes: ORANGE
 Reduces nitrate to nitrite, except E. agglomerans and due to skatole)
Erwinia
 Aerogenic, except Shigella
o LIA: -/+: Klebsiella, Serratia, Providencia,
Salmonella
o H2S production: SPACEd; LIA: SACEd
 Urease production:
o Rapid producer – Proteus, Providencia,
Morganella
B. Urease Test:
o Late urease producer – Citrobacter, Enterobacter,
 Christensen’s urea agar / Stuart’s urea broth
Klebsiella, Yersinia, Serratia
 Indicator: phenol red
 Acid -yellow; alkaline -red

Urea Hydrolysis

 Result: Negative
(left), positive (right)
C. MRVP Shigella, Citrobacter
 MRVP broth (peptone glucose broth) K/A SCiPPY Providencia, Proteus,
 Glucose fermented to pyruvatein 2 pathways Yersinia
- Mixed acid pathway (MR Test) K/K P Pseudomonas
- Butyleneglycol pathway (VP test)
 METHYL RED: production of lactic, acetic, and formic Escherichia coli
acid (+) result: RED  IMViC: + + - -
 VP: production of acetoine (acetylmethylcarbinol)  UTI – 90 %
 Sepsis, meningitis
40% KOH a naphthol
Acetoine dimethyl RED (+)  Diarrhea:
o ETEC
Methyl Red Reactions o EPEC
o EIEC
o EHEC
Result: o EAEC
 Negative
(left) ETEC
 Positive  Produces enterotoxin that mediates secretion of water
(right) and electrolytes
 Watery stool
 Traveller’s diarhea or “Turista”
Voges Proskauer Reactions  Montezuma’s revenge in Mexico
 New Delhi belly in India
 Bangkok ranch in Thailand
Result:  Only grows on BAP
 Negative
(right) EPEC (Enteropathogenic Escherichia coli)
 Positive  Non-invasive, no toxin produced
(left)  Causes nosocomial infections among children
 Watery stool with mucus but no RBC

EIEC (Enteroinvasive Escherichia coli)

 Invades intestinal epithelium
D. Citrate  Responsible for E. coli dysentery
 determines if the org utilizes citrate as sole source of  Watery stool with mucus, RBC and Neutrophils
carbon
 Medium: SCA (green-slant) EHEC (Enterohemorhagic Escherichia coli)
 Indicator: BTB (acid-yellow; alkaline-blue)  Cytotoxic against vero cell lines (derived from African
green monkey kidney cells
 HUS and colitis
Result:  Source: poorly cooked meat “hamburgers”
 Negative  Complications: MHA, Thrombocytopenia
(right)
 Positive EHEC 0157:H7 Strain
(left)  SMAC:
 Sorbitol MacConkey Agar
 Sorbitol is substituted for lactose
 Indicator: Neutral red
E. Motility o Acid – red
 Medium: semi-solid o Alkaline – yellow
 Motile –growth away from stab line  Colorless colonies
 Non-motile –growth only in the stab area  Non-sorbitol fermenter (NSF)
 Other strains: pink colonies

EAEC (Enteroaggregative Escherichia coli)

Result:  Adhere to human cells
 Negative  The exact pathogenic mechanism is unknown
(middle)
 Positive KLEBSIELLA-ENTEROBACTER GROUP
(left and Genera:
right)  Klebsiella
 Enterobacter
 Serratia
F. ONPG Common Characteristics:
 Orthonitrophenyl B-D-galactopyranoside  Increased gas in TSI
 Test for late lactose fermenter  Positive in Voges-Proskauer
 Detects B-galactosidase enzyme present in LLF
1. KLEBSIELLA
B-galactosidase  IMVIC: - - + +
ONPG O-nitrophenyl(yellow) (+)
 All are LF, except K. ozaenae and K. rhinoscleromatis
TSI REACTIONS IN ENTEROBACTERIACEAE  Colonies are mucoid due to large capsule
Serratia, Escherichia,  Non-motile
A/A SEEK o K. pneumoniae – “Friedlander’s bacillus”
Enterobacter, Klebsiella
Pneumonia, abscess and meningitis
Citrobacter, Arizona,
A/A, H2S CAP o K. ozaenae – purulent sinus infection
Proteus
o K. rhinoscleromatis – granuloma of the nose
Citrobacter, Arizona,
K/A, H2S CASE and oropharynx
Salmonella, Edwardsiella
K. pneumoniae 6. SALMONELLA
 TSI – A/A with gas  All members are NLF
 Citrate (+)  Motile, except S. pullorum and S. gallinarum
 VP (+)  Serotyping:
 Indole (-) o Based on char “O” and “H” antigen
 MR (-)  S. typhi – enteric fever (typhoid) ( - + - - )
 EMB, Mac, XLD – mucoid  S. cholerasuis – bacteremia
 Neufeld Quellung (+)  S. typhimurium – enterocolitis/gastroenteritis
 S. enteritidis - gastroenteritis
1. KLEBSIELLA
 K. oxytoca  IMVIC + - ++ Typhoid Fever
 K. rhinoscleromatis  K/A with gas, VP (-) Indole (-)  Specimen:
Citrate (-), MR (+) o 1st week – blood
 K. ozonae  MR(+), Citrate +/- o 2nd week – urine/stool
 K. aerogenes  A/A with gas, MR (-), VP (+), citrate o 3rd week – serological (Widal, Typhidot,
(+) Salmonella typhi IgG-IgM)
 WIDAL’s
2. ENTEROBACTER o Ab titer “O” > 1:160 – indicates active
 IMVIC: - - + + infection
 Motile o Ab titer “H” > 1:160 – past
o E. cloacae – most isolate of Enterobacter infection/immunization
(nosocomial) o Increased Ab titer “Vi” or “K” – carrier
o E. sakazaki – yellow pigment that intensifies at
250C S. typhi Other Salmonella spp.
o E. tylorae – lactose negative, ONPG (+)  TSI – K/A with  K/A with gas with
o E. gergoviae – strong urease producer small amt of gas H2S
o E. aerogenes – LF  ++- -  -+-+
o E. haffniae – LLF to NLF  Lysine  Lysine
decarboxylase (+) decarboxylase (+)
3. SERRATIA
 IMVIC: - - + + 7. ARIZONA
 Produces 3 enzymes:  Formerly Salmonella enteritica subgroup arizonae
o DNAse (distinguished from Salmonella being LLF and growth
o Lipase on malonate medium)
o Gelatinase  Associated with reptiles and birds
 S. marcescens/ S. rhubidea – produces red pigment  A/A with gas; or K/A with gas
(Prodigiosin)  -+-+
 S. odorifera – rancid potato odor  Lysine decarboxylas (+)

4. HAFNIA 8. CITROBACTER
 Haffnia alvei – characteristic isolate  C. freundii – diarrhea, UTI
 Resembles genus Enterobacter  C. diversus – neonatal meningitis, UTI
o Late Lactose Fermenter  C. amalonatus – UTI
o Citrate negative  A/A with gas sometimes K/A due to LLF
 Differ from Serratia in:  -+-+
o DNAse (-)  Lysine decarboxylase (-)
o Lipase (-)  Urease (+)

5. SIGELLA 9. PROTEUS
 Non-motile  NLF, Lysine deaminase (+)/ TSI – K/A with H2S
 NLF  Swarming growth is observed
 H2S (-)  Motile with burnt gun powder odor
 TSI – K/A  Rapid urease producer
 IMViC: v+ - -  Antigen is used in Weil Felix Test for Rickettsial
 Lysine decarboxylase (-) diseases
 Urea (-)  OX-2 and OX-19 (P. vulgaris)
 Shigellosis – Bacillary dysentery  OX-K (P. mirabilis)
 EMB, MAC, SSA – transparent  P. vulgaris – NF of intestinal tract
 XLD, HEA – green to blue-green  P. mirabilis – hospital and community acquired
pneumonia, UTI wound infection and septicemia
GROUP Organism Catalase Lactose  P. retgerri – nosocomial
 P. myofasciens – isolated from gypsy moth larvae (not
S. dysenteriae yet reported in humans)
A - -
(Shiga)  P. morganii (now P. morganella)– summer diarrhea in
B S. flexneri (Strong’s) + - children
C S. boydii (Boyd) + -
D S. sonnei + + 10. PROVIDENCIA
 IMVIC: + + - -
GROUP Organism ONPG Mannitol  Motile, closely related to Proteus in biochem
S. dysenteriae reactions.
A - -  Urease (-) to differentiate from Proteus
(Shiga)
B S. flexneri (Strong’s) - +  TSI – K/A
C S. boydii (Boyd) - +
11. MORGANELLA
D S. sonnei + +
 IMVIC: + + - -
 Formerly P. morganii
 Lysine deaminase (+)
  Urease (+)  Cultural characteristics:
 TSI – K/A with gas  Organisms can be readily cultured on NA,
BAP, MAC and Cetrimide agar plate.
12. EDWARDSIELLA  Grow well on 37-42°C, its growth in 42°C
 Edwardsiella tarda helps differentiate it from other
 With 148 serotypes based on 49 “O” Ag and 47 “H” Pseudomonas spp.
Ag  On BAP – large, flat, β-hemolytic colonies
 Closely resembles E. coli with a feathered edge and ground glass
 Found in cold and warm blooded animals (reptiles, appearance
fish, frogs and turtles)  Colonies tend to spread giving off a
characteristic odor resembling over-riped
 Biochemical reactions: grapes (grape-like fruity odor) which is due to
 TSI – K/A with H2S a substance aminoacetophenon.
 Lysine dearboxylase (+)  BAP with 5% Sheep’s blood large, flat, β-
 IMVIC ++- - hemolytic colonies with a feathered edge
 Motility (+) and ground glass appearance
 NLF
 H2S(+)

13. YERSINIA
 Y. pestis – causes plague
 Bubonic (lymph nodes)
 Pneumonic/Pandemic/Black death (lungs)
 Septicemic plague (Shwartzman phenomenon)
 Safety pin appearance due to bipolar bodies (Giemsa  Pigments:
and Wayson stains)  Most strains produce pyocyanin, a blue, water-
 Vector: Xenopsylla cheopsis (rat flea) soluble, chloroformextractable pigment seen on
 Optimal temp is 28°C uncolored media like
 Pathogenic properties: pesticin (bacteriocin)  Mueller Hinton Agar
 NA (mucoid); XLD (very small colonies)  Pseudomonas (P) agar
 This charcteristic is unique to Pseudomonas
A. Yersinia pestis aeruginosa, thus called “BLUE PUS AGENT”
 NLF  Some strains produce other pigments:
 TSI – K/A o Pyoverdin (fluorescein) – yellow to green
 MViC - + - - water-soluble pigment
 Urease (-) o Pyorubin – red
 TX: Streptomycin, Chloramphenicol, Tetracycline o Pyomelanin – brown to black

B. Y. enterocilitica (Yersinosis)  Virulence factors:

 enterocolitis characterized by diarrhea, abdominal  Presence of glycocalyx
pain and fever  Pili/fimbriae – for adherence
 Med: CIN (Cefsoludin Irgasan Novobiocin)  Endotoxin
 With Bull’s eye colonies  Extracellular enzymes – proteases, hemolysins, etc
 TSI – A/A  Exotoxin A – causes tissue necrosis
 Urease (+)  Exotoxin S – play a role in necrotic injury
 VP (+)  Cytotoxin and enterotoxin

NON-FERMENTATIVE GRAM NEGATIVE BACILLI  Disease produced:

Non-Fermentative Gram-Negative Rods  Pseudomonas aeruginosa inhabits soil, water and
 This group of organisms is differentiated from causes disease in humans with impaired host
Enterobacteriaceae in the way they utilize carbohydrates. defenses.
 They do not ferment sugars (in the absence of air) but  Burn wound infections: traumatic and operative
utilize oxidatively producing tiny amount of acid. wound infections
 This group includes:  Nosocomial infections like pneumonia (especially in
 Pseudomonas cystic fibrosis patients), UTI, endocarditis,
 Burkholderia osteomyelitis
 Acinetobacter  Eye infections seen in contact lens wearer
 Stenotrophomonas  Dermatologic infections
 Alcaligenes
 Flavobacterium  Laboratory diagnosis
 Eikenella  Flagellar stain/ Gram-stain
 Chromobacterium  Culture
o Selective media
PSEUDOMONAS AERUGINOSA  Pseudosel agar – containing
 Morphology cetrimide (cetrimethyl ammonium
 Aerobic, motile with single polar flagellum bromide)
 Gram (-) rods that occur singly, in pairs, and  Irgasan
occasionally in chains o Media enhancing fluorescent pigment
 Produces extracellular slime layer, similar to production
a capsule usually seen in mucoid strains  Pseudomonas F agar
isolated from patients with cystic fibrosis  GNF (Glucose N2- Fluorescein)
 Frequently possess pili that promote agar
attachment to host cell surface  Flo agar
  Biochemical Tests o Hikojima – Japan
o TSI – K/K with metallic sheen on the slant
surface EL TOR VS. CLASSICAL
o Open OF (+) and close OF (-) utilizing No RBC hemolysis With RBC hemolysis
glucose oxidatively Negative agglutination to Positive agglutination to
chicken RBC chicken RBC
o Oxidase and catalase (+)
VP (-) VP (+)
o Unable to oxidize lactose, sucrose and
Polymyxin susceptible Polymyxin resisatant
maltose
o Unable to decarboxylate lysine and ornithine,
VIBRIO
able to hydrolyze arginine  Enrichment med: Alkaline Peptone Water
o Serotyping of O antigen  Selective Med: TCBS
o Bacteriophage typing  Indicator: BTB
o Pyocin typing – for epidemiologic studies o V. cholerae - yellow colonies
o V. alginolyticus - yellow colonies
 Treatment o V. parahemolyticus - green colonies
 Aminoglycosides (amikacin, gentamycin and
tobramycin) CAMPYLOBACTER
 Gram-negative rod, S-shape / Sea-gull’s wing
 Extended spectrum penicillins (carbenicillin)
 Microaerophilic (5 % O2 , 10 % CO2 , 85 % N2
 3rd generation cephalosporin (ceftazimide and  Motile (monotrichous), DARTING MOTILITY in
cefoperazone) Darkfield microscopy and wet preparation
 Quinolones and carbapenems  Culture: Skirrows and Campy BAP
 42-43°C (optimum temperature)
HAEMOPHILUS
 Gram-negative rod, non-spore-forming Man:
 Oxidase (+), increase CO2 , blood-loving
 C. coli
 Requires X and V factor  C. jejuni
Gastritis and
o X factor – HEMIN (a heat-stable production diarrhea
of hemoglobin degradation) Animal:
o V factor – NAD (coenzyme I), heat-labile,
produced by some bacteria and yeast.  C. fetus – abortion (no growth at 42 C)

HELICOBACTER
Organism X factor V factor D-ALA
 H. pylori – formerly known as Campylobacter pylori
H. Influenzae   -
 Gram-negative, motile (4-6 polar flagella)
H. parainfluenzae X  +  Optimum temp: 35-37°C
H. hemolyticus   -  Habitat: stomach, since it produces a strong urease
H. parahemolyticus X  + enzyme
H. aphrophylus X X +  Responsible for peptic ulcer and duodenal ulcers.
H. paraprophylus X  +
H. aegypticus   - LEGIONELLA
H. ducreyi  X -  Gram-negative, non-spore-forming rod
 Source:
o Natural: ponds, creeks
HEMOPHILUS o Artificial: aircon
 Satellitism phenomenon  L pneumophila – Legionnaire’s disease
o Satellitism  L. michadei – Pittsburge pneumonia
 Development of colonies around  L. bozemanni – Wiga’s agent of pneumonia
Staphylococcal streak indicated that  Culture Med: BCYE (Buffered charcoal yeast extract)
organism produces NAD.
 (+) H. influenza BORDETELLA
 Gram-negative, non-sporeformer
Haemophilus aegypticus  Non-motile, except for B. bronchiseptica
 Koch’s week bacillus
 causes a pink-eye conjunctivitis ORGANISM UREASE NITRATE
PRODUCTION
Haemophilus ducreyi B. pertussis - -
 Causes CHANCROID or soft chancre
B. parapertussis + -
 A venereal disease char by painful ulcers in the
genitalia B. bronchoseptica + +
 Gram-negative rod in school of fish arrangement
 The smallest pathogenic bacteria
ORGANISM OXIDASE MOTILTY
Haemophilus influenzae
 Pfieffer’s bacillus B. pertussis + -
 Has 6 serogroups (a-f) but most common is b B. parapertussis - -
 Biotypes I – VIII B. bronchoseptica + +
 Only those encapsulated are virulent
 Produces a DEW-DROP colonies

VIBRIO B. pertussis
 Gram-negative rod, non-spore-former, motile with
 Whooping cough
monotrichous flagella
 Spx: nasopharyngeal swabs or washings
 Oxidase positive
 Selective culture media:
 Halophilic, except for Vibrio cholerae and Vibrio
o Bordet-Gengou agar – potato glycerol blood
mimicus
agar
Vibrio cholerae o Regan Lowe – consist of horse blood,
 Produces cholera toxin (choleragen) charcoal and antibiotics
 Vibrio O1 – rice watery stool  growth – MERCURY DROPLET
 String test positive (org. + Na deoxycholate  string) COLONIES
 Serogroups:
o Ogawa – India (EL TOR)
o Inaba – Philippines (CLASSICAL; EL TOR)
BRUCELLA  Vector: Pediculus humanus

 Gram-negative, non-spore-former B burgdorferi

 Obligate aerobe
 Causes: MALTA / UNDULANT FEVER  Lyme disease
 3-4 weeks incubation period  Vector: Ticks – Ixodes damini, I.pacificus. I. ricinus
 Culture Media:  Stages of infection:
o Brucella agar – contains pancreatic digest of o Stage I: erythema chronicorn migrans w/
casein and peptic digest of animal tissue and bull’s eye lesion
Castañeda bottle. o Stage II: organism disseminated to the blood
o Stage III: chronic stage with neurological
Organism Source Urease Thionin Fuchsin abnormalities and arthritis
inhibition inhibition o Culture Media: Kelly’s Medium
o Serological Test: WESTERN BLOT – gold
B. Cattle + - - standard
abortus
B. Sheep/goat + - - LEPTOSPIRA
milliensis
 Tightly twisted with one or both ends bent like a hook
B. suis Pig + - +
 L. biflexa – could be found in soil but non-pathogenic
B. canis Dog - - +  L. interrogans – (leptospirosis) parasitic to vertebrae;
Infection involves the liver kidneys and CNS.

PASTEURELLA VARIETIES of L. interrogans

 P. multocida  Var. icterohemorrhagiae  Weil’s disease

 Gram-negative, non-spore-forming  Var. canicula  Infectious jaundice
 Normal flora of dogs/cats in oral cavity, resp. tract and  Var. autumnalis  Fort Bragg fever
GIT  Var. hebdomadis  Seven-day fever
 In animals: SHIPPING FEVER  Var. mitis/pomonas  Swineherd’s disease
 In man: PASTEURELLOSIS – obtain from direct  Var. grippotyphosa  Marsch fever
contact of wound in animals
TREPONEMA
FRANCISELLA
 Tightly coiled resembling a corkscrew
 F. tularensis  Treponema pallidum – Venereal disease
 Gram-negative rod, non-spore-former o SYPHILIS
 TULAREMIA – in animals transmissible to man o Italian / French disease
 Man: TICKS (vector) o Great pox / Evil pox
 Cultute media: Cystine Blood Glucose Agar  MOT:
o Sexual
GARDNERELLA o Mother-fetus
o Parenteral
 Formerly Corynebacterium vaginalis
 Gram-negative rod, non-spore-former STAGES OF SYPHILIS
 Causes BACTERIAL VAGINOSIS
 Vaginal discharge: FISH AMINE ODOR  Primary
 Lab Tests: o Lesion: HARD CHANCRE
o Cytology: Clue cells – EC studded with the  Secondary
organism o Lesion: CONDYLOMA LATA
o Whiff/Smith Test: discharge + 10 % KOH  Tertiary
Result: fishy amine odor o Lesion: GUMMA
 Culture: Human blood bilayer medium will not grow on
BAP PRIMARY SYPHILIS
 Hard Chancre on the site of inoculation and
Streptobacillus moniliformis multiplication of the organism
 Dx: dark-field microscopy
 Gram-negative rod  (+) positive coiled organisms with corkscrew motility
 Diseases:
o RAT BITE FEVER – from bite and scratch of
animals SECONDARY SYPHILIS
o HAVERHILL FEVER – ingestion of  Systemic dissemination of organisms characterized
contaminated milk by fever, lymph adenopathy, and pharyngitis
 Condyloma lata–a flat lesions resembling warts in
SPIROCHETES moist areas of the body
 Dx: Dark-field microscopy, Serological test
 Coiled and motile organisms
 Periplasmic flagella
TERTIARY SYPHILIS
Genera:  Lesion: Gumma/ Gummata–granulomatouslesion in
o Borrelia skin, bones and subcutaneous tissue
o Leptospira  Cardiovascular involvement
o Treponema  Neurosyphilis
 Dx: Serological test
BORRELIA
 Tx: Penicillin
 Flexibly twisted resembling a bent or stretched spiral
 Microaerophilic  Subspecies of T. pallidum
o B. recurrentis 1. T. pallidum subsp. pertenue
o B. burgdorferi  YAWS: a chronic non-venereal disease of the skin
o B. pankeri  MOT: direct contact with infected skin lesion
o B. hermsii
2. T. pallidum subsp. endemicum
B. recurrentis
 BEJEL: endemic or non-venereal
 Louse-borne relapsing fever  1°-oral cavity; 2°-oral mucosa; 3°-skin, bone,
 Fever: 2-6 days – relapse after few days or weeks nasopharynx
3. T. pallidum subsp. carateum
 PINTA: ulcerative skin disease
 direct contact with infected skin lesion

CHLAMYDIA
 Obligate intracellular parasite
 Elementary bodies –metabolically active
particulate which will be transformed to Reticulate
bodies inside the cell (secondary organization)
 Reticulate bodies –infectious particles; binary
fission
 Dx:
 Culture media: Mc Coy’s–derived from mouse cell
lines
 Serological Test
 Staining –Giemsa/Iodine –look for Chlamydial
inclusions which appear as purple (giemsa) or
brown (iodine)
 Chlamydia psittaci–causative agent of Psittacosis
or Ornithosis from contaminated aerosol fomites
 C. pneumoniae–TWAR strain (Taiwan Acute
Respiratory Strain) which causes respiratory
infections
 C. trachomatis
 A, B, Ba, C–endemic trachoma strains
 D, E, F, G, H, I, J, K –non-gonococcal urethritis
 L1, L2, L3 strain –causes LGV
(Lymphogranuloma Venereum)

MYCOPLASMA
 Smallest free-living bacteria
 Pleomorphic (variation in shape) –no cell wall
 M. pneumoniae–Eaton’s agent of pneumonia
o Primary Atypical pneumonia (PAP) –dry
cough instead of exudative cough
o Walking Pneumonia
 Dx:
o Culture –Shephard’s agar, E agar, A7B agar
(fried egg appearance)
o Serological Test –for the presence of cold
agglutinins (anti-I association)
 Genital Mycoplasma
o M. hominis, Ureaplasma urealyticum

RICKETTSIA
1. Ehrlichia
2. Coxiella
3. Rochalimea
 All were transmitted by insect vector, except Coxiella
burnetti(aerosol)
 All could not survive outside the animal host (vector)
except C. burnetti

 Weil Felix Test–uses Proteus antigen

 OX2 and OX19 –Proteus vulgaris
 OX-K –Proteus mirabilis

Species Disease Vector

Rocky mountain
R. rickettsi Ticks
spotted fever
R. akari Rickettsial pox Mites
Australian Tick
R. australis Ticks
Typhus
R.
Scrub typhus Chigger bite
tsutsugamushi
Lice
Endemic typhus
Flying squirrels
R. prowazeki Sporadic typhus
Reactivation of
Brillzinser dse
latent infection
Endemic or
R. typhi Fleas
Murine typhus
R. quintana Trench fever Lice
Ehrlichia Ehrlichosis Ticks
C. burnetti Q fever Aerosol