Bacteriology

Medical Technology Licensure Examination (REVIEWER)
MICROBIOLOGY & PARASITOLOGY JOHN NEEDHAM (1748)
MICROBIOLOGY - He observed that boiled mutton broth

eventually became cloudy with
- from the word “micro” means small, and
microorganisms after pouring it into a flask
“biology” means the study of life
sealed and sealed tightly
- the study of organisms that individually are too
- He proposed that organic matter possessed a
small to be seen by the naked eye.
“vital force” that could give rise to life
INTRODUCTION
LAZZARO SPALLANZANI (1729 – 1799)
ROBERT HOOKE
- He improved the previous experiments of
- Observed a thin slice of cork and reported to Needham by heating the broth placed in a
the world that life’s smallest structural units sealed jar
were “little boxes” or “cells” - He observed that no growth took place as long
as the flasks remained sealed
LUCRETIUM (98 – 55 BC) and GIROLAMO FRACASTORO - He proposed that air carried microorganisms
(1478 – 1553) to the culture medium and that might be the
- They suggested that a disease was caused by reason for the growth of organisms present
“invisible creatures” already in the medium
- He concluded that microorganisms from the
FRANCESCO STELLUTI (1625 and 1630) air probably had entered Needham’s solutions
- He made the earliest microscopic observations after they were boiled
on bees and weevils using a microscope LAURENT LAVOISIER
probably supplied by Galileo
- He showed the importance of oxygen to life
HISTORY OF MICROBIOLOGY
RUDOLF VIRCHOW (1858)
ANTON VAN LEEUWENHOEK (1632 – 1723)
- He challenged spontaneous generation with
- The “first true microbiologist” the concept of “BIOGENESIS”
- The first person to observe and describe
microorganisms accurately THEODORE SCHWANN (1810-1882)
- Described microorganisms as “ANIMALCULES” - He observed that no growth occurred in a flask
- He used his self-made single lens microscope containing nutrient solution after allowing air
with 50-300x magnification to study to pass through a red-hot tube
protozoans and bacteria - Described the idea of fermentation
- The FATHER OF PROTOZOOLOGY and
BACTERIOLOGY GOERG FRIEDRICH SCHRODER and THEODORE VON
DUSCH
ARISTOTLE (384 – 322 BC)
- They observed that no growth occurred after
- He mentioned that simple invertebrates could allowing air to pass through sterile cotton wool
arise from “spontaneous generation” placed in a flask of heat-sterilized medium
FRANCESCO REDI (1626 – 1697) LOUIS PASTEUR (1822-1895)
- He demonstrated that maggots do not arise - He resolved the issue of spontaneous
spontaneously from decaying meat generation
- His results were a serious blow to the long-held - He stated that microorganisms are indeed
belief that large forms of life could arise from present in the air and can contaminate
non-life seemingly sterile solutions, however, the air
itself does not create microbes
PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT 1|Page
- He showed that microorganisms can also be o The microorganism must be present in
present in non-living matter every case of the disease but absent
- He stated that microbial life can be destroyed from healthy organisms
by heat (basis of aseptic technique) o The suspected microorganism must be
- He provided that microorganisms cannot isolated and grown in a pure culture
originate from mystical forces present in non- o The same disease must result when
living materials the isolated microorganism is
- He also described that certain microorganism inoculated into healthy host
known as “yeast” converts sugar to alcohol in o The same organism must be isolated
the absence of air (fermentation) again from the diseased host
- Proposed “PASTEURIZATION”
FANNIE EILSHEMIUS HESSE
FERDINAND COHN
- He suggested the use of agar as a solidifying
- He observed that no matter how long some agent
flasks were boiled, they always produced
RICHARD PETRI
certain growth (heat resistant bacterial spores)
- He developed the petri dish
JOHN TYNDALL (1829 – 1893)
MARTINUS BEIJERINK and SERGIE WINOGRADSKY
- He showed that dust carry germs which
contaminates sterile broth - Developed the enrichment-culture technique
- Developed “TYNDALLIZATION” and the use of selective media
IGNATZ SEMMELWIES (1816-1865) EDWARD JENNER (1749-1823)
- He demonstrated that routine hand washing - He experimented on how people can be
can prevent the spread of disease protected against small pox
- He collected scrapings from cowpox blisters
JOSEPH LISTER (1827-1912)
and inoculated a healthy volunteer with the
- He developed the antiseptic system of surgery cowpox material by scratching the person’s
- He introduced British surgery to hand washing arm with a pox-contaminated needle
and the use of phenol as an antimicrobial
EMIL VON BEHRING
agent for surgical wound dressings
- He demonstrated the use of phenol for - Prepared antitoxins for diphtheria and tetanus
treating surgical wounds and also sprayed
phenol over the surgical area PAUL EHRLICH
ROBERT KOCH (1843-1910) - Discovered salvarsan (606th drug) for

treatment of syphilis
- He established the first proof that bacteria
indeed cause diseases (germ theory of disease) ALEXANDER FLEMING
- He discovered Bacillus anthracis as causative - Discovered penicillin
agent of anthrax
- He discovered Mycobacterium tuberculosis HOWARD FLOREY and ERNST CHAIN
- He was the first to culture bacteria on boiled - made the purification process for penicillin
potatoes, gelatin and used meat extracts and
protein digests for culture NAMING AND CLASSIFYING MICROORGANISMS
- He developed culture media for observing
TAXONOMY
growth of bacteria isolated from human body
- KOCH’s POSTULATE: - An area of biologic science comprising three
distinct, but highly interrelated disciplines that
2|Page PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT

include classification, nomenclature and BIOSAFETY IN MICROBIOLOGY
identification
CLASSIFICATION OF BIOLOGIC AGENTS
- It is a formal system of organizing, classifying
and naming living things based on the 1. BIOSAFETY LEVEL 1 AGENTS
similarities and differences of genotypic and - include those that have no known
phenotypic characteristics potential for infecting healthy people
- used in laboratory teaching exercises of
CLASSIFICATION SYSTEM
students
- Ex. B. subtilis, M. gordonae
2. BIOSAFETY LEVEL 2 AGENTS
- agents acquired by ingestion;
percutaneous or mucous membrane
exposure
- include all the common agents of
infectious diseases
- personnel handling these agents should
have received immunization
- Ex. HIV, B. anthracis, Y. pestis, Toxoplasma,
Salmonella and Shigella
PROKARYOTES VS EUKARYOTES VS ARCHAEA 3. BIOSAFETY LEVEL 3 AGENTS
- potential agents for aerosol transmission;
ARCHAEA lethal pathogens
- From the word “archaois” meaning ancient - air movement must be controlled so as to
- Bacteria-like cells but don’t contain contain the infectious materials
peptidoglycans - Ex. M. tuberculosis, systemic fungi, F.
- They do not contain nucleus and membrane tularensis, Brucella spp, St. Louis
bound organelles encephalitis virus
- May stain as gram variable in various shape 4. BIOSAFETY LEVEL 4 AGENTS
- The structure of their cell envelope and - agents that are dangerous and exotic;
enzymes allows them to grow and survive causing life-threatening infections
under extreme environment conditions - maximum containment and
- EX: Methanogens, Halobacterium decontamination of all personnel and
materials before leaving the area are
observed
- aerosol transmission with pressure is
possible
- Ex. Arbovirus, Arenavirus, Filovirus, Small
pox
CATEGORIES OF POTENTIAL INFECTIOUS AGENTS OF

BIOTERRORISM
1. CATEGORY A AGENTS
- agents that pose the greatest public health
threat
- these are easily transmitted and highly
infectious
2. CATEGORY B AGENTS
- agents with moderate morbidity and low
mortality
- these are not as easily transmitted as ▪ 2 TYPES
category A agents • Class IIa – fixed
3. CATEGORY C AGENTS opening
- are the emerging pathogens • Class IIb – variable
sash opening
BIOSAFETY CABINET
- a devise that encloses a working area in such a

way as to protect workers from aerosol
exposure to infectious disease agents
o Class III cabinets

▪ provide the most protection
to the worker
- Three classes: ▪ air. coming into and going out
o Class I cabinets of the cabinet is sterilized
▪ allow room (unsterilized) air (HEPA) and the infectious
to pass into the cabinet and material within is handled
around the area and material with rubber gloves that are
within, sterilizing (HEPA) only attached and sealed to the
the air to be exhausted are cabinet
open-fronted with negative
pressure (ventilated cabinets)
▪ use for BSL 2 and 3 agents
STERILIZATION AND DISINFECTION
SEPSIS
o Class II cabinets
- Bacterial presence at a pathogenic level
▪ aka laminar flow BSC
▪ sterilize air (HEPA) that flows ASEPSIS
over the infectious material as
- Absence of significant contamination
well as air to be exhausted
- Aseptic technique minimizes contamination
▪ used for BSL 2 and 3 agents
ANTISEPSIS
- Chemical destruction of vegetative pathogens STERILIZATION

on living tissue
- Freeing of an article from all living organisms,
SANITIZATION including viruses, bacteria and fungi and their
spores
- Lowering microbial counts on eating and
- Uses
drinking utensils to safe levels
o Instruments and materials used in
ANTISEPTICS penetration into normally sterile body
parts
- Chemicals that kill microorganisms on living o Media, reagents and eqpt used in
skin or mucous membranes laboratory practices
BACTERICIDAL METHODS OF STERILIZATION
- Chemical agents capable of killing bacteria - Heat
- Similarly, agents that are virucidal, fungicidal - lonizing Irradiation
or sporicidal are agents capable of killing these - Filtration
organisms - Gases
BACTERIOSTATIC - Liquids
- Chemical agents that inhibit the growth of STERILIZATION BY HEAT

bacteria but do not necessarily kill them DRY HEAT
MICROBIAL CHARACTERISTICS AND CONTROL - Dry heat
o kills by oxidation (slow, uneven
penetration)
- Incineration
o 1000-1500 °C, pathological waste,
surgical dressings, sharps, needles,
other clinical wastes
- Flaming
o Scalpels, neck of flasks
- Red heat
o Inoculating loops, wires
- Hot air ovens
o 160 °C for one hour
o Glassware, oily fluids, powder
- Microwave oven
ANTISEPTICS vs DISINFECTANTS o Not reliable due to variable heat
ANTISEPTICS MOIST HEAT
- Used on skin and mucous membranes to kill - Moist heat

microorganisms o Kills by protein coagulation
- Not for use on inanimate objects (denaturation of enzymes)
o requires lower temperatures and
DISINFECTANTS shorter times, but the moisture must
- Used to kill microorganisms on inanimate penetrate the pathogens to be
objects effective
- Not for use on skin or mucous membranes
- Boiling
o At 100 °C at sea level - UV Radiation
o kills many vegetative cells and viruses o Low energy, non-ionizing radiation
within 10 minutes with poor penetrating power that is
lethal to microorganisms under
optimum conditions
o Air, water, thin films and surfaces such
as laboratory safety cabinets
- Gases
o Formaldehyde
o To disinfect hospital rooms or
laboratory safety cabinets
- Filtration
- Pasteurization o Air
o Destroys pathogens (Mycobacterium ▪ Supplied to OTs,
tuberculosis, Salmonella typhi, etc.) pharmaceutical clean rooms
without altering the flavor of the food and transplant centres
o does not sterilize (63 C for 30 sec) ▪ Extracted from laboratories
o Higher temperature short time that are handling dangerous
pasteurization applies higher heat for pathogens
a much shorter time (72°C for 15 sec) o A properly installed HEPA (High-
- UHT treatment Efficiency Particulate Air) filter
o An ultra-high-temperature, very short achieves 99.997% arrestance of
duration treatment particles of 0.5 pm or larger size
o 140 °C for 3 sec is used to sterilize dairy - Chemicals
products o Factors influencing the performance
- Tyndallization of chemical disinfectants
o Intermittent exposure at 100°C o Concentration and stability of agent
o Principle: One exposure kills o Accessibility of microorganisms
vegetative organisms, between o Temperature and pH
heatings the spores become o Presence of organic or other protein
vegetative forms which get killed (interfering) substances
during subsequent heating o Alcohols
o Gelatin media, media containing ▪ Isopropanol, ethanol and
sugars industrial methylated spirits
have optimum bactericidal
METHODS OF DISINFECTION
activity at a conc. of 70-90%
- Heat • Limited activity
- Moist heat against mycobacteria
- Dry heat and are not sporicidal
o Method of first choice • Washed
o Leaves no toxic residues thermometers and
o Washing laundry or eating utensils in trolley tops
water at 70-80°C for a few minutes ▪ Alcohol-based formulation
would kill most of the vegetative with chlorhexidine or
organisms povidone-iodine are good
o Exposure to boiling water for 20 min- choices for hand disinfections
highly effective disinfection, used
where sterilization is not available

o Aldehydes ▪ H,O, - Limited application for
▪ Glutaraldehyde treatment of wounds
• Broad spectrum o Surface active agents
action against ▪ Detergents and QACs
vegetative (Quarternary Ammonium
microorganisms Compounds)
• Flexible endoscopes ▪ CPC and Benzalkonium
that cannot be Chloride
sterilized or • Cleaning dirty
disinfected by heat wounds, hand
▪ Formaldehyde washing and skin
o Biguanides disinfection
▪ Chlorhexidine • Limited antimicrobial
• Skin and mucous activity against
membranes vegetative and no
• Less active against activity against spore
Gram-negative forms
bacteria
PROPER HANDWASHING
(Pseudomonas and
Proteus species)
• Limited virucidal,
tuberculocidal and
sporicidal activity
o Halogens
▪ Hypochlorites
• These broad
spectrum,
inexpensive and
chlorine-releasing
disinfectants are of
choice against viruses
such as Hepatitis Band PHYSICAL ANTIMICROBIALS
C and HIV viruses
• Bleaching activity
• Table tops and
discard pots
▪ Iodine
▪ Povidone-iodine
• Skin disinfection
and pre-
operative
preparation of
skin
o Oxidizing agents and H,O,
▪ Chlorine dioxide and
H, O, have good
antimicrobial properties
but are corrosive to skin
and metals
CHEMICAL ANTIMICROBIALS
CULTURE AND CULTURE MEDIA

iii. Ex. Brain Heart Infusion (BHI),
MAIN CONTENT
Trypticase Soy Broth (TSB),
1. Carbon Thioglycollate
2. Nitrogen b. Semi-Solid Medium
3. Sulfur i. contains 0.5% to 1% agar
4. Phosphorus ii. used for observation of
5. Hydrogen motility
6. Oxygen iii. Ex. Sulfide Indole Motility
7. Buffers (SIM) medium
8. Inhibitory agents (optional) c. Solid Medium
i. contains 2% to 3% agar
TYPES OF CULTURE ii. Ex. Triple Sugar Iron (TSI) agar,
1. Pure Culture Mac Conkey Agar, Blood Agar
- composed of only one species Plate (BAP)
2. Mixed Culture 2. According to Composition
- composed of more than one species a. Synthetic or Defined Medium
3. Stock Culture i. a medium where all the
- composed of several species contained in components are known to
a separate culture media user
- used for academic and industrial purposes ii. used for research purposes
b. Non-Synthetic or Complex Medium
CLASSIFICATION OF CULTURE MEDIA i. composed of some unknown
1. According to Consistency substances (peptones, meat
a. Liquid Medium and yeast extracts)
i. contains 0% agar ii. very useful for isolation of
ii. allows that growth of aerobes, bacteria
anaerobes and facultative iii. Ex. Nutrient broth, TSB and
anaerobes MAC

c. Tissue Culture ii. Ex. Hektoen Enteric Agar,
i. used for obligate intracellular MAC, Xylose Lysine
organisms Desoxycholate (XLD) Agar
ii. Ex. W138, HeLa 229 cells, and
INOCULATION TECHNIQUES
Mc Coy Cells
3. According to Medium is Dispensed STREAKING METHODS
a. Plated medium
i. weigh - dissolve - sterilize – - most commonly performed inoculation
dispense technique
b. Tube Medium 1. Simple streak
i. slant, butt and butt & slant 2. Radial streak
ii. Weigh - dissolve - dispense - 3. Overlap streak
sterilize 4. Multiple streak
4. According to Use 5. Multiple interrupted
a. Simple Media/General 6. Simple Interrupted streak
Purpose/Supportive Media PLACEMENT OF FLUID SPECIMEN or SWABS INTO
i. media without added CULTURE BROTH
supplement
ii. media that support growth of - broth can be inoculated by placing a few drops
most non-fastidious bacteria of the liquid specimen into the tube or placing
iii. Composition: meat and the swab into the broth
soybean extracts STABBING THE MEDIUM
iv. Examples: Nutrient Agar (NA),
Trypticase Soy Broth (TSB) - technique is applied to semi-solid medium
b. Enrichment Broth - also used to inoculate streptococci into BAP
i. contains specific nutrients or
STAB AND STREAK METHOD
supplements to sustain
growth of certain organisms - used to inoculate organisms into “slant and
ii. Ex. Alkaline Peptone Water butt medium”
(APW)
POUR PLATE METHOD
c. Enriched Media/Non-Selective Media
i. media with added - technique where the specimen is added to
supplements necessary for molten agar, mixed and then allows the
growth of fastidious medium to solidify
organisms
ii. Ex. Blood Agar Plate (BAP) and BACTERIAL CELL STRUCTURE
Chocolate Agar Plate (CAP)
d. Differential Media
i. media that allow visualization
of metabolic differences
between groups of organisms
ii. Ex. MAC and BAP
e. Selective Media
i. media incorporated with
antibiotics, dyes or chemicals 1. CELL ENVELOPE
to inhibit growth of other - the outermost layer
organisms while promoting - composed of a cell wall and plasma
growth of desired organisms membrane

CELL WALL/MUREIN LAYER/PEPTIDOGLYCAN 3. Generation of ATP
4. Cell motility
- a rigid structure that maintains the shape
5. Mediation of chromosomal
of the cell
segregation during replication
- composed of disaccharide-pentapeptide
6. Housing molecular sensors that
subunits
monitor chemical and physical
- FUNCTIONS OF CELL WALL
changes in the environment
1. To prevent bacterial cells from
2. CELLULAR APPENDAGES
rupture
- plays a role in causing infection and
2. Serves as a point of anchorage for
laboratory identification
flagella
- can vary among bacterial species
3. Determines staining
- composed of capsule, flagella,
characteristics of species
fimbriae/pilli
a. GRAM POSITIVE CELL
WALL CAPSULE/GLYCOCALYX/SLIME LAYER
i. very thick
- immediately exterior to the murein layer
peptidoglycan
of gram-positive bacteria and outer
layer
membrane of gram-negative bacteria
ii. contains teichoic
- composed of high-molecular weight
acid
polysaccharides
b. GRAM NEGATIVE CELL
- FUNCTION: prevents phagocytosis and
WALL
dessication
i. contains
periplasmic space FLAGELLA
ii. has outer
membrane aside - a complex structure composed of protein
from inner FLAGELLIN
membrane - responsible for locomotion
- ARRANGEMENT OF FLAGELLA
1. Atrichous: without flagellum
2. Monotrichous: single flagellum on
one end
3. AMPHITRICHOUS: single flagellum
on both ends
4. LOPHOTRICHOUS: tuff/group of
flagella on one end
5. AMPHILOPHOTRICHOUS: tuff of
PLASMA MEMBRANE flagella on both ends
- deepest layer of the cell envelope 6. PERITRICHOUS: spread over the
- a phospholipid bilayer with embedded whole surface
proteins that surrounds the cytoplasm PILLI/FIMBRIAE
vital for metabolism
- FUNCTIONS: - hair-like, proteinaceous structures that
1. Transport of solutes into and out extend from the cell membrane into the
of the cell external environment
2. Housing enzymes involved in - TWO TYPES:
outer membrane synthesis, cell 1. Common/somatic pilli: adhesins
wall synthesis, and the assembly that help bacteria attach to host
and secretion of extra cytoplasmic cell surfaces
and extracellular substances
10 | P a g e PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT
- are cationic or have positively charged
2. Sex pilli: essential for genetic groups that bind to negatively charged
transfer or conjugation process molecules like nucleic acids and proteins
3. CELL INTERIOR - EX. Methylene blue, basic fuchsin, crystal
- structures and substances that are violet, safranin and malachite green
bounded internally by the cytoplasmic 2. ACIDIC DYES
membrane - are anionic dyes or possess negatively
- composed of cytosol, polysomes, charged groups that bind to positively
inclusions, nucleoid, plasmids and charged cell structures
endospores - EX. Eosin, rose bengal and acid fuchsin
CYTOSOL STAINING TECHNIQUES
- contains thousands of enzymes 1. SIMPLE STAINING

- site for protein synthesis - a single stain is used
- it is directed towards coloring the forms
INCLUSION BODIES
and shapes present
- serve as energy source (food reserve) - EX. Malachite green, methylene blue,
- mainly composed of polysaccharide crystal violet
- TWO COMMON TYPES OF INCLUSIONS 2. DIFFERENTIAL STAINING
1. GLYCOGEN – storage form of - it divides bacteria into separate groups
glucose - it is directed towards coloring components
2. POLYPHOSPHATE GRANULES – a of those elements present
storage form of inorganic - EX. Gram stain and acid-fast stain
phosphates
GRAM STAIN
NUCLEOID
- the most commonly used differential stain in
- the bacterial chromosome microbiology laboratory
- consists of a single, circular chromosome
THREE GRAM STAIN REACTION THEORIES:
PLASMIDS
1. Mg RNA theory
- other genetic elements that exist 2. Benian theory
independently in the cytosol 3. Stearn and Stearn (law of magentism)
- located in the cytoplasm and serve as a site
TECHNICAL ERRORS IN GRAM STAINING
for the genes that code for antibiotic
resistance and toxin production 1. If the crystal violet is rinsed too vigorously
before it is complexed with iodine, it will be
ENDOSPORES
washed away and leave poor or no staining of
- the resistant structure gram-negative bacteria
- small, dormant structures that develop 2. If the decolorization is too vigorous or
inside the bacterial cell as a means of prolonged, the gram-positive complex will be
survival against harsh environmental removed and the normally gram-positive
condition bacteria will not be stained
3. If the decolorization is insufficient, organism
STAINING PROCEDURE may appear falsely gram positive
2 CLASSES of IONIZABLE DYES 4. If the safranin is left on the slide for a long
period, the gram-positive complex will be
1. BASIC DYES leached from the gram-positive cells
- are commonly used dyes in bacteriology 5. Failure to leave the safranin in place for
- sufficient time will result in failure to stain the
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gram-negative bacteria and background pneumophila, HSV, Varizella-zoster virus, CMV,
materials Adenovirus, and Respiratory viruses
EXCEPTIONS IN GRAM STAINING NEGATIVE STAINING
1. Organisms that exist almost exclusively with - utilized to demonstrate the presence of diffuse
host cells capsule surrounding some bacteria
EX. Chlamydia - excellent technique to study bacterial gas
2. Organisms that lack cell wall vacuoles and viral morphology
EX. Mycoplasma and Ureaplasma - also used to study cells sensitive to heat fixing
3. Organisms with insufficient dimension to be - EX. India ink and Nigrosin dye
resolved by the light microscope
SPECIAL STAINS:
EX. Spirochetes
1. Dyar stain: cell wall
ACID FAST STAIN
2. Hiss, Gin, Anthony and Welch: capsular stain
- used to stain bacteria that have high lipid 3. LAMB, Neisser, Albert and Ljubinsky:
content in their cell wall Metachromatic granules
- PRINCIPLE: mycolic acids render the cells 4. Dorner, Wirtz and Conklin, Schaeffer-Fulton:
resistant to decolorization, even with acid endosporse
alcohol decolorizers. Thus, acid fast 5. Gray, Leifson, Fisher and Conn: flagella
microorganisms retain the primary stain and 6. India ink/Borris Method and Nigrossin:
are colored red and non-acid fast organisms Capsule (yeast)
are blue or green color 7. Feulgen stains: DNA
- ACID FAST STAINING METHOD 8. Fontana Tribondeau and Levadite Silver
1. Ziehl-neelsen (hot method) Impregnation: 8Spirochetes
2. Kinyoun’s (cold method)
MICROBIAL NUTRITION AND GROWTH
3. Pappenheim’s method
4. Baugarten’s method BACTERIAL REQUIREMENTS
- ACID FAST STAINING PROCEDURE
1. Heat fix the slide 1. According to oxygen requirement
2. Flood smear with carbolfuchsin and a. OBLIGATE AEROBES
steam the slide gently for 1 minute by ▪ organisms that require oxygen
flaming from below the rack. Do not for growth
permit the slides to boil or dry out ▪ Ex. Bordetella, Brucella,
3. All the stain to remain on the slides for Mycobacteria, Pseudomonas
addition 4-5 minutes without heat b. OBLIGATE ANAEROBES
4. Rinse with water ▪ organisms that strictly does
5. Decolorize with 3% acid alcohol for 2 not require the presence of
minutes and rinse with water oxygen; they die in the
6. Flood slide with methylene blue or presence of oxygen
malachite green for 1 minute ▪ Ex. Clostridium and
7. Rinse and air dry Bacteroides
c. FACULTATIVE AEROBES
DIAGNOSTIC ANTIBODY or DNA PROBE-MEDIATED ▪ anaerobic organisms that can
STAINING live in the presence of oxygen
d. FACULTATIVE ANAEROBES
- it is directed specifically at identification of an
▪ aerobic organisms that can
organism
live in the absence of oxygen
- it is used for the specific identification of
▪ Ex. Staphylococcus species
selected pathogens such as Chlamydia
trachomatis, Bordetella pertussis, Legionella
e. AEROTOLERANT
▪ organisms that can survive in b. THERMOPHILE/HYPERTHERMOPHILE
the presence of oxygen but ▪ organisms that grow well at
will not be able to perform 50˚C to 125˚C
metabolic processes unless ▪ Ex. Stearothermophilus
placed in anaerobic c. MESOPHILE
environment ▪ organisms that grow between
▪ Ex. Propionibacterium and 20˚C to 45˚C
Lactobacillus ▪ clinically significant organisms
f. MICROAEROPHILES d. EXTREMOPHILE
▪ organism that requires small ▪ organisms that are able to live
amount of oxygen for growth at unusual conditions like
▪ Ex. HACEK group absence of oxygen, increased
g. CAPNOPHILES temperature and below
▪ organisms that require 5-10% earth’s surface
carbon dioxide and ▪ Ex. Bacillus infernus
approximately 15% oxygen 4. According to pH requirement
▪ Ex. Neisseria species a. ACIDOPHILE
2. According to nutritional requirement ▪ organisms that require pH
a. ACCORDING TO CARBON SOURCE <5.5
i. AUTOTROPHS - they use ▪ Ex. Sulfolubus, Picrophilus,
carbon dioxide as sole source Acontium
of carbon b. NEUTROPHILE
ii. HETEROTROPHS - they use ▪ organisms that require pH 6.5
reduced, preformed, organic to 8.5
molecules from other ▪ Ex. Clinically significant
organisms bacteria
b. ACCORDING TO ENERGY SOURCE c. ALKALOPHILE
i. PHOTOTROPHS - organisms ▪ organisms that require >8.5
that use light as energy source ▪ Ex. Bacillus alcalophilus and
ii. CHEMOTROPHS - organisms Natrobacterium
that use the energy produced 5. According to Ionic strength requirement
by the oxidation of organic or a. OSMOPHILE
inorganic compounds ▪ organisms that require
c. ACCORDING TO ELECTRON SOURCE increased osmotic pressure
i. LITHOTROPHS - they reduce b. HALOPHILE
organic molecules ▪ organisms that live in high salt
ii. ORGANOTROPHS - they concentration
require organic substances for c. HALOTOLERANT
growth and multiplication. All ▪ organisms that can survive in
bacteria that invade the high salt concentration
human body falls under this 6. Others
group a. Moisture
3. According to temperature requirement ▪ important for bacterial growth
a. PSYCHROPHILE/CRYOPHILE b. Pressure
▪ organisms that grow well at ▪ 600 to 1100 atm pressure
<20˚C ▪ Ex. Photobacterium,
▪ Ex. Listeria monocytogenes Shewanella and Colwellia
and Yersinia enterocolitica

c. Growth factors TEST TO DIFFERENTIATE MICROCOCCEAE FROM
▪ substances required by STREPTOCOCCUS
fastidious organisms for their
CATALASE TEST
growth and multiplication
- Reagent used: 3% hydrogen peroxide (H202)
BACTERIAL GROWTH CURVE
- Positive result: effervescence (bubbling)
LAG PHASE/PERIOD OF REJUVENESCENCE o Micrococcaceae: positive (+)
o Streptococcus spp: negative (-)
- the period wherein there is no cell division
- the start of biosynthesis although there is no
increase in cell mass
- the phase of adjusting to new environment
LOG PHASE/EXPONENTIAL PHASE
- period wherein microorganisms are actively TESTS TO DIFFERENTIATE STAPHYLOCOCCUS FROM

growing and dividing MICROCOCCUS
- cellular production is most active during this
period
- the phase wherein microorganisms are utilized
in physiological and biochemical testing
- the phase wherein microorganisms are
sensitive to radiation and antimicrobial agents
STATIONARY PHASE/PLATEAU PHASE
- period wherein there is balance between cell

division and dying organisms
- the number of viable microorganisms remain
constant
- the phase where metabolic activities of
surviving cells slow down and nutrients are
becoming limited
- the phase wherein dead debris is starting to
accumulate
DEATH PHASE/DECLINE PHASE
- period wherein there is cessation of bacterial

STAPHYLOCOCCI SPECIES
growth
- the number of cell death exceeds the number - Genus name is derived from Greek work
of living microorganisms staphle meaning “bunches of grapes”
- period wherein there is loss of nutrients and - Gram positive cocci in clumps
increased amount of toxic waste - Belongs to family of Micrococcaceae
- non-motile, non-spore forming and glucose
GRAM POSITIVE COCCI
fermenter

Staphylococcus aureus - Lipase (fat-splitting enzyme)
o Essential for survival in sebaceous
- most virulent species of Staphylococci
areas of the body
- β-hemolytic on BAP
- DNAse and phosphatase
- Halophilic (can grow in presence of 7.5-10%
o Destroys DNA
NaCl)
- β-lactamase
- Produces a golden yellow pigment
o It breaks down penicillin and other β-
(staphyloxanthin)
lactam drugs
DISEASE AND INFECTIONS - Enterotoxins (heat stable)
o Acts as neurotoxins that stimulate
- Toxin-induced diseases vomiting
o Scalded Skin Syndrome (SSS), or
Ritter’s Disease;
o Toxic Shock Syndrome (TSS)
- UTI
- Acute bacterial endocarditis
- Cutaneous infections (folliculitis,
furuncles/boil, carbuncles, impetigo)
- Enterotoxin F
o Stimulates production of large amount
of cytokines and causes almost all
cases of menstruating-associated TSS
VIRULENCE FACTORS - Leukocidins/Panton-valentine leucocidin
(cytolytic toxin)
- Catalase o Attacks and kills the WBC and prevents
o A heme enzyme that catalyzes the phagocytosis
decomposition of H202 - Hemolysin (cytolytic toxin)
o H2O2 – H2O + O2 o Causes anemia – make iron available
- Coagulase for bacterial growth
o Coagulates the fibrinogen to fibrin - Exfoliatin A and B
o Promotes formation of fibrin layer o Causes the epidermal layer to slough
around staphylococcal abscess off
thereby protecting the bacteria from - Protein A
phagocytosis o Antiphagocytic by competing with
o 2 types neutrophils for the Fc portion of
▪ cell-bound opsonins
coagulase/clumping factor
▪ Unbound coagulase/free DIFFERENTIAL TESTS
coagulase - Coagulase test (2 methods)
- Hyaluronidase o Slide method
o spreading factor enzyme ▪ screening test
o Enhances invasion and survival in ▪ detects cell bound
tissue; breaks down hyaluronic acid coagulase/clumping factor
present in the intracellular ground ▪ (+) result: clot/coagulum
substance of connective tissues formation
resulting to spread of bacteria ▪ other slide coagulase positive
- Staphylokinase organisms: S. lugdunensis, S.
o Fibrinolytic activity (dissolves fibrin) schleiferi
o Tube method Staphylococcus epidermidis (former S. albus)
▪ definitive test
- Normal flora of skin
▪ detects free/unbound
- Common contaminant of medical equipment
coagulase
(prosthetic heart valve implants, catheters)
▪ procedure:
- Secretes poly-gamma-DL-glutamic acid:
• inoculate a tube
provides adherence to devices
containing plasma
- Common nosocomial infections
and incubate at 35˚C
- Non hemolytic on BAP
for
• 1-4 hours Staphylococcus saprophyticus
• (+) result:
- Associated with community-acquired UTI in
clot/coagulum
young, sexually active females
formation
- Non hemolytic on BAP
• if no clot appears after
4hrs, incubate the STREPTOCOCCI SPECIES
tube at room
- Belong to family Streptococcaceae
temperature for 20
- Commonly found as part of human
hours
- normal flora but can cause life threating
▪ other tube positive coagulase
infection once they invade normally sterile
organisms: S. hyicus, S.
sites of the body
intermedius, S. delphini
- Gram positive cocci in chains or pairs
- Mannitol Salt Agar
- Facultative anaerobes; some species are
o Contains 1% mannitol + 7.5% NaCl
capnophilic
o pH indicator: phenol red
o (+) S. aureus: colonies surrounded by CLASSIFICATION OF STREPTOCOCCI
yellow halo
- Tellurite Glycine Agar - Academic/Bergey’s Classification
o S. aureus: jet black colonies o Based on temperature requirement
- Polymyxin sensitivity test:
o S. aureus: resistant
o Other Staphylococcus spp.: sensitive
- Smith and Brown Classification

o Based on hemolytic patterns
▪ Alpha-hemolytic
• S. pneumoniae
▪ Beta-hemolytic
- Voges-Proskauer test
• S. pyogenes, S.
o It differentiates S. aureus from S.
agalactiae, S.
Intermedius
dysagalactia subsp.
o (+) pink color: S. aureus
Equimilis and S.
- DNAse Test
anginosus group
o Medium: DNA medium
▪ Gamma-hemolytic
o Reagent: methyl green
• E. fecalis
o (+) result: clearing of the dye
- Lancefield Classification (antigen o allows the bacteria to move from the
serogrouping) clotted area (spread of infection)
o Based on extraction of C carbohydrate - Hyaluronidase
from the streptococcal cell wall o Spreading factor
o Mostly significant in classifying and - Pyrogenic toxins
identifying β-hemolytic streptococci o super antigens
o Group A, B, C, D, F, G, …. T o formerly known as erythrogenic toxin
o exotoxin B – degrades proteins;
Group A Streptococci
mediates rash in scarlet fever
- Pathogenic to man
INFECTIONS AND DISEASES
- Acquired thru contaminated droplets by cough
or sneeze - Pharyngitis/tonsilitis (strep throat)
- Colonies are small, translucent and smooth; - Scarlet Fever (Scarlatina)
well-defined β-hemolytic o Caused by the release of pyrogenic
- Species: S. pyogenes (“fever-producing exotoxins
bacteria; flesh-eating bacteria – involves o Cardinal signs: diffused red rash on the
deeper tissue and organs) upper chest to the trunk and to the
extremities and “strawberry red
VIRULENCE FACTORS
tongue”
- M-protein o Susceptibility Test: Dick’s test
o principal virulence factor ▪ (+) result: erythema or
o attached to the peptidoglycan redness of the test site
o antiphagocytic o Diagnostic test: Schultz Charlton
o for adherence to mucosal cells ▪ (+) result: blanching
- Protein F phenomenon – rash fade
o mediates epithelial cell attachment - Skin infections
- Lipoteichoic acid o Cellulitis
o bacterial adherence to the respiratory o Impetigo
epithelium o Erysipelas
- Hyaluronic acid and capsule
o weakly immunogenic
o prevents opsonized phagocytosis
o masks its antigen
- Hemolysins
o Streptolysin O (oxygen labile)
▪ responsible for subsurface
- Rheumatic Fever
hemolysis
o characterized by fever, inflammation
▪ highly antigenic
of heart, joints and blood vessels
o Streptolysin S (oxygen stable)
- Acute Glomerulonephritis (AGN) or Bright’s
▪ responsible for surface
disease
hemolysis
o inflammatory disease of the renal
▪ non antigenic
glomeruli, results from deposition of
- DNAse
antigen-antibody complexes in the
o 4 types: A, B, C and D (antigenic
kidneys
enzymes)
- Streptococcal TSS
o it lowers viscosity of exudates
o a condition which the entire organ
- Streptokinase
system shuts down
o it causes lysis of fibrin clots

- Hippurate Hydrolysis Test

LAB DIAGNOSTIC TESTS:
o reagents: sodium hippurate +
- Bacitracin Disc Test/ Taxo A ninhydrin
o differentiates S. pyogenes from other o (+) result: S. agalactiae- purple color
β-hemolytic Strep after ninhydrin rgt
o (+) result: Group A- susceptible
- Sulfamethoxazole and trimethoprim (SXT) test
o (+) result: Group A- resistant
- L-pyrrolidinyl-α-naphthylamide (PYR) test
o detects pyrrolidonyl-arylamidase
enzyme
o (+) result: bright/cherry red color
Group B Streptococci
- Part of the normal flora of female genital tract Purple: + for Hippurate hydrolysis
and lower GIT
- Can cause infections to fetus during passage GROUP C Streptococci
through the birth canal - Organisms are recovered from URT, vagina and
- Grayish white, mucoid colonies with small skin of humans
zone of β-hemolysis - Possess M protein just like the group A
- Species: S. agalactiae streptococci
INFECTIONS AND DISEASES: - Animal pathogens
- Species: S. dysagalactiae subsp equisimilis, S.
- Pneumonia equi subsp. zooepidermicus
- Meningitis
- Neonatal sepsis GROUP D Streptococci (S. bovis/non-enterococci)
- Postpartum infection - produces α-prime hemolysis
- Osteomyelitis - S. bovis is no longer a valid species name;
- UTI based on DNA studies, S. equinis and S. bovis
- endocarditis are the same
LAB DIAGNOSTIC TESTS: - often isolated in blood cultures of individuals
with GI carcinoma
- CAMP Test (Christie, Atkins, and Munch-
Peterson test) ENTEROCOCCI
o differentiates S. agalactiae from other - Belong to family Streptococcaceae
β-hemolytic strep - Formerly known as group D Streptococci
o (+) result: arrow head β-hemolysis - Natural inhabitants of the intestinal tracts of
near growth or bowtie appearance humans and animals
- Not highly pathogenic but can cause
nosocomial infections
- Can grow in extreme conditions (alkaline pH,
can grow at 45˚C and salt solution
- Can exhibit α, β, γ- hemolysis
- Species: E. fecalis (most common), E. faecium,
E. avium, E. gallinarum, E. durans, E. raffinosus

TESTS TO DIFFERENTIATE GROUP D - Bacteremia
NONENTEROCOCCUS FROM ENTEROCOCCUS SPP - Endocarditis
- Peritonitis
TEST TO DIFFERENTIATE S. PNEUMPNIAE FROM

VIRIDANS GROUP
Streptococcus pneumoniae
- Aka Diplococci/pneumococci
- Considered normal flora of 25-50% of the URT
of children
- Most common cause of bacterial meningitis in
Abiotrophia and Granulicatella
adults
- Gram positive cocci in pairs, oval or lancet - Formerly known as “nutritionally variant
shape streptococci” (NVS)
- Young colonies: α-hemolytic mucoid, dome- - Pyridoxal (vit B12) dependent, thiol-
shaped glistening colonies dependent (cysteine) and symbiotic
- Old colonies: α-hemolytic mucoid, flat colonies streptococci
with depressed center (nail-head appearance) - Part of human oral and GI normal flora
- Principal virulence factor: capsular - Opportunistic pathogens of low virulence
polysaccharide - Gram variable and pleomorphic forms
- Appears “satellite” around an organism that
INFECTIONS AND DISEASES
produces pyridoxal (E. coli, Klebsiella spp,
- Lobar pneumonia Enterobacter spp and yeast)
o shows bloody, rust-tinged sputum
STREPTOCOCCUS-LIKE ORGANISMS
o Results from the disturbance of the
normal defense barriers Aerococcus
o Predisposing factors: alcoholism,
anesthesia, malnutrition - Common airborne bacterium
- Meningitis - Resembles as viridans streptococci on culture
- Otitis media but similar to staphylococci microscopically
- Halophilic
SUMMARY OF BIOCHEMICAL TESTS OF - Species: A. viridans – bile esculin and PYR (+)
STREPTOCOCCI A. urinae – bile esculin and PYR (-)

Gemella
- Can be transmitted via sexual contact; infected
- Similar to colony morphology and habitat of
mother to newborn during birth (if baby
viridans group
passed through the birth canal)
- Easily decolorize on gram stain (may appear
- Leading cause of STI
gram negative cocci in pairs, tetrads, clusters
- Gram negative intracellular diplococci
or short chains
- On culture: small, tan, translucent and raised
- Species: G. haemolysans, G. morbiliorum, G.
after 24-48hrs of incubation
bergeriae and G. sanguinis
- Requires iron for growth
Lactococcus - Glucose fermenter
- Principal virulence factor: common pili
- Previously classified as group N. streptococci
- Physiologically similar to enterococci but do CLINICAL INFECTIONS
not produce acid from carbohydrates
- Gonorrhea
Leuconostoc o meaning “flow of seed” and “brothel”
o an acute pyogenic infection of non-
- They have irregular coccoid morphology ciliated columnar and transitional
- Share several phenotypic and biochemical epithelium with short incubation
characteristics with Lactobacillus, viridans period of 2 to 7 days
Streptococci, Pediococcus and Enterococcus o patients may be asymptomatic
- Species: L. citreum, L. cremoris, L. dextranicum, especially for females
L. lactis o symptoms: purulent discharge, lower
Pediococcus abdominal pain, dysuria and vaginal
bleeding
- Can be misidentified as viridans streptococci or
enterococci
- Grow at 45˚C
- Isolated from individuals who have GI
abnormalities or have undergone GI surgery
GRAM NEGATIVE COCCI
NEISSERIA
- Obligate aerobic
- Non-motile, non-hemolytic
- Purulent urethritis (males) and cervicitis
- Gram negative diplococci with coffee of kidney
(females)
bean shape
o if untreated may lead to sterility and
- On culture: small, gray-white opaque, convex
perihepatitis (fitz-hugh-curtis
and glistening colonies
syndrome)
- Capnophilic (2-8% CO2)
- Pharyngitis
- Carbohydrate fermenters
o chief complaint of symptomatic
- Oxidase (+)
oropharyngeal infections
- Catalase (+) except for N. elongata
- Anorectal infections (rectal gonorrhea)
- Natural habitats: mucous membrane and
- Conjunctivitis (opthalmia neonatorum)
urogenital tracts
o gonococcal eye infection passed to
Neisseria gonorrheae newborns during vaginal delivery
through an infected birth canal
- Never considered as normal flora
- Purulent arthritis
- Can be found in urogenital tract, anorectal
area, oropharynx or conjunctiva
Neisseria meningitidis
LABORATORY DIAGNOSIS - Aka meningococci

- Causative agent of epidemic meningococcal
- Gram stain
meningitis/meningococcemia/cerebrospinal
o appearance of intracellular gram-
fever/spotted fever
negative diplococci is diagnostic - > 5
- Intracellular/extracellular gram-negative
PMNs/field but no bacteria = non
diplococci encapsulated strains
gonococcal urethritis
- On culture: mucoid, bluish gray colonies (BAP);
o extracellular gram-negative diplococci
small, tan mucoid colonies (CAP)
= avirulent forms
- May be found commensal inhabitant of the
- Culture (confirmatory)
URT
o Thayer Martin Agar (TMA)
▪ CAP with enrichment CLINICAL INFECTIONS:
supplement and antibiotics
- Meningococcemia
(vancomycin, colistin and
o meningitis associated with
nystatin)
Meningococci in blood
o Modified Thayer Martin Agar (MTM)
o accompanied by appearance of
▪ TMA with additional
petechiae (rash)
trimethoprim lactate
o may lead to DIC
o Martin Lewis Medium (MLM)
- Waterhouse-friderichsen syndrome
▪ antibiotics: vancomycin,
o hemorrhage in adrenal glands
colistin, anisomycin,
- Conjunctivitis
trimethoprim lactate
- Pneumonia
o New York City Agar (NYC)
- Sinusitis
▪ antibiotics: vancomycin,
colistin, trimotheprim lactate
and ampothericin B
- Oxidase test
- Superoxol test
o reagent: 30% H2O2
o Differentiates N. gonorrheae from
other Neisseria spp
o (+) result: effervescence LABORATORY DIAGNOSIS
- Immunologic tests
o Fluorescent Antibody Test - Gram stain
▪ uses monoclonal antibodies - Culture
that recognize epitopes on the - Oxidase test
principal outer membrane
protein (Por) of N. gonorrheae - Immunologic tests
o Coaglutination o latex agglutination
▪ confirms biochemical o conterimmunoelectrophoresis
identification - CHO utilization test
o Molecular assays o ferments glucose and maltose
▪ detects gonococcal antigen or - Gamma-glutamyl aminopeptidase test
nucleic acid directly NON-PATHOLOGIC NEISSERIA
- CHO utilization test
o N. gonorrheae ferments glucose only N. flavescens
- a yellow pigmented neisseria species

- has ability to grow on BAP and CAP at 22˚C
DIFFERENTIAL TESTS FOR PATHOGENIC NEISSERIA
AND M. CATARRHALIS
N. lactamica
- commonly found in the nasopharynx of infants

and children
- β-galactosidase (+)
N. sicca
- on culture, it has a dry, wrinkled, adherent and

breadcrumbs ENTEROBACTERIACEAE
- like colonies - Gram negative, non-spore forming, facultative
N. elongata anaerobe bacilli
- Most are normal flora of GIT except
- “rod-shaped” gram negative cocci Salmonella, Shigella, Yersinia
- weakly positive or negative catalase test - Catalase (+) except Shigella dysenteriae
CARBOHYDRATE UTILIZATION TEST - Motile with peritrichous flagella except
Klebsiella, Shigella and Yersinia
- Non encapsulated except Klebsiella and
Enterobacter
- All members ferment glucose and reduce
nitrate to nitrite
Moraxella catarrhalis ANTIGEN DETERMINANTS
- Formerly Branhemella catarrhalis - Used for serological identifications

- Resembles as Neisseria o SOMATIC “O” ANTIGEN
- Normal flora of the URT ▪ heat stable
- Opportunistic pathogen ▪ located in the cell wall
- 3rd most common cause of otitis media and ▪ for E. coli and Shigella
sinusitis in children serotyping
- Fastidious, encapsulated and non-motile o FLAGELLAR “H” ANTIGEN
- Most isolates produce β-lactamase ▪ heat labile
- Small gram-negative cocci that tend to grow in ▪ found in the flagellum
pair with adjacent sides flattened ▪ for Salmonella serotyping
- On culture: smooth, opaque, gray to white o CAPSULAR “K” ANTIGEN
colonies with “hockey puck” appearance ▪ heat labile polysaccharide
▪ covers the O antigen
LABORATORY DIAGNOSIS ▪ found as K1 antigen of E. coli
and Vi antigen of S. enterica
- Gram stain
subsp. Enterica serotype Typhi
- Culture
o growth on BAP at 22˚C and NA at 35˚C Escherichia coli
- Oxidase test
- Catalase test - Part of the normal bowel flora of humans and
- DNAse test may also inhabit female genital tract
- Butyrate Esterase test - It is a primary marker of fecal contamination in
o (+) blue color – M. catarrhalis water purification
o specimen: eye or ear culture - Leading cause of nosocomial infection – UTI
- Exhibits green metallic sheen on EMB

- Virulence factors: endotoxin, common pili, K1 - IMViC: - - + +
antigen - Exhibits fish eye colonies on EMB
- TSI: a/a + -
- IMViC: + + - -
- E. coli strains:
o Enteropathogenic E. coli (EPEC)
o Enterotoxigenic E. coli (ETEC)
o Enteroinvasive E. coli (EIEC)
o Enterohemorrhagic E. coli (EHEC)
Cronobacter sakazakii
- Formerly known as Enterobacter sakazakii

- A pathogen of neonates causing meningitis
and bacteremia, often coming from powdered
infant formula
- Produces a yellow pigmentation that
intensifies at 25˚C
PANTOEA
KLEBSIELLA SPECIES Pantoea agglomerans
Klebsiella pneumoniae - Formerly known as Enterobacter agglomerans

- Causes nosocomial outbreak of septicemia due
- Common name: Friedlander’s bacillus to contaminated IV fluids
- Most common isolates of Klebsiella - It shows “triple decarboxylase negative”
- Causative agent of community acquired reaction
pneumonia (currant jelly-like sputum)
- Virulence factor: polysaccharide capsule SERRATIA
- String test (+) - Opportunistic pathogen
- Neufeld Quellang (+) - DNAse, lipase and gelatinase (+)
- TSI: a/a + - - ONPG (+)
- IMViC: - - + + (except for K. oxytoca: + - + +) - TSI: k/a + -
ENTEROBACTER - IMViC: - - + +
- Opportunistic infections: UTI, RT and wound Serratia odorifera

infections - Produces musty-pungent odor or “potato-like
- TSI: a/a + - odor”
Serratia marcescens ERWINIA
- Most clinically significant species - A plant pathogen and are not significant in
- Produces a pink to red pigment (prodigiosin) at human infections
25˚C
CITROBACTER
PROTEUS
Citrobacter freundii
- Isolated in urine, wound and ear infections
- Can be isolated in diarrheal stool cultures
- Rapid urease producer
- Associated with endocarditis in IV drug users
- Human pathogens: P. mirabilis and P. vulgaris
- IMViC: - + - +
- Exhibits “swarming phenomenon” and “burnt-
- TSI: k/a + +
gun powder odor”
- PAD test (+) Citrobacter koseri
- IMViC: P. mirabilis : - + - -; P. vulgaris: + + - -
- TSI: k/a + + - Formely known as C. diversus
- Causes nursery outbreaks of neonatal
meningitis and brain abscesses
- IMViC: + + - +
- TSI: k/a + -
SALMONELLA
- Most serious pathogenic enterobacteria for

humans, causing enteric fever (typhoid fever)
and acute gastroenteritis (food poisoning)
- Acquired by ingestion of contaminated animal
PROVIDENCIA
food products or improperly cooked poultry,
- One of the causes of nosocomial outbreaks milk, eggs and dairy products
involving burn units - May also be transmitted by human carriers
- Species: P. rettggeri (only urease +), P. stuartii, - Virulence factors: fimbriae and enterotoxin
P. alcalifaciens - LDC (+)
- PAD test (+) - IMViC: - + - + except S. typhi (- + - -)
- IMViC: + + - + - TSI: k/a + + except S. typhi (k/a - +)
- TSI: k/a - -
GENERAL CATEGORIES OF SALMONELLA INFECTION
MORGANELLA
- Gastroenteritis
- Same biochemical reaction with P. vulgaris o One of the most common forms of
except citrate (-) food poisoning
- Species: M. morganii o Salmonella strain: S. enterica subsp.
- PAD test (+) Enterica
- IMViC: + + - - o S. typhimurium: associated with
- TSI: k/a + - peanut butter outbreak
o Infective dose: 106 bacteria
EDWARDSIELLA o symptoms: nausea, vomiting, fever
- It has been isolated from cold-blooded and and chills, watery diarrhea and
warm-blooded animals abdominal pain
- Species: E. tarda (human pathogen) - Bacteremia
- LDC (+) o occurs with or without extraintestinal
- IMViC: + + - - foci of infection caused by
- TSI: k/a + + nontyphoidal Salmonella (S.

typhimurium, S. paratyphi, S. o Complications: ileus (obstruction of
cholerasuis) the intestine), seizures and HUS
o characterized by prolonged fever
- Enteric fever
o aka typhoid fever
o causative agent: S. typhi
o febrile disease that results from the
ingestion of contaminated food from
infected individuals or carriers YERSINIA
o symptoms: malaise, anorexia,
lethargy, myalgia, and continuous Yersinia pestis
frontal dull headache - Aka plague bacillus
o characteristic “red spots” appears - Considered class A bioterrorism agent
during the 2nd week of fever - Only member of Enterobacteriaceae that is
o complications: necrosis in the gall transmitted through bite of an infected flea
bladder (necrotizing cholecystitis) and (Xenopsylla cheopsis)
Peyer’s patches - Non-motile
SPECIMENS FOR SALMONELLA IDENTIFICATION - Causative agent of bubonic plague - black
death or 6th century pandemic
- blood- 1st week of infection - Inclusions: bipolar bodies
- Stool- 2nd week of infection - Appears closed safety pin appearance after
- Urine- 3rd week of infection wayson’s stain or methylene blue stain
- Exhibits “stalactite pattern” in broth cultures
SHIGELLA
- TSI: k/a - -
- Most inert Enterobacteriaceae
- Not a member of the normal GIT flora
- An intracellular organism – they multiply
within the cells of the colon epithelium
- Transmitted through fecal-oral routes
- No animal reservoir
- Virulence factor: shiga-toxin
- Antigenic structure: O antigen
Yersinia enterocolitica
- Specimen: rectal swab, stool
- Non-motile - Most commonly isolated species of Yersinia
- TSI: k/a - - - Causative agent of enterocolitis (waterborne
- IMViC: - + - - gastroenteritis)
- Motile at 22˚C but non-motile at 35˚C
DISEASE
- Has the ability to survive in cold environment
- Bacillary dysentery - Exhibits bull’s eye colonies in CIN agar
o Caused by S. dysenteriae type 1 - Ferments sucrose and mannitol
o Characterized by acute inflammatory - Grows best at 25-30˚C
colitis and bloody diarrhea - IMViC: - + - -
o Infective dose: <200bacilli (highly - TSI: k/a - -
communicable)
o In young children, rectal prolapse
occurs due to excessive straining
o Symptoms: fever, chills, abdominal
cramps, painful bowel movement and
tenesmus
LABORATORY DIAGNOSIS
- Culture
o EMB (Eosin-Methylene Blue Agar)
▪ CHO: lactose
o MAC (MacConkey Agar)
▪ contains crystal violet and bile
salts
▪ CHO: lactose
- TSI (Triple Sugar Iron)
▪ pH indicator: neutral red
o sugar ratio: 10:10:1
o HEA (Hektoen-Enteric Agar)
(lactose:sucrose:glucose)
▪ inhibitor: bile salts
o pH indicator: phenol red
▪ CHO: lactose, sucrose and
o H2S indicator: ferrous sulfate and
salicin
sodium thiosulfate
▪ pH indicator: bromthymol
blue
▪ H2S indicator: ferric
ammonium citrate
o SSA (Salmonella-Shigella Agar)
▪ CHO: lactose
▪ pH indicator: neutral red
▪ H2S indicator: ferric citrate
▪ Salmonella: non-LF, H2S (+)
colorless colonies with black
center
▪ Shigella: non-LF, H2S (-)
colorless colonies without
black center
- LIA (Lysine Iron Agar)
o detects lysine deamination and
decarboxylation
o pH indicator: bromcresol purple
o H2S indicator: ferric ammonium
citrate - SIM (Sulfide-Indole-Motility)
• alkaline slant/alkaline butt (K/K) o (+) sulfide: black color formation
(-) lysine deamination o (+) indole: pink to wine colored ring
(+) lysine decarboxylation after the addition of kovac’s reagent
• alkaline slant/ acid butt (K/A) o (+) motility: spread out/movement
(-) lysine deamination away from the stab line
(-) lysine decarboxylation - Methyl Red
o organisms that produce enough acid
• red slant/acid butt (R/A)
form glucose fermentation will
(+) lysine deamination
overcome the neutralizing effect of
(-) lysine decarboxylation
the buffer
o mixed acid production: lactic acid,
acetic acid, formic acid, succinic acid
- Voges Proskauer
o detects acetoin
o reagent: 40% KOH and α-naphthol
o positive result: red complex
- Citrate Utilization test
o determines if an organism can utilize
citrate as sole source of carbon - Urea Hydrolysis Test (Christensen’s Method)
o pH indicator: bromthymol blue o used to determine the ability of an
o (+) result: intense blue organism to produce the enzyme
urease, which hydrolyzes urea
o (+) result: change in the color of slant
from orange to magenta
o (+) organism: Proteus and Morganella
- Malonate utilization test

o determines if an organism is capable
of utilizing malonate as sole source of
carbon
o (+) result: blue color
- Orthonitrophenyl β-galactosidase (ONPG)
o determines whether an organism is a
slow or late lactose fermenter (LLF) or
true non-lactose fermenter (NLF)
o (+) result: yellow color
- Gelatin liquefaction
- Phenylalanine Deaminase (PAD)
o determines the ability of a baterium
o used to determine the ability of an proteases that hydrolyze gelatin and
organism to oxidatively deaminate
liquefy solid gelatin medium
phenylalanine to phenyl pyruvic acid
o (+) organism: Serratia
o (+) result: green colored complex

NONENTERIC GASTROINTESTINAL PATHOGEN - Acquired through ingestion of raw oysters and
fish (tilapia)
VIBRIO SPECIES
Vibrio alginolyticus
- Comma or curved bacillus
- Facultative anaerobe; monotrichous flagella - The least pathogenic Vibrio for humans
- Found in brackish or estuarine water and - A strict halophile (1-10% NaCl)
marine water or salt water - Can be an occupational hazard (fishermen and
- Acquired through consumption of raw or sailors)
undercooked seafoods - Infections: eye, ear and wound infections
- Halophilic except V. cholerae and V. mimicus
DIAGNOSIS
- Non-lactose fermenters except V. vulnificus
- Gram stain
Vibrio cholerae
o gram negative, straight or slightly
- Causative agent of cholera curved rods “curved or comma-
- Has a rapid darting or shooting-star motility shaped bacilli”
- Culture: smooth, medium to large colonies - Culture
with a greenish hue (BAP) o Media: TCBS and APW
- Virulence factor: cholarogen (cholera toxin) o Sucrose fermenters (yellow colonies):
- String test positive V. cholerae and V. alginolyticus
- Subgroups: Nonsucrose fermenters (green
o V. cholerae O1 colonies): V. mimicus, V. vulnificus and
o V. cholerae O139 V. parahemolyticus
o V. cholerae non-O1
- String test
o differentiates Vibrio species from
Aeromonas species
- CHOLERA
o Rgt: 0.5% Na desoxycholate
o An acute diarrheal disease that is
(+) result: lysis of cells (vibrio)
spread mainly through contaminated
= releases DNA, which can
water
then be pulled up into a string
o Acquired through ingesting
using an inoculating loop
improperly preserved food like
seafoods, AEROMONAS
o milk and ice cream
o Hallmark: rice watery stool - Found in fresh water, estuarine and
chlorinated water, isolated from meat
Vibrio vulnificus products
- Not part of the human flora
- Commonly referred to as the “lactose-
- Facultative anaerobe
positive” vibrio
- Responsible for a variety of diseases among
- Second to V. cholerae in terms of producing
warm- and cold-blooded animals (“red leg”
serious type of Vibrio-associated infection
disease in frogs)
- Causes primary septicemia and wound
infections
- In humans, it causes a nebulous syndrome - Secretes toxin antigenically similar to cholera
known as the “traveler’s diarrhea” similar to toxin
ETEC - Slow growing, fastidious and asaccharolytic;
- Culture: darting motility
o CIN – “bull’s eye colonies” apron-like - Optimum growth: 42ºC
pattern - Microscopy: curved or seagull-winged shaped
o BAP – large, round, raised, white and
DIAGNOSIS
opaque colonies
o MAC – lactose fermenters - Microscopy
- Biochemical tests: o seagull-wing shaped
o Oxidase positive - Culture
o Catalase positive o Media: CAMPY-BAP, Skirrow’s media
o Glucose fermenters and charcoal cefoperazone
o Motile with single polar flagellum desoxycholate agar (CCDA)
o Can grow at 4ºC to 42ºC
CAMPYLOBACTER SPECIES
- Motile by a single polar flagellum; non-spore HELICOBACTER SPECIES

forming
- Grown in 5-10% oxygen(microaerophilic) - Found in the GIT of mammals and birds
- Oxidase positive - Motile with bipolar flagella
- Most recognized antecedent cause of Guillain- - Microaerophilic
Barre syndrome - Most species have strong urease activity
- Also an animal pathogen (cattle and swine) - Microscopy: gram negative spiral-shaped
causing sterility and abortion organisms (S-shaped) rods resembling
- Microscopy: faintly staining gram-negative, campylobacter
small, curved or S-shaped rod - Culture: gray with translucent colonies
- Culture: gray, flat, glistening, irregular with a - Oxidase and catalase (+)
“tailing effect along the streak line” colonial Helicobacter pylori
growth
- Primary habitat: human gastric mucosa
Campylobacter jejuni (antrum and fundus)
- Most common cause of bacterial - motility allows this organism to escape acidity
gastroenteritis worldwide of the stomach
- Acquired through eating contaminated - Urease (+)
chicken and turkey DISEASES
- Invades epithelium of the small intestine
causing inflammation - Peptic ulcer
- Type B gastritis
- Gastric carcinoma
DIAGNOSIS - Culture:
o BAP: Dome-shaped, gray to whit
- Gram’s stain
colonies with double zone of
- Culture
hemolysis
o may require more than 5 days
o Litmus Milk Medium: “stormy
incubations
fermentation of milk”
o the presence of hemin may stimulate
- Virulence factors: α-toxin and enterotoxin
growth
- Microscopic examination:
o Media: CAP, MTM, Skirrow’s agar
o Boxcar-shaped bacilli
o Transport media: Stuart’s media
o Spores are seldom seen, but it is oval,
- Other tests:
central to subterminal
o Urea breath test and PCR
- Biochemical tests:
GRAM POSITIVE BACILLI o Lecithinase positive
o Nagler test positive
ANAEROBIC o Reverse CAMP test positive
- Clostridium
AEROBIC
- Bacillus
- Corynebacterium
- Listeria
- Erysipelothrix
- Lactobacillus
- Mycobacterium CLINICAL INFECTIONS
CLOSTRIDIUM SPECIES - Gas gangrene/myonecrosis (“eating sore”)

o a life-threatening destruction of
- Obligate anaerobic, gram-positive, spore- muscle and other tissues; necrotizing
forming rod infection of skeletal muscle
- Its toxin usually gain access to the body o caused by α-toxin (lecithinase
through ingestion or via wounds that have enzyme/phospholipase C)
been contaminated with soil o accompanied by bullae, pain, swelling,
- Motile with peritrichous flagella Serous, discharge, discoloration and
- Non-encapsulated tissuenecrosis
- Carbohydrate fermenters
- Common species:
o C. perfringens
o C. botulinum
o C. tetani
o C. difficile
Clostridium perfringens
- Formerly known as Clostridium welchii

- Most commonly isolated member of Clostridia
in blood cultures - Food poisoning/enteritis necrotans (“pig-bel”)
- Causes outbreak after ingestion of o characterized by ingestion of
contaminated milk and gravy enterotoxin in contaminated food
- Produces deoxyribonuclease o improperly stored food allows
germination of the spores and growth
of vegetative bacteria
o s/s: diarrhea (foul-smelling stool) and - TETANUS (devil’s grin)
crampy abdominal feeling o characterized by risus sardonicus or
o 2 types trismus (lock jaw/distorted grin)
▪ Type A food poisoning o occurs when the organism enters an
▪ Type C food poisoning open wound and elaborates the
potent toxin that mediates
Clostridium tetani
generalized muscle spasms
- Aka tack head bacillus o s/s: muscular rigidity (jaw, neck and
- A soil and environmental inhabitants lumbar region), difficulty in
- Endospores are found in hospital swallowing, rigidity of the abdomen,
environments, in soil and dust, and in the feces chest, back and limbs
of many farm animals o Incubation period: 3-21 days
- Culture:
o Heavy “smooth swarming”
anaerobically but slow growing
o BAP: colonies are with matte surface
with narrow zone of β-hemolysis
- Microscopic Examination:
o Spore is located terminally and
swollen – “drumstick/lollipop/tennis
racket” appearance Clostridium botulinum
- Aka canned good bacillus

- Characterized by presence of oval and
subterminal spore; β-hemolysis on BAP
- Virulence factor: botulism toxin (neurotoxin)
- most potent toxin known to man
- selectively cleaves the synaptic vesicle
membrane protein synaptobrevin, thus
preventing exocytosis and release of
neurotransmitter acetylcholine
- Virulence factor: tetanospasmin (neurotoxin) - BOTULISM (flaccid paralysis)
o an endopeptidase that selectively o double or blurred vision, impaired
cleaves the synaptic vesicle membrane speech, difficulty in swallowing,
protein synaptobrevin weakness and paralysis
o causes tension or cramping and ▪ Foodborne botulism - results
twisting in skeletal muscle from ingestion preformed
surrounding the wound and tightness toxin in nonacidic vegetables,
of the jaw muscles preserved food, meat-based
- Biochemical tests: food or mushroom foodstuffs
o Motile ▪ Infant botulism - an actual
o Gelatinase and indole positive infection caused by ingesting
o Lipase negative the organism from honey or
via breast feeding

- Culture:
o BAP - Colonies are flat, non-hemolytic
Clostridium difficile
irregular with swirling projections that
- Normal flora of GI of about 5% of individuals resembles “medusa head” or “beaten
- Present occasionally on the hands of hospital egg white”
personnel o EYA – inverted fir tree appearance
- Culture: - Microscopic examination:
o CCFA – yellow, ground glass colonies o Gram positive large, encapsulated and
o BAP – γ-hemolytic that fluoresce square ended rod with unstained
chartreuse under long wave UV light central spore – “bamboo fishing rod”
with “horse stable” or “barnyard” odor appearance
- Produces glutamate dehydrogenase
Bacillus anthracis
- Virulence factors: toxin A (enterotoxin) and
toxin B (cytotoxin) - Virulent Factors:
- Clinical Infections: o D-glutamic acid capsule
o Antibiotic-associated diarrhea o protects the organism from
o Pseudomembranous colitis phagocytosis
- Protein enterotoxins (EF, PA, and LF)
BACILLUS SPECIES
o PA and EF: results to edema
- Gram positive rod shaped, spore-forming o PA and LF: results to death
aerobically
CLINICAL INFECTIONS
- Motile with peritrichous flagella except B.
anthracis - ANTHRAX
- Can survive in extreme environmental o disease affecting primarily farm
conditions animals
- Catalase positive o 3 types:
- Glucose fermenters ▪ Cutaneous anthrax
- Can hydrolyze starch • acquired thru skin
- Common species: cuts and abrasions
o Bacillus anthracis • characterized by
o Bacillus cereus “black eschar”
o Bacillus subtilis ▪ Pulmonary anthrax or
o Bacillus thuringiensis Woolsorter’s disease
• acquired when spores
Bacillus anthracis
are inhaled into the
- Not part of normal human flora pulmonary
- Its endospores can remain viable in soil and parenchyma
animal products for decades • resembles as upper
- Non motile respiratory tract
- Halophilic (7% NaCl) infection
- Can grow in low pH (<6) • may progress into
- Growth factor: thiamine (B1) chest edema,
- Lecithinase positive cyanosis, coma and
- Can ferment glucose death
- Susceptible to penicillin ▪ Gastrointestinal anthrax
- Virulence factors: D-glutamic acid capsule and • spores are inoculated
protein exotoxins (edema factor, protective into a lesion on the
antigen and lethal factor) intestinal mucosa
following ingestion
Bacillus subtilis
- Aka hay bacillus

• s/s: abdominal pain,
- A common laboratory contaminant
nausea, vomiting,
- Halophilic organism (7% NaCl)
bloody diarrhea
- May cause eye infection among heroin addict
- Ferments mannitol, xylose, and arabinose
- Culture:
o Large, flat, dull with ground glass
appearance; may be β-hemolytic; may
be pigmented (pink, yellow, orange or
brown)
CORYNEBACTERIUM SPECIES
- Majority are found as normal skin flora and

Bacillus cereus mucous membranes of humans and animals
- Non-motile, non-spore forming, non-
- Aka fried rice bacillus encapsulated; pleomorphic rods
- Can be part of the normal fecal biota - Catalase and oxidase positive
- Motile and resistant to penicillin - Can ferment glucose and maltose except: C.
- Ferments salicin urealyticum and pseudodiththeriticum
- Lecithinase positive - Common species:
- Culture: o C. diphtheriae
o BAP – large, feathery, spreading, o C. jeikeium
frosted-glass o C. urealyticum
o β-hemolytic colonies o C. pseudodiphtheriticum
- Virulence factors: enterotoxins, cerelysin,
phospholipase C and pyogenic toxin Corynebacterium diphtheriae
CLINICAL INFECTIONS - Aka diphtheria bacillus/kleb loeffler’s bacillus

- Aerobic or facultative anaerobe; pleomorphic
- Diarrheal type rods
o associated with ingestion of - Inhabits human nasopharynx and considered
contaminated meat or poultry not part of human flora
products, and vegetables - Acquired through contaminated respiratory
o incubation period: 8-16 hours (long droplets or direct contact with infected
incubation) cutaneous lesions (hand-to-mouth)
o s/s: abdominal pain, watery diarrhea - Enriched medium is recommended for growth
without fever - Catalase positive
o due to heat-labile enterotoxin - Can reduce nitrate to nitrite
- Emetic type - Non motile
o associated with ingestion of - Virulence factor: diphtheria toxin (130 ng/kg
improperly stored fried rice/reheated BW – lethal dose)
rice
o incubation period: 1-6 hours (short CLINICAL INFECTIONS
incubation) - DIPHTHERIA
o s/s: abdominal cramps and profuse o an acute, contagious disease
vomiting characterized by the production of a
o due to heat stable enterotoxin systemic toxin and a false membrane
lining of the mucous membrane of the

throat leading to respiratory
obstruction
o due to diphtheria toxin
o 2 forms
▪ Respiratory form
acquired thru droplet
infection or hand-to-mouth
contact
▪ Cutaneous form (Velat
sore/Barcoo rot)
toxin is absorbed
systematically
- Gram stain
o highly pleomorphic gram positive
short or slightly curved rods with - Toxigenicity Test
rounded ends o Animal inoculation (in vivo) test
o cells are arranged in palisades or in ▪ uses guinea pigs
pairs forming X, V, Y and L formations o Elek’s test (in vitro test)
resembling “chinese letters” ▪ uses immunodiffusion
- Culture principle
o BAP, CTBA, Tinsdale Agar and Loeffler ▪ (+) result: 4-5 mm fine
Serum Agar precipitin lines at a 45º angle
o to differentiate 3 biotypes of C. to the streaks
diphtheriae
Corynebacterium jeikeium
- A common skin flora of hospitalized individuals

- A lipophilic species
- Common cause of diphtheroid prosthetic valve
endocarditis in adults
- Obligate aerobe
- Urease positive
- Schick’s test - Reduces nitrate to nitrite
o susceptibility test and hypersensitivity - Culture:
test o Non-hemolytic; appear as large
o intradermal injection of toxoid colonies when supplemented with 1%
o Positive reaction: - result: check for Tween 80
erythema after 6 days

Corynebacterium pseudodiphtheriticum ▪ Disease in pregnant women
• causes spontaneous
- Aka hoffman’s bacillus
abortion and stillbirth
- A normal flora of human nasopharynx
• s/s: flu-like illness;
- Causes UTI and cutaneous wound infections in
fever; headache;
immunocompromised patients
myalgia
- Urease positive
▪ Disease in newborn
- Nitrate positive
(granulomatous
Corynebacterium urealyticum infantiseptica)
• associated with
- One of the most frequently isolated clinically
intrauterine infection
significant corynebacterial
due to aspiration of
- A urinary pathogen
infected amniotic
- Strict aerobe
fluid
- Lipophilic
• leads to meningitis
- Shows resistance to several β-lactam
▪ Disease in
antibiotics
immunocompromised host
- Catalase positive
- Urease positive
• through ingestion of
- Culture
contaminated cheese,
o Non-hemolytic pinpoint, white,
coleslaw, ice cream,
smooth colonies
hot dogs and
Listeria monocytogenes processed meat
- Both human and animal pathogen; also LABORATORY DIAGNOSIS

isolated from crustaceans, ticks and flies
- Motility test
- Motile with characteristic “tumbling motility”
o Hanging drop/wet mount
- aerobic or facultative anaerobic, non-spore
▪ tumbling motility at room
forming
temperature
- Catalase positive
o Semi solid media (SIM)
- Has the ability to survive within phagocytes
▪ umbrella-shaped or inverted
- Optimal temperature: 30-37ºC
christmas tree pattern at
- Can grow at 4ºC
room temperature but not at
- Culture:
37ºC
o BAP – small, smooth, translucent
- Culture
grayish-blue colonies surrounded by
o enhanced growth on Mc Bride
narrow zone of β-hemolysis
medium
CLINICAL INFECTIONS o cold enrichment technique is used to
enhance recovery from clinical
- LISTERIOSIS
specimens
o serious infection primarily of
- Biochemical test
neonates, pregnant women and
o Glucose fermenter
immunocompromised hosts
o Catalase and motility test positive

o Hippurate hydrolysis and Bile Esculin SMALL PLEOMORPHIC GRAM-NEGATIVE BACILLI
hydrolysis positive
HAEMOPHILUS spp.
o Halophilic (6.5%)
o Voges proskauer and Methyl red - Derived from the Greek words meaning “blood
positive lover”
- Normally inhabit URT of humans H. ducreyi
Erysipelothrix rhusiopathae
- Obligate parasites on the mucous membranes
- The only species in the genus that cause - Fastidious and nonmotile; non-spore forming;
human disease; not part of the human flora capnophilic and facultatively anaerobic
- Transmitted to humans from animals by - Catalase and oxidase positive
contact with infected animal excreta, blood, - Culture: BAP - Moist, smooth, convex colonies
and flesh
Haemophilus influenzae
- Aerobic or facultatively anaerobic; non-spore
forming, non-motile; non-hemolytic - Transmitted by person to person by
- Microscopic examination: contaminated respiratory droplets
o Thin, pleomorphic rod; has tendency - Main cause of meningitis in children
to form long filaments arranged in - The only member of the genus that produce
singly, short chains or in V shape IgA protease
- Some strains are encapsulated (serotypes a-f)
CLINICAL INFECTIONS
- Nonencapsulated strains are part of the
- Septicemia normal flora of URT
- Endocarditis - Requires both factor X and V
- Erysipeloid - Culture: Translucent, convex, tan mucoid
o a localized skin infection that colonies with “mousy or bleach-like odor”
resembles streptococcal erysipelas
Haemophilus ducreyi
o lesions are seen on the hands or
fingers through inoculation of - Is not part of human flora
organism - Commonly seen in socioeconomically
o s/s: painful, swollen, slightly elevated, disadvantaged population
spreading purplish-red zone - Causative agent of “chancroid or soft chancre”
- Requires only factor X
- Microscopy: short bacilli – school of fish
- Culture arrangement
o Gelatin stab: appear like “test tube - Culture:
brush”/”bottle-brush” like growth o CAP - Transparent, small, flat, smooth,
- Biochemical tests nonmucoid, tan or yellow colonies
o (-): catalase; oxidase; esculin o Saline suspension – difficult to pick up;
hydrolysis; nitrate reduction; VP and clumpy appearance
urease
o (+): H2S production in TSI medium;
Lactobacillus acidophilus
- Normal flora of the mouth, gastrointestinal

tract and vaginal canal
- Nonpathogenic and has little clinical
significance
- Cultured on: tomato juice agar

Haemophilus aegypticus - Culture
o must contain X and V factor
- Aka Koch-Weeks bacillus
o Media: CAP, thioglycollate, BHI
- Genetically related to H. influenzae
- Porphyrin Test (delta-aminolevulinic acid Test)
- Observed in conjunctivitis exudates from
o test differentiating the heme-
Egyptians by Koch in 1883
producing species of Haemophilus
- Causative agent of “pinkeye conjunctivitis”
o Principle: based on the ability of the
enzyme to convert the substrate delta-
aminolevulinic acid (D-ALA) into
porphyrins or porphobilinogen,
intermediates in the synthesis of X
factor
- Specimens: CSF, sputum, genital lesion/ulcer,

joint fluid, vaginal swab, abscess drainage,
swabs from conjunctiva, bronchial wash and
blood Cardiobacterium hominis
o Consideration:
▪ CSF samples should be - Infects the aortic valve
centrifuged - Shows “false gram positive” reactions in parts
▪ Immediate transport and of the cells
processing are vital for - Microscopy:
isolation o BAP - “rosette” formation, swellings
▪ Ulcer should be cleaned with and filamentous
sterile gauze premoistened o Yeast extract – “sticklike structures”
with sterile saline Eikenella corrodens
▪ Cotton swabs premoistened
with phosphate-buffered - The corroding bacilli
saline - Causes mixed infection from bites or clenched
- Gram stain: fist wounds
o small, gram negative, pale pink - Assacharolytic organisms
coccobacilli to long filament arranged - Lysine decarboxylase positive
singly or in groups - Culture:
H. Ducreyi – “school of fish” o Yellow colonies; characteristic “pitting
appearance or corroding” the agar with sharp odor
H. Influenzae – presence of halos of bleach
due to capsule Kingella kingae
- Most pathogenic species of the genus

- May grow on gonococcal media (TMA) and
may ressemble N. gonorrheae
- Have the tendency to resist decolorization
- Microscopy:
o Gram negative plump rods to
coccobacilli with squared ends in pairs
or short chains
BRUCELLA SPECIES BORDETELLA SPECIES
- Aka Bang’s bacillus - Obligate aerobes, gram negative coccobacilli,

- Important human and animal pathogens – non-carbohydrate fermenters
infect human through contact with infected - Non motile except B. bronchiseptica
animals and animal products (milk) - Contains bipolar metachromatic granules
- A category B select biological agent - Replicates on ciliated respiratory epithelial
- Strict aerobes, intracellular parasites, class III cells of humans
pathogens - Catalase (+)
- Localize in tissues rich in erythritol - Growth factors: nicotinic acid, cystein and
- Nonmotile, nonencapsulated; some requires methionine
CO2 for growth - Virulence factors: pertusis toxin, adenylate
- Catalase and oxidase (+) cyclase, 10tracheal cytotoxin, and
- Rapid urease producers dermonecrotic toxin, pertactin and fimbriae
- Assacharolytic
NONFERMENTATIVE GRAM-NEGATIVE BACILLI
- Microscopy: Small, coccobacilli that appears
singly, in pairs or short chains with - Motile except Burkholderia mallei
“sandpaper” appearance - Oxidase positive
- Culture: small, convex, smooth, translucent, - Non-lactose fermenter
nonhemolytic, slightly yellow colonies that - Do not ferment carbohydrate by enzymatic
may become brownish with age reaction but oxidative method – produce very
weak acids as end products
DISEASE
- OF medium result: yellow (+) in the open tube;
- Brucellosis blue-green (-) in the closed tube
- Undulant fever - TSI reaction: K/K - -
- Malta fever
PSEUDOMONAS SPECIES
ROUTES OF INFECTION
- Obligate aerobes, non-spore forming gram
- Ingestion of unpasteurized and contaminated negative bacilli
milk or cheese from infected animals - Motile with polar flagella
- Inhalation of organisms - Most commonly isolated non-fermentative
- Penetration of ocular or oral mucosa bacilli
- Direct inoculation into the blood stream - Metabolism is respiratory and never
through abrasion in the skin or vaccination fermentative
- Can be found in cosmetics, swimming pools,
hot tubs, and inner soles
- Gram stain - Microscopic: straight and slender gram-
o carbol fuchsin should be substituted negative rod
for safranin to improve staining
Pseudomonas aeruginosa
- Culture
o Media: BAP, Trypticase Soy Agar, - Agent of “blue pus”
castaneda medium - Non-spore forming, monotrichous organism
- Most commonly isolated species of the genus
in clinical specimens
- Acquired through ingestion of contaminated
food or water, exposure to contaminated
medical devices and penetration of wounds

- Culture: flat spreading pigmented colonies; β- ACINETOBACTER
hemolytic with fruity “grape-like” or “corn-
- Obligate aerobic and nonmotile
tortilla” odor
- 2nd frequently isolated non-fermenters
- Virulence factors: endotoxin, pili, exotoxin A,
- Has been isolated from hospital equipment,
capsule, lecithinase, elastase and protease
foodstuff, soil and water
- Clinical Significance:
- Microscopy: plump, gram-negative
o Leading cause of nosocomial
coccobacilli; appear in pairs
respiratory tract infections
- Culture:
o 3rd most common cause of gram-
o MAC – purplish colonies
negative bacillary bacteremia
o BAP – gummy colonies
o 3rd most common cause of nosocomial
infections
o Catalase (+)
o Pseudomonas aeruginosa
o Oxidase (-)
DISEASES o Optimum temperature: 30-35ºC
o Optimum pH: 5.5-6.0
- Ecthyma gangrenosum
- Swimmer’s ear, Stenotrophomonas maltophila
- Hot tub or jacuzzi syndrome (necrotizing skin
- 3rd most commonly isolated non-fermentative
rash)
gram-negative bacilli
- Infections of the nail beds
- Has been isolated from plant materials, water,
- Lung infections in cystic fibrosis patients
milk, frozen food and sewage
DIAGNOSIS - Can contaminate blood-drawn equipment and
disinfectants
- (+) gluconate production
- Associated with “pseudoinfections”
- (+) arginine dihydrolase (ADH)
- Microscopy: short to medium size, gram
- (+) acetamide and citrate utilization
negative straight rods
- Can grow at 42ºC
- Culture:
o BAP – lavender green to light purple
pigment with “ammonia smell”
o BHI agar with tyrosine – brown
pigment
o Catalase positive
o Esculin positive
o Gelatin hydrolysis positive
o DNAse and LDC positive
o Oxidase negative
Pseudomonas fluorescens and Pseudomonas putida
BURKHOLDERIA spp.
- Isolated from contaminated blood products,
- Not considered part of normal human flora
cosmetics, hospital equipment, urine and
- Generally non-pathogenic
respiratory specimens
- Involves human contact with heavily
- Linked to transfusion-associated septicemia
contaminated medical devices
- Can grow at 4ºC
- Microscopic: medium sized gram-negative
- Can produce acid from xylose
straight rods
- Gelatin hydrolysis:
- Grows well on BAP and CAP
P. fluorescence (+)
- Biochemical tests: catalase and oxidase (+)
P. putida (-)

Burkholderia cepacia Reviewer Notes:
- A low-grade nosocomial pathogen

- Has been isolated from irrigation fluids,
anesthetics, nebulizers, detergents and
disinfectants
- Culture: Grows well on most culture media but
lose viability on BAP in 3-4 days
o BAP – non-fluorescing and
nonwrinkled yellow colonies
- Biochemical test: weak, slow, positive oxidase
reaction
o (+) LDC and ONPG
Burkholderia mallei
- Agent of Glander’s or Farcy disease

- A potential bioterrorism
- Oxidase production is variable
- Has variable growth in MAC
Burkholderia pseudomallei
- Agent of melioidosis (vietnamese time bomb

disease)
- Isolated in muddy soil, streams and surface
water such as rice paddies
- Can survive within phagocytes
- Culture: dry, wrinkled, deep pink colonies with
“earthy odor”

Bacteriology

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Medical Technology Licensure Examination (REVIEWER)

MICROBIOLOGY & PARASITOLOGY JOHN NEEDHAM (1748)

MICROBIOLOGY - He observed that boiled mutton broth

ROBERT KOCH (1843-1910) - Discovered salvarsan (606th drug) for

2|Page PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT

CATEGORIES OF POTENTIAL INFECTIOUS AGENTS OF

- a devise that encloses a working area in such a

o Class III cabinets

STERILIZATION AND DISINFECTION

- Chemical destruction of vegetative pathogens STERILIZATION

- Chemical agents that inhibit the growth of STERILIZATION BY HEAT

ANTISEPTICS MOIST HEAT

- Used on skin and mucous membranes to kill - Moist heat

6|Page PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT

CULTURE AND CULTURE MEDIA

8|Page PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT

PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT 9|Page

CYTOSOL STAINING TECHNIQUES

- contains thousands of enzymes 1. SIMPLE STAINING

EXCEPTIONS IN GRAM STAINING NEGATIVE STAINING

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LOG PHASE/EXPONENTIAL PHASE

- period wherein microorganisms are actively TESTS TO DIFFERENTIATE STAPHYLOCOCCUS FROM

STATIONARY PHASE/PLATEAU PHASE

- period wherein there is balance between cell

DEATH PHASE/DECLINE PHASE

- period wherein there is cessation of bacterial

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- Smith and Brown Classification

PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT 17 | P a g e

- Hippurate Hydrolysis Test

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TEST TO DIFFERENTIATE S. PNEUMPNIAE FROM

PREPARED AND COMPILED BY: ANGELO DEL ROSARIO, RMT 19 | P a g e

GRAM NEGATIVE COCCI

LABORATORY DIAGNOSIS - Aka meningococci

- a yellow pigmented neisseria species

- commonly found in the nasopharynx of infants

- on culture, it has a dry, wrinkled, adherent and

Moraxella catarrhalis ANTIGEN DETERMINANTS

- Formerly Branhemella catarrhalis - Used for serological identifications

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- Formerly known as Enterobacter sakazakii

KLEBSIELLA SPECIES Pantoea agglomerans

Klebsiella pneumoniae - Formerly known as Enterobacter agglomerans

- Opportunistic infections: UTI, RT and wound Serratia odorifera

- Most serious pathogenic enterobacteria for

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- Malonate utilization test

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- Motile by a single polar flagellum; non-spore HELICOBACTER SPECIES

CLOSTRIDIUM SPECIES - Gas gangrene/myonecrosis (“eating sore”)

- Formerly known as Clostridium welchii

- Aka canned good bacillus

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- Aka hay bacillus

- Majority are found as normal skin flora and

CLINICAL INFECTIONS - Aka diphtheria bacillus/kleb loeffler’s bacillus

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- A common skin flora of hospitalized individuals

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- Both human and animal pathogen; also LABORATORY DIAGNOSIS

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