Introduction to BACTERIOLOGY

I. HISTORY
1. LUCRETIUS and GIROLAMO FRACASTORO (1478-1553)
• Suggested that diseases were caused by “invisible living creatures”
2. ANTON VAN LEEUWENHOEK (1632-1723)
• “first true microbiologist”
• “Father of Bacteriology and Protozoology”
• used the term “animalcules” or “beasties”
3. JOHN NEEDHAM (1731-1781)
• Boiled mutton broth eventually became cloudy after pouring it into a flask that was then sealed
tightly
• Organic matter possessed a “vital force” that could give rise to life
4. LAZZARO SPALLANZANI (1729-1799)
• improved the previous experiments of Needham by heating the broth placed in a sealed jar
and observe no growth took place
• Concluded that microorganisms from the air probably had entered Needham’s solutions
5. FRANCESCO REDI (1626-1697)
• Invalidated the long-held belief that life forms could arise from non-living things (abiogenesis)
• Maggots could not rise spontaneously from decaying meat
6. RUDOLF VIRCHOW (1821-1902)
• Challenged the doctrine of spontaneous generation with the concept of biogenesis
7. LOUIS PASTEUR (1822-1895)
• Disproved the doctrine of spontaneous generation
• Improved the wine-making process (fermentation and pasteurization)
• Developed vaccines for anthrax and rabies
8. ROBERT KOCH (1843-1910)
• First to show irrefutable proof that bacteria indeed cause diseases
• Discovered Bacillus anthracis and Mycobacterium tuberculosis
• developed Culture Media for observing growth of bacteria isolated from human body
• Koch’s Postulates:
- The microorganism must be found in abundance in all organisms suffering from the
disease, but should not be found in healthy organisms.
- The microorganism must be isolated from a diseased organism and grown in pure culture.
- The cultured microorganism should cause disease when introduced into a healthy
organism.
- The microorganism must be reisolated from the inoculated, diseased experimental host and
identified as being identical to the original specific causative agent.
• Exceptions to Koch’s Postulates:
- Many healthy people carry pathogens but do not exhibit symptoms of the disease. These
carriers may transmit the pathogens to others who then may become diseased.
- Some microbes are very difficult or impossible to grow in vitro in artificial media. (Viruses,
rickettsia, chlamydia, M.leprae, T. pallidum)
- Introducing a pure culture to the experimental animal, the animal must be susceptible to
that of the pathogen. Many animals are resistant to the specific pathogen and most
pathogens are species-specific.
- Use of human volunteer are difficult to find and ethical considerations limit their use.
- Certain pathogens develop only when an opportunistic pathogen invades a weekend host.
II. DIVISION OF MICROBIOLOGY
1. PARASITOLOGY: study of parasites, their hosts, and the relationship between them.
2. MYCOLOGY: study of fungi, including their genetic and biochemical properties, their taxonomy
 and their use to humans as a source for tinder, medicine, food and entheogen, as well as their
dangers, such as poisoning or infection.

Prepared by: Ansharimar C. Balagosa, RMT

3. PHYCOLOGY “algology”
• From the greek word phykos, meaning “seaweed” 
• the scientific study of algae, often is regarded as a sub-discipline of botany
4. VIROLOGY: study of viruses submicroscopic, parasitic particles of genetic material contained in a
protein coat and virus-like agents.
5. BACTERIOLOGY: study of bacteria- involves the identification, classification, and
characterization of bacterial species
III. TAXONOMY
- Comprises 3 distinct areas: Classification, Nomenclature and Identification
- formal system of organizing, classifying and naming living things
1. Classification
• DOMAIN- Bacteria and Archaebacteria
• KINGDOM- similar phyla; similarities of DNA and RNA
• PHYLUM- similar classes
• CLASS- similar orders
• ORDER- similar families
• FAMILY- similar genera
• GENUS- various species with common characteristics
• SPECIES- basic group or collection of bacterial strains with common physiologic and genetic
features
• SUBSPECIES (“subsp.”)- species which are subdivided based on the ff. phenotypic
differences:
- SEROTYPE/ serovarieties (“serovar”): based on serologic differences
- BIOTYPE/ biovarieties (“biovar”): based on biochemical differences
2. Nomenclature
Staphylococcus aureus
Staphylococcus aureus
staphylococci
IV. PROKARYOTIC CELL
1. CYTOPLASMIC STRUCTURES
• No nucleus
• Genome: single circular chromosome
• Ribosomes: consists of RNA and protein
• With CYTOPLASMIC GRANULES
• Some bacteria produce ENDOSPORES in response to harsh environmental condition
2. CELL ENVELOPE STRUCTURES
• Contains Plasma Membrane (PM)
• Contains Cell wall and some do not
3. CELL WALL
a. GRAM POSITIVE
• Very thick protective peptidoglycan (murein) layer
• Peptidoglycan is consist of alternating N-acetyl-d-glucosamine (NAG) and N-acetyl-d
muramic acid (NAM) and crosslink with peptide bridges
• Teichoic acid: exterior of the cell anchored to the peptidoglycan
• Lipotechoic acid: exterior of the cell anchored to the PM (plasma membrane)
b. GRAM NEGATIVE
• Composed of 2 layers
 - Inner layer: much thinner peptidoglycan layer than gram (+) bacteria
- Outer layer: contains proteins, phospholipids and lipopolysaccharide (LPS)
✓LPS 3 regions: antigenic O- specific polysaccharide, core polysaccharide and Lipid A
(endotoxin)
✓Lipid A: responsible for producing fever and shock to pxs. infected with gram (-)
bacteria

Prepared by: Ansharimar C. Balagosa, RMT

• Periplasmic space: between the outer and the inner membrane and encompassing the thin
peptidoglycan layer
- with a gel-like matrix containing nutrient-binding proteins and degradative and
detoxifying enzymes
c. ACID FAST
• Mycobacteria and Nocardia
• have gram (+) cell wall but also contain a waxy layer of glycolipids and fatty acids
(MYCOLIC ACID) — 60% of cell wall’s exterior
• mycolic acid is a strong “hydrophobic” molecule making it difficult to stain with Gram stain
and is best stain with ACID FAST STAIN
d. ABSENCE of CELL WALL
• Mycoplasma and Ureaplasma
• lack a cell wall and contain STEROLS in their cell membranes; are seen in various
shapes 4. SURFACE POLYMERS
• Various pathogenic bacteria produce CAPSULE and SLIME LAYERS
5. CELL APPENDAGES
• FLAGELLA: organ of locomotion; exterior protein filaments that rotate and cause bacteria to be
motile
6. CELL APPENDAGES
• PILI: “conjugation pili”; nonmotile, long, hollow protein tubes that connect two bacterial cells
and mediate DNA exchange
• FIMBRIAE: nonflagellar, sticky, proteinaceous, hairlike appendages that adhere some bacterial
cells to one another and to environmental surfaces
V. BACTERIAL GENETICS
- The mechanism by which genetic information is changed and exchanged among bacteria:
a. MUTATION
• a change in the original nucleotide sequence of a gene or genes within an organism’s genome
(genotype)
b. RECOMBINATION (HOMOLOGOUS RECOMBINATION)
• genes are transferred or exchanged between homologous regions on 2 DNA molecules •
occurs when a portion of the genetic material that originates from one bacterial cell (donor) is
transferred into a second bacterial cell (recipient)
• Mechanism of Gene Transfer
- TRANSFORMATION
✓uptake and incorporation of naked DNA into a bacterial cell
- TRANSDUCTION
✓transfer of bacterial genes by a bacteriophage ( virus-infected bacterium) from one cell
to another
- CONJUGATION
✓transfer of genetic material from a donor bacterial strain to a recipient strain
✓the donor strain produces a sex pilus, which binds to the recipient cell and brings the
 two cells in close contact
VI. MICROBIAL GROWTH & NUTRITION
a. Major nutritional need for growth:
• Source of CARBON (for making cellular constituents): 50%
• Source of NITROGEN (for making proteins): 14%
• Source of ENERGY (ATP, for performing cellular functions)
b. Nutrient requirements:
• Carbon
- Autotrophs (lithotrophs)
- Chemolithotrophs
- Heterotrophs (organotrophs)
• Growth factors
- Prototrophics: do not require an exogenous source of growth factor

Prepared by: Ansharimar C. Balagosa, RMT

- Auxotrophics: require the addition of growth factor to culture media

• Ionic strength
- Halophilic: requiring High
Salt
concentrations
• Carbon dioxide (3-10%)
- Capnophiles: requiring High
CO2
concentrations
• Moisture
- Humidophiles: requiring increased
Moisture content
• Oxygen
c. Physical requirements:

VII.BACTERIAL GROWTH CURVE

1. a lag phase (Phase of Rejuvenation/ Physiologic Youth)
• during which bacteria are preparing to divide (very active
metabolically)
• adaptation to their new environment
• little or no multiplication
2. a log phase (Exponential/ Logarithmic Phase)
• during which bacteria numbers increase logarithmically
• balanced growth - maximal rates of cell division & mass increase
3. a stationary phase (Phase of Equilibrium/ Plateau Phase)
• in which nutrients are becoming limited and the numbers of
bacteria remain constant
 • rate of cell production = rate of cell death
4. a death phase (Phase of Decline)
• when the number of nonviable bacterial cells exceeds the number
of viable cells
• complete cessation of multiplication occurs

VIII.BACTERIAL MORPHOLOGY
1. Cocci (spherical)
• General rule: All cocci are gram positive except Neisseria, Branhamella, Veilonella
2. Bacilli (rod-shaped)
• General rule: All bacilli are gram negative except Bacillus, Listeria, Clostridium, Corynebacterium,
Erysipelothrix, Lactobacillus, Mycobacterium
3. Spirals

Prepared by: Ansharimar C. Balagosa, RMT

 7METHODS of STUDYING BACTERIA

I. SPECIMEN COLLECTION & PROCESSING

- GOAL of the specimen collector: MAINTAIN THE VIABILITY OF THE ORGANISMS WITH MINIMAL
CONTAMINATION
- BASIC PRINCIPLES:
a. If possible, collect the specimen in the ACUTE PHASE OF THE INFECTION and BEFORE
ANTIBIOTICS ARE ADMINISTERED.
b. Select the CORRECT ANATOMIC SITE FOR COLLECTION of the specimen.
c. Collect the specimen using the PROPER TECHNIQUE and SUPPLIES WITH MINIMAL
CONTAMINATION from normal biota.
d. Collect the APPROPRIATE QUANTITY of a specimen.
e. PACKAGE THE SPECIMEN in a container or transport medium designed to maintain the viability
of the organisms and avoid hazards that result from leakage.
f. LABEL the specimen with: SPECIFIC ANATOMIC SITE, DATE AND TIME OF COLLECTION,
PATIENT’S NAME and an UNIQUE ID NUMBER.
g. TRANSPORT the specimen to the laboratory PROMPTLY or make provisions to STORE THE
SPECIMEN in an environment that will not degrade the suspected organism/s.
h. NOTIFY the laboratory in advance if unusual pathogens or agents of bioterrorism are suspected.
- Collection procedures:
a. Containers for specimens should be STERILE except for stool, which can be collected in CLEAN,
LEAKPROOF CONTAINERS.
b. SWABS are NOT RECOMMENDED for collection.
• appropriate for specimens from the UPPER RESPIRATORY TRACT, EXTERNAL EAR, EYE
and GENITAL TRACT
• tips of swab may contain: COTTON, DACRON or CALCIUM ALGINATE
• SWAB COLLECTION SYSTEMS are available that provide TRANSPORT MEDIA and
protect the specimen from drying.
c. For LESIONS, WOUNDS and ABSCESSES:
• area should be CLEANSED to eliminate as much of the commensal flora as possible
• collect from the ADVANCING MARGIN of the lesion and should be collected by NEEDLE
ASPIRATION rather than by swab
• aspirated material should be placed into a STERILE TUBE or TRANSPORT VIAL
- URINE
• CLEAN-CATCH MIDSTREAM URINE SPECIMEN
• FIRST MORNING is preferred.
• First, cleanse the external genitalia.
• Container: Sterile wide mouth glass or plastic jar with tight - fitting lids.
• Processing: within 2 HOURS AFTER COLLECTION
- SPUTUM
• Often collected for the diagnosis of BACTERIAL PNEUMONIA.
• FIRST EARLY MORNING SPECIMEN is preferred.
• Patient should rinse mouth with WATER and expectorate with the aid of a DEEP COUGH directly into
a STERILE CONTAINER (expectorated sputum).
• 1 specimen: for BACTERIAL LOWER RESPIRATORY TRACT INFECTION
• 3 separate early morning specimens: if FUNGAL or MYCOBACTERIAL INFECTIONS are
suspected • Induced sputum: specimens collected through aerosol induction
• Laboratory should be informed of whether the specimen is EXPECTORATED or INDUCED. •
Gram stain of EXPECTORATED SPUTUM should reveal >10 EPITHELIAL CELLS and >25 WHITE
BLOOD CELLS per low-power field
- STOOL
• Detection of GASTROINTESTINAL PATHOGENS
• RECTAL SWAB can be submitted as long as fecal material is visible.

Prepared by: Ansharimar C. Balagosa, RMT

 • Bacterial infection: 3 specimens- once a day for 3 days
• Parasite infection: 3 specimens within 10 days
• Excrete DIRECTLY INTO THE COLLECTION DEVICE
• STOOL to PRESERVATIVE RATIO- 1:3
• Container: Clean wide mouth with tight fitting leak proof lid
II. TRANSPORTATION
- Primary goal in the TRANSPORTATION of specimens to the
laboratory: MAINTAIN THE SPECIMEN AS NEAR TO ITS
ORIGINAL STATE AS POSSIBLE WITH MINIMAL
DETERIORATION AND TO PREVENT RISK TO THE
SPECIMEN HANDLER
- Specimen should be transported IDEALLY WITHIN 30
MINUTES OF COLLECTION, PREFERABLY WITHIN 2 HOURS.
- If transport is delayed, the specimen can be maintained by
storage under certain conditions or with the use of
PRESERVATIVES, ANTICOAGULANTS, TRANSPORT or
HOLDING MEDIUM, or CULTURE MEDIUM.
- PRESERVATIVES
• 2 specimen in which preservative can be use: URINE and
STOOL
- URINE
• BORIC ACID: maintain accurate urine colony counts at RT for
24 hours and useful for collection at distant locations
- STOOL
• If not transported IMMEDIATELY, it can be REFRIGERATED ( if
the delay is longer than 2 hours, specimen can be added to
CARY-BLAIR TRANSPORT MEDIA)
• Stools for C. difficile toxin assay should be collected
WITHOUT PRESERVATIVE & can be REFRIGERATED; if the
delay is longer than 48 hours, the specimen should be
FROZEN at -70°C
- ANTICOAGULANTS
• used to prevent clotting of specimens, including blood, bone
marrow and synovial fluid
• SODIUM POLYANETHOL SULFONATE (SPS): most common
anticoagulant used for microbiologic specimens;
concentration must not exceed 0.025% wt/vol
• SPS:Blood ratio (1:5 to 1:10)
• HEPARIN: often used for viral cultures and for isolation of
Mycobacterium spp. from blood
- HOLDING or TRANSPORT MEDIA
• these usually contain substances that do not promote multiplication of microorganisms but ensure
their preservation
• STUART’s or AMIE’s TRANSPORT MEDIA: commonly used
• JEMBEC SYSTEM (contains selective agar and a CO₂-generating tablet): specimens for N.
gonorrhoeae
- UNACCEPTABLE SPECIMENS and SPECIMEN REJECTION
• information on the requisition DOES NOT MATCH the information on the specimen label •
specimen is NOT SUBMITTED IN THE APPROPRIATE TRANSPORT CONTAINER or container is
LEAKING
• INADEQUATE QUANTITY
• specimen transport time is >2 HOURS and the specimen HAS NOT BEEN
PRESERVED • specimen is in FORMALIN
• specimen is DRIED UP
 Prepared by: Ansharimar C. Balagosa, RMT

III. SMEARS
a. Smears from swab
b. Smears from thick, liquids, or semisolids
c. Smears from thick, granular, or mucoid
materials
d. Smears from thin fluids
IV. GRAM STAINING
- MICROSCOPIC EXAMINATION
• CHRISTIAN GRAM developed GRAM
STAIN
- STAINING
• imparts an artificial coloration to the smear
materials that allows them to be inspected
using the magnification provided by a
microscope
• category: SIMPLE STAINS, DIFFERENTIAL
STAINS and PROBE-MEDIATED STAINS
a. SIMPLE STAINS
✓directed toward coloring the FORMS
and SHAPES present
b. DIFFERENTIAL STAINS
✓coloring specific components of the elements
present
c. PROBE-MEDIATED STAINS
✓directed specifically at identification of an
organism
V. CULTURE PREPARATION
- CULTURE MEDIA
• any material containing the necessary nutritional and
environmental requirements for bacterial growth
• CLASSIFICATION according to: PHYSICAL STATE/
CONSISTENCY, COMPOSITION, HOW THE
MEDIUM IS
DISPENSED AND USE
a. According to PHYSICAL STATE/ CONSISTENCY:
✓LIQUID- without any trace of agar or solidifying
substances (e.g. nutrient broth)
✓SEMISOLID- contains 0.5-1.5% of agar (e.g. SIM)
✓SOLID- contains 2-3% of agar (e.g. BAP, TSI)
b. According to COMPOSITION:
✓SYNTHETIC- exact chemical composition of the ingredients are known (e.g. commercially
prepared culture media)
✓NON-SYNTHETIC- precise composition of some or all of the nutritive substances used are
known (e.g. meat extract broth)
✓TISSUE CULTURE MEDIA- contains living tissue (e.g. embryonated egg for viruses)
c. According to HOW THE MEDIUM IS DISPENSED:
✓PLATED- distributed in sterile petri dishes
✓TUBED- distributed in sterile test tubes (types: SLANT, BUTT, BUTT/SLANT)
d. According to USE:
✓NONSELECTIVE- growth of most non-fastidious microbes (e.g. SHEEP BLOOD AGAR)
✓SELECTIVE- growth of 1 type/group of microbes; it may contain certain inhibitory substances
 ✓DIFFERENTIAL- allow grouping of microbes based on different characteristics demonstrated
on the medium
- DIFFERENTIAL & NONSELECTIVE- e.g. SHEEP BLOOD AGAR
- DIFFERENTIAL & SELECTIVE- e.g. MacConkey AGAR
✓ENRICHED- contain growth enhancers that are added to non-selective agar to allow fastidious
organisms to flourish (e.g. CAP)

Prepared by: Ansharimar C. Balagosa, RMT

- CULTURE MEDIA STORAGE, the following are the shelf life or

prepared media when stored in a cool, dark place:
• TUBE with COTTON-WOOL PLUG: 3 WEEKS
• TUBE with LOOSE CAP: 2 WEEKS
• CONTAINER with SCREW CAP: 3 MONTHS
• PETRI DISHES, if sealed in PLASTIC BAGS: 4 WEEKS
- SPECIMEN PREPARATION before isolation
• PUS, URINE, SPUTUM, SPECIMEN IN SWABS: inoculated directly
into the media
• Large volumes (PERITONEAL, PLEURAL): concentrated to increase
the recovery of bacteria- CENTRIFUGATION AND FILTRATION is
performed
- Isolation techniques
- CULTURE INCUBATION
• Most bacteria cultures are incubated at 35°C- 37°C
• Some bacteria are CAPNOPHILES: uses CANDLE JAR, CO2
INCUBATOR
• Some bacteria are MICROAEROPHILES: uses JARS or BAGS
- INCUBATION TIME
• Most bacterial cultures are held for 48-72 HOURS
• Culture for ANAEROBES and BROTH CULTURES may be held for 5-7
DAYS
VI. GROSS COLONY CHARACTERISTICS
a. Hemolysis
• hemo: red blood cells , lysis: dissolution or break apart
• it is the lysis of the RBCs surrounding or underneath the colony caused by enzymatic or toxin activity
of bacteria
b. Size
• described as LARGE, MEDIUM, SMALL, PINPOINT
c. Form/Margin
• described as FILAMENTOUS, IRREGULAR, SMOOTH, ROUGH or RHIZOID
d. Elevation
• determined by tilting the culture plate and looking at the side of colony
• may be FLAT, RAISED, CONVEX or DOME, UMBILICATE, UMBONATE
e. Color
f. Density
• determined by looking through the colony while using transillumination
• may be TRANSPARENT, TRANSLUCENT or OPAQUE
g. Consistency
• determined by touching the colony with a loop
• may be BRITTLE, CREAMY, DRY, WAXY
h. Pigment
 • pigment production is an inherent characteristic of a specific organism (e.g. Pseudomonas
aeruginosa & Serratia marcescens)
i. Odor
• determined when the lid of the plate is removed and the odor dissipates into the surrounding
environment (e.g. Staphylococcus aureus & Pseudomonas aeruginosa )

Prepared by: Ansharimar C. Balagosa, RMT

 STAPHYLOCOCCI

I. GENERAL CHARACTERISTICS
- non-motile, catalase-producing, gram (+) cocci
- Greek word “staphle”, which means bunch of grapes
- smear: exhibit spherical cells (singly, pairs or clusters)
- colonies: medium-sized, β hemolytic colonies that are creamy or white & rarely gold
- members of the family Staphylococcaceae
- resemble some members of the family Micrococcaceae, such as the genus Micrococcus: catalase
producing, coagulase (-), gram (+) cocci
II. Staphylococci VS. Micrococci
a. CHO OXIDATION FERMENTATION TEST
• indicator: bromthymol blue
• medium: Hugh & Leifson tubes
• OPEN TUBE: without Vaseline to produce an
environment with Oxygen within the tube
• CLOSED TUBE: with Vaseline to produce an
environment without Oxygen within the tube
• (+) yellow (with growth)
• (-) green (without growth)
b. MODIFIED OXIDASE
• Principle: Micrococcus organisms possess CYTOCHROME C in the cytochrome oxidase system,
while Staphylococci do not.
• Reagent: 6% tetramethylphenylenediamine hydrochloride in dimethyl sulfoxide or Microdase Disk
(with impregnated Modified oxidase reagent)
• Expected Results:
- (+): Dark-blue
- (-): No color change
c. LYSOSTAPHIN SUSCEPTIBILITY TEST
• Principle: Lysostaphin is an endopeptidase that cleaves the glycine-rich peptide linkages,
rendering the cells susceptible to osmotic lysis (lysostaphin susceptible). The interpeptide bridge
of Micrococcus does not contain glycine.
III. Staphylococcus aureus
- most clinically significant species of staphylococci
- facultative anaerobe and exhibits GOLDEN YELLOW PIGMENTATION due to the action of the pigment
STAPHYLOXANTHIN
- VIRULENCE FACTORS
a. ENTEROTOXINS
• are acid-stable & heat stable (stable at 100°C for 30 minutes)
• 9 serologically distinct enterotoxins have been identified (A to E, G to J)
• A: food poisoning
• B: staphylococcal pseudomembranous colitis
• C & D: contaminated milk
b. TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1)
• formerly referred to as Enterotoxin F
• only toxin that is SYSTEMATIC
• common: MENSTRUATION WOMAN using TAMPONS
c. EXFOLIATIVE TOXIN (Epidermolytic Toxin)
• causes the epidermal layer of the skin to slough off and is known to cause
STAPHYLOCOCCAL SSS
 d. CYTOLYTIC TOXINS
• Α HEMOLYSIN: disrupts smooth muscle in blood vessels

Prepared by: Ansharimar C. Balagosa, RMT

• Β HEMOLYSIN (SPHINGOMYELINASE C): “hotcold lysin”, acts on sphingomyelin in the
plasma membrane of RBC
• δ HEMOLYSIN: destroy surfactants in cell membrane (like detergent) affecting RBC and is
considered less toxic to cells than α & β hemolysin
• γ HEMOLYSIN: toxic to WBC which causes lysis to the cell and often found only in association
with Panton-Valentine Leukocidin (PVL)
e. PROTEIN A
• ability to bind to the Fc portion of IgG
f. ENZYMES
• COAGULASE: conversion of fibrinogen to fibrin
• STAPHYLOKINASE (fibrinolysis): dissolves fibrin clot and may enable release of bacteria thus
beginning the spread of infection
• LIPASE: hydrolyze lipids in plasma & skin (causes boils, carbuncles & furuncles)
• HYALURONIDASE: “spreading factor”, hydrolyze hyaluronic acid present in connective tissue
causing the spread of infection
• DNAse: hydrolyze DNA
• β- LACTAMASE (Penicillinase): hydrolysis & inactivation of penicillin
IV. Staphylococcus epidermidis
- former name is Staphylococcus albus
- infections caused are predominantly hospital acquired such as PROSTATIC HEART-VALVE
ENDOCARDITIS
- BIOFILM PRODUCTION
V. Staphylococcus saprophyticus
-
associated with UTIs IN YOUNG WOMEN

- in urine cultures, S. saprophyticus may be found in low numbers (<10,000 colony-forming units/ mL)
and still be considered SIGNIFICANT
VI. BIOCHEMICAL CHARACTERISTICS
1. CATALASE TEST
• Staphylococci VS. Streptococci
• reagent: 3% H₂O₂
• (+) result is EFFERVESCENCE/ Bubble Formation
• (+) Staphylococci
• (-) Streptococci
2. COAGULASE TEST
• Staphylococcus aureus VS. CoNS
• 2 forms: cell bound coagulase (“clumping factor”)
free coagulase
(“staphylocoagulase”)
• (+): Staphylococcus aureus
• (-): CoNS ( Staphylococcus saprophyticus &
Staphylococcus epidermidis)
• SLIDE COAGULASE TEST
- for detecting CELL BOUND COAGULASE
- Test: Rabbit Plasma + Organism
 - Control: NSS + Organism
- (+) MACROSCOPIC CLUMPING in 10 seconds
- (-) NONE, proceed to tube test to confirm
• TUBE COAGULASE TEST
- for detecting FREE COAGULASE
- sterile test tube with 0.5 mL RABBIT PLASMA then inoculate colonies to give MILKY
SUSPENSION
- incubate 35°C for 24 hrs.
- (+) COAGULUM/ CLOT FORMATION
3. NOVOBIOCIN SUSCEPTIBILITY TEST
• PURPOSE: differentiate S. saprophyticus from S. epidermidis

Prepared by: Ansharimar C. Balagosa, RMT

• PRINCIPLE: after incubation with 5µg NOVOBIOCIN, S. saprophyticus is not inhibited by the antibiotic
while S. epidermidis is susceptible
• RESULTS:
- Susceptible: zone is >16mm
- Resistant: zone is <16mm
4. BETA-LACTAMASE (CEFINASE) TEST
• detect β lactamase production
• CEFINASE DISK is impregnated with the CHROMOGENIC
CEPHALOSPORIN NITROCEFIN
• (+): yellow to RED
• (-): NO COLOR CHANGE
5. DNA HYDROLYSIS/ DNAse TEST
• detect the ability of an organism to hydrolyze DNA with DNAse
6. Growth on MANNITOL SALT AGAR
• inhibitor: 7.5% NaCl
• CHO: MANNITOL
• pH indicator: PHENOL RED
• Mannitol fermenter: “yellow halo: (Staphylococcus aureus)
• Non-mannitol fermenter: CoNS (Staphylococcus saprophyticus & Staphylococcus epidermidis) Prepared
by: Ansharimar C. Balagosa, RMT
 STREPTOCOCCACEAE

I. GENERAL CHARACTERISTICS
- Streptococcus & Enterococcus spp. belong to the family Streptococcaceae
- members of both genera are CATALASE (-), GRAM (+) COCCI
- smear: usually arranged in PAIRS or CHAINS, cocci in pairs (lancet shaped)
- BAP: grayish, pinpoint with mucoid colonies
II. CLASSIFICATION OF Streptococci & Enterococci

III. Streptococcus pneumoniae VS. Viridans

a. Mouse Virulence Test
b. Inulin Fermentation Test
c. Bile Solubility Test
• Principle: Bile or a solution of bile salt (e.g.
sodium desoxycholate) rapidly lyses
pneumococcal colonies.
• Expected Results:
- (+): Broth clearing
- (-): Turbid
d. Optochin Test (Taxo P)
• Principle: This test is used to determine
the
effect of Optochin (ethylhydrocupreine
hydrochloride) on an organism.
Optochin
lyses pneumococci but not alpha
 streptococci.
• Expected Result:
- (+): greater than or equal to 14mm zone
of inhibition- using 6mm disk
(SUSCEPTIBLE)
- (-): no zone of inhibition (RESISTANT)
- Equivocal: any zone of inhibition <14mm is questionable for pneumococci, the strain will be
confirmed using BILE SOLUBILITY
e. Neufeld-Quellung Reaction (Capsular Swelling)
• Principle: Test is performed by mixing on a slide a loopful of emulsified sputum with a loopful of
ANTI-PNEUMOCOCCAL SERUM and METHYLENE BLUE
• Result:
- (+) reaction: capsule appears SWOLLEN due to a change in refractive index which in turn is
due to a serological reaction
IV. Streptococcus pneumoniae
- a.k.a DIPLOCOCCUS or PNEUMOCOCCUS
- BAP: α hemolytic & mucoid colonies
- young colonies: “DOME-SHAPED”
- old colonies: DIMPLE SHAPED or DONUT SHAPED

Prepared by: Ansharimar C. Balagosa, RMT

- colonies may closely resemble colonies of VIRIDANS STREPTOCOCCI

- SKIN TEST
• also known as FRANCIS TEST
• used to detect antibodies against pneumococci
- VIRULENCE FACTORS
a. POLYSACCHARIDE CAPSULE
• principal virulence factor for its pathogenesis
b. ENZYMES
• Neuraminidase: degrades surface structure of host
• Proteases: facilitate bacterial colonization on mucosal surfaces by eliminating
immunoglobulins (IgA, IgG and IgM)
c. AUTOLYSIN
• facilitates the release of Pneumolysin O & other toxic proteins or inflammatory substances
from cells
d. PNEUMOLYSIN O
• O₂ sensitive toxin & cytolytic for cells
- DISEASE ASSOCIATION
a. BACTERIAL PNEUMONIA (“LOBAR pneumonia”)
• “rust- tinged” sputum
b. RECURRENT OTITIS MEDIA
• usually affects children under 3 yrs. old
c. BACTERIAL MENINGITIS
• affects ADULTS
V. Viridans streptococci
- constituents of the normal microbiota of the upper RT, female genital tract and GT
- are fastidious organisms, with some strains requiring CO₂ for growth
- MOST COMMON ISOLATE: Streptococcus mutans
- most common cause of SUBACUTE BACTERIAL ENDOCARDITIS
- 3 MAIN INFECTIONS cause by Viridans
a. DENTAL INFECTIONS
• bind to the teeth & ferment sugars, which produces ACID & DENTAL CARIES
(cavities) b. ENDOCARDITIS
• dental manipulation send showers of these organisms in the blood system
c. ABSCESSES
• most frequently cause by Streptococcus intermedius group (S.intermedius, S.constellatus &
S.anginosus) which are microaerophilic
VI. Streptococcus pyogenes (Group A)
- VIRULENCE FACTORS
a. M PROTEIN
• attached to the peptidoglycan of the cell wall & extends to the cell surface
• causes the streptococcal cell to resist phagocytosis
• play a role in adherence of the bacterial cell to mucosal cells
b. STREPTOLYSIN O (SLO)
• O₂ labile, highly antigenic, subsurface hemolysis on BAP
c. STREPTOLYSIN S
• O₂ stable, non-antigenic, surface hemolysis on BAP
d. STREPTOKINASE
• lysis of fibrin clots
e. ERYTHROGENIC EXOTOXINS
• “pyrogenic toxins”
• EXOTOXIN B: a cysteine protease that causes SCARLET FEVER (erythematous sandpaper-like
rash)
- DISEASE ASSOCIATION
a. POSTSTREPTOCOCCAL SEQUELAE

Prepared by: Ansharimar C. Balagosa, RMT

• PHARYNGITIS or Tonsilitis: “STREP THROAT”
• Rheumatic Fever
• Acute Glomerulonephritis: “BRIGHT’S DISEASE”
b. SCARLET FEVER (“Scarlatina”)
• Cardinal Signs: diffuse red rash on the upper chest & spreads to trunk & extremities AND
“strawberry colored tongue”
• Susceptibility Test: DICK’S TEST
✓used to detect Erythrogenic Toxin
✓(+) result: ERYTHEMA/ REDNESS ON SITE
• Diagnostic Test: SCHULTZ- CHARLTON TEST
✓used to diagnose whether the skin rashes are
due to
scarlet fever or not
✓(+) result: “BLANCHING PHENOMENON”
c. SKIN INFECTIONS
• Cellulitis
• ERYSIPELAS
• Necrotizing fasciitis: “GALLOPING GANGRENE” or “FLESH-EATING BACTERIA SYNDROME”
- IDENTIFICATION TESTS
 a. BACITRACIN TEST (TAXO A)
• a presumptive test which differentiates Streptococcus pyogenes from other
Streptococci • Principle: Based on the selective inhibition of the growth of S.pyogenes by
a paper disc containing 0.02-0.04 UNITS OF BACITRACIN
• Result:
✓(+): any zone of inhibition around the disk
✓(-): no zone of inhibition
b. PYR TEST
• detect the organisms ability to hydrolyze the
substrate L
pyrrolidonyl-beta-napthylamide
VII.Streptococcus agalactiae (Group B)
- VIRULENCE FACTORS
a. Capsule made of SIALIC ACID
• most important virulence factor
• prevents phagocytosis
b. CAMP FACTOR
• a.k.a PROTEIN B
• pore forming protein secreted by the organism
- DISEASE ASSOCIATION
• most common etiologic agent of NEONATAL SEPSIS and NEONATAL MENINGITIS
• most infections of infants occur in the FIRST 3 DAYS AFTER BIRTH, usually WITHIN 24
HOURS - IDENTIFICATION TESTS
a. SODIUM HIPPURATE HYDROLYSIS
• use to determine whether a microorganism, by action of
the enzyme HIPPURATE HYDROLASE can hydrolyze
sodium hippurate to benzoic acid & glycine
b. CAMP (Christie, Atkins & Munch-Peterson) TEST
• differentiate S.agalactiae from other Streptococci
• PRINCIPLE: CAMP factor is produced by S.agalactiae
which has the ability to act synergistically with the beta
hemolysin produced by S.aureus to produce even more
potent hemolysis
• RESULT:
• (+) “ARROW HEAD” SHAPED ZONE OF ENHANCED HEMOLYSIS
- CULTURE MEDIA
a. TODD-HEWITT BROTH: 10 µg/mL COLISTIN and 15 µg/mL NALIDIXIC ACID

Prepared by: Ansharimar C. Balagosa, RMT

b. StrepB Carrot Broth (SCB): produced ORANGE or RED PIGMENT even after 6 HOURS OF
INCUBATION
VIII.Enterococcus spp. (Group D)
- consists of gram (+) cocci that are natural inhabitants of the intestinal tracts of humans & animals - no
establish virulence factors
- commonly identified species in clinical specimens: Enterococcus faecalis and Enterococcus faecium -
frequent causes of NOSOCOMIAL INFECTIONS
Prepared by: Ansharimar C. Balagosa, RMT
The Brightfield Compound Microscope
• also known as the Compound Light Microscope
• an optical microscope that uses light rays to produce a dark image against a bright
background • specimens used are prepared initially by staining to introduce color for easy
contracting characterization
• proper way to cary the microscope: always carry a microscope with one hand holding the arm and one
hand under the base

1. Eyepiece (Ocular lens) - it has two eyepiece lenses at the top of the microscope which focuses the
image from the objective lenses. this is where you see the formed image from, with your eyes 2.
Objective lenses - are made up of six or more glass lenses, which make a clear image clear from the
specimen or the object that is being focused
3. Fine adjustment knob and the coarse adjustment knob - two focusing knobs found on the
microscopes’ arm, which can move the stage or the nosepiece to focus on the image. their function
is to ensure the production of a sharp image with clarity
4. Stage - found just below the objectives and this is where the specimen is placed, allowing movement
of the specimen around for better viewing with the flexible knobs and it is where the light is focused
on
5. Stage control - adjusts the position of the mechanical stage horizontally in the X and Y plane, in
order to position the slide so that a portion of the specimen is under the objective
6. Stage clips (slide holder) - hold the slide in place on the mechanical stage
7. Condenser - mounted below the stage which focuses a beam of light onto the specimen 8. Arm -
sturdy metallic backbone of the microscope, used to carry and move the microscope from one place to
another
9. Illuminator (light source)
10. Light intensity control (rheostat) - knob used to adjust the amount of light that reaches the specimen
or slide from the base illumination
11. Nosepiece - has about two to five objective lenses with different magnifying power. it can move
round to any position depending on the objective lens to focus on the image
12. Diaphragm - controls the diameter of the beam of light that passes through the condenser

Basic Apparatus & Equipment in Microbiology

1. PPE
2. Petri Dish - a shallow cylindrical, round glass that is used in laboratories to culture different
microorganisms and cells
3. Test tubes - are slender containers that hold and mix or store materials that are used in scientific
experiments and research
4. Test tube rack - apparatus used in a laboratory to hold and transport test tubes during experiments
or while examining cultures
5. Inoculating loop & needle - consist of a handle with a terminal loop or needle which is designed to
pick up and transfer a small quantity of inoculum (typically 1 to 10μL) from a donor culture to the
growth medium of choice
6. Glass rod - a piece of laboratory equipment used to mix chemicals and liquids for laboratory
purposes
7. Beaker - are useful as a reaction container or to hold liquid or solid samples
8. Erlenmeyer flask - are used to contain liquids and for mixing, heating, cooling, incubation, filtration,
storage, and other liquid-handling processes
9. Wash bottle - is a squeeze bottle with a nozzle, used to rinse various pieces of laboratory glassware,
such as test tubes and round bottom flasks
10. Glass slide - is a thin, flat, rectangular piece of glass that is used as a platform for microscopic
specimen observation
11. Staining rack - hold multiple microscopic slides simultaneously for the most efficient staining
processing
 Prepared by: Ansharimar C. Balagosa, RMT
12. Sterile cotton swab - are generally used for biological sample collection to avoid sample
contamination, and for medical use to avoid infection
13. Biological safety cabinet - is a primary engineering control used to protect personnel against
biohazardous or infectious agents and to help maintain quality control of the material being worked
with as it filters both the inflow and exhaust air
14. Autoclave - is used not only to sterilize liquid substances such as prepared media and saline
(diluents) solutions, but also to sterilize glassware’s, when required
15. Microbiological incubator - maintains a constant temperature specifically suitable for the growth of a
specific microbe
16. Fridge (refrigerator) - serves as a repository for thermo labile chemicals, solutions, antibiotics, serums
and biochemical reagents at cooler temperatures and even at sub-zero temperatures (at less than
0°C)
17. Weighing scale - are used to measure the weight of an item
18. Hot plate - is used to heat chemicals and reagents
19. Magnetic stirrer - is used to dissolve such substances easily and quickly
20. Microscope - are used for visual observation of morphology, motility, staining and fluorescent
reactions of bacteria
21. Alcohol lamp - it is a gas-fueled single open flame and is commonly used for processes like
sterilization and heating
22. Automated bacteria identification system - an instrument used for automatic computer-assisted
identification of bacteria
Prepared by: Ansharimar C. Balagosa, RMT