BACTERIOLOGY

M.A.D.M, RMT, MLS (ASCPi) 2019

Species – Most specific and exclusive taxon used

Identification – delineation of key features which uses relevant and useful information
o Provide standardize groupings
o Share similar phenotypic traits
o Identified using similar methods

Phenotypic Genotypic
 Color of growth  Antibiotic resistance
 Bacterial cell stage
 Bacteria on slide
 Fermentation of lactose

BACTERIAL COMPONENTS
1. Cytoplasm: interior of the cell

2. Pili/Fimbriae: hair-like extensions that extend into the environment

- 2 types:
 Common pili
 Sex/conjugate pili

3. Flagella: connected to the cellular envelope. Used for usual LOCOMOTION of bacteria
- Different types:
 Atrichous
 Monotrichous
 Amphitrichous
 Lophotrichous
 Peritrichous
- Flagellar stains
a. LEIFSON
b. FISHER AND CONN
o Endoflagella: Spirochetes – Borrelia, Leptospira, Treponema

4. Capsules: composed of polysaccharide or protein layers

 Anti-phagocytic – defense mechanism
 Slime layer – loosely arranged material
o BIOFILM: accumulation of organism embedded in a polysaccharide matrix
 Capsular stains
o HISS STAIN
o INDIA INK/ NIGROSIN

5. Endospores (Resting cells): highly refractile bodies in cell; highly resistant to dessication, heat and
chemical agents
 Organisms with spores: Bacillus, Clostridium
 Spore stains:
a. Schaeffer and Fulton
b. Doemer’s
c. Wirtz & Conklin

6. Cell Wall/ Murein Layer: external wall of most bacteria

Gram positive Gram negative

Thicker cell wall Thinner cell wall
Periplasmic space – found only in gram negative
organisms. Contains murein layer. Collection and
enzymatic degradation of nutrients.

EXOTOXINS ENDOTOXINS
High concentration in fluid media; not associated Consists of LPS; released at cell lysis or death and
with the production of fever capable of inducing fever
Heat-sensitive except : Staphylococcus enterotoxin Heat-resistant
Converted to toxoids Not converted to toxoids
TSS, Diphtheria, Clostridium toxins UTI, Typhoid

_____________________ - Test to detect Bacterial Endotoxin (Gram negative) in body fuids and surgical
instruments
 BACTERIAL GROWTH CYCLE
1. Lag phase
 Period of adjustment

2. Log phase
 exponential phase
 most metabolically active and
 sensitive to antimicrobials

3. Stationary phase
 plateau, bacterial death due to:
a. Lack of nutrients
b. Development of unfavourable pH
c. Accumulation of toxin

4. Phase of decline
 viable decrease

TYPES OF ORGANISMS:
 Normal flora/Commensals: Colonizers or resident flora
 Pathogenic organsims: Infectious/Disease causing
 Nosocomial pathogen: Hospital-acquired
 Opportunistic pathogen: Colonizes the skin but is capable of causing infection under the
appropriate conditions

TYPES OF FIRES AND FIRE EXTINGUISHERS

Fire type Composition of fire Extinguishing materials Extinguisher

Class A Class A Wood, paper, clothing Water (pressurized) and dry
chemicals
Class B Class B Flammable organic materials Dry chemicals, carbon dioxide,
foam or halon
Class C Class C Electrical (Computer Fires) Dry chemicals, carbon dioxide
or halon
Class D None Combustible metals Sand or dry powder
Class ABC Dry Chemicals
Class E Arsenal fires (ammunition) Usually allowed to burn out
nearby materials protected

Class K Class K Grease, oil and fats Liquid designed to prevent

splashing and cool the fire

MOST LABORATORIES USE: __________________________

SPECIMEN COLLECTION REQUIREMENTS FOR SPECIFIC SITES

I. Respiratory Tract
 Throat cultures, nasopharyngeal cultures and specimens from the oral cavity.
 Rejected: saliva, oropharyngeal secretions, sinus drainage (lower RT contamination)
 SPUTUM – detection of bacterial pneumonia/tuberculosis
o Early morning- preferred
o Expectorated- patient should rinse the mouth with water and expectorate with the aid of
a deep cough directly into a strerile container
o Induced sputum- through aerosol induction, patient breathes aerosolized droplets of a
solution that stimulates cough reflex
o DSSM - ________________________
~ Test used by DOTS in diagnosing TB
~ Most widely used means for diagnosis of pulmonary TB and is available in most
primary healthcare laboratories at health-center level. (WHO)
 The most frequent cause of bacterial pharyngitis is _________________
 Epiglottitis is caused by is __________________

Early-onset pneumonia Late-onset pneumonia

Carries good prognosis Carries bad prognosis
Contracted within the first 4 days of hospitalization Contracted after 5 days or more of hospitalization
Usually cause by antiobiotic sensitive bacteria MDR organism is the cause
 II. Gastrointestinal Tract
- Diagnose the cause of gastroenteritis
- Gastric aspirate: Collect in early morning before patient eats or gets out of bed. Most gastric
aspirates are on infants or for AFB.
- Gastric biopsy: ____________

III. Urine
- Diagnose upper or lower UTI
- ___________________: safer, less traumatic; specimen of choice for bacterial culture
- 1 uL = 0.001 mL of urine (# of colonies x 1000)
- ___________________ : Indicates infection
- ___________________ : Most common cause of UTI (90% , worldwide)
- ___________________ : Cause of UTI in sexually active young females
- Specimens for Bacterial Culture
a. CATHETERIZED SPECIMEN – most commonly requested for bacterial culture
b. Midstream
c. Suprapubic
- CATHETER
 Straight catheter (in and out) IV CATHETERS, PINS
-clean Do not culture Foley catheters; IV
catheter into bladder; allow first 15 mL to pass catheters are cultured quantitatively
-plate (1:100 & 1:1000) by rolling the segment back and forth
 Indwelling (Foley) across agar with sterile forceps four
-disinfect times; _____________ are
-aspirate _______ of urine with needle and syringe associated with clinical significance
-plate (1:1000)

IV. Blood
 BACTEREMIA
 SEPTICEMIA
- “4S” Anticoagulants
1. ________________________
2. ________________________
3. ________________________
4. ________________________
- Aerobic and anaerobic blood culture: Green, orange, yellow
- Adult: _________ (Ratio: 1:10)
- Pediatric: _________
- Collection considerations:

BACTEC/BACTALERT TIMING OF COLLECTION ANTISEPSIS

Why blood cultured bottles are agitated in these Because the timing of intermittent sepsis is Skin site is defatted (fat removal) with 70% isopropyl
machines? unpredictable, it is generally accepted that alcohol and an antiseptic is applied to kill surface and
_________ should be space _________ subsurface bacteria.
The agitation(rocking motion) of the bottles apart.
increases oxygenation of the broth, enhancing the As part of ongoing quality assurance, laboratories that
detection of microorganisms in the bottles and recover contaminants at rate greater than >3% should
Overall, ________________ cultured was suspect improper phlebotomy techniques and should
decreasing the time to detect growth. Therefore, more critical to increasing organism yield institute measures to educate the phlebotomists in
the purpose is for ________________________ than timing proper skin preparation methods.

V. Cerebrospinal Fluid
- Subject to cytocentrifigutaion
- Diagnosis of meningitis
- If CSF is not processed immediately, it can be stored at _____________
- Plated on:
o Routine: _________________
o SHUNT: _________________
o *In general: __________________

VI. Genital Tract

- Sexually-transmitted diseases
FEMALES
Bartholin cyst anaerobic transporter (disinfect skin before collection); aspirate fluid ; consider
chlamydia and GC culture

Cervix swab moistened with Stuart’s or Amie’s medium (remove mucus before collection) ;
do not use lubricant on speculum (swab deeply into endocervical canal)
 Cul-de-sac anaerobic transporter; submit aspirate

Endometrium anaerobic transporter; surgical biopsy or transcervical aspirate via sheathed

Urethra Swab moistened with Stuart’s or Amie’s medium (remove exudate from urethral
opening); collect discharge by massaging urethra against pubic symphysis or insert
flexible swab 2-4 cm into urethra and rotate swab for 2 seconds; collect at least 1
hour after patient has urinated

Vagina Swab moistened with Stuart’s or Amie’s medium or JEMBEC transport system
(remove exudate); swab secretions and mucous membranes of vagina.

Culture is not recommended for the diagnosis of bacterial vaginosis; Inoculate

selective medium for GROUP B Streptococcus (LIM broth) if indicated on pregnant
women. Do gram stain for BV and check for presence of curved, gram-negative rods
indicative of Mobiluncus spp.

MALES
Prostate Swab moistened with Stuart’s or Amie’s medium or sterile, screw-cap tube (clean
glans with soap and water; collect secretions on swab or in tube

Urethra Swab moistened with Stuart’s or Amie’s medium or JEMBEC transport system; insert
flexible swab 2-4 cm into urethra and rotate for 2 seconds or collect discharge on
JEMBEC transport system
-for Chlamydia, Mycoplasma, and Gonorrhea

VII. Wound and Abscesses

- Pus aspirates, irrigation fluids and swabs of purulent drainage from the dermis
- Needle and syringe aspiration
o Superficial : swab
 Presence of mixed gram positive and gram negative organisms may indicate
occurrence of normal flora, it should be added to _________________________
o Deep : aspirate from wall/excision
 Wash granules and emulsify in saline
- Specimen characteristics of ANAEROBIC INFECTIONS:
1.) ________________ 2.) ________________ 3.) _________________

VIII. Conjunctival specimens

- Conjunctival infections
- Clinicians instill local anesthesia
- Specimen of choice for corneal ulcer: ____________________
- Inoculated on BA, CA, SDA, 7H10, Ana, Thio
- Detection of Acanthamoeba spp; HSV, Chlamydia trachomatis & fungi; T. solium

IX. Stool
- Routine culture: Salmonella, Shigella, Campylobacter, Vibrio, Aeromonas, Plesiomonas, Yersinia
and Escherichia coli O157:H7 (follow up shiga-toxin assay by CDC)
- Inoculated on BAP, MAC, XLD, HEA, Campy, EB
- Stain for fecal leukocytes: _____________________
- O&P
o Outpatient : Collect 3 specimens every other day at a minimum

o Inpatient: Daily specimens collected for three days

o If parasites are suspected: 3 stool specimens collected within 10 days- should be

sufficient for microscopic detection of ova and parasite

o If patient has received antiparasitic compounds: Wait for 7-10 days

 Barium – appears as white chalky substance and masks the appearance of
parasites under the microscope
 Iron, Kaopectate, Metronidazole, Milk of Magnesis, Peptobismol, or Tetracycline

o Intestinal amoebiasis and giardiasis: 6 stool specimens in 10 days

- Rectal swab: Swab placed in enteric transport medium (Cary-Blair). Insert the swab 2.5 cm past
anal sphincter, feces should be visible. Consider Vibrio, Y. enterocolitica, E. coli O157:H7

- Note: Do not perform routine STOOL CULTURES for patients whose:

 length of stay in the hospital _______
 admitting diagnosis was not diarrhea
 THESE PATIENTS SHOULD BE TESTED FOR _____________
X. Hair, Nails or Skin Scrapings (Fungal culture)
- Place in clean, screw-top tube
- For nails or skin wipe with 70% alcohol
Hair: Collect hairs with intact shaft and follicle
Nails: Send clippings of affected area (use sterile scissors or scalpel)
Skin: Scrape skin at leading edge of lesion (use scalpel or blade

SPECIMEN TRANSPORT AND PROCESSING

 _______________ - transport medium used if a delay is anticipated

o Methylene blue: Acts as _____________
o Thioglycollate: Acts as a _____________ to permit survival of anaerobic bacteria
o Sodium glycerophosphate: To buffer calcium chloride

 Cary-Blair medium – stool specimens (enteric pathogens)

 Calcium alginate/ Dacron swabs – preferred

 Viral Transport Medium – all swabs for viral testing

INITIAL SPECIMEN PLATTING AND IDENTIFICATION METHODS

 Microscopic Examination of Clinical Specimens

 Gram Stain
 Selection and Inoculation of Primary Media
 Broth
 Agar

Classifications of Culture Media

General Isolation Media Support the growth of moist non-fastidious
(Supportive Media) bacteria
Nonselective Isolation BAP Contain nutrients and supplements
Media (Enriched/Nutritive CAP
Media)
Differential Media Provide distinct colonial appearance of
microorganisms. The medium which allows the
growth of more than one microorganisms of
interest but with morphologically distinguishable
colonies
Enrichment Broth Selenite Inhibit the growth of one organism while
Thioglycollate enhancing that of another by providing nutrients
When a substance is added to a liquid medium
which inhibits the growth of unwanted bacteria and
favors the growth of wanted bacteria
Selective Media HEA Contain agents that inhibit the growth of all
SSA bacteria except those that are sought
XLD When a substance is added to a solid medium
which inhibits the growth of unwanted bacteria but
permits the growth of wanted bacteria
Antibiotic Media Selective for a specific group of bacteria through
the addition of specific antibiotics

Components of BAP Chocolate Agar (CAP)

 Trypticase soy agar - Essentially the same as blood agar except that during preparation the
RED BLOOD CELLS are LYSED when added to molten agar base.
 Brucella agar or - The cell lysis provides for the release of intracellular nutrients such as:
 haemoglobin, hemin (X factor)
 BHI with 5% sheep blood  nicotinamide adenine dinucleotide (NAD or V factor) into the
agar for utilization by fastidious bacteria

ISOLATION OF BACTERIA FROM SPECIMENS: To enhance isolation of bacterial colonies, the loop
should be flamed for _______________ between the streaking of each subsequent quadrant.
 SEMI-QUANTITATIVE GRADING PROCEDURE FOR BACTERIAL
ISOLATES ON GROWTH MEDIA
Number of colonies visible in each quadrant
Score (growth) #1 (1st #2 #3 #4
quadrant)
1+ : Rare <10
2+ : Few or <10 <10
light
3+ : Moderate >10 >10 <10
4+ : Many, >10 >10 >10 >5
heavy

INCUBATION
1. Mesophilic
- Most pathogenic bacteria

2. Strict aerobes/ Obligate aerobes

- Require 0.03% CO2 and 21% O2 for survival

3. Strict anaerobes/ Obligate anaerobes

- Require 5-10% CO2, 80-90% N2, 0% O2
- Candle Jar (Gas Pak Jar)
 CO2 concentration: 3% CO2
 Catalyst: Palladium pellets
 Envelope: generates H2 and CO2 when water is added

Oxygen +
Oxygen -

4. Capnophilic
- 5-10% CO2, 15% O2
- Neisseria gonorrhoeae, Haemophilus spp.

5. Microaerophilic
 Increased CO2 requirement (8-10%), decreased O2 requirement (5-10%)

6. Facultative anaerobes
 Can survive with or without O2

METHODS OF STERILIZATION AND DISINFECTION

a. Physical methods of STERILIZATION

1) Incineration
 Most common method of treating infectious waste
 Burned to ashes at 870-980 C
 Safest method to ensure that no infective materials remain in samples or containers
when disposed
2) Moist heat
 Steam under pressure; 121 C, 15 psi, 15 min
 Fastest and simplest physical method
 Sterilize biohazardous waste and heat-stable objects
3) Dry heat
 Requires longer exposure times and higher temperatures than moist heat
4) Filtration
 Method of choice for heat-labile objects: antibiotic solutions, toxic chemicals,
radioisotopes, vaccines and carbohydrates
 Filtration of air is accomplished using HEPA filters designed to remove organisms larger
than ____ um
5) Ionizing (gamma) radiation
 Composed of short wavelength and high-energy gamma rays

 Most common chemical sterilant: _________________

 Sterilants for HEPA filters- 1.________________
2.________________
 Cold sterilization-1. _________________
2. _________________
b. Physical methods of DISINFECTION
1) Pasteurization
 Kills food pathogens without damaging the nutritional value or flavour adding
microorganisms
2) UV radiation
 Nonionizaing radiation
 UV rays are long wavelength and low energy do not penetrate well, and organisms must
have direct surface exposure, such as the working surface of a BSC, for this form of
disinfection to work
3) Boiling
 Kills vegetative bacteria at 100C

c. Chemical methods of DISINFECTION

1) Alcohols
 Ethyl or isopropyl alcohol (nonsporicidal)
2) Aldehydes
 Generally not used due to irritating fumes
3) Halogens
 Chlorine and iodine
o Household bleach
o Iodophore
o Tincture
4) Heavy metals
 1% silver nitrate
5) Quaternary Ammonium Compounds
 Inactivated by organic materials
6) Phenolics
 Amphyl

BIOLOGIC SAFETY CABINETS

BIOSAFETY LEVELS
BSL 1 No known potential to cause disease
E.g.

BSL2  Common agents of infection

E.g.

BSL3  Infectious aerosols

E.g.

BSL4  Exotic agents

E.g.

OBSERVATION OF GROWTH CHARACTERISTICS

- Size, Form, Elevation, Margin, Surface, Form of margin, Consistency, Density, Color and
Pigmentation
- __________________ - Grape-like, tortilla chips or fruity gum odor with serrated edges,
confluent growth colonies

PRELIMINARY BIOCHEMICAL TESTS

 Carbohydrate Utilization
- Fermentation
- Positive result: _______________
- Glucose fermentation test - pH indicator: ____________
o Oxidizer :
o Fermenter :

 Catalase
- Enzyme which breaks down hydrogen peroxide into water and oxygen
- 30% H2O2 reagent (Bailey & Scott)
- Positive result: _______________
o Weak bubbles: ____
o Inadvertently inoculated from BAP, weak bubbles are ____
- Helpful in differentiation of two gram positive cocci:
_________________ & _________________

 Cytochrome Oxidase
- Oxidase positive bacteria are able to transfer electrons to oxygen through aerobic bacterial
respiration systems
- Positive result: _______________
- Modified oxidase/Microdase test: __________ Oxidase Test: If an iron-containing
- When performing oxidase test, the ff. can be done: wire is used to transfer growth, a
i. Put a drop of reagent on the colony ________________ reaction may
ii. Rub the colony on a filter paper strip and add a drop of reagent result. Therefore, platinum wire or
iii. Rub the colony on a piece of filter paper containing the reagent wooden sticks are recommended.

 Coagulase
- An enzyme which converts fibrinogen to fibrin
- _____________ produces bound and free coagulase
o Bound coagulase/CF: ________________
o Free coagulase/CRF: ________________
- _____________ : gold standard plasma
- Positive result: after 4 hours

 Spot Indole
- Can be performed on bacteria grown only on media that contain tryptophan (SBA & Choc Agar)
- Positive result: _________
- Indole test: (+) _________

 PYR hydrolysis
- Differentiate gram-positive cocci: _____________ & _____________
- Positive result: _________
 TYPES OF HEMOLYSIS
Hemolytic Pattern Description
Alpha Incomplete or partial hemolysis
Beta Complete or clear zone of hemolysis
Gamma No lysis
Alpha-prime Wide zone of beta hemolysis surrounding the the
small zone of alpha hemolysis

MICROSCOPY, STAINING AND TRADITIONAL METHODS OF EXAMINATION

MICROSCOPY
A bright-field microscopy is
 Bright-field microscopy
easily adapted for dark-field
- Specimen’s image appears dark against a brighter background microscopy by replacing the
condenser with a dark-field
 Phase-contrast microscopy condenser that contains an
- Light beams are deflected by different thickness of an object opaque disk.

 Darkfield microscopy
- Specimens appear luminous against a background of little or no light

 Fluorescent microscopy
- Uses dyes, fluorophores which absorbs light in UV range, fluoresce and then emit light of a
greater wavelength
- Fluorochroming: direct chemical interaction between fluorescent dye and component of
bacterial cell

Direct Examination Methods

o Saline mount
o Hanging drop
o Iodine mountz
o Potassium Hydroxide
o India ink

Staining Methods
o Simple stains
o Differential stains
o Methylene blue

GRAM STAIN
- first introduced by Hans Christian Gram (1800s)
- bacterial morphology and gram reaction

Reagents Gram positive (+) Gram negative (-) Control organisms used in Gram Staining:
Primary stain Violet Violet (+) Positive control: S. aureus/[Link]
Mordant Violet Violet (-) Negative control: E. coli
Decolorizer Violet Colorless
(_________________)
Secondary stain Violet Pink/Red

Gram Stain Reporting *Satisfactory or unsatisfactory for culture:

Bacteria WBCs, RBCs, Epithelial cells
Many 4+ 10-20/OIF 25 or greater/LPF
Moderate 3+ 6-10/OIF 10-25/LPF
Few 2+ 3-5/OIF 2-10/LPF
Rare 1+ <10 identified on complete smear <2/LPF
None

Grade PMNs/LPF *A score of 0 or less indicates lack of inflammation / too much saliva
0 <10 Grading:
1+ 10-25
2+ >25
1+ *mucus
Grade Squamous Epithelial cells/LPF
-1 10-25
-2 >25
 Organisms found in sputum:
 “ASH larva”
 eggs of Paragonimus spp
 Echinococcus granulosus hooklets
 Entamoeba histolytica
 Entamoeba gingivalis
ACID-FAST STAINS - Mycobacteria  Trichomonas tenax
 Cryptosporidium spp
Ziehl-neelsen Kinyoun Acid-fast org Non-acid fast org  Possibly microsporidia
Primary stain Carbol fuchsin Red Red  Candida spp.
Mordant Steam/heat Tergitol/phenol Red Red
Decolorizer AcidAcid
Fastalcohol Red
Smear Reporting (WHO/CDC) Colorless
Secondary stain
FUCHSIN STAIN Methylene blue/malachite green
FLUOROCHROME Red
FLUOROCHROME Blue/green
REPORTING
(250x) (450x)
0 0 0 No AFB seen FLUORESCENT
1-2/300 fields 1-2/30 fields 1-2/70 fields Doubtful; request STAIN
another specimen o Rhodamine-
1-9/100 fields 1-9/10 fields 2-18/50 fields 1+ auramine:
1-9/10 fields 1-9/field 4-36/10 fields 2+ Mycobacteria
1-9/field 10-90/field 4-36/field 3+
>9/field >90/field >36/field 4+ o Acridine orange:
selectively binds
to ________________
-nonspecific: cannot distinguish G(+) from G(-)

o Calcofluor white: binds to the ____________ of fungi

-bleaching agent
-apple green fluorescence

FUNGAL STAIN
o Lactophenol Cotton Blue (LPCB):

o Methenamine Silver:

o Periodic Acid-Schiff (PAS):

ANTIBODY-CONJUGATED STAINS
o Fluorescein-conjugated
 Ex. DFA for Legionella pneumophila & Bordetella pertussis

o Enzyme-conjugated
 HPO, viral detection

AUTOMATION, IMMUNODIAGNOSTICS & MOLECULAR METHODS

Automated Identification Systems in Microbiology
 Colorimetry – measure color change
 Nephelometry – light scattering
 Fluorometry – fluorescence

Automated Systems for Identification

 MicroScan System
 Sensitire system
 BD phoenix

 MALDI-TOF __________________________________________
- A biophysical method that significantly reduces the time required to specifically
identify fungal organisms/all organisms

 VITEK Sytem
- Monitor optical density reading (GP/GN card)

Immunochemical methods to detect microorganisms

a Precipitin test : detects soluble antigens

o Double immunoelectrophoresis/ Ouchterlony
o Counterimmunoelectrophoresis : Immunoelectrophoresis + electrical current
o Nephelometry : light scattering
 b Agglutination
o Particle Agglutination
o Latex Agglutination
o Hemagglutination
o Coagglutination
o Liposome-enhanced agglutination

c Immunofluorescence
o Direct immunofluorescence (DFA)
o Indirect immunofluorescence (IFA)
o Specimens that contain yeasts, certain bacteria, mucus, leukocytes with Fc receptors
for antibody can cause non-specific binding = false positive immunofluorescence

Interpretation of Fluorescence Intensity using FITC

Negative NO apple-green fluorescence
1+ FAINT yet unequivocal apple-green fluorescence
2+ Apple-green fluorescence
3+ BRIGHT apple-green fluorescence
4+ BRILLIANT apple-green fluorescence

d Enzyme Immunoassay
o Solid-phase immunoassay
o Membrane-bound solid-phase immunoassay

ACUTE AND CONVALESCENT PHASE SPECIMENS

o Acute phase specimen – collected ______________________

o Antibody Titer – reciprocal of the highest dilution of patient’s serum in which antibody is detected
o Convalescent phase specimen – collected ____________________

*** _________________ an increase in the patient’s titer of two doubling dilutions (e.g. from a
positive result of 1:8 to a positive result of 1:32) is considered to be diagnostic of current infection.
(Bailey & Scott)

ACUTE INFECTION CHRONIC INFECTION

*Latent
Sign *S/S Symptom

IMMUNOSEROLOGICAL APPLICATIONS

PRECIPITIN TESTS: detects the formation of fine precipitate that denotes the reaction of soluble
antigen with antibody

FLOCCULATION TEST: Special type of ________________

NON-VENEREAL TEST
RPR VRL

VENEREAL TEST (agglutination tests)

Detect specific Treponema Antibodies

1. Treponema pallidum immobilization (TPI)

- GOLD standard
- drawback : uses live motile Treponema

2. Fluorescent Treponemal Antibody Adsorption Test (FTA-ABS)

 - most popular, most specific

3. Microhemeagglutination Assay (MHA-TP)

- uses RBC with sensitized Treponema antigen where Treponema antibody attaches =
agglutination

4. T. pallidum Indirect Hemeagglutination (TPHA)

- uses sensitized RBCs that aggregate when exposed to positive patient serum

5. T. pallidum Particle Agglutination (TP-PA)

- utilizes gelatin particles sensitized with T. pallidum subsp. pallidum antigens
- serum samples are diluted in a microtiter plate and sensitized gelatin particles are
added
- the presence of specific antibody causes the gelatin particles to agglutinate and form a
flat mat across the bottom of the microdilution well in which the test is performed

6. Particle Gel Immunoassay (PaGIA)

- contains recombinant antigens for the detection of T. pallidum antibodies in the
patient’s serum or plasma

MOLECULAR TECHNIQUES IN MICROBIOLOGY

i. Nucleic Acid Probes

 Nucleic Acid/genetic probes – short, specific sequences of single-stranded DNA or RNA.
Their purpose is to identify one or more sequences of interest within the nucleic acid

ii. Amplification Methods

 PCR (Polymerase Chain Reaction) – most widely used method to amplify the target
nucleic acid ; detects ________________
o ______________- invented the PCR in 1985 and later nick named
_________________
o Conventional PCR involves 25-50 repetitive cycles with each cycle comprising
three sequential reactions

Three-step process of PCR:

Denaturation - Heating of the sample to 94 C; Activate the tag polymerase

Annealing - Conducted at 50-58 C or higher

Extension - Tag (Thermus aquaticus) DNA polymerase – enzyme commonly used for
primer extension, which occurs at 72 C. It can withstand the denaturating
temperature of 94 C through several cycles

 Signal amplification – uses multiple enzymes and many layers of probes, which reduce
background noise and enhance detection
o Branched DNA (bDNA) – solid phase sandwich hybridization that uses multiple
sets of synthetic probes
o Hybrid capture assays – solution hybridization antibody capture method with
chemiluminiscence detection of the hybrid molecules

 Target Amplification – an enzyme or multiple enzymes produce copies of the target

nucleic acid
o Primers – short sequences of nucleic acid which are selected to anneal or
hybridize to a particular nucleic acid target ; requires 2 primers to amplify target
DNA fragment

Components of PCR
Template DNA Serves as target for PCR
Template for PCR __________ !!!
Oligonucleotide primers Used to start synthesis of new strands of DNA
Thermostable DNA Synthesizes new strands polymerase of DNA
Magnesium Chloride Required by DNA polymerase
Essential cofactor for tag polymerase
Buffer Ensures proper conditions and pH for DNA polymerase
Deoxynucleotides Used by DNA polymerase to synthesize new DNA
Thermal cycler Instrument that heats and cools PCR cycle steps
Minimize time lag requiredd
 *Restriction Fragment Length Polymorphisms (RFLPs) – involve the use of restriction
endonuclease enzymes, followed by electrophoresis of the DNA segments.
_______________ technique

*Nucleic Acid Sequencing – determine the nucleotide sequence of a nucleic acid

segment from an infectious agent

*Bacterial cells genetically evolve by:

1. Recombination with plasmids, transposons, and other bacterial chromosomes
2. Mutation and recombination
3. Use of the mechanisms of transformation, transduction and conjugation

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Categories and Types of Antibiotics
Categories Mode of activity Examples
β-lactam Antibiotics Inhibit cell wall synthesis Penicillins, cephalosporins,
aztreonam, carbapenems,
vancomycin
Alter bacterial cell membranes Disrupts cell membrane Bacitracin, polymixin, B and E
(colistin)
Inhibit protein synthesis Aminoglycosides, streptomycin,
gentamicin, tobramycin, amikacin,
tetracyclines, macrolides,
clindamycin, chloramphenicol
Folic acid synthesis inhibitors Completely inhibits folic acid Sulphonamides
synthesis
DNA gyrase activity inhibitors Interferes with DNA synthesis Quinolones
Bacterial enzyme/ protein Inhibition of bacterial enzymes
Nitrofurantoin
inhibitors or protein synthesis
DNA-dependent RNA Rifampin
polymerase inhibitor
Alter DNA synthesis metronidazole
____________________ - group of enzymes that convert the beta-lactam ring of the
penicillins and cephalosporins and imipenem into inactive forms

Mechanisms of Beta-lactam resistance:

1. 3.
2. 4.

DISK DIFFUSION SUSCEPTIBILITY TESTING

o Kirby-Bauer Disk Diffusion Test Relationship between MIC and ZOI:
 Mueller-Hinton Agar “The size of a zone of inhibition in a
 Bacterial Inoculum KB test is INVERSELY related to the
o __ MIC, which is the amount of antibiotic
o __ required to prevent bacterial growth in
o __ an overnight culture
 Antibiotics
 Results: Susceptible/sensitive, Intermediate or Resistant
 Minimum Inhibitory Concentration (MIC) – The minimum concentration of antimicrobial
agent needed to prevent visually discernible growth of a bacterial or fungal suspension
 Minimum Bacteriocidal Concentration (MBC) – The minimum concentration of
antimicrobial agent needed to yield a 99% reduction in viable colony-forming units of a
bacterial or fungal suspensions

[Notes]

Antibiotic Disc : Starting in the (high) center of the plate, the instrument deposits the highest
concentration of antibiotic, and from that point drug application proceeds to the (low) periphery of the
plate.

Strip : The strip in the center is impregnated with a gradient of antimicrobial drug, with the highest
concentration at the TOP OF THE STRIP
*Swarming area, discontinuous growth, poor growth or tiny colonies near the end of the zone SHOULD
BE IGNORED when interpreting the zone of a motile, swarming organism such as Proteus spp.
GRADIENT DIFFUSION TESTING

Breakpoint : Level of drug achievable in serum

- Refers to antimicrobial concentration in serum associated with optimal therapy using the
customary dosing schedule. An organism is susceptible if the MIC is at or below the breakpoint

SYNERGY TESTING
Synergy The activity of the antimicrobial combination is substantially greater
than the activity of the single most active drug alone
Indifference The activity of the combination is no better or worst than the single
most active drug alone
Antagonism The activity of the combination is substantially less than the activity of
the single most active drug alone (an interaction to be avoided)

ESBL (___________________________) - a plasmid-mediated mechanism involving genes that code for

beta-lactamase
3 Principles of Specific Method to Determine Bacterial Resistance (Beta-lactamase and Cefinase)

1. _____________________
Positive result:

2. _____________________
Positive result:

3. _____________________
Positive result:

AmpC – mediate resistance to cephalothin, cefazolin, cefoxitin, cephalosporinase enzyme

Carbapenemase – first found in __________________

o Modified Hodge Test – Escherichia coli is plated onto MHA, either ertapenem or meropenem disk
is placed in the center of the plate
o If carbapenemase is present: __________________
o If carbapenemase is absent: __________________

D TEST – Inducible Clindamycin Resistant (+)

(+) Flattening of Cd zone adjacent to E zone to give “D” pattern.
_____ gene results in resistance to erythromycin and inducible or constitutive resistance to clindamycin ;
______ gene results in resistance to erythromycin but susceptibility to clindamycin

Supplemental Methods for Detection of Antimicrobial Resistance

Test Purpose Result
Oxacillin agar screen Detection of staphylococcal resistance to Growth= RESISTANT
penicillinase-resistant penicillins (e.g. No growth = SUSCEPTIBLE
oxacillin, methicillin, nafcillin)
Vancomycin agar screen Detection of enterococcal resistance to Growth= RESISTANT
vancomycin No growth = SUSCEPTIBLE

Aminoglycoside screens Detection of acquired enterococcal high- Growth= RESISTANT

level resistance to aminoglycosides that No growth = SUSCEPTIBLE
would compromise synergy with a cell wall
active agent (e.g. ampicillin or
vancomycin)
Oxacillin disk screens Detection of streptococcus pneumonia Inhibition zone ≥ 20 mm: penicillin susceptible
resistance to penicillin
Inhibition zone  ≤19 mm: penicillin resistant,
intermediate or susceptible; further testing
by MIC method is needed

D test Differentiate clindamycin resistance Blunting of clindamycin zone to give “D” pattern,
among S. aureus resulting from efflux indicating inducible clindamycin resistance
(msrA gene or MSLB resistance)
GRAM-POSITIVE COCCI

Staphylococcus
 Gram-positive cocci in tetrads or clusters
 Non-motile, nonspore forming
 Most species are facultative anaerobe
 Medium-sized, raised, creamy colonies on blood agar or CAN with white, cream or golden
pigmentation called _______________
 Salt-tolerant – Staphylococcus Micrococcus
o able to grow in 7.5% - 10% Catalase + +
NaCL Aerobic Growth + +
 Mannitol Salt Agar (MSA) Anaerobic Growth + -
 CHO: Manitol Glucose Utilization (media) FERMENTER OXIDIZER
Modified oxidase - +
 pH indicator: Phenol Red
Benzidine - +
 MF: ____________ Resistant to lysostaphin & - +
 NMF: ___________ furazolidone
Resistant to bacitracin + -

Staphylococcus aureus
- Gram positive cocci in clusters
- Medium to large, raised colonies on SBA and CAN with cream to golden yellow pigmentation
- B-hemolytic on SBA
- Coagulase positive (Bound and Free coagulase)
- Latex Agglutination Test
- DNAse positive
- Associated with skin and soft tissue infections, pneumonia (most prevalent opportunistic bacterial
pathogen causing cystic fibrosis in <10 year old patients), osteomyelitis, septicaemia,
endocarditis, food-borne disease and toxic shock syndrome
- Most common cause of adult joint infection (Ciulla)
- Most common cause of food poisoning in the US

Coagulase-Negative Staphylococci (CoNS)

Staphylococcus epidermidis
- White, creamy colonies, non-hemolytic
- Normal flora of the skin and mucous membranes of humans and other animals
- Associated with prosthetic valve endocarditis and can colonize prosthetic devices
- NOVOBIOCIN _______________

Staphylococcus saprophyticus
- Major cause of UTI in young women (especially in sexually active ones)
- Non-hemolytic
- NOVOBIOCIN _______________

Streptococcus
- Gram positive cocci in pairs or chains
- Catalase-negative
- Small pinpoint and translucent or clear colonies
- Facultative anaerobe
- Non-motile, nonspore forming
- Require supportive or enriched media (blood agar) for growth

Species Lancefield Bacitracin PYR CAMP Hippurate Bile VP

Antigen Hydrolysis Esculin
S. pyogenes A S + - - - -
S. agalactiae B R - + + - -
S. dysagalactiae C&G R - - - V -
subsp. dysagalactiae &
subsp. equisimilis
S. equi C&G R - - - - -
subsp, equi &
subsp. zooepidemicus
S. anginosus A, C, F, G R - - - + +
 Streptococcus pyogenes (Group A Streptococcus)
- Causes bacterial pharyngitis, skin infections (Erysipelas), and other invasive diseases.
Associated with complications or sequelae such as Rheumatic Heart disease and Acute
glomerulonephritis
- Most common cause of bacterial pharyngitis (Strep throat)
- Scarlet fever
- Skin infections or pyoderma, impetigo, cellulitis, wound infections and erysipelas
- BACITRACIN ________________
- PYR ________________
*** Purpose of stabbing: Stabbing the inoculating loop vertically into the agar after streaking the
blood agar plate allows subsurface colonies to display hemolysis caused by streptolysin O
 SLO : Subsurface hemolysis
 SLS: Surface hemolysis

Streptococcus agalactiae (Group B Streptococcus)

- Medium sized, flat and translucent or opaque colonies
- Narrow zone of B-hemolysis or non-hemolytic
- CAMP _____________
- Hippurate hydrolysis _______________
- Bacitracin resistant; SXT resistant
- PYRase negative
- Bile-esculin hydrolysis negative
- Todd-Hewitt broth
- Skin infections or pyoderma, impetigo, cellulitis, wound infections and erysipelas. Causes
NEONATAL MENINGITIS

Viridans Streptococci
- A-hemolytic on SBA
- Most are normal flora of human oropharynx, GI, and female genital tracts
- Important agents of subacute bacterial endocarditis
- S. mutans- dental carries

Nutritionally Variant Streptococci (NVS) : Abiottrophia & Granulicatella

- Viridans strep that require cysteine or ________________ for growth
- Normal flora of the human oral cavity and have been implicated as endogenous agents in
endocarditis

Streptococcus bovis
- Bile-esculin positive
- PYR-negative, does not grow in 6.5% NaCl
- Either a-hemolytic or nonhemolytic on SBA

Streptococcus pneumoniae
- Gram positive diplococcic in lancet or bullet shape and form chains
- Requires 5-10% CO2
- A-hemolytic on SBA
- OPTOCHIN TEST (Taxo P/ P-disc): __________________________
- Bile solubility
- Capsular swelling test
- Diseases attributed include pneumonia, bacteremia, sinusitis, otitis media and meningitis
- Most common cause of bacterial community-acquired pneumonia
- Leading cause of otitis media in infants and small children
- Rust-tinged sputum; LOBAR PNEUMONIA

Enterococcus
- Able to grown in 40% bile and hydrolize esculin
- E. faecalis – causes almost 80-90% of human enterococcal infections
- E. faecium – 5-10%
- Normal flora of GI tract, skin, oral cavity and gut
- Intrinsically resistant to AMINOGLYCOSIDES. It is important to identify high-level aminoglycoside
resistace (HLAR) to provide appropriate therapy

Other Catalase-Negative Streptococci-like Organisms

 Rare human pathogens:
o Aerococcus viridans
o Leuconostoc (MRS Broth)
o Pediococcus
 o Gemella

Differentiation of Group D Streptococci and Enterococci

Bile esculin 6.5% NaCl broth PYRase Penicillin
Group D + + + R
Enterococcus
Streptococcus + - - S
bovis group D
non-
Enterococcus
Not group D - - -

GRAM-NEGATIVE COCCI
Neisseria
 Gram-negative diplococcic with adjacent ends flattened, resembling tiny coffee or kidney beans in the
Gram stain. Except for ______________: the only human species that is rod shaped.
 Obligate aerobes but preferred to be capnophilic
 Pathogenic Neisseria are very fastidious
 All Neisseria are OXIDASE POSITIVE (+)
 All Neisseria are CATALASE POSITIVE (+) except for N. elongate
 Pili: hairlike structures on the bacterial cell that enable the bacteria to bind to human cells

Neisseria gonorrhoeae
 Agent of gonorrhoea, an acute pyogenic infection mainly of the mucous membranes of the
endocervix in females and the urethra of males
 _________________ - extragenital infection, a conjunctivitis acquired by newborns from an
infected mother during delivery
 This organism can also cause DGI, endocarditis and gonococcal arthritis
 Produces acid from glucose
 Specimen to be collected and processed
o Females: endocervical specimens
o Males: urethral

Culture Media for N. gonorrhoeae

1. Thayer-Martin Identification of N. gonorrhoeae
- Vancomycin Gram stain – Gram-negative
- Colistin intracellular diplococci.
- Nystatin NEVER REPORT: “Presence of N.
2. Modified Thayer-Martin gonorrhoeae in gram stain. CULTURE
- Vancomycin IS CONFIRMATORY.
- Colistin
- Nystatin
- __________________
3. Martin-Lewis
- Vancomycin
- Colistin
- Trimethoprim Lactate
- __________________
4. New York City (also supports growth of _________________)
- Vancomycin
- Colistin
- Trimethorprim Lactate
- __________________

Neisseria meningitidis
 Etiologic agent of meningitis and meningococcemia
 May be carried asymptomatically in nasopharynx
 Isolate on SBA or CA
 Produces acid from glucose and maltose
 Associated to:
___________________: uncontrollable clotting within the bloodstream
___________________: adrenal gland haemorrhage
 Nasopharyngeal specimen

Moraxella (Branhamella) catarrhalis

 Formerly considered to be non-pathogenic member of the normal flora of the upper RT
 Nonpigmented colonies, opaque, gray and smooth. Nonhemolytic when grown in SBA
 Oxidase (+) but distinctly different from Neisseria spp. Due to its ________________ property
 DNAse (+) positive
 ______________ disk (+) positive