Microbiology - Bacte, Myco, Viro
Microbiology - Bacte, Myco, Viro
Microbiology - Bacte, Myco, Viro
MICROBIOLOGY
( BACTERIOLOGY, MYCOLOGY, VIROLOGY)
Prof: Charles U. Arconado, RMT, MAEd, ASCP, AMT, DTA, IFBA PC, 9th Placer
3. Virology 4%
a. General characteristics, transmission and diseases
b. Collection, transport and examination of clinical specimens
BACTERIOLOGY
I. Classification
• the organization of microorgnisms that share similar morphologic, physiologic, and
genetic traits into specific groups or taxa
1. Morphology (4 Bacterial Morphology)
a. Cocci
b. Bacilli
c. Coccobacilli
d. Spiral (Leptospira, Borrelia, Treponema)
2. Physiology
3. Genetic make-up
II. Nomenclature
• the naming of microorganisms according to established rules and guidelines
• microorganisms are named based on:
a. Scientist who discovered
b. Disease they cause
c. Where the organism was first isolated
III. Identification
• is the process by which a microorganism’s key features are delineated
• Microorganisms can be identified based on their biochemical activities.
Ex.:
1. Staphylococcus aureus is known to coagulase positive.
2. Staphylococcus spp. is known to be catalase positive.
3. Enterobacteriaceae is fermenter of carbohydrates.
Genotype – relate to the organism’s genetic make-up, nature of the organism’s genes and constituent
nucleic acids.
Phenotype – based on features beyond the genetic level and include readily observable characteristics
and those characteristics that may require extensive analytic procedures to be detected.
Terminologies:
• Strain
• Biovars
• Morphovars
• Serovars
Prokaryotes
> ”Pro” meaning before and “karyon” meaning nucleus, nut, kernel or corn
> These are organisms that do not contain a true nucleus (instead, nucleoid) surrounded by nuclear
membrane, characteristic of lower forms such as bacteria.
> They do not contain organelles and all functions take place in the cytoplasm or cytoplasmic membrane
(the counterpart of cytoplasm in eukaryotic cells is nucleoplasm)
1. Cell Wall
• Protection or support – prevents rupture
• Serves as an anchorage for flagella (organ for locomotion)
• Gives shape to bacteria
• Its thickness is the basis of gram stain
• Another name: peptidoglycan / murein layer
• Composition: disaccharides, pentapeptides
• Some are made up of teichoic acid and lipoteichoic acid
GRAM GRAM
CODE KEY STEPS REAGENT/S DURATION/TIME
POSITIVE NEGATIVE
V Primary/Initial Staining CRYSTAL 1 MINUTE (rinse) Violet/Purple Violet/Purple
VIOLET
I Mordanting IODINE 1 MINUTE (rinse) Violet/Purple Violet/Purple
A Decolorization/Differentiation ACETONE Quick ON & Violet/Purple COLORLESS
ALCOHOL Rinse
S Counterstaining SAFRANIN 30 seconds Violet/Purple RED/PINK
RED OR (rinse)
“O”
KEY STEPS
ACID NON-ACID
CODE (MODIFIED KINYOUN REAGENT/S DURATION/TIME
FAST FAST
METHOD)
CARBOL
C Primary/Initial Staining RED RED
FUCHSIN 5 MINUTES
CHEMICAL: (rinse)
T Mordanting RED RED
TERGITOL
ACID ALCOHOL Continuous drop
A Decolorization/Differentiation (1% H2SO4 in until washing is RED COLORLESS
70% Ethanol) colorless (rinse)
METHYLENE 30 seconds
M Counterstaining RED BLUE
BLUE (rinse)
Acid-fast organisms:
*Mycolic acid / hydroxymethoxy acid – the substance unique in the cell wall of AFO
• Mycobacteria
• Nocardia
• Coccidians
Atypical microorganisms – Organisms that have characteristics different from the other organisms
-e.g. Rickettsia, Chlamydia, Mycoplasma and Ureaplasma
2. No Cell Wall:
• They contain sterols in their cell membrane (like fungi).
a. Mycoplasma
b. Ureaplasma
Functions:
• Generate ATP
• Site of photosynthesis
• Site of respiration
• Regulates osmotic pressure
• Transports solute
1. Glycogen
2. Carboxysomes – Cyanobacteria (produces pigment), Nitrifying bacteria, Thiobacilli
3. Gas vesicle (bacterias that are usually can be found beneath earth surface) – Halobacteria,
Cyanobacteria, Thiothrix
4. Cyanopophysin
5. poly-β-hydroxybutyrate
6. Metachromatic granules / volutin / polyphosphate granule
Corynebacterium diptheriae - Babes-Ernst Granules
Mycobacterium tuberculosis - Much’s Granules
Yersinia, Pasteurella, Brucella - Bipolar Bodies (if stained with Wayson’s stain shows “safety-pin
appearance)
Actinomyces, Nocardia - Sulfur Granules
e. Endospores
- bacterial structues that resist extreme environmental condition
- dipicolinic acid + calcium = calcium dipicolinate
- Bacillus, Clostridium
Terminal Spore – Clostridium tetani
Central Spore – Bacillus anthracis
Subterminal Spore – Clostridium botulinum
- Sporogenesis/Sporulation – process of spore production
- Germination – the end of the spore’s dormant stage
4. Cell Appendages
a. Glycocalyx
b. Flagellum
c. Pili / Fimbriae
A. Glycocalyx
- outward complex of polysaccharides
- attachment of bacteria to the cell
- resists phagocytosis / dessication
B. Flagellum
- organ for locomotion
*Taxis – is the movement of the bacteria toward or away from a particular stimulus
> Protein component: flagellin
MOTILE NON-MOTILE
1. Alcaligenes 1. Bacillus anthracis
2. Bacillus cereus 2. Bordetella
3. Bacillus subtilis 3. Brucella
4. Campylobacter 4. Clostridium perfringens
5. Clostridium botulinum 5. Corynebacterium
diptheriae
6. Clostridium tetani 6. Erysipelothrix
7. Escherichia coli 7. Haemophilus
8. Listeria 8. Mycobacterium
9. Proteus 9. Pasteurella
10. Providencia 10. Staphylococcus
11. Pseudomonas 11. Streptococcus
12. Salmonella 12. Shigella
13. Vibrio 13. Klebsiella pneumoniae
14. Neisseria
Eukaryotic Cell:
>meaning “true nucleus”
> organisms whose cells have a true nucleus bounded by a nuclear membrane within which lie the
chromosomes.
> cells of higher plants and animals, fungi and protozoa; morphologically more complex and larger than
prokaryotes
1. According to O2 requirement
a. Aerobic bacteria
>use oxygen and grow well in room air
> Obligate aerobic
- Bordetella
- Brucella
- Pseudomonas
- Haemophilus
b. Anaerobic bacteria
> Obligate anaerobic – is an organism that strictly does not require the presence of oxygen; die in the
presence of oxygen.
- Bacteroides
- Clostridium
- Actinomyces
> Facultative anaerobic- organisms that do not require oxygen but grows better in the presence of
oxygen
- Escherichia coli
- most of the pathogens
B. As to energy source
> Phototroph – organisms that use light
> Chemotroph – organisms that use the energy produced by the oxidation of organic or
inorganic compounds.
C. As to electron source
> Lithotroph – they reduced inorganic molecules
> Organotroph – they require organic substances (CHO, CHON, lipids) for growth and
multiplication; all bacteria that inhabit the human body fall into this group.
4. As to temperature requirement
35-37οC – is the optimum temperature for most bacteria
C. Thermophilic – 50-60οC
> Bacillus stearothermophilus (indicator of autoclave)
> Thermus aquaticus
D. Hypothermophilic – 80-113οC
> Sulfolobus
> Pyrococcus
> Pyrodictium
E. Extremophilic – prokaryotes that are able to live at unusual conditions like absence of
oxygen, increased temperature and below earth’s surface
> Bacillus infernus
*Thermal Death Time – time required to kill the bacteria at constant temperature
*Thermal Death Point – temperature required to kill the bacteria at constant time
e.g. Pasteurization: 63οC – 15mins.
5. As to pH requirement
A. Acidophilic – pH 0-5.5
> Lactobacillus acidophilus
> Sulfolobus
> Picrophilus
> Acontium
B. Neutrophilic – pH 5.5-8.0
> E. coli
> most of the pathogenic bacteria
C. Alkalinophilic – pH 8.5-11.5 (ex. bile salt)
> Vibrio
> Bacillus alcalophilus
> Natronabacterium
*diagnostic laboratory media (agar) for bacterial isolation are usually adjusted to a final pH between 7.0
& 7.5.
6. As to moisture requirement
– bacteria love moist environment (humidophilic)
7. As to pressure requirement
> Barophilic – organisms that grow rapidly in the presence of high pressure 600 to 1100 atm.
pressure
Growth
Generation – doubling replication
BACTERIAL GENETICS
3 Major Aspects:
• Organization and structure of genetic material
• Replication and expression of genetic information
• The mechanisms by which genetic information is changed and exchanged among bacteria
3. The mechanisms by which genetic information is changed and exchanged among bacteria
Recombination – it occurs when a portion of the genetic material that originated from one bacterial cell
(donor) is transferred into a second bacterial cell (recipient).; it involves a number of binding proteins,
with the RecA protein playing a central role
1. Transformation
- involves recipient cell uptake of free DNA released into the environment when another bacterial cell
(donor) dies and undergo lysis.
Ex.: Griffith’s experiment
2. Transduction
- DNA of 2 bacteria will come together to form other traits
- this process is mediated by viruses that infect bacteria (bacteriophage)
3. Conjugation
- it involves cell-to-cell contact
- the sex pilus / transposon (donor) establishes a conjugative bridge that serves as the conduit for DNA
transfer from donor to recipient cell.
- the plasmid (independent circular DNA, nonchromosomal element) may be transferred by conjugation,
but not all plasmids are capable of conjugative transfer.
ANTIGENIC SHIFT – a major genetically determined change in the antigenic character of a pathogen
which may result to not being recognized by the host’s immune system; responsible for most of the
pandemic infections
ANTIGENIC DRIFT – minor antigenic changes as a result of mutation in pathogen strains and facilitate the
pathogen avoid host immune responses; responsible for most of the epidemic infections
BACTERIAL METABOLISM
> Glycolysis – first stage in carbohydrate metabolism; oxidation of glucose to pyruvic acid; is the
major route of glucose metabolism in most cells
> Kreb’s Cycle – an enzyme system which convert pyruvic acid to carbon dioxide in the presence of
oxygen with accompanying release of energy, which is trapped in the form of ATP molecules
Substrate: acetyl CoA
Final electron acceptor: O2 (aerobic)
- Homolactic fermenters – utilize the glycolytic pathway and the enzyme, lactate dehydrogenase directly
reduce almost all the pyruvate to lactate.
- Heterolactic fermenters – they produce substances other than lactate.
Sterilization – it refers to the removal of all forms of life, including bacterial spores
Disinfection – it refers to the removal of pathogenic forms only
• Physical Method
• Chemical Method
I. Physical Method
A. Application of heat / high temperature
B. Filtration
C. Low / cold temperature
D. Dessication / lyophilization
E. Osmotic pressure
F. Radiation
B. Autoclave
> steam under pressure
> fastest and simplest method of sterilization
> can't kill Hepatitis A virus
a. 121οC, 15-20 minutes, 15 psi – materials in the lab
b. 132οC, 30–60 minutes, 15 psi – for decontaminating medical wastes
BioIogical indicators: Bacillus stearothermophilus or Clostridium PA 3679
D. Inspissation
> for sterilization of materials with high protein content such as LJ (Lowenstein-Jensen) medium of
Mycobacterium tuberculosis
> thickening via evaporation
> 70-80οC for 2 hours (3 consecutive days)
DECIMAL REDUCTION TIME – it refers to the time in minutes to reduce the bacterial population or
spores by 90% at a specified temperature
- widely used in food industry (Food Microbiology)
B. Filtration – method of choice for sterilization of vaccines, radioisotopes and toxic chemicals
1. Depth filters – consist of fibrous / granular material
e.g. asbestos
2. Membrane filters (circular filters) – they are composed of cellulose acetate / polycarbonate (0.1mm
thick)
- used to sterilize antibiotics and oil products
a. Liquid filtration
b. Air filtration – it uses high efficiency particulate air (HEPA) filter (remove organisms larger than
0.3µm)
c. Critical sterilizing – for parenteral equipments and IV drugs (0.22µm membrane filters are used)
d. 0.45µm membrane filters – filtration of fungi, molds and protozoa
E. Osmotic Pressure
> the use of increase osmotic pressure or high concentrations of salts and glucose can cause death of
bacteria
PLASMOLYSIS – as water leaves the cell, the protoplasm shrinks away from the rigid cell wall
F. Radiation – when it passes through the cells, it creates H+ ions and free radicals and some
peroxidases, which in turn cause different intracellular damage
BioIogical indicator: Bacillus pumilus
1. Ionizing radiation
- it causes mutation in DNA and produce peroxides
- it is used for sterilization of syringe and needles
e.g. ƴ ray (1,500-2,500 radiation), x-rays and sunlight
- it has shorter wavelength
2. Non-ionizing radiation
- it causes damage to cellular DNA by producing thymine dimers
- it is used to disinfect operating rooms and laboratories
e.g. UV light (10-400 nm)
- 260nm – most lethal
- it has longer wavelength
PC = highest dilution of disinfectant that will kill organism at a given time / highest dilution of phenol
that will kill organism at a given time
PATHOGENESIS OF INFECTION
A. Infection – involves the growth and multiplication of microorganisms that result in damage to the
host
B. Disease – a specific illness or disorder characterized by a recognizable set of signs and symptoms
attributable to hereditary infection, diet or environment.
5. Endotoxins
- secreted or excreted by dead bacteria
- toxicity is due to lipid A (LPS)
- found on outer membrane
- heat stable (121οC)
- from G(-)
- it stimulates the fever center (Hypothalamus) ------> Interleukin I
- low toxic / antigenic
- synthesized directly by chromosomal genes
EXOTOXIN ENDOTOXIN
Representative Diseases:
Gas gangrene, tetanus, Typhoid fever, UTI,
botulism, diphtheria, meningococcal infection,
scarlet fever meningitis
Effects On Hosts:
Destroys particular part of Clotting (DIC), fever,
the host’s cell hypotension, shock and
death
ROUTES OF TRANSMISSION
1. Airborne Transmission
• Droplet nuclei – are the small residues from the evaporation fluid from larger droplets and are
light enough to remain airborne for long periods.
2. Transmission by food and H2O
• Infections occurs via the Fecal-oral Route
3. Close Contact
4. Arthropods
5. Cuts and bites
Epidemiology – the study of occurrence, distribution, and causes of disease and injury
1. Carrier – is a person or animal who harbors and spreads a microorganism that causes disease but who
does not become ill
a. casual / acute / transient carrier – harbors the microorganism temporarily for a few days or
weeks
b. chronic carrier – remains infected for a relatively long time, sometimes throughout life
(typhoid bacillus).
c. convalescent carrier - an individual who has recovered from infection but continues to harbor
large numbers of the pathogen.
d. active carrier – is an individual who has an overt clinical case of the disease.
2. Endemic – when an organism or disease is constantly present in a population; it is indigenous to a
geographic area or population
3. Epidemic – when a disease affects significantly large number of people at the same time in a
geographic area; influenza is a classic example of an epidemic.
4. Pandemic – epidemic over a large area affecting tens of millions of people.
5. Incidence Rate – the number of times a new event occurs in a given period.
6. Incubation Period – the time between exposure to a pathogen and the onset of symptoms; difficult to
determine because individuals often hae difficulty pinpointing the date or time of exposure – an
individual may be infectious during this period.
7. Morbidity Rate – the rate at which an illness occurs; a measure of the infectiousness of an organism;
is the number of cases of a disease in a specified population during a defined time interval.
8. Mortality Rate – is the number of deaths due to a disease in a population.
9. Reservoir – source of an infection, may be a person, animal or something in the environment.
STAINS
CAPSULE:
Hiss
Anthony
Nigrossin
Gins
ENDOSPORES:
Dorner
Heat and acetic acid
Wirtz-Conklin
Schaeffer-Fulton
METACHROMATIC GRANULES:
LAMB (Loeffler’s Alkaline Methylene Blue)
Alberts
Neisser’s
Ljubinsky
FLAGELLA:
Gray
Leifson
Fischer-conn
SPIROCHETES:
Levaditi
Silver impregnation
Warthin-Starry
Fontana Tribondeau
ANTIMICROBIALS (ANTIBIOTICS)
Classifications:
1. Natural drugs – produced by bacteria or fungi
2. Semisynthetic drugs – these are chemically modified natural drugs with added extra chemical groups
3. Synthetic drugs – chemically produced drugs
• MIC (Minimal Inhibitory Concentration) - lowest concentration of drugs that inhibits the
microorganism
• MBC (Minimal Bactericidal Concentration) – lowest concentration of drugs that kills the
microorganism
- AKA: MLC (Minimal Lethal Concentration)
Therapeutic index
• Is the ratio of the toxic dose to the therapeutic dose
increase TI = more effective
Action of antimicrobials
• Inhibiting cell wall synthesis
• Inhibiting protein synthesis
• Inhibiting nucleic acid synthesis
• Destroying the cell membrane
• Inhibiting essential metabolites
Notes to remember:
• Vancomycin – inhibits translocation and elongation of peptidoglycan; a glycopeptide; cup-
shaped molecule; structurally similar to teicoplanin drug
• Isoniazid - acts only on growing cells; can be either be bactericidal or bacteriostatic
• Penicillin - against D-alanyl-D-alanine (peptidoglycan)
• Penicillin G - destroyed by stomach acids (parenterally)
• Penicillin V - not destroyed by acid (orally)
• Penicillinase-resistant penicillin – Methicillin, Nafcillin, Oxacillin
• Cephalosporins – Cephalothin, Cefoxitin, Ceftriazone, Cephalexine, Cefixime, Cefoperazone
• Ampicillin - effective against G(+) and G(-); not destroyed by acid
• Carbenicillin - effective against Pseudomonas and Proteus; not destroyed by acid
• β-lactamase – the enzyme present with some bacterias responsible for Penicillin-resistance
• Bacitracin is not considered as a strict antibiotic.
• Β-lactamase test
1. Chromogenic cephalosporin test
(+) result: pink/red
2. Iodometric test
- uses iodine and penicillin
(+) result: colorless
3. Acidimetric test
- uses phenol red and penicillin
(+) result: yellow
Notes to remember:
• 30’s ribosome inhibitor - misreading of mRNA; interferes w/aminoacyl-tRNA
• 50’s ribosome inhibitor - inhibition of peptidyl transferase; inhibits peptide chain elongation
• Tetracycline - liver and kidney damage; yellowing of the teeth of children
• Chloramphenicol - Depression of bone marrow (aplastic anemia); leukopenia (toxic side effect)
• Aminoglycosides - cyclohexane ring (deafness); loss of balance (toxic)
• Macrolide antibiotics - Erythromycin, Clindamycin, Azithromycin
*Others
• Anti-TB Drugs- RIPES
- Rifampicin
- Isoniazid (INH)
- Pyrazinamide
- Ethambutol
- Streptomycin
*Anti-Fungal Agents
• Polyenes (Amphotericin B)
• Azole (Clotrimazole, Ketoconazole, Fluconazole)
2. Macrodilution
- broth volume: >10mL
Turbidity Standard:
> 0.5% Mc Farland Turbidity Standard
99.5mL of 1% H2SO4 + 0.5mL of 1.175% BaCl2
Notes to Remember:
• The diameter of the zone of inhibition around disk is measured in millimeters using a caliper
• The zone of inhibition is inversely related to MIC – the larger the zone of inhibition, the lower
the MIC
• MHA containing 5% sheep’s blood is used for testing streptococci and other fastidious
organisms
D. E test
• Is a dilution test based on the diffusion of a continuous concentration gradient of an
antimicrobial agent from a plastic strip into an agar medium.
• (+) result: ellipse of growth inhibition
• An alternative susceptibility test for fastidious bacteria (S. pneumoniae and H. influenzae).
Bacteriology Instruments
1. Bacteriology Incubator
• Set at 35 +/- 2 C
• Standard incubation period for aerobic culture: 18-24 hours
• Standard incubation period for anaerobic culture: 24-48 hours
2. Durham tube
• Used in Water Bacteriology
• Gas detector
3. Inoculating needles
• Composition: nichrome or platinum
• Measurement: Not longer than 5 cm
4. Cotton swab
• Carrier state
• Toxic to Neisseria but good for viruses
5. Tuberculin syringe
• Mantoux test – one of the skin tests for M. tuberculosis
6. Pasteur pipette
• Type of pipette to transfer liquids
Biosafety Levels
• BSL-1 (Biosafety Level 1) – suitable for work involving well characterized agents that are
not known to cause disease in immune-competent adult humans, and prevent minimal
potential hazard to personnel and the environment. Risk group contains biological
agents that pose low risk to personnel and the environment.
Ex.: Escherichia coli strain K12, Mycobacterium gordonae, Bacillus subtilis,
Agrobacterium radiobacter, Aspergillus niger, Bacillus thurigiensis, Lactobacillus
acidophilus, Micrococcus luteus, Neurospora crassa, Pseudomonas fluorescens, Serratia
marcescens.
• BSL-2 (Biosafety Level 2) – builds upon the practices, procedures, containment
equipment and facility requirements of BSL-1. BSL-2 is suitable for work involving
laboratory both human and animals infected with agents associated with human disease
and pose moderate hazards to personnel and the environment. It also addresses
hazards from ingestion as well as from percutaneous and mucous membrane exposure.
Risk group contains biological agents that pose moderate risk to personnel and the
environment.
Ex.: Influenza virus, HIV, Lyme disease, Yersinia pestis, Bacillus anthracis, Streptococcus
pneumonia, Salmonella cholerasuis, COVID-19 positive non-respiratory specimens
• BSL-3 (Biosafety Level 3) – involves practices suitable for work with laboratory both
human and animals infected with indigenous or exotic agents, agents that present a
potential for aerosol transmission, and agents causing serious or potentially lethal
diseases. BSL-3 builds upon the standard practices, procedures, containment
equipment, and facility requirements of BSL-2. This biosafety level practice is required
FAMILY MICROCOCCACEAE
• Staphylococci
• Micrococci
• Stomatococcus (animals)
• Planococcus (animals)
Diagnostic Tests
1. Gram staining:
STAPHYLOCOCCUS - G(+) cocci in clusters
MICROCOCCI - G(+) cocci in tetrads (by 4) / sarcina (by 8)
Enzyme: hemolysin (bacterias which has this enzyme has the ability to cause anemia)
5. Catalase Test
- Differentiates Staphylococcus and Streptococcus
Reagent: 3% H2O2
(+) result: bubbling formation / effervescence / gas bubbles
Additional Notes:
*Stomatococcus – Modified Oxidase (-), Lysostaphin (R) and Furazolidone (R)
*Staphylococcus is more resistant to lysozyme (destroys peptidoglycan)
7. Coagulase Test
Staphylococcus aureus
- Protein A – cell wall, anti-phagocytic, virulence
- β hemolytic colonies
- normal flora of skin
- associated w/ foreign things (e.g. tampon)
- #1 wound infection; #1 osteomyelitis (septic arthritis)
- cause skin infections: boils, carbuncles, furuncles, folliculitis, cellulitis, impetigo, SSS (Scalded Skin
Syndrome), bacteremia, endocarditis
- pigment: cytochrome / lipochrome / staphyloxanthin (golden yellow or yellow orange pigment)
- inhibited by bile
- nitrate reduction test (+), gelatin hydrolysis test (+), novobiocin susceptible
- MRSA (Methicillin-Resistant Staphylococcus aureus) – grows in 35◦C with 2% NaCl; Treatment:
Vancomycin
- Culture media: BAP, Vogel-Johnson medium, Chapman medium, Tellurite glycine medium, P agar, PEA
(Phenyl Ethanol Agar), Columbia CNA (Colistin-Nalidixic Acid) agar
8. DNase test
Detects DNase (deoxyribonuclease) – degrades DNA
Bacterias which are DNase test (+): “SMASH VSS”
-Serratia marcescens
-Moraxella catarrhalis
-Aeromonas
-Streptococcus pyogenes
-Helicobacter pylori
-Vibrio cholerae
-Stenotrophomonas maltophilia
-Staphylococcus aureus
Medium: DNA medium
• Two methods:
a. Toluidine blue – (+) = pink zone
Methyl green – (+) = clear zone
b. HCl precipitation – no precipitation after 1N HCl when DNase (+)
(+) = clear zone; (-) = no clearing
S. epidermidis – causes endocarditis (associated with Prosthetic Heart Valve Surgery infection)
- normal flora of skin
- Novobiocin SUSCEPTIBLE
- non-hemolytic
- blood culture contaminant
S. saprophyticus – causes UTI in young women; Specimen for diagnosis: urine
- Novobiocin RESISTANT (less than 16mm)
FAMILY STREPTOCOCCACEAE
I. Lancefield grouping
A. Streptococcus pyogenes
B. Streptococcus agalactiae
C.
Streptococcus dysagalactiae
Streptococcus equisimilis
Streptococcus equisimilus
Streptococcus equi
Streptococcus zooepidimicus
D.
Enterococcus
Enterococcus faecalis
Enterococcus faecium
Enterococcus avium
Enterococcus ducans
VRE (Vancomycin-Resistant Enterococcus)
Non-Enterococcus
Streptococcus bovis – Colon Cancer
Streptococcus equinus
Laboratory Diagnosis:
1. Gram staining: G(+) cocci in pair / chain
S. pneumoniae – G(+) diplococci; “lancet-shape”; "dome-shape colonies”
2. Growth on BAP – white pinpoint colonies
3. Catalase (-)
Diseases:
– leading flesh-eating bacteria (necrotizing fasciitis)
- Impetigo
- Pharyngitis
- RHD (Rheumatic Heart Disease) / RF (Rheumatic Fever)
- AGN (Acute Glumerulonephritis)
- Scarlet fever
- Erysipelas
- Wound, burn
- Toxic Shock Syndrome, pyoderma
- Tonsillitis / Strep throat
- Puerperal sepsis
Group C, F, G
– rarely cause disease
- β-hemolytic
- Bacitracin resistant
- SXT sensitive
- Pneumonia
- UTI
- Bacteremia
- Cellulitis
Group D
– rarely cause disease
- UTI
- wound infection
Enterococcus Non-
Enterococcus
6.5% NaCl (+) (-)
PYRase test (+) (-)
Pencillin R S
Growth at (+) (-)
45◦C
Growth (+) (-)
At 10◦C
Sodium- (+/-) (-)
Hippurate
Hydrolysis test
S. pneumoniae
- Normal flora of nasopharynx and oropharynx
- has capsule (virulence factor)
- not included in the Lancefield grouping
- α hemolytic colonies
- causative agent of Lobar pneumonia / pneumococcal pneumonia / Diplococcus pneumonia (#1
community acquired pneumonia in the Philippines)
- rust-colored sputum
- #1 adult bacterial meningitis
- #1 otitis media
Viridans
- Normal flora of upper respiratory tract, gastrointestinal tract and genitourinary tract
- not included in the Lancefield grouping
- α hemolytic colonies
– SBE (Subacute Bacterial Endocarditis)
>>>MUSCISM
Streptococcus mutans – dental plaques / caries
Streptococcus uberis
Streptococcus salivarius
Streptococcus constellatus
Streptococcus intermedius
Streptococcus sanguis
Streptococcus mitis (mitior)
Francis Test – detects pneumococcal antibody; skin test for S. pneumoniae infection
Dick’s Test – detects erythrogenic antigen; skin test for S. pyogenes infection; (+) result: redness
Schultz-Charlton Test – a skin test and immunity test for scarlet fever that uses antitoxin to the
erythrogenic toxin of S. pyogenes subcutaneously; a positive reaction is blanching of the rash (rash fade)
in the area around the injection site.
Neisseria
Gram (-) diplococci EXCEPT Neisseria elongata
Has pili
Obligate aerobe
Fastidious organism
Capnophilic (requires 5-10% CO2)
Non-motile
Catalase (+) except N. elongata
Oxidase (+)
Superoxol catalase test (+)
JEMBEC system – transport system for Neisseria
Pigmented Neisseria – Neisseria subflava, N. flavescens
Neisseria gonorrhoeae
• Kidney (coffee) bean shaped in PMNs
• Oxidase positive and ferment glucose
• Virulence – “pili”
Diseases:
- Gonorrhea
- Drip, Epididymitis (Male)
- Cervitis, Salphingitis (Female)
- Anorectal gonorrhea (Homosexuals)
- Opthalmia neonatorum - blindness
- Pharyngitis (acquired through oral sex)
- Fitz-Hugh Curtis
- PPNG (Penicillinase Producing Neisseria gonorrhoeae)
Neisseria meningitidis
• Normal flora of nasopharynx
• Virulence factors: capsule and endotoxin
• Serotypes: A, B, C, Y, W135 (Capsular Antigens)
• Mode of transmission: respiratory droplets
Moraxella catarrhalis
- Former names: Neisseria catarrhalis or Branhamella catarrhalis
- Gram (-) diplococci
- Oxidase positive, reduce nitrate to nitrite, DNase positive (best test to differentiate from other
Moraxella spp.)
- Tributyrin Hydrolysis (Butyrate Esterase Disc Test) = (+)
- Assacharolytic
– rarely cause infection
- Pneumonia and other upper respiratory tract infections
- Bacteremia
- 3rd cause of otitis media (middle ear infection)
Cultivation:
*Grow well on CAP
*No growth on BAP except N. meningitidis
*GC (Gram Negative Cocci) Agar – AST media
*Thayer-Martin Agar
*Modified Thayer-Martin Agar
*New York City Agar (Mycoplasma hominis and Ureaplasma urealyticum can also grow)
*Martin-Lewis Agar
Composition:
Base medium: CAP
• Thayer-Martin Agar - VCN
Antibiotics:
Vancomycin - inhibits G(+)
Colistin - inhibits G(-)
Nystatin - inhibits fungi
• Modified TMA – VCNT (Vancomycin, Colistin, Nystatin, Trimethoprim lactate)
• NYC Agar – VCAT (Vancomycin, Colistin, Amphotericin B, Trimethoprim lactate)
• Martin-Lewis Agar – VCAT (Vancomycin, Colistin, Anisomycin, Trimethoprim lactate)
Characteristics of Anaerobes
Brick red fluorescence Prevotella,
Porphyromonas
Red fluorescence Veilonella
Pitting of agar Bacteroides ureolyticus
Double zone of hemolysis Clostridium perfringens
Swarming; Assacharolytic Clostridium tetani,
Clostridium septicum
Molar tooth colonies, Actinomyces israelli
sulfur granules
Breadcrumb colony Fusobacterium nucleatum
Horse manure odor (CCFA) Clostridium difficile
Enterobacteriaceae
• Gram (-) rods
• All are motile w/ peritrichous flagella EXCEPT Klebsiella, Shigella
• Facultative anaerobe
• Ferments GLUCOSE
• Often produce gas = cracks, spacing, bubbles, elevation, splits medium
• All are catalase (+) EXCEPT Shigella dysenteriae
• Oxidase (-) EXCEPT Vibrio, Plesiomonas shigelloides, Aeromonas
• All can reduce nitrate to nitrite EXCEPT Erwina, Pantoea agglomerans
Antigenic structure
K Ag– capsular antigen; heat labile
*Salmonella – ViAg (virulent antigen)
O Ag– somatic or cell wall Ag; heat stable; IgM
- reacts to H antibodies
H Ag– flagellar antigen; destroyed by alcohols, heat denaturation; IgG
Manner of reporting:
A = Acid = yellow
K = Alkaline = red
TSI reactions:
A/A = 2-3 sugars fermented
K/A = glucose fermented
K/K = no sugar fermented
Manner of reporting:
A = Acid = yellow
K = Alkaline = purple
R = red
LIA reactions:
❖ K/K = LDA(-), LDC(+)
❖ K/A = LDA(-), LDC(-)
❖ R/A = LDA(+), LDC(-)
IMViC Test
4. Indole test
Detects: Tryptophanase
Substrate: Tryptophan
Product: Indole
Medium: SIM (Sulfide Indole Motility) medium, Tryptophan broth or slant
Indicator: Kovac’s reagent / Erlich’s reagent / PDAB (paradimethylaminobenzaldehyde)
(+) result: red ring
(+) control: Escherichia coli; (-) control: Klebsiella pneumoniae
b. Voges-Proskauer test
Principle: Butylene glycol of glucose fermentation
Detects: acetoin (acetylmethylcarbinol)
Reagent:
α-napthol and KOH (Barrits Method)
α-napthol and 40% KOH in creatine (Coblentz Method)
(+) result: red
(-) result: salmon / brown / yellow
(+) KESH (Klebsiella, Enterobacter, Serratia and Hafnia)
Note: Most Enterobacteriaceae bacteria have opposite MR/VP results
*(NH4)2CO3 is alkaline
pH indicator: phenol red
(+) result: pink
(-) result: yellow
Decarboxylase Testing
Lysine Ornithine Arginine
K. (+) (-) (-)
pneumoniae
K. oxytoca (+) (-) (-)
E. aerogenes (+) (+) (-)
(Klebsiella
aerogenes)
E. gergorvae (+) (+) (-)
E. cloacae (-) (+) (+)
E. sakazakii (-) (+) (+)
Hafnia alvei (+) (+) (-)
P. (-) (-) (-)
agglomerans
Escherichia
I – (+)
M - (+)
V - (-)
C - (-)
• TSI: A/A; LOA: +, +, -; Greenish metallic sheen on EMB; yellow colonies on XLD
• MUG (Methyl Umbelliferyl β-D-Glucoronide) Test
o Uses UV light
(+) result: electric blue fluorescence
(-) result: no electric blue fluorescence
(+) for: All Escherichia spp. except E. coli O157:H7
Diseases:
- #1 cause of UTI
- Gram negative sepsis
- #2 meningitis in infants (Escherichia coli K1)
- Diarrheal disease
- Nosocomial infection, wound infection, bacteremia, pneumonia
Biotypes:
1. ETEC – Enterotoxigenic Escherichia coli
- produces LT (heat labile) and ST (heat stable) enterotoxins
- Causative agent of Montezuma revenge (Turista or Traveller’s diarrhea)
- causes “Childhood diarrhea”
- produces Cholera-like toxin (Consistency of the stool: rice watery stool)
- stimulates adenylcyclase
- O6, O8, O25
Klebsiella
I - (-)
M - (-) NON-motile
V - (+)
C - (+)
• TSI: A/A; LIA: K/K
• LOA: +, -, -; Urease and malonate (+); MAC – mucoid colonies; Lactose Fermenter
• Causes pneumonia (currant jelly-like sputum), wound infection, meningitis, UTI
• has mucoid polysaccharide capsule
Enterobacter
I - (-)
M - (-) Motile
V - (+)
C - (+)
• TSI: A/A; LIA: K/K
• LOA: +, -, -; Urease (-) except E. gergoviae
- causes opportunistic infections, upper respiratory tract infections, UTI, wound infections and
septicemia
- Enterobacter cloacae complex are the predominant species; it includes Enterobacter cloacae,
Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii
- Enterobacter sakazaki – produces yellow colonies at 25οC
- Enterobacter gergovae – the only species of Enterobacter that is urease (+)
Serratia
Known to produce these three enzymes:
- DNase
- Lipase
- Gelatinase
Salmonella
- non-lactose fermenter
- sources of infection: H2O, dairy products, poultry, pet house
- causing: Typhoid fever, Systemic infection, Enterocolitis, Enteric fever (non-tissue invasive)
- Black colonies on SSA
- Selenite F and Tetrathionate broth (+)
- BGA (Brilliant Green Agar) – all Salmonella spp. are cultured except S. typhi
- BSA (Bismuth Sulfite Agar) - S. typhi only
- TSI: K/A + H2S, LDC: (+), LOA: +, +, +, LIA: K/K, Indole (-), Blood culture (+)
- Aerogenic except Salmonella typhi and Salmonella gallinarum
- Motile except Salmonella gallinarum and Salmonella pullorum
- S. paratyphi (H2S and LDC negative)
Serotypes:
A Salmonella paratyphi A
B Salmonella paratyphi B
C Salmonella cholerasuis (paratyphi C)
D Salmonella typhi
*Salmonella typhi - most important species; 1st week – blood and urine, 2nd week – stool; best
specimen: bone marrow; causes meningitis, osteomyelitis
*Salmonella paratyphi A and B - paratyphoid fever
*Salmonella cholerasuis – known to cause bacteremia / septicemia
*Salmonella typhimurium (enteritidis) – known to cause enterocolitis / gastroenteritis (poultry)
*Other Salmonella spp. – food poisoning
Salmonella arizonae
- LLF
- related to Salmonella
- TSI: A/A + H2S, ONPG (+), LIA: K/K
SHEWANELLA SALMONELLA
H2S (+) (+)
NLF colorless colony colorless colony
Sucrose (+) (-)
Shigella
- Common name: Inert Bacteria
- non-lactose fermenter
- non-motile, colorless on SSA, acetate (-), TSI: K/A, LIA: K/A, H2S (-), LDC (-), Indole (+), Blood culture (-)
- LOA: -, -, - except S. sonnei (ornithine +), related to E. coli but acetate (+)
- causes Bacterial dysentery (tissue invasive; produces bloody stool with mucus)
- Specimen: fresh stool with mucous flecks or rectal swab of ulcer
- Related to E. coli
- natural habitat is limited to primates and humans
Group Catalase ONPG Mannitol
type (O (same
Antigen) pattern in
Ornithine)
Shigella
A (-) (-) (-)
dysenteriae
Shigella
B (+) (-) (+)
flexneri
Shigella
C (+) (-) (+)
boydii
Shigella
D (+) (+) (+)
sonnei
Citrobacter
- Citrate (+) Gram negative bacilli
- TSI: A/A; H2S +/-; LIA: K/A; ONPG: +
- causes UTI and sepsis
- resembles Salmonella (LDC +)
Proteus
- TSI: K/A + H2S
- Lysine deamination positive; LIA: R/A
- Rapid urease (+)
- LOA: -, -, - except P. mirabilis (ornithine positive)
- PAD test (+)
- produce infections in the intestinal tract
- #2 cause of UTI, bacteremia, nosocomial infections, renal stone (triple phosphate calculi)
- Dienes phenomenon – when two identical Proteus cultures are inoculated at different points on the
same plate of non-inhibitory medium, the resulting swarming of growth coalesce without signs of
demarcation
Providencia
- Lysine deamination positive; LIA: R/A
- Rapid urease (+) except Providencia alcalifasciens
- LOA: -, -, -
- PAD (+)
- Indole (+)
- normal flora
- can cause UTI and other infections
- resistant to antibiotics
TSI Urease Ornithine
P. stuartii K/A (+/-) (-)
P. rettgeri K/A (+) (-)
P. K/A (-) (-)
alcalifasciens
Morganella morgani
- TSI: K/A
- Lysine deamination positive; LIA: R/A
- Rapid urease (+)
- PAD (+)
- Indole (+)
- Ornithine (+)
Edwardsiella tarda
- can be isolated from cold and warm blooded animals
- most infections associated to human including diarrhea wound andbacteremia
- TSI: K/A + gas + H2S (Like Salmonella)
- IMVC: ++-- (Like E. coli)
- Lactose (-)
- Lysine decarboxylase (+)
Yersinia pestis
- Common name: Plague Bacillus
- V and W Antigens
- non-motile, urease (-) and ornithine (-)
- causative agent of Bubonic, Pneumonic, Septicemic Plague, Famine
- causative agent of Black Death (bioterrorism agent)
- infection of wild rats / rodents
- Produced pandemic infections (millions of people were involved)
- Common vector: Xenopsylla cheopsis (rat flea)
- in broth culture, it shows stalactite pattern
- Inclusion bodies: Bipolar bodies (safety pin appearance)
- Stain: Wayson
Yersinia enterocolitica
- causes zoonotic infections through ingestion of unpasteurized milk
- causes enterocolitis, fever, diarrhea, abdominal pain, arthritis, appendicitis and erythema nodosum
- also causes bacteremia
- #1 blood bag contaminant
- Culture medium: CIN (Cefsulodin-Irgasan-Novobiocin) medium
- shows bull’s eye appearance / colony
- Aeromonas: CIN (+) but Oxidase (+)
- oxidase (-); motile at 22◦C but not at 35◦C; cold enrichment at 4◦C
Yersinia pseudotuberculosis
- LOA: -, -, -; Urease (+);
- causes Mesenteric Lymphadenitis and septicemia
- animal pathogen (pseudotubercles)
Y. Y. Y.
pestis enterocolitica pseudotuberculosis
Motility (-) (+) (+)
Urease (-) (+) (+)
Ornithine (-) (+) (-)
Sucrose (-) (+) (-)
Vibrio
- Sources of infection: seafoods, shellfish
- facultative anaerobe
- has more than 1 chromosome
- comma shape (has monotrichous or polar flagella)
- All are oxidase (+) except V. mitschnikovii
- non-sucrose fermenters except V. cholerae and V. alginolyticus
- catalase (+) and indole (+), glucose fermenter
- O129 Sensitivity Test (susceptible)
- All are halophilic EXCEPT: Vibrio cholerae and Vibrio mimicus (8% NaCl negative)
- Alkalinophilic, LOA: +, +, -, nitrate reduction (+)
- String test (+)
*String test
Reagent: 0.5% Sodium desoxycholate
(+) result: string formation
(+) for: Vibrio
(-) for: Aeromonas & Pleisomonas
Vibrio alginolyticus
- causes wound infections (acquired from marine bodies of water); gastroenteritis
- halophilic (8% NaCl positive)
- sucrose fermentation positive (TCBS: yellow colonies)
Vibrio parahemolyticus
- gastroenteritis (seafoods)
- gas formation associated w/ wound infections
- halophilic (8% NaCl positive); Indole (+)
- sucrose fermentation negative (TCBS: green colonies)
- Kanagawa positive; LOA: +, +, -
Vibrio mimicus
- Clinical significance: gastrointestinal infections
- NON-halophilic
- sucrose fermentation (-)
Vibrio vulnificus
- halophilic (8% NaCl positive)
- TCBS: green colonies
- Disease: Septicemia and wound infections
Pseudomanas spp.
- Oxidase (+), motile, TSI = K/K; O-F Test: Yellow (O), Green (F); causes opportunistic infections
(environmental)
Pseudomonas aeruginosa
- Former name: Burkholderia pyocyaneus
- oxidase (+), MAC (+), catalase (+), nitrate reduction (+), LDC (-), TSI = K/K; O-F Test: Yellow (O) Green
(F), Acetamide (+), Citrate (+)
- obligate aerobic
- motile (monotrichous flagella)
- causes opportunistic infections (environmental)
- does not ferment carbohydrates
- Odor: Sweet, Grapelike, Tortilla, Corn chips odor due to 2-aminoacetophenone
- Grows well at 37οC
- Grows better at 42οC - differentiates Pseudomonas aeruginosa from P. fluorescens and other CAMP
test (+) organisms
- Produces green-blue pigment
> Blue: pyocyanin
> Green: pyoverdin
> Red: pyorubin
> Fluorescence: fluorescein (also P. putida and P. fluorescens)
- Culture medium: Cetrimide agar / Pseudocel agar
- causes infection of burns (#2)
- causes meningitis, UTI, pneumonia, sepsis, otitis media, endocarditis, contaminant in contact lens care
solutions
- complicates Cystic Fibrosis patients (#1)
- causative agent of Swimmer’s Ears (otitis media)
- causative agent of Ecthyma gangrenosum / Pseudomonas dermatitis / “blue pus” – skin lesion or
wound infection
- causative agent of Whirlpool dermatitis / “Jacuzzi” Hot Tub Syndrome / Businessman’s syndrome -
acquired from hot tub
- resistant to disinfectants (that’s the reason why it is considered to be the #1 cause of nosocomial
infections)
- #1 ICU (Intensive Care Unit) isolate, #1 NFO isolate
Burkholderia mallei
- Former name: Pseudomonas mallei
Burkholderia pseudomallei
- Disease: Melioidosis, Glander’s like infections (pneumonia) or Vietnamese Time Bomb disease
- Common name: Whittmores Bacillus
- “Vietnamese Time Bomb Disease”
- Wrinkled colonies on Ashdown medium; O-F Test: +/- (lactose oxidizer)
- Grows at 42◦C
- Motile (lophotrichous flagella)
OTHER NFO
Burkholderia cepacian *#2 complication of Cystic
Fibrosis; pneumonia,
sepsis; oxidase and LDC (+)
*Motile (lophotrichous
flagella); yellow on OFPBL
(Oxidative Fermentative
Polymixin B Bacitracin
Lactose)
*Earthy odor / Dirt-like
odor; pink colonies on
MacConkey (lactose
oxidizer)
Stenotrophomonas *Oxidase (-); DNase (+)
maltophilia *Lavender green colonies
Shewanella putrefasciens *TSI: K/K + H2S; Oxidase
(+)
Acinetobacter *#2 NFO
*Oxidase (-), catalase (+),
non-motile, growth on
MacConkey, LDC (+)
*Mistaken as Neisseria
(Difference: Oxidase +);
drug-resistant; UTI,
wound, diarrhea
*Acinetobacter anitratus -
(oxidizer); Other name:
Herella vaginocola
*Acinetobacter lwoffi –
(non-oxidizer); Other
name: Mima polymorpha
Alkaligenes faecalis *Asaccharolytic = O/F
Test: (-/-)
*Oxidase, catalase and
MacConkey (+); Apple like
“fruity” odor
*UTI, wound, diarrhea;
motile (peritrichous
flagella)
Moraxella lacunata / *Causes
Morax axenfield Blepharoconjunctivitis;
oxidase and catalase (+)
PARVOBACTERIA
• Gram negative, fastidious coccobacilli
1. Haemophilus
2. Bordetella
3. Brucella
4. Francisella
5. Pasteurella
Haemophilus
- encapsulated, nonmotile, nonsporeforming, fastidious Gram (-) coccobacilli
- facultative anaerobe
- most are Oxidase (+)
- Catalase (+)
Culture media:
> Enriched CAP + 5% CO2 (made with horse’s blood; for fastidious organisms)
> BAP w/ X factor
> Levinthal
> Fildes
X- V- Β- D-ALA /
factor factor hemolysis porphyrin
H. influenzae (+) (+) (-) (-)
H. (-) (+) (-) (+)
parainfluenzae
H. hemolyticus (+) (+) (+) (-)
H. (-) (+) (+) (+)
parahemolyticus
H. aegypticus (+) (+) (-) (-)
H. aphrophilus (-) (-) (-) (+)
H. (-) (+) (-) (+)
paraaphrophilus
H. ducreyi (+) (-) (-) (-)
Notes to remember:
• Porphyrin test (X-factor) – Delta-aminolevulinic acid (ALA) to Protoporphyrin (Porphyrin) = red
fluorescens (+)
• All Haemophilus spp. that requires V-factor shows satellitism phenomenon with Staphylococcus
aureus or Candida albicans.
• S. aureus and C. albicans produces V-factor.
Haemophilus influenzae
- Gram negative coccobacilli
- Common name: Pfieffer’s Bacillus
- Virulence factors: capsule, IgA protease, LPS and pili
- causes respiratory system infections, major cause of acute epiglottitis, cystic fibrosis, otitis media,
conjunctivitis, pneumonia, sepsis
- has 6 serotypes; the most common and most severe is Serotype B (HiB)
- associated with meningitis of <5 y/o (3rd cause of bacterial meningitis)
- Selective medium: Horse Blood Bacitracin Agar
- shows grayish, drew drop colonies with mousy odor
- exhibits sattelitism around S. aureus (BAP)
- Beta-lactamase (+) control (Cefinase Disc Test)
Haemophilus aegypticus
- Common name: Koch Week’s Bacillus
- Diseases: pink eye / conjunctivitis, Brazilian purpuric fever
Haemophilus ducreyi
- causative agent of chancroid or soft chancre
- causes venereal infections – isolated from genitalia
- Direct exam: school of red fish
- Culture medium: CAP with vancomycin
Campylobacter
- Gram stain: curved, gram negative rod (Seagull wings appearance)
- oxidase and catalase positive
- microaerophilic (5% O2, 10% CO2, 85% N2)
- associated with gastroenteritis, diarrhea, animal abortion (zoonotic)
- its motility is because of its 1 flagella
- Optimum temp.: 42-43οC; indoxyl acetate (+)
- Culture media: Camphy’s medium, Skirrow medium, Butzler medium, CBAP (Campylobacter Blood
Agar Plate)
- Type of motility - darting motility
Representative species:
Campylobacter jejuni – Guillain-Barre syndrome
Campylobacter coli
Campylobacter fetus
*Suturella wadworthiensis – resembles Campylobacter
Helicobacter
- Gram stain: curved, gram negative rod
- associated w/ duodenal and gastric infections (peptic ulcer, peptic cancer, gastritis)
- Motile (4-6 flagella; sluggish motility)
- Natural habitat: stomach
- Optimum Temp.: 36-37οC
- CAMP test (+)
- Specimen for culture: tissue biopsy of stomach
- oxidase (+), catalase (+) and microaerophile
- Urea Breath Test (+) – best test to differentiate from Campylobacter; rapid urease (+)
- Culture medium: Urea agar
- Representative species: Helicobacter pylori
Bordetella
- encapsulated, obligate aerobe, Gram (-) coccobacilli
- non motile EXCEPT: Bordetella bronchiseptica
Culture media:
• Bordet-Gengou agar
AKA: PBGA (Potato-Blood-Glycerol Agar)
Composition:
-potato
-glycerol
-blood
• Regan-Lowe agar
-most preferred medium (best) for the isolation of Bordetella pertussis
AKA: CCBA (Charcoal-Cephalexin Blood Agar)
Composition:
-amphotericin
-cefalexin
-horse blood
-charcoal
• Jones Kendrich
Brucella
- causative agent of Brucellosis, Undulant fever, Malta fever, Mediterranean fever, Gibraltar fever,
Cyprus fever, Bang’s disease, animal abortion, endocarditis, laboratory acquired infection
- Non-motile (no capsule), obligate aerobe, zoonotic, Gram (-) coccobacilli
- Special growth requirement: Erythritol
- normal flora of urinary tract and GIT of sheeps and goats
CO2 Urease
Basic
(5- H2S Thionine
Fuchshin
10%)
B. abortus (+) (+) inhibited Growth (+)
(Bang’s
disease)
B. (-) (-) Growth Growth (+)
melitensis
B. suis (-) (-) Growth Inhibited (+)
B. canis (-) (-) Growth Inhibited (+)
Culture media: TSB (Trypticase Soy Broth), W medium, Castaneda (biphasic medium)
Specimen: blood / bone marrow
Cuture incubation: 3-4 weeks
Legionella
Pasteurella
- Gram negative coccobacilli
- capsulated, non-motile with bipolar bodies (“safety pin appearance”)
- oxidase (+), catalase (+), glucose (+), ornithine (+), indole (+), urease (+)
- grow on BAP but not in MacConkey
- acquired from bites and scratches of cats and dogs (zoonosis)
- causes wound infections (animal bite wound), pneumonia, endocarditis, meningitis, arthritis
- agent of shipping fever in cattles
- Representative species: Pasteurella multocida
• causes Pasteurollosis
• Etymology:
“multo” - many
“cida” - killings
Francisella
- Special growth requirement: Cysteine and cystine
- Gram (-) coccobacilli, non-motile (capsulated), urease (-), MAC (-), aerobe, catalase (+), oxidase (-),
beta-lactamase (+)
- Mode of transmission:
• Direct transmission: rodents, primarily rabbits
• Indirect transmission: ticks and deerfly
- Representative species: Francisella tularensis – causative agent of Tularemia (fever), Ohara fever,
Market men’s disease, Rabbit fever, Water –trapper fever, laboratory acquired infection
- Culture media: Blood Glucose Cysteine Medium, PCA (Peptone Cysteine Agar), CHA (Cysteine Heart
Agar)
Gardnerella
Gardnerella vaginalis
- Disease: bacterial vaginosis
- G (variable) coccobacilli
- oxidase and catalase negative
- Former names: Haemophilus vaginalis and Corynebacterium vaginalis
- Characteristic of vaginal discharge: foul-smell grayish vaginal discharge
*Amsel-Nugent scoring – scoring being used
- Cytology exam (Paps smear): clue cells
- Whiff or Sniff test
- Vaginal discharge + 10% KOH
- (+) fishy amine like odor
- Culture medium: HBT (Human Blood w/ Tween 80) medium, V agar, Columbia CAN (Colistin-Nalidixic
Acid Agar)
Streptobacillus moniliformis
- normal flora of respiratory tract of rodents
- acquired from bites and scratches of rodents
Streptobacillus moniliformis
Spirilium major – spiral organisms
Spirilium minor (minus) – spiral organisms
Capnocytophaga
- oral flora
- Capnophilic (requires 5-10% CO2)
- filamentous / fusiform rod
- motility: gliding motility (spreading colonies)
- Disease: Periodontal disease
- Nitrate reduction test and Esculin hydrolysis test (+)
Calymmatobacterium granulomatis
- encapsulated, safety-pin appearance with Donovan bodies (using Giemsa stain)
- Disease: Donovariosis or Granulomatous inguinale
- closely related to Klebsiella
- Former name: Klebsiella granulomatis
Chromobacterium violaceum
- MAC: non-lactose fermenter
- habitat: soil, H2O
- Characteristic: violet colored colonies (due to “violacein” pigment) with ammonium cyanide odor
Cardiobacterium hominis
- normal flora of respiratory tract of humans
- rare cause of Endocarditis
HACEK
- causes SBE (Subacute Bacterial Endocarditis)
- requires carbon dioxide; BAP (+) but MAC (-)
H – Haemophilus aphrophilus
A – Actinobacillus (Aggregatibacter) actinomycetemcomitans
C – Cardiobacterium hominis
E – Eikenella corrodens
K – Kingella kingae
Bacillus
- sporeformers
- found in soil
- sporulate aerobically
- catalase (+)
Bacillus anthracis
- Gram (+) rods in chains
- Virulence factors: spore, exotoxin and capsule (Composition: D-glutamate) – McFadyean’s reaction (+)
- NON-motile, spore-forming, zoonotic
- most virulent species of Bacillus
- causative agent of Anthrax
*Cutaneous anthrax - Black Eschar (a form of macule or malignant pustule)
*Pulmonary anthrax - Wool-Sorter’s Disease (occupational hazard)
*Gastrointestinal anthrax – gastroenteritis; bloody diarrhea; Mode of transmission: ingestion
- Catalase (+)
- colonies: Medusa head colonies, inverted pine tree appearance or colonies with swirling phenomenon
in BAP
- Gram stain: Bamboo pole appearance (square ends)
- String of Pearls Test: MHA or BAP w/ penicillin (0.05 units) – (+) result: susceptible to penicillin
- Selective medium: PLET (Polymyxin-Lysozyme-EDTA-Thallous acetate) medium
- Skin test / serologic precipitation test: Ascoli test
Bacillus cereus
- Common name: Fried Rice Bacillus
- Source of infection: cereal, rice grains, milk
- Mode of transmission: food ingestion
- causes gastroenteritis
- Virulence factors: spore and exotoxin (cholera-like toxin)
B. anthracis B. cereus
Catalase (+) (+)
test
Lecithinase (+) (+)
Capsule (+) (-)
Motility (-) (+)
Hemolysis on γ hemolysis Β hemolysis
BAP
Growth at 45◦C (-) (+)
Bacillus subtilis
- Gram (+) rods in chain with central spore
- most common laboratory contaminant
- most common BI (Biological Indicator)
- Common name: Hay’s bacillus
- causes conjunctivitis or eye infection in heroin addicts
- BAP: large, flat, dull β-hemolytic colonies (Ground glass appearance)
- bacteria used in Guthrie Bacterial Inhibition Test for PKU (Phenylketonuria)
Bacillus stearothermophilus
- one of the BI used in autoclave (aside from Clostridium PA 3679)
- associated with flat sour spoilage
- no gas formation
Clostridium
- sporeforming, obligate anaerobic Gram (+) rods (must be cultivated in a gas pak jar)
- Catalase (+)
- Habitat: human and animal
- All Clostridium spp. are saccharolytic except C. tetani and C. septicum
3 types of Clostridium:
1. Neurotoxic – C. tetani and C. botulinum
2. Histotoxic – C. perfringens and C. septicum
3. Enteric – C. difficile
Clostridium perfringens
- Common name: Box Car Shape Bacillus
- Former names:
1. Bacillus aerogenes
2. Clostridium welchii
- Encapsulated, non-motile
- Common source of infection: wound contaminated with soil
- associated with sulfhemoglobin
- Diseases:
1. Gas gangrene (myonecrosis)
2. Food poisoning (enterotoxins)
3. Necrotic enteritis
Laboratory diagnosis:
6. Chopped meat medium= produces gas + growth
7. Lecithinase or Nagler reaction test (+)
8. Reverse CAMP test (+)
- Base medium: BAP
Clostridium botulinum
- Common name: Canned Good Bacillus, Pigbel Bacillus
- causes food poisoning (due to ingestion of home-made canned goods)
- also causes wound botulism – due to wound contaminated of its spores
- Classic sign of botulism: flaccid paralysis
- causative agent of “Floppy Baby or Honey Bee Syndrome” - infant botulism
- toxin: botulism toxin – a powerful neurotoxin (0.1µL is already fatal); heat labile toxin; blocks the
release of acetylcholine; the most potent exotoxin
- its toxin is used in botox
- Spores: subterminally located, spherical
- Lipase: (+)
Clostridium tetani
- Common name: Tack-head Bacillus
- causative agent of Tetanus
- Signs of tetanus: Lock jaw, Risus sardonicus (Devil’s grin), spastic paralysis, opisthotonus (arching of the
back)
- Assacharolytic
- found in soil
- Mode of transmission: Traumatic implantation of contaminated materials
- exotoxin: tetanospasmin and tetanolysin – causes spasm or contraction; binds to ganglioside receptors
& inhibit neurons in CNS
- Spores: terminally located, round (tennis racket or drumstick appearance)
Vaccines:
*Tetanus toxoid
*DPT
*anti-tetanus
Clostridioides difficile
- Former name: Clostridium difficile
- Family: Peptostreptococcaceae
- Colon flora
- “difficile” means difficult to identify
- causative agent of AAPC (Antibiotic Associated Pseudomembranous Colitis) / Pseudomembraneous
colitis – a form of diarrhea associated with long-term antibiotic therapy (usually Clindamycin)
- Spores: subterminally located, spherical
- Laboratory diagnosis: direct detection of its toxins in stool by EIA or cytotoxin assay
- culture: CCFA (Cycloserine-Cefoxitin-Fructose Agar) - Yellow colonies with horse’s manure odor
Corynebacterium diptheriae
- Pleomorphic gram (+) rods
- Gram stain: club shape, Chinese letter character appearance or palisade (L, V, X, Y or Z-shape)
- Common names: Kleb Loeffler’s bacillus, Bull’s Neck Bacillus, Elek agent
- Causative agent of diphtheria
Culture Media:
10. LAMB medium (Loeffler’s Alkaline Methylene Blue) medium – enhancement of
metachromatic granules
a. Loeffler’s serum slant
b. Pai slant medium / Pai coagulated egg medium (gray-black colonies)
11. Tellurite agar, modified tellurite agar, potassium tellurite medium - gray to
black colonies
12. BAP
13. Clauberg medium
14. MacLeod’s medium
15. Tinsdale medium - black colonies with brown halo
16. CT-BAP (Cystine Tellurite BAP) - gun metal gray colonies
Notes to remember:
- C. diptheriae culture is similar to C. pseudotuberculosis and C. ulcerans.
- Potassium tellurite present in tellurite medium inhibits normal flora.
DIPTHEROIDS
*Diptheroids – Corynebacterium spp. which are normal flora; all Corynebacterium spp. except C.
diptheriae
• Corynebacterium jeikeium
- Common name: JK Bacillus
- Urease (-), Nitrate reduction (-), Glucose fermenter
- resistant to most antibiotic therapy
- causes Pneumonia, Endocarditis, Peritonitis
• Corynebacterium pseudodiptheriticum
- Common name: Hoffman’s Bacillus
- oral flora
- Urease (-), Nitrate reduction (+/-), non-CHO fermenter
– causes throat infections
• Corynebacterium xerosis
Listeria monocytogenes
- Gram (+) bacilli; Catalase (+); β-hemolysis on BAP; CAMP test (+) – black hemolysis
- Gelatin medium: inverted christmas tree
- found on cold environment / enrichment (4◦C)
- found also in soil
- Major source of infection: food (cheese, coleslaw etc.)
- Diseases: Meningitis, Sepsis, Food poisoning, Fetal abortion, Still birth (Perinatal Listeriosis or
Granulomatis Infantseptica)
- Motility: Tumbling Motility (room temperature)
For demonstration of motility:
> hanging drop method (living state, wet mount)
> semisolid medium (SIM) – shows umbrella-like pattern
- Culture: McBride medium
- Virulence Test: Anton’s test or Ocular Virulence Test
> detects Listeriolysin O (O2 labile hemolysin)
> it involves the usage of rabbit
> (+) result: purulent discharge conjunctivitis in rabbit
Erysipelothrix rhusiopathiae
- Gram (+) bacilli; non-motile
- Catalase (-)
- the only Gram (+) bacteria that is H2S (+)
- Disease: erysipeloid or Diamond Skin Disease - cutaneous inflammation of hands or fingers
- Veterinary and occupational hazard (e.g. fish handlers, butcher’s cut)
- Gelatin medium: test tube brush appearance
Listeria Erysipelothix
monocytogenes rhusiopathiae
Catalase (+) (-)
Motility (25◦C) (+) (-)
Hemolysis Beta Alpha
Vogues (+) (-)
Proskauer
H2S production (-) (+)
Bile esculin (+) (-)
hydrolysis test
Hippurate (+) (-)
hydrolysis test
Gluconate (+) (-)
Media McBride BAP
medium, cold
enrichment
Salicin (+) (-)
Lactobacillus cassei
*Shirota strain
*the good bacteria in Yakult and yogurt
*produces: lactic acid
Kurthia bessonnii
- found in soil
- causes opportunistic infections
Rothia
- normal flora in mouth (can cause endocarditis)
- causes opportunistic infections like abscess and toothache
Rhodococcus equi
- pleomorphic (rod-cocci) and show pink colonies after 24 hours
- CAMP test (+) with S. aureus = β-hemolytic
Nocardia spp.
- partially (modified) acid fast - uses 1% H2SO4
- Urease (+), gram positive branching rod; cause pneumonia
- Casein hydrolysis: Nocardia asteroides (-); Nocardia brasiliensis (+)
- Grow on media without antibiotic
Arcanobacterium haemolyticum
- β-hemolysis on BAP; lipase and lecithinase (+)
- Reverse CAMP (+)
Mycobacterium tuberculosis
- Common name: Koch’s Bacilli
- causative agent of Pulmonary Tuberculosis (PTB)
- slender, slightly curved, G(+) rod
- diameter: 0.2-0.4 µm
- length: 1-4 micra
- obligate aerobe, require 5% CO2 for growth
- Virulence factors: cord factor and sulfatides
Resistant to:
1. Drying - (because of Much’s granules)
Susceptible to:
1. Sunlight
2. Autoclave
3. Pasteurization
4. Boiling (10 mins.)
Pathogenesis:
middle lower lungs ------> may include macrophages (phagocytosis) ------> fibroid cells ------>
granulomatous lesion (granuloma) seen on X-ray ------> calcify (Ghon’s complexes)
Anti-TB Agents:
1ο Rifampicin
Isoniazid
Pyrazinamide
Ethambutol
Streptomycin
2ο Ethionamile
Capreomycin
Ciprofloxacin
Ofloxacin
Kanamycin
Cycloserive
Rifabutin
Specimen of choice: 2 sputum samples (every morning for 2 consecutive days) Source: DOH 2013
*To lessen exposure - wash the used inoculating loop with sand
In order for a sputum specimen to be acceptable for culture, this criteria must be met:
• < 10 squamous epithelial cells and > 25 PMNs
Media Criteria:
1. It must always contain malachite green.
2. It must always be egg-based or protein-based (The media for Mycobacterium tuberculosis is
not ideal for autoclaving, instead use pasteurization, Arnold’s sterilization or
inspissation)
3. It must be suitable for heavily contaminated specimens.
Stains:
1. Ziehl-Neelsen
2. Kinyoun’s
3. Fite Faraco - secondary stain: hematoxylin
-used for staining tissues obtained from biopsy (AFS for M. leprae)
4. Truant’s – also known as Auramine-Rhodamine stain
5. Spergler’s – AFS for color-blinded person
(+) result: black bacilli
OTHER MYCOBACTERIA
M. bovis - TB of the cattle; intestinal tuberculosis (BCG)
M. avium - Chicken’s / Bird's TB
M. avium-intracellulare complex - Battey Bacillus; AIDS TB
M. kansasii - Yellow bacillus – produces yellow pigment
M. gordonae - Tap water bacillus
M. marinum - causes Swimming pool granuloma
M. terrae - Raddish bacillus
M. ulcerans - Inert bacillus-like (negative in most biochemical tests); causative agent of Buruli ulcer
M. gastri - J bacillus
M. phlei – provide CO2
M. smegmatis – confused with MTB in urine
M. xenopi – hot and cold water taps
M. africanum – pulmonary TB (Africa)
M. genavensi – disseminated infections in AIDS, BACTEC (+)
M. paratuberculosis – Crohn’s disease
M. fortuitum – rapid grower in MAC
Group I:
Photochromogens - produces pigment when exposed to light (photoreactive)
• Visible colonies after 10-21 days
Mycobacterium marinum
Mycobacterium asiaticum
Group II:
Scotochromogens - produces pigment in the dark and light
• Visible colonies after 10-21 days
Group III:
Non-photochromogens - non-pigmenters
• Visible colonies after 10-21 days
Group IV:
Rapid growers - visible colonies after 3-7 days
Mycobacterium fortuitum-chelonae complex
Mycobacterium phlei
Mycobacterium smegmatis
Mycobacterium abscessus
Mycobacterium mucogenicum
5. Arylsulfatase test
Tripotassium phenolphthalein disulfide / sulfate acted upon by arylsulfatase to produce free
phenolphthalein
(+) result: pink / red
(+) for: Group IV or Rapid Growers like Mycobacterium fortuitum-chelonei
6. Pyrazinamidase test
Pyrazinamide ------> pyrazinoic acid (pink)
8. Urease
(+) result: red/pink
M. bovis
M. scrofulaceum
M. gastri
M. tuberculosis M. bovis
Niacin test (+) (-)
Nitrate (+) (-)
reduction test
TCH test (-) (+)
SPIROCHETES
ORDER SPIROCHAETALES
Gram (-) spiral bacteria
long, slender
corkscrew-like motility or spinning motility
Unusual morphologic features:
1. axial filaments / axial fibrils - organ for locomotion, flagella-like
2. outer sheaths - covering
3. insertion disk - plate-like structure where fibrils are attached
Microscopes for demonstration:
1. Darkfield Microscopy
2. Phase-contrast Microscopy
3. Fluorescence Microscopy
TREPONEMA
BORRELIA
LEPTOSPIRA
*Jarisch Herxheimer Reaction (JHR) is a transient clinical phenomenon that occurs in patients infected by
spirochetes who undergo antibiotic treatment.
Treponema
- looks like a “turning thread”
- 6-10 axial filaments
- 1 insertion disk
- Catalase (-)
- Not culturable on agar medium
- Obligate intracellular organism
- Tissue culture: rabbit’s testicles
- Easily killed at 42οC (can serve as therapy to Treponema infected patients)
- It is visible in whole blood and plasma products up to 24 hours
Treponema pallidum
- causative agent of “syphilis”
- fine, spiral organism w/ 2 periplasmic flagella
- microaerophilic (requires 3-5% O2)
- Mode of Transmission: sexual contact
intact skin penetration
cross placenta (vertical transmission)
- it has remarkable tropism (attraction)
- resistant to drying / chemical disinfectants
- Generation time: 30 hrs.
- Drug of choice: Penicillin
- Salvarsan (Drug 606) – first drug that was invented for the cure of Syphilis
- Wasserman test - first serological test that was invented for the diagnosis of Syphilis
- Syphilis was carried from the New World (Americas) to Old World (Europe) by the voyage of Cristopher
Columbus crew. (Old world → New world: Small pox)
Clinical manifestations:
*Syphilis - disease of perivascular area and blood vessels; chancre (NON-tender, painless lesion)
*AKA: French disease, Italian disease, Spanish disease, Great Pox, Great Immitator
*causes fever, sore, arthritis, headache, rash (in palm and sole) and GUMMAS
Stages:
• Primary Syphilis - Hunterian Chancre / Hard Chancre
- 3-6 wks. after infection
- at the site of inoculation
• Secondary Syphilis – Condylomata Lata
- 2-24 wks. after it has sufficient number
- fever, malaise, myalgia, loss of appetite, generalized skin rashes
• Latent Stage
- subclinical (no signs and symptoms)
- best stage to identify and to perform serological tests
Laboratory Diagnosis:
Skin Lesion - Dark Field Microscopy
Fluorescent Microscopy
Phase Contrast Microscopy
1. Microscopy – search for motile spirochetes with corkscrew motility using dark-field microscope with
400X objective
Stains:
Levaditi Silver stain
Silver impregnation stain
Fontana Tribondeau stain
Warthin-Starry stain – best
2. Serology
a. Non-treponemal tests – - non-specific; cross-react with other diseases
• RPR – Rapid Plasma Reagin
• VDRL - Venereal Disease Research Laboratory
• TRUST – Toluidine Red Unheated Serum Test
b. Treponemal tests - - specific; true antibodies
• TPHA - Treponema Pallidum Hemagglutination test
• MHA-TP - Microheme Agglutination Assay to Antibodies for T. pallidum
• FTA-Abs Fluorescent Treponemal Antibody-Absorption test
• HATTS – Hemagglutination Treponemal Test for Syphilis
Stages:
1ο - Mother yaws
2ο - Daughter yaws
3ο - Gangrosa / granuloma
Treponema carateum
Causative agent of:
• Pinta
• Carate
• Maldel
• Pinto
• Azul
Manifestation:
Pinta - papule of the skin
Borrelia
- Common name: Blood Spirochetes
- causes infection in the blood and meninges
- 3-10 loose coils
- actively motile
- 30-40 axial filaments
- 2 insertion disks
- Catalase (-)
- microaerophilic
- Diagnosis: Wright’s-Giemsa stained blood
- culturable
Borrelia recurrentis
- Causative agent of: Epidemic Relapsing Fever or European Relapsing Fever
- agent of louse-borne infections
- Vector: Head louse (Pediculus humanus capitis)
- Humans are the only reservoir
Borrelia hermsii, Borrelia turicatae, Borrelia dutoni, Borrelia parkeri, Borrelia anserina
- causative agents of Endemic Relapsing Fever or American Relapsing Fever
- Vector: Soft ticks (Ornithodorus)
Borrelia burgdorferi (Common name: Sensu stricto), Borrelia afzeli, Borrelia garinii
- Revised name of Borrelia burgdorferi: Boreliella burgdorferi
- causative agents of Lyme’s Disease
*first discovered in Old Lyme Connecticut, USA
- Vector: Ticks (Ixodes dammini)
- Reservoir hosts: deer, mouse, rats, rodents
Clinical manifestations:
1. Relapsing fever
2-10 episodes of fever / relapses
2-15 days
- fever, myalgia, headache, arthralgia
2. Lyme’s infection - acute recurrent inflammatory disease of the joints
- inflammation and swelling (e.g. ankle)
Laboratory diagnosis:
1. Microscopic examination
- specimen of choice: blood, bone marrow
Leptospira
- tightly coiled, thin, flexible
- Catalase (+)
- obligate aerobe
- often seen as chain of cocci (darkfield microscope)
- “question mark appearance”
- spiral with hook ends
- shed on renal tubules of certain animals (acquired from animal urine)
- reservoir hosts: rats, dogs
- can survive in slight alkaline H2O for weeks
- can be cultivated in an artificial medium
- can be cultivated in vivo - hamster, white rat, guinea pig
Representative spp.
Leptospira interrogans icterohemorrhagica - pathogenic type; Generation time: 16 hrs.
Leptospira biflexia - non-pathogenic type
Clinical significance:
Leptospirosis / Weil’s disease (Infectious Jaundice)
1. Anicteric phase
• meninges - septicemic (3-7wks.)
• Hallmark: Aseptic Meningitis
2. Icteric phase
• liver, kidney, vascular area dysfunction
Mode of Transmission:
- traumatized skin penetration
- entry through mucous membrane / conjunctiva
Symptoms:
- vomiting
- headache
- stomach ache
Laboratory diagnosis:
Specimen: 1st week – blood, CSF; 2nd week - urine
1. Microscopic exam - motile Leptospira
2. Culture
a. Fletcher’s – animal serum + fatty acid (incubated at 30◦C for 6 weeks)
b. Stuart’s enriched w/ rabbit plasma
c. Bovine serum
d. Noguchi’s
h. EMJH (Ellinghaussen-McCullough-Johnson-Harris) - incubated 4-6 weeks in a dark room
*urine is cultured immediately
Anticoagulants: Oxalate, Heparin
3. Serodiagnosis
a. MAT (Macroscopic Agglutination Test) – screening test
b. MIT (Microscopic Agglutination Test) – confirmatory test; gold standard test for Leptospira
Serum + Antigen (Live Leptospira) = Agglutination using Darkfield microscope (positive result)
Chlamydia
- known for its inclusion bodies
*Infectious particle: Elementary bodies
*Metabolic active particles: Reticulate bodies
- Former name: Bedsonia (formerly classified as large viruses)
- Gram (-) like cell wall
- Replication: Binary fission
- Obligate intracellular parasite
- needs energy in the form of “ATP”
Laboratory diagnosis:
1. McCoy cells – best culture medium for Chlamydia
2. Cytology – demonstration of inclusion bodies using iodine or giemsa stain
a. Glycogen inclusion bodies (Halberstadter-Prowazeik) – Chlamydia trachomatis
Stain: Iodine
b. Non-glycogen inclusion bodies (Levinthal-Cole-Lillie) – Chlamydia psittaci
Stain: Giemsa
3. Serokit – DFA (detects Chlamydia antigen)
4. PCR / NAAT – gold standard
5. Frei Skin Test
Chlamydia pneumoniae
Chlamydia trachomatis
- causes STD
- #1 NGU (Non-Gonococcal Urethritis) and #1 PID (Pelvic Inflammatory Disease)
- causes TRIC (Trachoma Inclusion Conjunctivitis) and Reiter’s syndrome
- acquired via direct contact
- Sensitive to sulfonamide
Subtypes:
A, B, Ba, C, TRIC agent – causes Endemic Trachoma (blindness), inclusion conjunctivitis
D to K – causes UTI, cervitis, urethritis, epididymitis
L1, L2, L3 – causative agent of (LGV) Lymphogranuloma venereum or Buboes - STD
Mycoplasma
- Pleomorphic w/o cell wall
- Special growth requirement: Sterol except Acholeplasma
- incubated aerobically with CO2
- Penicillin-resistant
Mycoplasma pneumoniae
- Mode of Transmission: Inhalation
- Common name: Eaton agent; PPLO (Pleuropneumonia-Like Organism)
- Disease: PAP (Primary Atypical Pneumonia) or Walking pneumonia
- manifested with dry cough sounded with crackles
- it causes pleuropneumonia of cattles
- Confirmatory test: Hemadsorption test, inhibition of growth by specific anti-sera
- grows on CAP
Mycoplasma hominis
- Common name: Genital Mycoplasma
- has large fried egg colonies
- Diseases: STD, post-abortal and post-partum fever, PID
Ureaplasma urealyticum
- Urease (+)
- Common name: T-strain (“T” means tiny)
- has TINY fried egg colonies
- Disease: NGU
- Special growth requirement: Sterol
- no haze in broth
Laboratory diagnosis:
1. Culture
a. Shepherd’s mom SP4/A7/A8
b. E agar
c. NYC agar
d. PPLO agar and Edward Hayflick’s agar – selective media for Mycoplasma pneumoniae (also grows on
CAP)
colonies: fried egg or mulberry appearance
2. Serology
M. pneumoniae - cold agglutinin (Anti-I); Hemadsorption test; inhibition of growth by specific antisera
(best)
3. Stain for Mycoplasma: Dienes stain
Laboratory diagnosis
*Serological test for Rickettsia: Weil Felix Test – detection of Rickettsial antibodies
Note: Rickettsia cross react with Proteus
(+) Rickettsia, Proteus
(-) Bartonella
*Special stain for Rickettsia: Gimenez-Macchiavello
*Culture medium for Rickettsia: embryonated egg, cell culture
Bartonella
- originally from: Rickettsia family
- short, G(-) rod
- fastidious organism
- oxidase (-)
- destroy RBC
- Grows best on:
1. BAP
2. CO-cell agar
MODE OF
HABITAT INFECTION
TRANSMISSION
rodent,
B.quintana human Lice Trench Fever
(uncertain)
Carrion’s
disease,
Verruga
B. Human
Sandflies Peruana (skin
bacilliformis (uncertain)
eruption),
Oroya fever
(anemia)
Cat Scratch
Disease
saliva, bite, (CSD),
B. henselae Cat scratch of Bacillary
cat angiomatosis,
Peliosis
hepatitis
saliva, bite, Cat Scratch
B.
Cat scratch of Disease
clarridgeiae
cat (CSD)
B.
Rat Fleas Endocarditis
elizabethae
Clinical Manifestations:
Bacteremia
Endocarditis
Lymphadenopathy
Pericarditis
Afipia felis
- acquired from cats
- specimen for diagnosis: CSF
- rarely cause infections
- medically important
CULTURE MEDIA
MYCOLOGY
Mycology Terms
Mycology Terms
1. Moulds: Multicellular fungi (22-25◦C; filamentous; culturable stage)
2. Yeasts: Single-cell fungi (37◦C; unicellular subunits of fungi; tissue-form of fungi)
3. Mycosis: Fungal infection
4. Systemic mycosis: Multiorgan infection caused by fungi
5. Opportunistic mycosis: Fungal disease that occur primarily in immunocompromised patients
DIMORPHISM
• Ability of fungi to convert from yeast (37οC) to mold (25οC) and vice versa
• C. albicans - in tissues (mold, yeast)
HYPHAE
- (singular hypha) Basic structural units of fungi
- branching, threadlike, tubular filaments that either be:
1. Septate - cross wall
2. Aseptate (coenocytic) - no cross wall
3. Hyaline - transparent hyphae
4. Dematiaceous- pigmented hyphae
Staining methods:
(1) Calcofluor white = staining for fungal cell wall; apple green or bluish white fluorescence
(2) Periodic Acid Schiff = fungi stained bright red or purplish red after hydrolysis to release aldehyde
which can combine with schiff reagent. The carbohydrate in the cell wall take the red stain.
(3) Grocotts Methenamine Silver = pathogenic fungi in tissues
(4) Lactophenol cotton blue = contains:
(1) Lactic acid
(2) Phenol
(3) Glycerin
(4) Cotton blue
(5) India ink
(6) Gram stain = all fungi are gram (+)
CLASSIFICATION OF FUNGI
A. OPPORTUNISTIC MYCOSES
f. Culture characteristics
1) C. albicans grows on most fungal media as well as sheep blood, chocolate, and eosin-methylene blue
agars.
2) On cornmeal agar with Tween 80, isolates produce chlamydospores.
3) Biochemical tests
a) A positive germ tube can be a presumptive identification of C. albicans; however, not all strains are
positive. C. dubliniensis is also positive and will form chlamydospores.
- Reagent for Germ Tube Test: Serum
- Produces germ tubes after incubation in serum for 2 hours at 37 C
b) Except for C. krusei, all Candida are urease negative. Not all strains of C. krusei are urease positive.
c) Candida spp. are inositol negative.
2. Cryptococcus neoformans
a. Causes cryptococcosis (Torulosis, European Blastomycoses, Buschke’s disease in brain, meninges and
lungs), which can produce a mild to moderate pulmonary infection; however, in the
immunocompromised patient, cryptococcosis can lead to systemic infections and meningitis.
Cryptococcosis is also associated with prostate and tissue infections.
b. C. neoformans can be acquired by contact with bat, pigeon, or other bird droppings, in addition to
contaminated vegetables, fruit, and milk.
c. Identifying characteristics for direct specimens
1) On Gram stain the yeasts appear in spherical form and are not of uniform size. (“star-burst pattern” in
CSF)
2) Hematoxylin and eosin stains are used to show capsules in tissue.
3) Direct antigen test for cryptococcal antigen: Performed on CSF and serum specimens
4) India ink – negative stain; seen as yeast cells surrounded by huge, clear capsules. It rarely forms
hyphae.
d. Culture characteristics
1) Brown to black colonies on Niger seed (bird seed) agar or caffeic acid agars
2) Only forms blastoconidia
3) Biochemical tests
a) Positive for urease and phenol oxidase
b) Inositol utilization
c) Negative for nitrate reduction
3. ASPERGILLUS
• Microscopically, appears as long, dichotomous non-septate hyphae / conidiophore in the tissues
that is swollen at the tip to form a vesicle
• A. fumigatus
4. Zygomycosis / Mucormycosis
• Causative agents: Rhizopus, Absidia, Mucor
• (Zygomycetes / Phycomycetes)
• transmission: inhalation of conidia
• tissue form: NONSEPTATE HYPHAE (coenocytic)
5. Penicillium spp.
• Spore: bearing hyphae characteristically form a “penicillus” or brush (brush like conidiophore)
6. Fusarium
Macroscopic: white at first, cottony or wooly frequently becoming pink or purple colony
I. SUPERFICIAL MYCOSES
17. Malassezia furfur
• Causes Ptyriasis (Tinea) versicolor, liver spots (disseminated infection for patients undergoing
lipid therapy)
• Hypo- or hyperpigmentation of skin (characterized by superficial browny scaly areas on person
• KOH: budding lipophilic yeast cells with short curved hyphae
• “spaghetti and meatballs appearance” (PAS);
• Needs lipids – (+) SDA w/ olive oil
2. Piedra agent
• Black piedra – Piedraia hortai
• White piedra – Trichosporon beigelii
3. Phaeoannelomyces werneckii
• Cause tinea nigra; Brownish spot (dark pigmentation)
• Dematicaeous – Moist, shiny-black and yeast like colonies
Trichophyton tonsuran
• Balloon shape microconidia; “Black dot”, endothrix hair invasion
• Common cause of Tinea capitis
2. MICROSPORUM
Microsporum canis
• Zoophilic (animals)
• Is an organism that can cause Tinea capitis in humans, and simple ringworm in pets
• Growth on rice grain medium, Spindle shape macroconidia
• Green to yellow fluorescence of ectothrix hair (Wood’s lamp +)
Microsporum gypseum
• Geophilic (soil), do not fluoresence on UVL (-)
• Oblong (ellipsoidal) macroconidia
• Causes ectothrix hair infection
Microsporum audouinii
• Anthropophilic (insects) fungus causing non-inflammatory infections of scalp and skin
• Most common cause of Tinea capitis in children
• Fluoresce yellow-green on Wood’s lamp (+)
3. EPIDERMOPHYTON
E. floccosum
• Club shape macroconidia (Dutch pants fuseaux)
• Tinea cruris and Tinea pedis
• SDA + chloromycetin – velvety, powdery greenish + actidion yellow and cottony center
Lab. Diagnosis
• 10% KOH method
- Specimen consist of scrapings of both skin, nails and hairs plucked from involved
areas.
- Specimens placed on a slide with a drop of 10-20% KOH, covered with a
coverslip and examined immediately and then after 20 mins.
- Used as digesting agent; digest keratinous tissue and facilitates observation of
any fungi present
• Culture on Sabouraud’s agar slants on mycosel, incubated for 1-3 weeks at room temperature
- Chloramphenicol and cycloheximide are the antibiotics which serve as inhibitory
agents present in Mycosel agar
• Wood’s lamp (UVL) – fluorescence (Microsporum)
• Treatment:
Local antifungal creams - miconazole,
Oral - griseofulvin, ketoconazole
Sporothrix schenckii
• Associated with asteroid bodies – they may appear as small, round to cigar-shaped, gram
positive budding cells (tissue-cigar shaped bodies); product of antigen-antibody reaction
• Mold form – Tear drop shaped conidia in “flowerette” or “sleeve” arrangement (flowerette
conidia)
• ROSE GARDENER’S DISEASE, SPOROTRICHOSIS, GARDEN’S DISEASE – a chronic granulomatous
infection when traumatically introduced into the skin (cord-like multiple subcutaneous nodules)
Chromoblastomycosis agents
• Old name: Chromomycosis
• Caused by dematiaceous fungi (brown pigment fungi)
• Type of sporulation – ID genus & spp.
• Distribution: world-wide, more common among bare-footed
Phialophora verrucosa – specialized conidiophores that are vase-shape (vase like). There are flask-
shaped or tubular phialides with flared tips. (or with cup-shaped collarettes)
Fonsecae pedrosoi – most common; most conidia form in short branching chains (acrotheca) with the
terminal cell budding to form a new conidium.
Cladosporium carrionii – with numerous elongate conidiophores that give rise to long flexous chains of
delicate conidia
• It usually infects the skin and the subcutaneous tissues (brown sclerotic bodies)
• Most lesions are painless unless complicated by bacterial super infection (cauliflower-like lesion)
• Colonies vary in pigmentation from olive-gray to brown to black (dark colonies w/ jet black
reverse)
Rhinosporidum seeberi
• Rhinosporidiosis
• Characterized by a friable, sessile or pedunculated polypoid masses on the mucous
membrane of the nose, pharynx, soft palate and conjunctiva in clinical specimen.
• The polypoid masses (tissue form) show round, thicked-walled (sporangium / sporangia) –
sac-like structure filled with endospores
Loboa loboi
• lesion: keloid-like subcutaneous nodule involving the extremities
• tissue: multiple budding cells in chain
C. SYSTEMIC MYCOSES
1. This fungal group is often acquired via inhalation (infective spores) and can disseminate to any
of the body's organ systems.
2. Most systemic fungi are dimorphic, exhibiting a nonmould (e.g., yeast) parasitic phase at 35-37°C and
a mould (or mycelial) saprobic phase at 25-30°C.
Ideal specimen: sputum
Primary infection: pulmonary involvement
3. Most systemic fungi are dimorphic, exhibiting a nonmould (e.g., yeast) parasitic phase at 35-37°C and
a mould (or mycelial) saprobic phase at 25-30°C.
4. Identifying characteristics
a. Identification is based on temperature and medium requirements and colony and microscopic
morphology.
b. Most systemic dimorphic fungi are very slow growers and require 3-7 weeks to grow.
c. Because the mould forms are highly infective, slants are used for culture.
d. Colonies are membranous and develop tan aerial mycelia.
e. Conidia identification is necessary in species identification.
f. Conversion of dimorphic fungi from the mould to yeast phase is confirmation that the fungus in
question is dimorphic.
5. Systemic dimorphic fungi
a. Blastomyces dermatitidis (blastomycosis)
1. BLASTOMYCES DERMATITIDIS
a. Blastomycosis is a respiratory infection that can affect the skin and bones. Infections are
acquired by inhalation of conidia or hyphae and can be mild to chronic.
b. The precise environmental location of this fungus is unknown. Outbreaks have occurred
following contact with moist environments such as streams and rivers and contact with decaying
vegetation. Cases in the U.S. occur most frequently in the Ohio and Mississippi River basins.
More cases occur in males than in females.
c. B. dermatitidis can be cultured from tissue or body fluids.
• North America Blastomycosis, Chicago Disease, Gilchrist’s disease – PNEUMONIA, SKIN
INFECTION
• tissue form - single-budding yeast with BROAD based (double countered)
• mold form: lollipop in appearance
• Culture medium with cycloheximide
• KOH method (tissue)
• Amphoterecin B
2. PARACOCCIDIOIDES BRASILIENSIS
a. Paracoccidioidomycosis is a chronic granulomatous disease of the lungs and skin that can
spread to the liver and spleen.
b. Mostly found in South America
c. Acquired by spore inhalation or ingestion
3. HISTOPLASMA CAPSULATUM
a. Histoplasmosis can be a fatal pulmonary infection but can also affect the spleen, liver,
kidneys, bone marrow, and heart.
b. Infection is acquired by spore inhalation from barns, chicken houses, and bat caves. H.
capsulatum has been associated with guano, in particular from starlings and bats.
c. Most infections occur in the southern and Midwestern U.S. and along the Appalachian
Mountains. The major risk factor for infection is environmental exposure.
VIROLOGY
Viral structure
• a nucleic acid genome (RNA or DNA)
• Capsid - a protective protein coat
• Envelope - lipid derived from host
a. Naked virus – Ether resistant
b. Enveloped virus – Ether sensitive
• Virion – complete virus particle; infectious unit of a virus
Replication (APUSAR)
1. Adsorption is attachment of the virus to a specific receptor on the host cell.
2. Penetration is entry of the virus into the host cell.
3. Uncoating occurs when there is either the separation of the capsid from the genome or
rearrangement of the capsid proteins exposing the genome for transcription and replication.
4. Synthesis or the eclipse period is the stage when the genetic material is replicated but intact virions
are not yet detectable.
a. Viral DNA or RNA serves as the template for mRNA production.
b. mRNA codes for viral protein and enzymes necessary for nucleic acid synthesis.
5. Assembly (maturation): Genetic material is assembled into a protein coat.
6. Viruses are then released from the host cell.
a. Cell lysis: Naked viruses lyse host cell and leave through a hole in the plasma membrane.
b. Budding: Intact virion pushes outward from a host's membrane. The membrane wraps around the
virion; the membrane is cleaved and then resealed around the virion, thus becoming the viral envelope.
Diagnostic Virology
• Electron Microscopy – viral morphology
• Light Microscopy - inclusion bodies
Electron Microscopy
1. Due to size, most individual virions can only be seen by electron microscopy. However, the poxviruses
are about the size of some small bacteria.
2. Electron microscopy is sometimes used to identify Norwalk viruses, astrovirus, calicivirus, and
coronavirus. Electron microscopy is expensive, requires expertise, and is usually not very sensitive. For
these reasons, electron microscopy is not commonly used.
*Stains: Negative stain, Gold stain, Silver stain, Phosphotungstic acid
Transport medium
• Stuart’s medium, Leibovitz-Emory
• Earles/Hanks BSS, amino acids, vitamins and bicarbonates, Penicillin-Streptomycin, phenol red
Characteristic CPE
*Use tissue culture to be demonstrated
• Rounding necrosis - Enterovirus
• Ballooning / Giant cell - HSV, VZV
• Grape-like cluster - Adenovirus
• Syncitium form – RSV, Measles, Rubella
• Hemagglutination / Hemadsorption – Influenza, Parainfluenza, Measles and Mumps
• Refractile, round cell - rhinovirus (33◦C)
• No CPE - Parainfluenzae, mumps, Influenza
DNA VIRUSES
Herpesviridae - Herpes simplex viruses types 1 and 2, varicella zoster virus, Epstein-Barr virus,
cytomegalovirus, human herpesviruses 6, 7, and 8
Adenoviridae – Adenovirus
Papillomaviridae – Papillomavirus
POXVIRUS
• Double stranded DNA enveloped, brick-shaped, complex virus
• largest virus
• Inclusions: Guarnieri bodies, vesicular skin lesion in the host
• Variola major virus – Smallpox (According to WHO, in 1980, the world is declared to be free from
smallpox)
• Variola minor virus - Alastrim
• Vaccinia – complication of vaccine against smallpox that ranged from milad reaction and fatal
encephalitis
Diagnosis:
• CPE on cell culture or pocks on chorioallantoic membrane (CAM)
HERPES VIRUSES
*Causes latent infection
Herpes simplex virus Type 1 - Gingivostomatitis in children and young adults, recurrent oral-labial
infection (cold sores/oral herpes/herpes labialis/fever blister), infection of the cornea (keratitis), herpes
encephalitis
-Latency site: Trigeminal ganglion
Herpes simplex virus Type 2 - Genital herpes (herpes genitalis), neonatal herpes
-Latency site: Sacral ganglion
Varicella zoster (Human herpesvirus 3) - Chickenpox (primary infection), shingles or zoster (reactivation)
-Latency site: Dorsal root ganglion
• Diagnosis:
1. Tzanck smear = HSV and VZV
2. ELISA = most widely used
• All adenovirus types except those of avian origin have a group reactive antigen, the hexon.
HEPADNAVIRUS
• Hepatitis B virus - #1 blood borne infection, Hepatocellular carcinoma
• Hepatitis D (Co-infection, Super-infection)
• Serological markers: HBsAg, HBeAg, anti-HBc, anti-HBe, anti-HBs
• Diagnosis: ELISA, PCR
RNA VIRUSES
Coronaviridae – Coronavirus
Togaviridae - Rubella virus and western, eastern, and Venezuelan equine encephalitis viruses
Flaviviridae - Yellow fever, dengue, St. Louis encephalitis, hepatitis C, and West Nile viruses
Enterovirus
1. Poliovirus (paralytic poliomyelitis)
• Transmission: fecal-oral route; respiratory droplets
• anterior horn cells of the spinal cord
• Vaccines:
SABIN - live attenuated virus vaccine (oral)
SALK – inactivated (dead) virus
2. Coxsackie A virus – usually associated with surface rashes called exanthems (Herpangina,
Myocarditis)
- Herpangina – fever with painful ulcers on the palate and tongue leading to problems swallowing and
vomiting
- Myocarditis – fever, chest pains, arrhythmia and even cardiac failure result
ORTHOMYXOVIRUS (MYXOVIRUS)
• HELICAL, SEGMENTED, ENVELOPED
• HEMAGGLUTININ (H) and NEURAMINIDASE (N)
• Antigenic changes:
• ANTIGENIC SHIFT - genetic reassortment - pandemic
• ANTIGENIC DRIFT - point mutation - epidemic
PARAMYXOVIRIDAE
• Single stranded RNA, helical, non-segmented, enveloped viruses
• Hemagglutinin (H), Neuraminidase (N), Fusion (F) Ag
Parainfluenza virus
• Laryngotracheobronchitis – common clinical manifestation of this virus
• Best specimens are nasopharyngeal aspirate / washing
• Culture: PMKC (Primary Monkey Kidney Cell), LLC-MK2 cell
• Presence can be demonstrated by hemadsorption, IIF (Indirect Immunofluorescence), EIA
• Treatment: aerosolized ribavirin; no vaccine
Mumps virus
• Mumps complications: Parotitis, Orchitis (20-30% of cases in post-pubertal males but rarely
sterility), Aseptic meningitis
• Virus can be cultured from saliva, urine or spinal fluid
• Diagnosis: IFA, HAI
• Culture - PMK, HEK, Embryonated egg
HENIPAVIRUS - ssRNA
1. NIPAH virus - Encephalitis (pig to man)
2. HENDRA virus - respiratory disease of horses
RUBELLA VIRUS
• German Measles (3-day rash), “blueberry muffin” baby
• Teratogenic virus (fetal defect)
• Direct exam by IF, EIA, HA test (sensitive)
FILOVIRIDAE - Filamentous
• MARBURG VIRUS and EBOLA VIRUS- causes HEMORRHAGIC FEVER
FLAVIVIRUS (ARBOVIRUS)
• Dengue fever virus (Vector: Aedes aegypti and Stegomyia mosquito) - acute hemorrhagic fever /
DHF (Dengue Hemorrhagic Fever), saddleback fever, breakbone fever, dengue syndrome, DSS
(Dengue Shock Syndrome)
4 serotypes: DEN-1, DEN-2, DEN-3, DEN-4
ADE (Antibody Dependent Enhancement) – causes the pathology of secondary infection; this
occurs when antiviral antibodies enhance viral entry into host cells, leading to increased
infectivity in the host cells
• St. Louis Encephalitis virus (Vector: Culex mosquito) – causes Encephalitis
• Yellow fever virus (Vector: Aedes mosquito) – causes Hemorrhagic fever, Yellow jack, Black
vomit, American plague)
Councilman bodies – necrotic masses appear in the cytoplasm of hepatocytes
• Japanese B Encephalitis virus (Vector: Culex mosquito) – causes Encephalitis
• West Nile Fever virus (Vector: Culex mosquito, Argosidae ticks) – causes Dengue syndrome,
Encephalitis
BUNYAVIRUS
• California encephalitis virus, La Crosse virus, Rift valley fever virus, Hantaan virus
CORONAVIRUS - icosahedral, club-shaped virus; look like a crown (“corona” latin = crown)
• Widespread in human and animals. It infects dogs, cats, poulty, cattles, rodents and pigs
• Known pathogen: HuCV (Human Corona Virus)
• SARS-CoV-1 – first erupted in Guangdong, China (Spring 2002)
• Immunity conferred by infection
• Disease caused: common colds, gastroenteritis
CALCIVIRUS
• Norwalk virus - adult diarrhea; causes viral gastroenteritis in adult
• Astrovirus (star-like virus) – described in relation to an outbreak of gastroenteritis (diarrhea)
Hepatotrophic viruses
• HAV (Picornavirus)
a. Infectious Hepatitis, Short-Incubation Hepatitis
b. HAV contains RNA, and the naked virion has an icosahedral shape. HAV ranges in size from 24
to 30 nm.
c. It is a member of the family Picornaviridae and the genus Hepatovirus.
• HBV (Hepadnavirus)
a. Long-Incubation Hepatitis
b. HBV contains partially double-stranded DNA. The complete virion has an envelope, ranges in
size between 42 and 47 nm, and is sometimes referred to as Dane particles.
c. The virus is unusual in that an RNA intermediate is required for replication of the genome. The
virus needs a viral-encoded reverse transcriptase for replication.
d. Member of the family Hepadnaviridae
• HCV (Flavivirus)
a. Post-transfusion Hepatitis, NANB Hepatitis
b. HCV is the most common cause of non-A, non-B (NANB) hepatitis. It is common worldwide.
c. Spread through contaminated blood products, organ transplants, renal dialysis, and
intravenous drug abuse
d. No vaccine currently exists for HCV.
Test of choice: HCV-RNA (Viral load)
• HEV (Calicivirus)
a. Water-borne Hepatitis
b. HEV is spread by the fecal-oral route, often in contaminated water. It is the most common
cause of hepatitis in some countries with poor sanitation.
• HGV (Flavivirus)
a. Blood-borne Hepatitis
b. It is in the same family, Flaviviridae, as HCV
SPECIAL TOPICS:
1. WATER BACTERIOLOGY
= E. coli, Coliforms
= Sodium thiosulfate – must be present in the bottle container of test samples of chlorinated
water because it neutralizes chlorine.
= Reported in MPN (Most Probable Number)
1. Presumptive Test
Lactose broth / Lauryl tryptose + water → incubate at 35οC for 24 hours = gas (+); 48
hours (-)
(+) Result: gas only
2. Confirmatory Test
EMB / Endo agar + inoculums (24 hours incubation) = colony
(+) Result: growth
3. Completed test
Lactose broth fermentation tube + organism → incubate at 35οC for 24-48 hours = acid +
gas (+)
(+) Result: gas and growth
Multiple Tube Fermentation Test
Gold standard test (5 test tubes)
Reported in MPN (Most Probable Number)
Positive: >1.1 MPN/100mL; Negative: <1.1 MPN/100mL
Stages of MTFT
Presumptive Test – Triple Strength Lactose Tube broth + H2O = (+) gas (Durham tube); (-) no
gas after 48 hours
Confirmatory Test for Total Coliforms – Brilliant green lactose broth = (+) gas
Confirmatory test for Fecal Coliforms = EC broth at 44οC = (+) gas
2. MILK BACTERIOLOGY
Pathogens: Salmonella, V. cholera, B. abortus, C. diptheriae, M. bovis, M. tuberculosis, B.
anthracis
Coxiella, FMDV (Foot-and-Mouth Disease Virus), Cowpox virus, S. pyogenes, Clostridium
QUICK FACTS
1. Nuclear or cytoplasmic membrane is the part of the virus in which the envelope is
acquired.
2. Eosinophilic cytoplasmic inclusions are elementary bodies of Poxvirus.
3. The incubation period for fungi that grow on blood is 28 days.
4. Dientamoeba fragilis and Trichomonas vaginalis have neither a cyst form.
5. The roundworm that inhabits small intestine and usually demonstrated as
rhabditiform larva is Strongyloides stercoralis.
6. Calcium alginate swab is used for specimens taken from nasopharynx for the isolation
of Corynebacterium diptheriae.
7. Staphylococcus aureus can be isolated from stool cultures by medium with 7.5% salt.
8. Methylene blue and malachite green are used as counter stains for acid fast staining.
9. Veilonella and Actinomyces are examples of anaerobic bacteria.
10. The positive result for bile solubility test is lysis of the semisolid media.
11. Neisseria meningitides does contain a capsule.
12. The epithet/species of an organism is written in a lower case form.
13. Corynebacterium diptheriae produces the Babes-Ernst granules.
14. To prepare an agar medium, put water first then the powdered agar. (Half water →
Agar → Half water)
15. The composition of an iodophore is iodine and detergent.
16. The composition of tincture of iodine is iodine and alcohol.
17. The type of plasma that is used for tube coagulase test is rabbit plasma or human
plasma.
18. Clostridium perfringens is lecithinase positive and bull’s eye hemolysis.
19. MIC (Minimum Inhibitory Concentration) refers to the lowest concentration of
drugs that inhibits bacterial growth.
20. MLC (Minimum Lethal Concentration) refers to the lowest concentration of drugs
that kills/destroys organism.
21. Gram-negative, coffee bean shaped diplococci with adjacent sides flattened is
descriptive of Neisseria spp.
22. Specimens from eyes, rectum, vaginal discharge, oral cavity and gastric fluid may
be appropriate for culturing Neisseria gonorrheae.
23. Corynebacterium diptheriae grows on cysteine tellurite agar.
24. Bordetella pertussis is the causative agent of whooping cough.
25. Bile salt inhibits gram positive bacteria and fungi in MAC agar.
26. Thrush is Candida albicans infection on mouth.
27. Strongyloides stercoralis possesses a rhabditiform larva in stool.
APOLLON QUESTIONS
Bacteriology 8, 26, 38, 48, 52, 54, 58, 62, 75, 77, 81, 83,
86, 91, 97, 114, 124, 148, 150, 151, 166,
170, 190, 196, 215, 220
Mycology 26, 30, 36, 38, 75, 76
Virology 1, 5, 7, 12, 17, 31
Parasitology 4, 5, 24, 28, 56, 78, 89, 94, 97, 99, 100,
108, 103, 114, 123, 125, 128, 129, 135,
141, 144, 146, 154
References:
• Bailey & Scott’s Diagnostic Microbiology Twelfth Edition by Betty A. Forbes, Daniel
F. Sahm and Alice S. Weissfeld
• Textbook of Diagnostic Microbiology by Connie R. Mahon
• Review Handbook in Diagnostic Bacteriology by Maria Teresa T. Rodriguez
• Success! In Clinical Laboratory Science by Anna P. Ciulla
Notes of:
• Ms. Alicia Aldave
• Mr. Emerson Ronald K. Bernardino
• Mr. Nathaniel Ranon