Microbiology - Bacte, Myco, Viro

* NLE * MIDWIFERY * LET * RAD TECH * CRIMINOLOGY * DENTISTRY * PHARMACY
MICROBIOLOGY
( BACTERIOLOGY, MYCOLOGY, VIROLOGY)
Prof: Charles U. Arconado, RMT, MAEd, ASCP, AMT, DTA, IFBA PC, 9th Placer
TABLE OF SPECIFICATION FOR MICROBIOLOGY

(MEDICAL TECHNOLOGY LICENSURE EXAMINATION)
1. Bacteriology 49%
a. Collection, transport, processing and staining of specimens
b. Culture media
c. Bacteria (Aerobes)
- Morphology and staining characteristics
- Cultural characterisitics
- Work-up for identification: Biochemical, Differential and Confirmatory
tests
- Serological / Molecular tests
- Susceptibility tests
- Bacteriologic examination of water, food, milk and utensils
d. Bacteria (Anaerobes)
e. Mycobacteria
f. Other bacteria with unusual growth requirements (Spirochetes, Chlamydia,
Mycoplasma, Rickettsia)
2. Mycology 4%
a. Collection, transport and examination of clinical specimens
b. Culture
3. Virology 4%
a. General characteristics, transmission and diseases
b. Collection, transport and examination of clinical specimens
4. Equipment and Instrumentation 5%

a. Manual
b. Automated
5. Quality Assurance and Safety 8%

a. Collection of specimen
b. Quality control
c. Safety of patients and staff
d. Safety in the workplace and environment
6. Parasitology 30%
TOTAL 100%
BACTERIOLOGY
BACTERIOLOGY – study of bacteria

• Taxonomy – is an area of biologic science comprising three distinct, but highly interralated,
disciplines that include classification, nomenclature, and identification.
• Classification:
MICROBIOLOGY by CUA Page 1

1. Kingdom
2. Division
3. Class
4. Order
5. Family
6. Genus
7. Species
• Anton van Leeuwenhoek - Observation of “little animals”; first to describe bacteria; invention of
microscope; Father of Microbiology
• Carl Von Linne – a Swedish botanist laid down the basic rules for taxonomic categories (binomial
system).
• The proper way to write a scientific name is by putting an underline or italicizing it.
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.

4. Example of bacterias that are lactose fermenters are E. coli, Enterobacter and Klebsiella.
5. Bacillus sporulates aerobically while Clostridium sporulates anaerobically.
6. Mycobacteria were once classified as fungi (eukaryotic), but now it is known to be bacteria
(prokaryotic).
7. Pseudomonas aeruginosa is known to produce green pigment.
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
1. Strain – is a population of organisms that is differentiated from populations within a particular

taxonomic category.
• e.g.
Coronavirus – common cold
o SARS (severe acute respiratory syndrome) = 2003
o MERS-CoV (Middle East Respiratory Syndrome-Corona Virus) = 2012
o SARS-CoV-2 = 2019
2. Biovars – are variant prokaryotic strains characterized by biochemical or physiological differences.

• e.g. Staphylococcus aureus is known to be a fermenter of mannitol. There are biovars of this
bacterium that cannot ferment mannitol.
3. Morphovars – are variant prokaryotic strains which differ morphologically.

• Enterobacteriaceae – Largest group of bacteria; have the largest number of bacteria which have
morphovars
• e.g. Escherichia coli are known to be bacilli. There are morphovars of those bacteria that are
coccobacilli.
4. Serovars – are strains with distinctive antigenic properties.
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)
I. Cell Envelope – outermost structure

A. Outer membrane = G (-)
B. Periplasm = G (-)
C. Cell wall = G (+), G (-)
D. Plasma membrane = G (+), G (-)
Gram Stain – General Rule
 All cocci are Gram (+), EXCEPT:

Neisseria
Moraxella / Branhamella
Veilonella
Megasphera

Acidaaminococcus
 All bacilli are Gram (-), EXCEPT: (BBMACCPELLN)
Bacillus
Bifidobacillus
Mycobacteria
Actinomyces
Clostridium
Corynebacterium
Propionebacterium
Erysipelothrix rhusiopathiae
Listeria monocytogenes
Lactobacillus
Nocardia
 All spiral organisms are Gram (-)
 All yeasts are Gram (+)
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
• Chemicals that can destroy the cell wall:

*Penicillin - inhibits transpeptidation (process of replicating peptidoglycan)
*70% alcohol - Contact time: 1 min.
*10% Chlorox / muriatic acid - ionic charge
*Lysol
G (+) Cell wall

• Composes of thick layer of peptidoglycan
• Consists of glycan chains of alternating N-acetyl-D-muramic acid and N-acetyl-D-glucosamine
• Teichoic acid [found only on G (+) cell wall] - contributes to the negative charge of the G(+) cell
wall; regulates cation
• Contains exotoxin
G (-) cell wall

• Composes of thin layer of peptidoglycan (can be found on the inner part of the cell envelope)
• protein, phospholipids, LPS (outer membrane)
*LPS - Lipopolysaccharide
A. lipid A – major constituent, toxic
B. core polysaccharide
C. antigenic O – specific polysaccharide for serologic identification
• LPS [found only on G (-) cell wall] - contributes to the negative charge of the G(-) cell wall;
regulates cation
• Contains endotoxin
• The periplasm is the specific area for transpeptidation
Comparison of a Gram positive and Gram negative Bacterial Cell Wall
CELL WALL COMPONENT GRAM POSITIVE GRAM NEGATIVE

Peptidoglycan Thick Layer Thin Layer

Periplasmic Space/Area Absent Present
Outer Membrane Absent Present
Teichoic Acid Present Absent
Lipoteichoic Acid Present Absent
Lipoproteins Absent Present
Lipopolysaccharide Layer Absent Present
Summary of Gram Staining Technique: Reagent/s and Reactions
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”
Summary of Acid Fast Staining Technique: Reagent/s and Reactions
KEY STEPS ACID NON-ACID

CODE REAGENT/S DURATION/TIME
(ZIEHL-NEELSEN METHOD) FAST FAST
CARBOL
C Primary/Initial Staining RED RED
FUCHSIN 4-5 MINUTES
PHYSICAL: (rinse)
H Mordanting RED RED
HEAT
ACID ALCOHOL
2 MINUTES
A Decolorization/Differentiation (3% HCl in 95% RED COLORLESS
(rinse)
Ethanol)
METHYLENE
M Counterstaining 1 MINUTE (rinse) RED BLUE
BLUE
KEY STEPS ACID NON-ACID
CODE REAGENT/S DURATION/TIME
(KINYOUN METHOD) FAST FAST
CARBOL
FUCHSIN 5 MINUTES
CHEMICAL: (rinse)
T Mordanting RED RED
TERGITOL

ACID ALCOHOL
2 MINUTES
A Decolorization/Differentiation (3% H2SO4 in RED COLORLESS
(rinse)
95% Ethanol)
METHYLENE 1-3 MINUTES
M Counterstaining RED BLUE
BLUE (rinse)
Summary of Modified Kinyoun Method for Partially/weakly Acid-Fast Organisms
KEY STEPS
ACID NON-ACID
CODE (MODIFIED KINYOUN REAGENT/S DURATION/TIME
FAST FAST
METHOD)
CARBOL
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
3. Plasma / Inner Membrane

• it is a lipoprotein that surrounds the cytoplasm
• It functions as mitochondria, Golgi complexes and lysosomes of eukaryotic cells.
Functions:
• Generate ATP
• Site of photosynthesis
• Site of respiration
• Regulates osmotic pressure
• Transports solute
a. Ribosomes (70S) – site of protein synthesis

b. Nucleoid
– circular double stranded naked DNA
- bacterial structure where the chromosome is located
- all bacteria have only 1 chromosome EXCEPT Vibrio spp. (have more than 1 chromosome)
c. Plasmids
– antibiotic resistance structure; exists independently of chromosome
- extrachromosomal double stranded DNA
- they sometimes disappear during cell division and they can transform bacteria to become pathogenic

1. large plasmid – responsible for the production of β-lactamase against β-lactam of penicillin
and oxacillin
2. small plasmid – resistant to tetracyclines, chloramphenicol and gentamicin
d. Inclusion Bodies –
1. Food reserve
2. Lessens the osmotic pressure
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
Types of Flagellar Arrangements in Bacteria

MONOTRICHOUS – single polar flagellum (figure a)
AMPHITRICHOUS – single or clusters of flagella at both poles (figure b)
LOPHOTRICHOUS – single tuft of cluster of flagella at a pole (figure c)
BILOPHOTRICHOUS – two tufts or clusters of flagella at both poles (figure d)
PERITRICHOUS – single flagellum randomly distributed around the bacteria
ATRICHOUS – no flagella
c. Pili and fimbriae

- attachment
- hairlike structure (2µm)
- Neisseria gonorrheae – w/o flagellum but with pili
a. Common pili – virulence factor / bacterial attachment or adherence
b. Sex pili (transposon) – gene transfer (conjugation process)
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
Bacteria with Endospores:

1. Bacillus
2. Clostridium
Bacteria with Capsule:

• Slimy layer
• Anti-phagocytic (can resist phagocytosis; factor for immunoevasion)
• Composition: polysaccharide capsule except B. anthracis (D-glutamate or D-glutamic acid)
1. Bacillus anthracis
2. Streptococcus pneumoniae
3. Haemophilus influenzae
4. Neisseria meningitidis
5. Klebsiella pneumoniae
6. Streptococcus agalactiae
Bacteria with Pili:

1. Neisseria
2. Pseudomonas
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

> contain many membrane-bounded organelles in which cellular functions are performed: endoplasmic
reticulum, golgi body, mitochondria, lysosomes and nucleus.
>more complex compared with prokaryotic cell
Characteristics of Prokaryotes Versus Eukaryotes
CRITERIA OF PROKARYOTE EUKARYOTE

DIFFERENTIATION
1. TYPICAL SIZE 0.2 - 2 μm in diameter 7 – 100 μm in diameter
0.5 – 5 μm in length > 10 μm in length
2. GENETIC MATERIAL CIRCULAR; complexed with RNA LINEAR; complexed with basic
**Except: Streptomyces spp. histones & other proteins
Borrelia burgdorferi
3. NUCLEAR BODY NUCLEOID NUCLEUS
4. CELL WALL Made up of PEPTIDOGLYCAN (aka With Cell Wall
murein or mucopeptide layer) in Plants: Cellulose & lignin
most bacteria. Algae: Glycan
Backbone structures: Fungi: complex carbohydrates
N-acetylglucosamine (NAG) (polymers) such as
N-acetylmuramic acid (NAM) chitin, mannan, glucan,
**Except: Mycoplasma, chitosan)
Ureaplasma, Sprioplasma, & Without Cell Wall: Animals &
Anaeroplasma Protozoans
4. CELL DIVISION/MEANS Asexual: BINARY FISSION – SOMATIC/BODY CELLS –
OF REPRODUCTION spontaneous splitting/division of Mitosis
bacterial cells given the optimum
growth requirements
**DOUBLING/GENERATION TIME SEX CELLS/GAMETES – Meiosis
– time/period required for a
bacterial cell to divide into two.
4. CYTOPLASMIC/PLASMA Membrane Phosphoplipid Bilayer Fluid Phosphoplipid Bilayer
MEMBRANE usually WITHOUT carbohydrates usually WITH carbohydrates
and sterols and sterols. Also has glycolipids
**Except: Mycoplasma & & glycoproteins
Ureaplasma – with sterols
5. MEMBRANE BOUND ABSENT PRESENT

ORGANELLES
• SITE OF PROTEIN Free Ribosomes Ribosomes in the Endoplasmic
SYNTHESIS reticulum
• SITE OF ENERGY Cytoplasmic/Plasma Membrane Mitochondria
PRODUCTION
• RIBOSOMAL SIZE
70S (consists of 50S & 30S 80S (consists of 60S & 40S
subunits) subunits)
BACTERIAL NUTRITION AND GROWTH
1. According to O2 requirement
a. Aerobic bacteria
>use oxygen and grow well in room air
> Obligate aerobic
- Bordetella
- Brucella
- Pseudomonas
- Haemophilus

- Mycobacterium
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
c. Microaerophilic – organisms that requires 2-10% oxygen for growth

- Campylobacter
- Helicobacter
- Treponema pallidum
d. Aerotolerant anaerobic – anaerobe that grows well in the presence of oxygen

- Lactobacillus
- Streptococcus pyogenes
- Enterococcus faecalis
Enzymes that inhibits the toxic effect of O2:

- SOD (Superoxide Dismutase)
- Catalase
2. According to CO2 requirement

- 0.03% CO2 = needed by aerobic bacteria
- Capnophilic bacteria – require 5-10% CO2
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria gonorrhea
3. According to nutritional requirement

A. As to carbon source
> Autotroph – they use CO2 as the sole source of carbon
> Heterotroph – they used reduced, preformed, organic molecules from other bacteria
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.
*Most of G(+) are chemoheteroorganotrophs.

*Most of G(-) are photoautolithotrophs.
*Saprophytes – require dead organic substances.

*Parasites – require organic substances from living tissues.
4. As to temperature requirement
35-37οC – is the optimum temperature for most bacteria

A. Psychrophilic / Cryophilic – grows well at 0◦C to a maximum of 15◦C (refrigerator)
> Listeria monocytogenes – coleslaw food poison
> Yersinia enterocolitica - #1 blood bag contaminant
B. Mesophilic – 20-45οC, most of the pathogenic bacteria grow at this temperature

> Escherichia coli
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
8. As to high salt concentration requirement

• Halophilic - requires increased concentration of sodium chloride (6.5 % NaCl)
> Staphylococcus aureus
> Vibrio
9. As to growth factor requirement

- purine, pyrimidine, amino acids
Growth
Generation – doubling replication

Generation time – time required for cell to double
BACTERIAL GROWTH CURVE
A. Lag phase / Period of rejuvinescence / Adjustment phase

- bacteria is adapting or adjusting to new environment
- increase in bacterial cell size, not in number
- No doubling cells
- Biosynthesis
- Resistant to antibiotics
B. Log phase / Exponential phase

- increase in growth rate (cell division)
- increase in bacterial cell number
- doubling cells
- Biochemical process
- Susceptible to antibiotics or antimicrobial agents
C. Plateau / Stationary phase

- No net growth
- balance between live bacterial cells and dying bacterial cells
- Evidence: drying of culture medium
D. Death / Period of Decline

- increase in death rate
- cessation of bacterial growth
- accumulation of toxic wastes
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
1. Organization and structure of genetic material

• The bacterial chromosome contains all genes essential for viability and exists as double
stranded, circular, naked DNA
• DNA – encodes genetic material, RNA
• A DNA sequence that encodes for a specific product (RNA or protein) is defined as a gene.
• All genes taken together within an organism comprise that organism’s genome.
• Genome organized in discrete elements is known as chromosomes.
2. Replication and expression of genetic material

• The process generally takes approximately 40 minutes in rapidly growing bacteria
A. Transcription – converts the DNA base sequence of a gene into a messenger RNA molecule; RNA
polymerase is the enzyme central to the transcription process.
B. Translation – involves protein synthesis; the genetic code with mRNA molecule is translated into
specific amino acid sequence that are responsible protein structure and function.
3. The mechanisms by which genetic information is changed and exchanged among bacteria
Mutation – a process whereby there is a change / exchange of genes

a. physical
b. chemical
c. gene exchange
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
ANABOLISM – there is a synthesis of complex molecules and energy production

CATABOLISM – the large complex molecules are converted to simpler, smaller molecules accompanied
by energy utilization
• Respiration – is an ATP-generating process in which molecules are oxidized and the final
electron acceptor is an inorganic molecule
Glucose ------> CO2 + H2O
> 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)

• Fermentation – it does not require oxygen, the use of Kreb’s cycle or an electron transport
chain; ATP is generated only during glycolysis, thus only few molecules are produced; it releases
energy from sugars or other organic molecules, such as amino acids and purines
Glucose ------> pyruvate ------> Lactic acid

Propionic acid
Ethanol
Formic acid
Final electron acceptor: organic matter (anaerobic)
• Oxidation (aerobic) – NFO (Non-Fermentative Organisms)
Glucose ------> acid
*Yeast and fungi ferment sugar to ethanol (wine).

*Pyruvate is converted to formic acid (Enterobacteriaceae).
*Pyruvate is reduced to lactic acid (Bacillus); used to make yogurt
- 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 AND DISINFECTION
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
A. Application of heat / high temperature

1. Moist heat procedure
2. Dry heat procedure
• Moist heat procedure

> it destroys microorganisms by coagulation or denaturation of protein
A. Boiling
> 100οC for 10-15 mins.
> it kills vegetative forms (non-sporulating bacteria) only
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
C. Fractional sterilization / Intermittent sterilization / Tyndallization

> Arnold’s Sterilizer
> 100οC for 30 minutes (3 consecutive days)
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)
E. Pasteurization / Partial sterilization

> it is used to sterilize vaccines, milk, dairy products and alcoholic beverages
> Phosphatase test – to detect success of Pasteurization
1. LTH (Low Temperature Holding)
> 63οC for 30 minutes
2. HTST (High Temperature Short Time)
> 72οC for 15 seconds
3. UHT (Ultra High Temperature)
> 140οC for 3 seconds
• Dry heat procedure

- kills microorganisms by denaturation of proteins
- it is utilized for the sterilization of oil products and powders
Biological indicator: Bacillus subtilis variation niger
A. Flaming
B. Oven
> it is used for glasswares
> 160-170οC; 1 ½ - 2 hrs.
C. Incineration
> most common method of treating infectious wastes, infected laboratory animal and for waste
disposal
> 300-400οC - materials in the lab; burning materials into ashes
> 870-980οC- medical wastes (hazardous materials)
D. Cremation
> to control the spread of infectious diseases / highly contagious diseases
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
C. Low / Cold Temperature

> considered bacteriostatic (inhibits growth of bacteria)
> 0-7οC – refrigerator temperature
*Listeria monocytogenes - lives best at 4οC; enhanced if refrigerated

*Syphilis (Treponema pallidum) - killed at 2-8οC for 72 hrs.

D. Dessication / Lyophilization - through drying or dehydration
*Bacteria that can resist dessication:
Neisseria gonorrhoeae (pili) – 1 hour
Mycobacterium tuberculosis (Much’s granules) - 2 months
Bacillus, Clostridium (spores) - 10 years
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
II. Chemical Method

ANTISEPSIS – it is usually applied topically to skin (antiseptic)
DISINFECTION – it is usually applied to inanimate objects (disinfectant)
Factors that affect the activity of chemicals / disinfectants

1. Type of organism present
2. Temperature and pH process
increase temperature = decrease potency of the chemical
3. The number of organisms present
4. The concentration of disinfectants
e.g. dil. HCl
5. The amount of organics present
6. The length of contact time
increase contact time = decrease effectivity
7. The type of H2O used for disinfection
e.g. distilled H2O (USPG)
A. Strong acids and strong alkalines – it coagulates proteins

B. Phenol and phenolic compounds
e.g. Lysol
Cresols
Amphyl – cleans and disinfects laboratory benchtops
Tuberculocidals - penetrates organic matter
C. Alcohol – it causes denaturation of CHON and dissolves lipids [cell wall of G(-)]
e.g. Fungicidals - sterol (lipid)
D. Halogen – it destroys microorganisms by oxidation
e.g. Chlorox / Sodium hypochlorite – 10-30 mins. (optimum time of contact); spillage disinfectant
Halozone – used to disinfect drinking H2O
Iodophore - iodine with detergent
Tincture of iodine - iodine with alcohol
E. Salt of heavy metals – it destroys microorganisms by precipitation or inactivation of cell proteins

1. AgNo3 (eye drop solution) – used as a prophylaxis against Opthalmia Neonatorum (blindness) - Credes
prophylaxis
2. CuSO4 – algicide
3. HgCl2 – no longer used as an antiseptic because it can cause carcinoma
F. Quarternary Ammonium Compounds (Quats)
1. Detergent – it disrupts cell membrane; non-tuberculocidal and non-sporicidal
e.g. disinfectants to lab benchtops and utensils
Zephiran (benzalkonium Cl and ceepryn)
Cetylpyridium chloride
2. Aldehydes – it causes inactivation of proteins and nucleic acid; sporicidal

e.g. formaldehyde – is used to sterilize HEPA filters
glutaraldehyde
cidex
G. Gas Sterilant – it is utilized for heat sensitive objects
Biological indicator: Bacillus subtilis variation globijii
1. Ethylene oxide – it destroys microorganisms by fusion with cell protein; it is both microbiocidal and
sporocidal
> 5-8 hours; 38οC
> 3-4 hours; 54οC
2. BPL (Betapropiolactone) - sera and vaccines; does NOT penetrate objects well
Disinfectant Screening Test

Phenol Coefficient (PC) – the highest dilution that kill the bacteria after 10 minutes
Increase PC = greater effectiveness
>1 = the disinfectant is more effective than phenol to S. aureus and S. typhii
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
PATHOGENESIS – the source or cause of an illness or abnormal condition

PATHOGENICITY – pertaining to the ability of a pathogenic agent to produce a disease
A. Infection – involves the growth and multiplication of microorganisms that result in damage to the
host
Types of infection according to distribution in the host

1. Local infection – signs and symptoms are confined to one area; infected wound, boils, abscess, acne
2. Focal infection – starts as a local infection and spread to other parts of the body; tooth infection,
tonsillitis, appendicitis, wound infection caused by C. tetani
3. Systemic infection – microbes are spread throughout the body
Bacteremia - bacteria in blood
Septicemia - bacteria multiply in blood
Pyemia - pus-producing organisms in blood
Toxemia - toxins in blood
According to extent of infection

1. Primary infection – initial infection causing the illness
2. Secondary infection – caused by opportunistic pathogen after primary infection has weakened host
immune system
3. Latent infection – clinically silent inside the body w/out any noticeable illness in host before suddenly
causing severe and acute infection
4. Mixed infection – caused by two or more organisms
5. Acute infection – those infection that develops and progress rapidly
6. Chronic infection – infections that develop slowly with milder but longer-lasting symptoms

AUTOGENOUS INFECTION – is caused by a microorganism from the microbiota of the patient
NOSOCOMIAL INFECTION – hospital acquired infection

1. UTI - 33%
2. Lung Infection (Pneumonia) - 15%
3. Surgical Site Infection -15%
4. Blood Stream Infection -13%
B. Disease – a specific illness or disorder characterized by a recognizable set of signs and symptoms
attributable to hereditary infection, diet or environment.
Classification of infectious diseases

• Communicable disease – spread from one host to another, directly or indirectly
• Contagious disease – spread easily from one person to another
• Non-communicable disease – not spread from one host to another
Classification of Disease as to occurrence

• Sporadic disease – occurs only occasionally
• Endemic disease – constantly present in a particular location or population
• Epidemic disease – many people acquire the disease in a particular location or population
• Pandemic disease – an epidemic that spans the world (world wide epidemics).
Effects of infectious disease

Symptoms – subjective feelings not obvious to an observer (pain, malaise)
Signs – objective changes that can be measured; fever, redness, swelling, paralysis
Syndrome - a group of signs and symptoms that are associated w/ a disease
e.g. AIDS
Phases of Infectious Diseases

• Incubation Period – the time between the exposure to a pathogenic organism and the onset of
symptoms of a disease
• Prodromal Period – first signs and symptoms of disease
• Clinical / Illness Period – peak of characteristic signs and symptoms of infection or disease.
• Decline Period – signs and symptoms begin to subside as host condition improves; the condition
of host deteriorates possibly to death
• Convalescent Period / Period of recovery – full recovery of surviving host
Predisposing Factors for Disease

a. Gender
b. Genetic factors
c. Climate and weather
d. Nutrition
e. Fatigue / stress
f. Environment
g. Lifestyle
h. Age
i. Occupation
PATHOGENS – microorganisms that cause infection and/or disease

1. True Pathogens – are able to invade the tissues of healthy individuals through some inherent ability
(power) of their own.
2. Opportunistic Pathogens – organisms that normally do not cause disease in their natural habitat in a
healthy person; they may cause disease if the host is weakened or if they enter a different part of the
body.
Ex.:
• Neisseria meningitidis - normal flora of the respiratory tract but causes meningitis
• E. coli – normal flora of the GIT but causes UTI

VIRULENCE - degree of pathogenicity
VIRULENT – pertaining to a very pathogenic or rapidly progressive condition
Factors Influencing Virulence

1. Toxic factors
TOXIGENICITY – ability to produce toxic substances
TOXINS – are poisonous substances produced by pathogenic microorganisms
2. Enzymatic factors
- Hyaluronidase
- Coagulase
- Leucocidin
- Collagenase
- Streptokinase
- Hemolysin
- Lecithinase
3. Cellular structure
Capsule - resists phagocytosis
Host Resistant Factors

1. Physical barriers – skin
2. Cleansing mechanism - nasal hairs, cough-sneeze reflex, cilia
3. Antimicrobial substances – lysozymes (destroy bacterial cell wall), bile salts (disrupt bacterial
membranes)
4. Indigenous microbial flora
a. transient flora - inhabit but do not multiply
b. resident flora - inhabit and multiply
5. Phagocytosis
a. PMN
b. macrophage
6. Inflammation – plays a reinforcement mechanism against microbial survival and proliferation in
tissues and organs
Signs: Tumor, Rubor, Calor, Dolor, Functio laesa
Components: phagocytes, complement system, coagulation system, cytokines
7. Immune response
*Immune system has a “memory” so that if a microorganism is encountered second or third time, an
immune mediated defensive response is immediately available.
2 Arms of Specific Immunity

• Humoral (antibody-mediated) immunity - B-lymphocytes
IgG - chronic infection
IgA - secretions
IgM - acute infection
IgE - allergy
IgD – found on the surface of a mature B-cell
• Cellular (cell-mediated) immunity – T-lymphocytes
ACTIVE IMMUNIZATION - life-long

• Is the protection of susceptible humans and domestic animals from communicable diseases by
the administration of vaccines (vaccination)
• Vaccines may be prepared from killed microorganisms, living, weakened (attenuated),
inactivated bacterial toxins (toxoids), purified macromolecules, recombinant vectors (modified
polio vaccine) or DNA vaccines.
PASSIVE IMMUNIZATION (artificially acquired passive immunity) – temporary

• Protection does not require participation of the recipient’s immune system.
• It is administered to individuals exposed to certain pathogens that cause diseases such as
botulism, diphtheria, hepatitis, measles, rabies and tetanus.

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
Infectious Agent Factors

1. Adherence - pili (fimbriae), surface polysaccharides
2. Proliferation
> secretory antibody, lactoferrin, lysozyme - prevents proliferation
3. Tissue Damage
4. Exotoxins
- secreted or excreted by living bacteria
- toxicity is due to simple proteins
- found outside the cell
- heat labile; inactivated at 60οC
- from G(+) and some from G(-)
- they can be cytotoxins, neurotoxins, enterotoxins
- usually produces no fever
- highly toxic / antigenic
- ex.: exotoxin of Clostridium botulinum (1 mg)
- carried by extrachromosomal genes
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
6. Invasion – the process of penetrating and growing in tissues

• Gonococcus – involves deep tissues
7. Dissemination – the spread
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

6. Zoonoses
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
• DNA – Acridine orange, Feulgen

• Bipolar bodies - Wayson
• Cell wall - Dyar
• Rickettsia - Macchiavelo, Gimenez
• B. anthracis – McFayden
• H. pylori – Toluidine blue
• Mycoplasma – Dienes
• Legionella – Dieterle Silver Stain
• Corynebacterium – Bismarck brown
ANTIMICROBIALS (ANTIBIOTICS)
Bacteriostatic agents – antimicrobial agents that inhibit bacterial growth

Bactericidal agents – antimicrobial agents that usually destroy/kill the organisms
Narrow spectrum – effective against a limited number of pathogens

Broad Spectrum – destroy different kinds of organisms
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
Characteristics of Good Antibiotics

1. must kill or inhibit the microorganism
2. must have selective toxicity
3. must be non-allergenic
4. must be stable
5. must remain in the specific body tissue
6. must kill the pathogen before mutation can happen
Action of antimicrobials
• Inhibiting cell wall synthesis
• Inhibiting protein synthesis
• Inhibiting nucleic acid synthesis
• Destroying the cell membrane
• Inhibiting essential metabolites
Cell Wall Inhibitors

• Bacitracin
• Cephalosporin
• Ampicillin
• Imipenem
• Penicillin
*Beta lactamase resistant penicillin

DOMNC
• Dicloxacillin
• Oxacillin

• Methicillin
• Nafcillin
• Cloxacillin
*Broad spectrum penicillin
TAPAC
• Tisarcillin
• Amoxicillin
• Piperacillin
• Ampicillin
• Carbenicillin
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
Protein Synthesis Inhibitors

30’s ribosome inhibitor:
• Tetracycline - bacteriostatic
• Aminoglycosides - bactericidal
> Streptomycin, Kanamycin, Amikamicin, Gentamicin, Tobramycin, Neomycin / Netilmicin, Fusidic Acid
50’s ribosome inhibitor:

• Bind to 23S rRNA
• Chloramphenicol – bacteriostatic
• Macrolide
• Erythromycin - bacteriostatic/cidal
• Clindamycin - bacteriostatic/cidal
• Lincomycin
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
Nucleic acid Synthesis Inhibitors

• Rifampicin

• Mitomycin
• Metronidazole - disruption of DNA; effective against anaerobic bacteria
• Novobiocin
• Nitrofurantoin
• Quinolones and Fluroquinolones (Ciprofloxacin, Nalidixic Acid, Norfloxacin, Levofloxacin) -
interfere w/ DNA gyrase and replication; highly effective against enteric bacteria (E.coli);
bactericidal
Essential Metabolite Inhibitors - inhibits enzyme activity
*Folic Acid Inhibitor

• Sulfonamides - high TI
• Trimethoprim – blocks tetrahydrofolate synthesis
• SXM (Trimethoprim- Sulfamethoxazole)
• Co-trimoxazole
• Dapsone
• Isoniazid – inhibits the synthesis of cord factor (mycolic acid)
*Others
• Anti-TB Drugs- RIPES
- Rifampicin
- Isoniazid (INH)
- Pyrazinamide
- Ethambutol
- Streptomycin
*Cell Membrane Inhibitors

• Polymixin B – used for G(-) bacteria (P. aeruginosa)
• Polymixin E (Colistin) – antibiotic ointment
*Anti-Fungal Agents
• Polyenes (Amphotericin B)
• Azole (Clotrimazole, Ketoconazole, Fluconazole)
ANTIMICROBIAL SUSCEPTIBILITY TESTING
A. Test Tube Dilution / Broth Dilution Method

• Involves challenging the organism of interest with antimicrobial agents in a broth environment
(Mueller-Hinton broth)
• Specific amount of antibiotic is prepared in a decreasing concentration in broth by serial dilution
techniques, and standard numbers of the test organism is inoculated.
• Standard inoculum size: 5 x 105 CFU/ml
• Can be used to determine MIC and MLC concentrations.
1. Microdilution
- broth volume: 0.05 -0.1 mL
- Schaedler’s broth, West Wilkins broth, BHI
2. Macrodilution
- broth volume: >10mL
*MH broth with 2% NaCl – to improve detection of oxacillin-resistant staphylococci
B. Agar Dilution Method

• The antimicrobial concentrations and organisms to be tested are brought together on an agar-
based medium rather than in a broth
• Standard inoculum size: 1 x 104 CFU/mL
• Shelf life of agar dilution plate is only one week for most antimicrobial agents.
• Reference method for testing anaerobes and N. gonorrheae

• Reference method for anaerobes: Brucella agar with laked blood and vitamin K (Wadsworth
method)
C. Disk Diffusion Method (Kirby-Bauer Test)

• Limited to aerobic and facultatively anaerobic bacteria.
• Filter paper disks impregnated with various antimicrobial agents of specific concentrations are
carefully placed on an agar plate previously inoculated with the bacterium being tested
• Standard inoculum size: 1.5 x 108 CFU/mL
• Susceptibility standard medium: Mueller Hinton Agar (MHA)
Turbidity Standard:
> 0.5% Mc Farland Turbidity Standard
99.5mL of 1% H2SO4 + 0.5mL of 1.175% BaCl2
1% H2SO4 + 1.175% BaCl ------> BaSO4 + HCl
> BaSO4 - causes TURBIDITY
Preparation of Pure Culture for Susceptibility Testing

a. Pure inocula (cultures) are obtained by selecting 4-5 colonies of the same morphology, inoculate
them into broth medium and incubate for 3-5 hours to achieve a turbid suspension.
b. Alternatively, 4-5 colonies 6-24 hours of age may be selected from an agar plate and suspended
in broth or 0.85% NSS to achieve a turbid suspension
c. Matching turbidity using the unaided eye is facilitated by holding the bacterial suspension and
MacFarland tubes side by side and viewing them against a black-lined background.
d. If the bacterial suspension initially does not match the standard’s turbidity, the suspension may
be diluted, or supplemented with more organisms, as needed.
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
Factors affecting Zone of Inhibition:

• Thickness of the susceptibility agar plate (4-6mm)
• If the agar is too thick, zone sizes would be smaller; if agar is too thin, zone sizes would be
larger.
• The amount of inoculum or test organism
• The growth rate of the test organism.
• Air at 35◦C-37◦C for most bacteria (16-24 hrs.)
• 5-7% CO2 for N. meningitidis
• Prolonged incubation may result in false resistance interpretation
• The pH of the medium (7.2-7.4)
• Decreased pH affects the activity of antimicrobial agents.
• Aminoglycosides and macrolides may lose potency; penicillin may have increase activity.
• The number of disk per plate.
• 12 disks / plate
• Placement of more than 12 disks may result in overlapping zones (erroneous results).
• The concentration of divalent cations (calcium and magnesium)
• Can affect testing of aminoglycosides and tetracyclines against P. aeruginosa
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).

> Serum Bactericidal Test
- Determines the activity of one or more antimicrobial agents present in serum against an organism
that has been recovered from the patient.
Equipment and Instrumentation

1. Phenotype Identification
a. Morphology
b. Biochemical tests
c. Rapid programs: API, Enterotube, Crystal ID
d. Automated programs: Vitek I and II (can perform both identification and AST)
2. Genotype identification: DNA-PCR
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

for agents that may cause serious disease (human, animal or plant) that is often
untreatable.
Ex.: Tuberculosis, Mycobacterium spp., Brucella, Francisella, molds, COVID-19 positive
respiratory specimens
• BSL-4 (Biosafety Level-4) - biologic agents which are classified as BSL-IV (Biosafety Level-
IV) post high risk but cannot be treated (viruses). Risk group contains biological agents
that usually produce very serious disease (human, animal or plant) that is often
untreatable.
Ex.: Ebola virus, most of the viruses
Risk Group Classification of Infectious Agents

Risk group – ranking a microorganism’s ability to cause injury through disease
• Risk Group 1 – agents not associated with disease in healthy adult humans.
• Risk Group 2 – agents associated with human disease that is rarely serious and for which
preventive or therapeutic interventions are often available.
• Risk Group 3 – agents associated with serious or lethal human disease for which
preventive or therapeutic interventions may be available (high individual risk but low
community risk).
• Risk Group 4 – agents likely to cause serious or lethal human disease for which
preventive or therapeutic interventions are not usually available (high individual risk and
high community risk).
BSC (Biological Safety Cabinet)

• BSC (Biological Safety Cabinet) or biosafety cabinet – a hood with high-efficiency particulate air
(HEPA) filters that provides personnel, environmental and/or product protection when
appropriate practices and procedures are followed.
• HEPA (High Efficiency Particulate Air) filter – exhaust system that removes particles equal to, or
greater than 0.3 microns, which essentially include all bacteria, spore and viruses, with an
efficiency of 99.97%. HEPA filters are effective at trapping particulates.
• Types of BSC:
BSC (Biologic Safety Cabinet) Class I – air velocity 75 linear feet/min; exhaust air thru HEPA filter;
product (culture) contaminant; biological safety cabinet that allows room air to pass through and
within the cabinet and only sterilizes the air exhausted
BSC (Biologic Safety Cabinet) Class II – air velocity 75-100 linear feet/min; vertical laminar airflow; no
product contamination; exhaust and recirculated air through HEPA filter; must for microbiology
laboratory and hospitals
BSC (Biologic Safety Cabinet) Class IIa – exhausts air inside the room; biological safety cabinet that
sterilizes the air that flows over the infectious material as well as air to be exhausted; also, it is the
most commonly used biological safety cabinet which has a fixed opening and 70% of the filtered air is
recirculated
BSC (Biologic Safety Cabinet) Class IIb – exhausts air outside the building (filters exhausted air
directed outside the building)
BSC (Biologic Safety Cabinet) Class III – supply and exhaust air thru HEPA filter; for BSL-IV (viruses)
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)

2. Growth on BAP 5% Sheep's blood
(Substitute: horse's blood, rabbit's blood)
Staphylococci - white pinhead colonies

Types of Hemolysis
• α hemolysis – incomplete / partial hemolysis (greenish zone)
• β hemolysis - complete hemolysis (clear or colorless zone)
• ƴ hemolysis - no hemolysis (no zone)
• α prime hemolysis / target hemolysis / double zone hemolysis – α – inner, β – outer (a small
zone of alpha hemolysis surrounded by zone of beta hemolysis after refrigeration)
Enzyme: hemolysin (bacterias which has this enzyme has the ability to cause anemia)
3. Growth on Loeffler’s Serum Slant

Purpose: enrichment of the pigment production of Staphylococcus
S. aureus - golden yellow pigment

S. citreus - lemon yellow pigment
S. albus - porcelain white pigment
4. Growth on MSA (Mannitol Salt Agar)

Inhibitor: 7.5% NaCl
Carbohydrate: Mannitol
pH indicator: phenol red
*acidic / positive result - yellow
*alkaline / negative result - red
S. aureus – yellow halo formation

S. epidermidis – pink colonies
S. saprophyticus – pink colonies
5. Catalase Test
- Differentiates Staphylococcus and Streptococcus
Reagent: 3% H2O2
(+) result: bubbling formation / effervescence / gas bubbles
3% 2H2O2 --- Catalase ---> 2H2O + O2
(+) Staphylococcus and Micrococcus

(-) Streptococcus
Tests to differentiate Staphylococcus and Micrococcus

*Staphylococcus – facultative anaerobe
*Micrococcus - obligate aerobe
PROPERTIES STAPHYLOCOCCUS MICROCOCCUS

1. Aerobic (+) (+)
growth
2. Anaerobic (+) (-)
growth
3. Lysostaphin S R
4. Bacitracin / R S
Taxo A (0.04U)
5. Modified (-) (+) [blue]
Oxidase
6. Glucose Fermentation > C Oxidation > O2
Utilization > Acidic > Alkalinic

AKA: O/F test
7. Furazolidone S R
Additional Notes:
*Stomatococcus – Modified Oxidase (-), Lysostaphin (R) and Furazolidone (R)
*Staphylococcus is more resistant to lysozyme (destroys peptidoglycan)
6. Modified Oxidase Test / Microdase Test

Reagent:
1% tetramethylparaphenylenediaminedihydrochloride in dimethylsulfoxide (DMSO)
(+) result: blue / purple (Micrococcus luteus)
(-) result: no color change
Indicator: bromthymol blue
*Alkalinic - blue
*Acidic - Yellow
7. Coagulase Test
Fibrinogen ------> Fibrin clot
> Most definitive test / determinant to S. aureus (+)
A. Slide Coagulase (Screening test)

Detects: cell bounded coagulase / clumping factor
Reagent: rabbit plasma / human plasma (must be obtained using EDTA top tube)
(+) result: clot/clumping
B. Tube Coagulase (Confirmatory test)

Detects: free coagulase
Reagent: rabbit plasma / human plasma (must be obtained using EDTA top tube)
(+) result: clot/clumping
Important rule: check every 30 mins.

Enzyme that can possibly dissolve clot: fibrinolysin / staphylokinase
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
Differentiation among Coagulase (+) Staphylococcus

SPECIES COAGULASE VP PYRase
(ACETOIN)
1.
Staphylococcus
(+) (+) (-)
aureus spp.
Aureus

2.
Staphylococcus (+) (-) (+)
intermedius
3.
Staphylococcus (+) (-) (-)
hyicus
3.
Staphylococcus (+) (+) (+)
schleiferi
Other Staphylococcus aureus-like bacteria

*Staphylococcus lugdunensis – Slide coagulase (+); PYRase (+)
*Staphylococcus haemolyticus – β-hemolytic; Coagulase (-)
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
9. Novobiocin Test (5µg)

Differentiates: Coagulase Negative Staphylococcus; Identification of Staphylococcus saprophyticus
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)
10. Staph A Coagglutination Test

• Staphylococcus aureus (Cowar strain I) with protein A as inert particles to which antibody (Fc
fragment) binds
• Detects specific antigens of S. pneumoniae, N. meningitidis, N. gonorrheoae and H. influenzae
11. Phosphatase Test

(+) S. aureus, S. epidermidis
12. Gelatin Hydrolysis Test

(+) S. aureus, S. epidermidis, S. saprophyticus
Pathogen Determinants of S. aureus

1. Protein A - inhibits phagocytosis
2. Coagulase – converts fibrinogen to fibrin clot
3. Staphylokinase / fibrinolysin - dissolves fibrin clot

4. Lipase - degrades lipids (cellulitis, boils); fat splitting enzyme
5. Hyaluronidase - degrades hyaluronic acid (cartilage); spreading factor
6. DNase (Deoxyribonucease) - degrades DNA
7. Exfoliatin (epidermolysin) - destroys stratum granulosum of skin (SSS - Scalded Skin Syndrome)
8. Leukocidin - destroys PMNs and monocytes; also known as Panton Valentine factor
9. Hemolysin - destroys RBC (anemia)
10. Enterotoxin – causes food poisoning and septic shock syndrome; makes Staphylococcus aureus the
#1 cause of food poisoning in the Philippines
11. TSST-1 (Toxic Shock Syndrome Toxin-1)
12. β-lactamase
13. Gelatinase
FAMILY STREPTOCOCCACEAE
Genus Streptococcus, Enterococcus

G(+) cocci in pairs/chains
Non-motile
Non-sporeformers
Facultative anaerobe
Catalase (-), white “pinpoint” colonies
Capnophilic (5-10% CO2); Medium of choice: Sheep’s Blood Agar Plate
Selective medium: PEA (Phenyl Ethyl Alcohol Agar)
Classification:
 Lancefield grouping – based on the antigens (polysaccharides or carbohydrates) present on the
surface of their cell wall
 Smith and Brown’s grouping – based on hemolytic reactions
 Bergey’s grouping – based on temperature
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
II. Smith and Brown’s grouping

α Hemolysis Streptococcus pneumoniae, Viridans,
some of group D
β Hemolysis A, B, C, some of D, F, G
ƴ Hemolysis Most of D
III. Bergey’s grouping

a. Pyogenic – pus-producing Streptococcus spp.
b. Lactic – Streptococcus spp. used in making dairy products
c. Enterococcus – normal flora of the gut

d. Viridans – normal flora of upper respiratory tract
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 (-)
Group A Streptococcus (S. pyogenes)

- M-protein – cell wall, anti-phogocytic, virulence factor
- Streptolysin O – O2 labile, Antigenic, anerobic, lytic
- Streptolysin S – O2 stable, Non-antigenic
- Erythrogenic toxin – Scarlet fever
- Streptokinase
- Hyaluronidase
- Sensitive to Bacitracin (Bacitracin / Taxo A positive control)
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 B Streptococcus (S. agalactiae)

- β-hemolytic
- normal flora of vagina and upper respiratory tract
- neonatal sepsis
- #1 neonatal meningitis
- pneumonia
- bacteremia
- resistant to Taxo A, Taxo P and SXT
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
4. Bile Esculin Hydrolysis Test

• Differentiates Group D from other Streptococcus; presumptive test for Group D
Medium: BEA (Bile Esculin Agar) – contains 40% bile and esculin
Indicator: Ferric Ammonium Citrate (reacts with esculetin)

Incubation: 35◦C for 30 minutes
40% bile + esculin ------> esculetin
(+) result: brown or black precipitate / blackening of agar

(+) for: Group D Streptococcus
(+) control: E. faecalis
(-) control: S. mitis
Tests to differentiate Group D Enterococcus and Non-Enterococcus
Enterococcus Non-
Enterococcus
6.5% NaCl (+) (-)
PYRase test (+) (-)
Pencillin R S
Growth at (+) (-)
45◦C
Growth (+) (-)
At 10◦C
Sodium- (+/-) (-)
Hippurate
Hydrolysis test
5. PYRase (Pyrrolidonylarylamidase) test

Substrate: PYR (L-pyrrolidonyl B-napthylamide)
PYR ---PYRase---> β-naphthalidamide
Color indicator: P-dimethylaminocinnamaldehyde

(+): red
(+) for: Group D (Enterococcus), Group A
6. CAMP (Christie-Atkinson-Munch-Peterson) test

Principle: CAMP factor of S. agalactiae enhances β-hemolysis (synergistic reaction to beta hemolysin of
S. aureus)
Base medium: BAP
(+) result: arrow-head zone of β-hemolysis
(+) for: S. agalactiae, Listeria monocytogenes, Helicobacter pylori
(+) control: Group B Streptococcus
(-) control: Group A Streptococcus
7. Sodium-Hippurate Hydrolysis Test

Indicator: Ninhydrin reagent (reacts to glycine)
Hippuric acid ------> benzoin + glycine

(+): purple / violet
(-): colorless / pink
(+) for: S. agalactiae
8. Bacitracin Susceptibility Test (Taxo A)

- best test to identify S. pyogenes
- test to differentiate Group A from Group B Streptococcus
(+) result: any zone of inhibition
(+) control: Group A Streptococcus
(-) control: Group B Streptococcus
9. LAP (Leucine Aminopeptidase) Test

Incubation: room temperature for 5 minutes
(+) result: red
(-) result: yellow / no color change
(+) for: E. faecalis and Pediococcus
(-) for: Leuconostoc
10. Pyruvate Broth Test

Incubation: 35◦C for 48 hours
(+) result: yellow
(-) result: green
(+) for: E. faecalis
(-) for: E. faecium
11. SXT (Sulfamethoxazole and Trimetoprim)
GROUP BACITRACIN SXT Also gives

positive
results to:
A S R PYRase
B CAMP,
R R Sodium-
hippurate
C, F, G R S
D BE, NaCl,
R R
(Enterococcus) PYRase
D (Non- R R BE
Enterococcus)
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)
Tests To Differentiate Streptococcus pneumoniae and Viridans

TEST STREPTOCOCCUS VIRIDANS
PNEUMONIAE
Mouse inoculation /
Dead Alive
virulence test

Inulin fermentation
(+)
(-)
Bile Solubility [(+)
(+) = due to
result: clearing of (-)
autolysis
the medium]
Optochin / Taxo P
[Composition:
S R
ethylhydroxycuprein
HCl; 5 µg]
Neufeld-Quellung
[detects capsular
(+) (-)
Ag; (+) result:
capsular swelling]
10. Optochin Disk Test (Taxo P)

- used to identify S. pneumoniae
- Composition: ethylhydroxycuprein HCl (5 µg)
(+) result: >14 mm zone of inhibition
(+) control: S. pneumoniae
(-) control: S. mitis
11. Bile Solubility Test

Incubate: 35◦C for 30 minutes
(+) result:
a. 10% sodium desoxycholate (BAP) = lysed colonies
b. 2% sodium desoxycholate (tube) = clear
(+) for: S. pneumoniae
(-) for: Viridans group, E. faecalis
(+) control: S. pneumoniae
(-) control: E. faecalis
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.
Nutrionally Variant Streptococcus

>requires cysteine for survival
also: Thiol, Vit. B6 (pyridoxine)
e.g. Streptococcus defectivus

Streptococcus adjacens
Abiotrophia spp.
*shows satelliting on agar
*Staph Streak Test (+)
Pediococcus / Leuconostoc (Streptococcus-like organisms)

• Vancomycin resistant test – differentiate Pediococcus and Leuconostoc (+) from
Streptococcus viridans (-)
TESTS Pediococcus Leuconostoc Aerococcus

Vancomycin R R S
Bile esculin + + +/-
PYR - - +/-
6.5% NaCl + + +

MRS broth (-) no gas (+) gas N/A
test
LAP (+) red N/A N/A
AEROBIC GRAM NEGATIVE COCCI
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
*Oxidase Test / Taxo N

- presumptive (screening) test for gram negative cocci
Reagent: 1% tetramethylparaphenylenediaminedihydrochloride
(+) result: purple
Taxo N -- cytochrome oxidase → indophenol blue
(+): Neisseria, Moraxella, Aeromonas, Pseudomonas

Techniques in performing the oxidase test:
a. Put a drop of reagent on the colony
b. Rub on a filter strip paper and add a drop of reagent
c. Rub the colony on a piece of filter paper containing the reagent
(+) control: Pseudomonas aeruginosa
(-) control: E. coli
*Superoxol Catalase Test

Reagent: 30% H2O2
(+) vigorous bubbling formation
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

Diseases:
- Meningitis
- Meningococcemia – can lead to DIC (Disseminated Intravascular Coagulation)
- Waterhouse-Friderichsen Syndrome – disease of the adrenal glands
Other Neisseria – rarely cause infection

- Wound infection
- Pneumonia
- UTI
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)
Carbohydrate Fermentation / Utilization Test

• Confirmatory test for Gram (-) cocci
• Medium: CTA (Cysteine Trypticase Agar)
• Indicator: phenol red
• (+) result: yellow
Glucose Maltose Sucrose Lactose
M. catarrhalis (-) (-) (-) (-)
N. (+) (-) (-) (-)
gonorrhoeae
N. (+) (+) (-) (-)
meningitidis
N. subflava (+) (+) (+/-) (-)
(secca)
N. lactamica (+) (+) (-) (+)
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)

ANAEROBIC BACTERIOLOGY
Collection: Needle aspiration

1) Reduced media – Anaerobic BAP, Schaedler, Bacteroides Bile Esculin
2) Laked Kanamycin Vancomycin BAP (LKVBA), Anaerobic PEA, Egg Yolk Agar, Chopped Meat,
Thioglycollate, Lombard Dowell Agar
METHODS TO PROMOTE ANAEROBIOSIS
• Gas Pak Jar or Mcintosh Fildes jar, Brewer, Torbal or any anaerobic jar
• Cooked meat medium / Chopped Cooked Meat Medium
• Anaerobic Glove Box & chamber
• PRAS – Pre-reduced Anaerobically Sterilized (Roll Tube of Hungate)
• Thioglycollate medium
indicator: resazurin red
boiling: drives off O2
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
Gram positive anaerobic bacilli

1. Actinomyces – fungus-like bacteria
a. Actinomyces bovis – lumpy jaw
b. Actinomyces israeli – draining sinus tract with sufur granules
2. Bifidobacterium dentium
3. Eubacterium lentum
4. Propionebacterium acnes (Cutibacterium acnes) - #1 blood culture contaminant
5. Lactobacillus (Doderlein bacillus)
6. Mobiluncus – causes bacterial vaginitis (also G. vaginalis)
Gram negative anaerobic bacilli

1. Bacteroides fragilis – needs 20% bile; KVC (Kanamycin, Vancomycin, Colistin) resistant; the most
abundant bacteria in the gastrointestinal tract
Culture medium: Bacteroides Bile Esculin medium
2. Porphyromonas asaccharolytica – black pigment, brick red fluorescence on ultraviolet light (UVL)
3. Prevotella melaninogenica – black pigment, brick red fluorescence on ultraviolet light (UVL)
4. Fusobacterium nucleatum – breadcrumb colonies, fusiform rod
5. Fusobacterium necrophorum – Vincent’s angina
6. Bacteroides ureolyticus – pitting of agar
Gram positive anaerobic cocci

1. Peptostreptococcus anaerobius – SPS sensitive, indole (-)
2. Peptostreptococcus asaccharolyticus
3. Peptostreptococcus niger (Staphylococcus like)
Gram negative anaerobic cocci

1. Veilonella parvula – fluoresce red UVL
2. Megasphera
3. Acidaaminococcus

GRAM NEGATIVE BACILLI
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
H2S producers – SPACE

H2S (+) result: blackening of medium
Salmonella
Proteus
Arizona
Citrobacter freundii
Edwardsiella
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
1. Growth on enteric media

a. EMB – Eosin-Methylene Blue
Inhibitor: Eosin Y, methylene blue
CHO: Lactose
Indicator: Eosin Y, methylene blue
LF: Red / purple
NLF: Colorless
b. MAC – MacConkey Agar
Inhibitor: Crystal violet, bile salt – anti-fungal and anti-Gram (+) substances
CHO: Lactose
Indicator: Neutral red
LF: Red / pink
NLF: Colorless
c. XLD – Xylose-Lysine-Desoxycholate Agar
Inhibitor: Bile salt
CHO: Xylose, lactose, sucrose
Indicator: Phenol red
LF: Yellow
NLF: Red
d. HEA – Hektoen-Enteric Agar
CHO: Salicin, lactose, sucrose
Indicator: Bromthymol blue
LF: Yellow
NLF: Green
e. DCA – Desoxycholate-Citrate Agar

CHO: Lactose
LF: Red / pink
NLF: Colorless
f. SSA – Salmonella-Shigella Agar
CHO: Lactose
LF: Red
NLF: Colorless
Results:
• Colorless colonies with black centers – Salmonella spp.
• Colorless colonies without black centers – Shigella spp.
g. BSA – Bismuth-Sulfite Agar
Inhibitor: Brilliant green
CHO: Glucose
Indicator: Bismuth sulfite
LF: Black colonies (Salmonella typhi)
h. TCBS – Thiosulfate-Citrate-Bile Salt-Sucrose Agar
CHO: Sucrose
LF: Yellow
NLF: Green
i. KIA – Krigler’s Iron Agar
CHO: Glucose, Lactose Sucrose + Iron
j. RDA – Russel’s Double Agar
CHO: Glucose, Lactose
2. TSI (Triple Sugar Iron)

CHO: Sucrose, Lactose and Glucose (Ratio = 10:10:1 respectively) + Iron
Slant: Sucrose and Lactose

Butt: Glucose
Also contains: Na thiosulfate

pH indicator: phenol red = red to yellow
H2S indicator: FeSO4 (ferrous sulfate) = black
Gas indicator = splits medium
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

3. LIA (Lysine Iron Agar)
Principle: test to detect if the bacteria has the ability to degrade amino acid lysine
pH indicator: bromcresol purple
H2S indicator: Ferric Ammonium Citrate = black
Gas detected: splits medium
a. Lysine deamination – slant
❖ Positive – red; Negative – purple
b. Lysine decarboxylation – butt
❖ Positive – purple; Negative – yellow
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
*Rapid Spot Indole Test

- used as screening test for indole production
- filter paper strips impregnated with p-dimethylcinnamaldehyde
(+) result: blue
5. MRVP (Methyl Red / Voges-Proskauer)

Medium: MRVP medium (peptone glucose broth)
a. Methyl Red Test

Principle: Mixed acid of glucose fermentation
>Lactic acid, Acetic acid, Formic acid, Succinic acid
Indicator: Methyl red
Acidic (pH below 4.4) – red

Alkalinic – yellow
(+) result: red
(-) result: yellow
(+) control: Escherichia coli; (-) control: Enterobacter cloacae
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
6. Citrate Utilization test

Medium: Simmon citrate agar (slanted)
Principle: to detect bacteria that utilizes citrate as a sole source of carbon
Alkalinic - Blue
Acidic - Green
Na citrate ------> NH4 + NH4OH
(+) result: Prussian blue / royal blue

(-) result: Green
(+) control: Klebsiella pneumoniae; (-) control: Escherichia coli
7. AMA (Acetate, Malonate, Acetamide Utilization Tests)

Principle: to detect bacteria that utilizes acetate / malonate / acetamide as a sole source of carbon
(+) blue
(-) green/yellow
Incubation: 37◦C for up to 7 days
Positive Control Negative Control

Acetate Escherichia coli Shigella flexneri
Malonate Salmonella Shigella
Acetamide Pseudomonas Stenotrophomonas
aeruginosa maltophilia
8. Urease Test (Urea Hydrolysis Test)

Medium: Christensen’s urea agar (slant) or Stuart’s urea broth
Urea ---Urease---> (NH4)2CO3 + NH3 + CO2
*(NH4)2CO3 is alkaline
pH indicator: phenol red
(+) result: pink
(-) result: yellow
a. Rapid urease = after 2 hours gives positive result

PPM
Proteus
Providencia
Morganella

b. Slow urease = after 4 hours gives positive result
CKEYS
Citrobacter
Klebsiella
Enterobacter gergovae
Yersinia
Serratia
9. Lactose Fermentation Test
a. RLF (Rapid Lactose Fermenters)

Has 2 enzymes:
• Β-galactosidase
• Lactose permease
Mnemonics: EKE
• Escherichia
• Klebsiella
• Enterobacter
b. LLF (Late Lactose Fermenters)

Has only 1 enzyme:
• Β-galactosidase
Mnemonics: S2HYCS
• Salmonella arizonae
• Shigella sonnei
• Hafnia
• Yersinia
• Citrobacter
• Serratia
c. NLF (Non-lactose Fermenters)

No enzyme!
Rule:
- All Salmonella are NLF except S. arizonae
- All Shigella are NLF except S. sonnei
- The rest of Enterobacteriacea that are not mentioned above
10. ONPG test

- rapid test to detect β-galactosidase
Bacteria which are positive: RLF (Rapid Lactose Fermenters) and LLF (Late Lactose Fermenters)
Substrate: (ONPG) orthonitrophenyl-β-delta-galactophyranoside
Product: orthonitrophenol
(+): yellow
(-): no change in color
(+) control: E. coli; (-) control: S. typhimurium
11. Decarboxylase test / LOA (Lysine, Ornithine, Arginine) test

Medium: Moeller’s decarboxylase medium
Indicator: bromcresol purple
Uses 4 test tubes:

LOAC
Lysine – decarboxylase → cadaverine
Ornithine – decarboxylase → putrescine
Arginine – dihydrolase → citrulline
Control

Note: Put mineral oil on top to make the environment anaerobic.
(+) result: purple
(-) result: yellow
LDC (+) control: K. pneumoniae
LDC (-) control: E. cloaceae
Decarboxylase Testing
Lysine Ornithine Arginine
K. (+) (-) (-)
pneumoniae
K. oxytoca (+) (-) (-)
E. aerogenes (+) (+) (-)
(Klebsiella
aerogenes)
E. gergorvae (+) (+) (-)
E. cloacae (-) (+) (+)
E. sakazakii (-) (+) (+)
Hafnia alvei (+) (+) (-)
P. (-) (-) (-)
agglomerans
12. PAD (Phenylalanine deaminase) test

Principle: test to detect if the bacteria has the ability to remove the amino group (-NH2) of phenylalanine
Medium: phenylalanine with 10% FeCl3
Product: PPA (Phenyl Pyruvic Acid)
Indicator: 10% FeCl3
(+) result: green
(+) for: Proteus, Providencia, Morganella
(+) control: P. vulgaris; (-) control: E. coli
13. Gelatin liquefaction (hydrolysis) test

Principle: test to detect if the bacteria is able to produce the enzyme gelatinase
Medium: Nutrient Gelatin Medium
(+) result: gel liquefaction
(-) result: gel solidification
Bacteria that give positive result: Serratia, Proteus
(+) control: Proteus vulgaris; (-) control: E. aerogenes
14. KCN (Potassium Cyanide) Broth Test

(+) result: turbid
(-) result: clear
(+) for: Klebsiella, Enterobacter, Proteus, Providencia, Citrobacter
15. Bile Esculin Hydrolysis Test

(+) for: Klebsiella pneumoniae
(-) for: Shigella flexneri
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

(+) control: E. coli
(-) control: P. aeruginosa
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
2. EIEC – Enteroinvasive Escherichia coli

- causes dysentery – painful passage of stool
- Causes Shigella-like infection or diarrhea (invasin)
- Sereny test – is a virulence test used to test the invasiveness of enteroinvasive Escherichia coli, Shigella
species and Listeria monocytogenes
- causes stool w/ RBC, pus and mucus
- O124, O143, O164
3. EPEC – Enteropathogenic Escherichia coli

- strain of E. coli that causes “infantile diarrhea” or diarrheal outbreaks in infants (pathogenicity island)
- Non-invasive and doesn’t produce toxin
- nosocomial infection
- causes watery diarrhea w/o RBC, pus and mucus
- O111, O114
4. EHEC – Enterohemorrhagic Escherichia coli

- Other name: VTEC - Verotoxin Escherichia coli
- Most virulent strain = 0157:H7
- produces verotoxin (first isolated from African green monkey; Shigella-like toxin)
- causes HUS - Hemolytic Uremic Syndrome, hemorrhagic colitis
- Sorbitol MacConkey (SMAC) = (-) colorless colonies
- MUG test = (-)
5. EAEC – Enteroaggregative Escherichia coli

- causes acute and chronic diarrhea (probably binding w/ pili
and aggregative adhesion fimbriae)
- produces ST like enterotoxin
- produces hemolysin
- the characteristics is not fully understood
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

- Representative spp.:
• Klebsiella quasipneumoniae subsp. similipneumoniae (Friedlander’s Bacillus) – former name is
Klebsiella pneumoniae; encapsulated; tend to sting
• Klebsiella ozaenae – causes purulent sinus infection; Malonate (-)
• Klebsiella rhinoscleromatis – causes nasal and pharyngeal infections; Malonate (+)
• Klebsiella oxytoca [the only spp. of Klebsiella that is indole (+)]
LDC (Lysine VP test and Indole test

Decarboxylase) Urease test
K. pneumonia (+) (+) (-)
K. oxytoca (+) (+) (+)
K. ozaenae (+) (-) (-)
K. (-) (-) (-)
rhinoscleromatis
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
> produces red pigment (prodigiosin)

> late lactose fermenter; LOA: +, +, -; Arabinose: (-); Citrate: (+); Drug-resistant
> common opportunistic pathogen in hospitalized patients (nosocomial infection)
> Causes UTI, bacteremia, pneumonia
Serratia marcesens
Serratia rubidae – arabinose and malonate (+); pink to red colonies
Serratia odornifera – produces colonies with potato odor
Serratia liquifascien – blood bag contaminant, arabinose (+)
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)

- Related to Citrobacter (but 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
Widal test - tube agglutination test

O - Somatic antigen; titer = 1:160 - active salmonella infection
H - Flagellar antigen; past infection / immunity (IgM)
Vi - Capsular antigen; carrier (IgG)
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 +)

Citrobacter freundii – the only species of citrobacter that is H2S (+); UTI, pneumonia, endocarditis
Citrobacter diversus – nursery outbreaks, neonatal meningitis
TSI Indole Malonate
C. freundii A/A + H2S (-) (+)
C. diversus A/A (+) (+)
and koseri
C. A/A (+) (-)
amalonaticus
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
- Proteus vulgaris – Indole (+)

- Proteus mirabilis – Indole (-); HIGHLY MOTILE, shows swarming phenomenon on BAP
- share specific polysaccharide w/ Rickettsia
Proteus vulgaris – source of OX2 and OX19
Proteus mirabilis – source of OXK (“K” means Kingsbury)
Providencia
- 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
- 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
- late-lactose fermenter
- bioterrorism agent
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 cholerae O1
- non-halophilic (1-3% NaCl); nitrate and indole positive (Cholera red test / Nitrosoindole test)
- alkalinophilic
- Disease: Cholera (causes rice watery stool; severe dehydration)
- produces choleragen
- Type of microscope for diagnosis: Dark-field microscope
- All strains are Polymixin B susceptible except El Tor
- Culture: Alkaline Peptone Water (6-8 hours), TTGA (Tellurite Taurocholate Gelatin Agar), TCBS
(Thiosulfate Citrate Bilesalt Sucrose; shows yellow colonies because it is sucrose fermenter)
- String test (+)
- Motility: shooting-star motility / rapid darting motility
SEROTYPE Country Anti- Anti-Inaba

/ SEROVAR Ogawa
Inaba Philippines (-) (+)
Ogawa India (+) (-)
Hikojima Japan (+) (+)
BIOTYPE / Classical El Tor

BIOVAR
Sheep RBC (-) (+)
hemolysis
Voges (-) (+)
Proskauer test
Polymixin B Susceptible Resistant
susceptibility
Agglutination of (-) (+)
chicken RBC
Lysis by (+) (-)
bacteriophage
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
Aeromonas and Plesiomonas

- Oxidase (+), catalase (+), indole (+), glucose fermenter with polar flagella
Vibrio Aeromonas Plesiomonas

NaCl (+) (-) (-)
requirement
Motility (+) (+) (+)
Oxidase (+) (+) (+)
O129 S R S
LOA +, +, - +, -, + +, +, +
DNase (-) (+) (-)
TSI A/A or K/A A/A + gas + A/A or K/A
H2S
Disease All cause diarrhea, wound and
septicemia
NON-FERMENTATIVE ORGANISMS (NFO)
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
*Pseudomonas aeruginosa acanthi – associated with wearing contact lenses (conjunctivitis)

*Pseudomonas fluorescens – fluorescein but not pyocyanin; no growth at 42◦C; gelatin hydrolysis (+);
transfusion associated sepsis / bacteremia (blood bag contaminant)
*Pseudomonas stutzeri – brown (buff colored) wrinkled colonies; 6.5% NaCl (+), Reduce nitrate to
nitrite, non-lactose oxidizer
Burkholderia mallei
- Former name: Pseudomonas mallei

- causative agent of Glander’s disease = a contagious zoonotic infectious disease that occurs primarily in
horses and similar animals. It can be contracted by humans.
- Mode of transmission: through contact with tissues or body fluids of infected animals
- The disease is characterized by purulent nasal discharge, nasal mucosal ulceration, lung lesions, and
ulcerating nodules along the subcutaneous lymphatics
- O-F Test: +/- (glucose, maltose, lactose)
- the only non-motile NFO
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 (+)

*Mistaken as Neisseria
(Difference: carbohydrate
fermenter)
*Assacharolytic (non-
fermenter of
carbohydrates);
MacConkey (-)
Chryseobacterium * Formerly called as
meningosepticum Flavobacterium
*Oxidase, DNase, gelatin
hydrolysis and indole (+)
*Causes neonatal
meningitis, sepsis; Yellow
colonies in BAP due to
pigment “flavin”; non-
motile
*MacConkey (-)
Eikenella corrodens *Fastidious
*Capnophile
*Normal flora of gingival
area and GIT (human bite
wound, “clenched fist”)
*Characteristic: pit /
corrode the agar
*Odor: Bleach-like odor
*SBE (Subacute Bacterial
Endocarditis) agent;
MacConkey and catalase (-
); oxidase (+)
Kingella kingae *Oxidase (+); catalase and
MacConkey (-)
*Cause SBE; part of the
HACEK group; pits the agar
*O-F Test (Oxidative-Fermentative) Test

Medium: Hugh and Leifson medium (1% glucose, 1% agar, low peptone)
• Acid (+): yellow
• Alkaline (-): green
Note: Add mineral oil on top of F (Fermentative) Tube
Results:
Oxidizer – O tube (yellow), F tube (green)
Fermenter – O tube (yellow), F tube (yellow)
Non-utilizer – O tube (green), F tube (green)
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 (+)

- Doesn’t grow on MAC
Culture media:
> Enriched CAP + 5% CO2 (made with horse’s blood; for fastidious organisms)
> BAP w/ X factor
> Levinthal
> Fildes
Special growth factor requirement:

X-factor = hemin
V-factor = NAD coenzyme I
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
Curved, gram negative rod / bacilli

1. Campylobacter

2. Helicobacter
3. Arcobacter
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
37◦ 42◦ Nalidix Cephaloth Hippura

C C ic in te
C. + + S R +
jejuni
C. coli + + S R -
C. fetus + - R S -
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

Composition:
-charcoal
-yeast extract
• Stainer and Scholte
• Casamino broth
Most ideal specimen: nasopharyngeal swab

colonies: Mercury droplet / Pearl-like appearance
Bordetella pertussis – etiologic agent of whooping cough
3 stages of whooping cough:

Cattarhal – flu like illness
Paroxysmal- repetitive coughing
Convalescence - recovery period
Bordetella parapertussis – causes flu-like symptoms

Bordetella bronchiseptica - inhabits the respiratory tract of dogs (kennel cough); infrequently inhabits
the respiratory tract of humans
UREA MOTILI NITRAT OXIDA MAC,

SE TY E SE BAP
B. pertussis (-) (-) (-) (+) (-)
B. (+) (-) (-) (-) (+)
parapertussi
s
B. (+) (+) (+) (+) (+)
bronchisepti
ca
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
Goats - Brucella melitensis

Cattles - Brucella abortus
Dogs - Brucella canis
Pigs - Brucella suis
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

- L. pneumophilia - Legionnaire’s disease, Pontiac fever, Broadstreet pneumonia
- Legionella micdadei - Pittsburg pneumonia
- Legionella bozemanni - Wiga’s agent of pneumonia
- Gram (-) coccobacilli; aerobic

- Special growth requirement: L-cystine and iron for growth
- acquired from air-conditioning and water cooling systems
- specimen: nasopharyngeal swab
- transport temp.: 4◦C; storage temp.: -70◦C
- medium: BCYE (Buffered Charcoal Yeast Extract; blue green colonies / cut glass colonies; best culture
medium), Faeley German medium
- Stain: Deiterle silver stain - dark brown to black colored bacilli
- Other laboratory diagnostic test: DFA (Direct Fluorescence Assay) – detects Legionella antigen
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

- also acquired through ingestion of contaminated milk
- shows String of beads / fluff balls in broth culture
- SPS sensitive
Streptobacillus moniliformis
Spirilium major – spiral organisms
Spirilium minor (minus) – spiral organisms
Diseases they cause:

1. Rat bite fever
2. Haver Hill Fever
3. Sudoku Fever
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
Oxidase Catalase Features

Haemophilus (+/-) (-) Do not require
aphrophilus X and V factors
Actinobacillus (-) (+) Star-like
(Aggregatibacter) colony; causes
actinomycetemcomitans granulomatous
disease in
animals; In
Gram-stained
smear, this
bacteria show
both bacillus
and coccus

form together,
known as
‘morse code’
appearances
(dots and
dashes
appearance)
Cardiobacterium (+) (-) Indole (+);
hominis Tear drop
shape
Eikenella corrodens (+) (-) Assacharolytic
Kingella kingae (+) (-) Twitching

motility
GRAM POSITIVE BACILLI
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 (-) (+)

Salicin (-) (+)
fermentation
test
Penicillin G Susceptible Resistant
Gelatin (-) (+)
hydrolysis test
PEA (-) (+)
Nagler test / lecithinase test

- Detects: alpha toxin, lecithinase C, phospholipase C
- Medium: EYA (Egg Yolk Agar), Mc Clung or Neomycin Egg Yolk
- (+) result: opalescence on agar w/o anti-toxin
- (-) result: NO opalescence on agar w/ anti-toxin
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

- (+) result: Target Hemolysis / Double Zone of Hemolysis / α prime
α-inner
β-outer
9. Can be cultivated in thioglycollate medium
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
Motility Lecithinase Lipase Lactose Glucose

C.
(-) (+) (-) (+) (+)
perfringens
C.
(+) (-) (+) (-) (+)
botulinum
C. tetani (+) (-) (-) (-) (-)
C. difficile (+) (-) (-) (-) (+)
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

- toxin: exotoxin – causes Pseudomembrane (grayish/white patches inside the mouth including tonsils
and pharynx)
- Non-motile, non-spore former, encapsulated, Sucrose (-); Nitrate reduction (+); Urease (-); Catalase (+);
DNase (+); Starch / glycogen hydrolysis (-); Glucose and Maltose fermenter
- Metachromatic granules / volutin (food storage): Babes-Ernst granules
- DPT (Diptheria, Pertussis, Tetanus) vaccine - injected at gluteus area
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.
Colonial Types (Biotypes):

1. Gravis - largest (1-2µm); gray, non-hemolytic, starch / glycogen fermentation (+)
2. Mitis – medium-sized; black, β-hemolytic, starch / glycogen fermentation (-)
Appearance: fried egg plasma
Odor: bleach-like odor
3. Intermedius – smallest (0.5µm); black, small, non-hemolytic
Toxigenicity (Virulence) Tests:

*serves as final diagnosis (confirmatory tests) for Diptheria toxin of C. diptheriae
1. In vivo (inside the body) – Guinea Pig Lethality Test
2. In vitro (outside the body) - Elek’s Plate Test – definitive test; immunodiffusion test; (+) result:
precipitation
Schick’s Test - susceptibility test (skin test) for diphtheria

- (+) result: redness
*Arthrobacter culture similar to Brevibacterium
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

- Urease (-), Nitrate reduction (+), Ferments glucose, maltose, sucrose
– causes conjunctival infections
• Corynebacterium acne – causes skin infections
• Corynebacterium minutissimum
– causative agent of Erythrasma - like the diaper rash of a baby; irritation in the superficial layer of skin
- shows “coral red fluorescence” on Wood’s light due to the presence of porphyrin
• Corynebacterium ulcerans
- Urease (+); Nitrate reduction (-); Starch / glycogen hydrolysis (+)
• Corynebacterium pseudotuberculosis
- Urease (+); Nitrate reduction (+/-); Starch / glycogen hydrolysis (-)
• Corynebacterium amycolatum
- most frequently recovered Corynebacterium spp. from human clinical material; it is part of the normal
skin microbiota
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 acidophilus
- normal flora of mouth, GIT and vagina (to maintain the acidity)
- NON-pathogenic (has little chain of infection)
- cultured on: Tomato Juice Agar (Rogosa’s agar)
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
Casein L-tyrosine Xanthine

hydrolysis hydrolysis hydrolysis
N. asteroides (-) (-) (-)
complex,
N. farcinica
or N. nova
N. (+) (-) (-)
brasiliensis
N. otitidis (-) (+) (-)
N. caviae (-) (-) (+)
Streptomyces (+) (+) (+)
or
Nocardiopsis
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)

2. Chemicals (such as 5% phenol)
*Conditions and their associated survival time:

when protected from sunlight: 24 hrs.
putrifying sputum: weeks
dried sputum: 6-8 mos.
droplets of dried sputum: 8-10 days
culture + sunlight: 2 hrs.
sputum + sunlight: 8-10 hrs.
Susceptible to:
1. Sunlight
2. Autoclave
3. Pasteurization
4. Boiling (10 mins.)
Mode of Transmission: air-borne droplets

Habitat: lungs, meninges, long bones and spinal column (causative agent of Pott’s disease)
Pathogenesis:
middle lower lungs ------> may include macrophages (phagocytosis) ------> fibroid cells ------>
granulomatous lesion (granuloma) seen on X-ray ------> calcify (Ghon’s complexes)
Skin test: PPD (Purified Purine Derivatives) / Tuberculin test

> heat killed with ammonium sulfate
route of administration: intradermally
(+) result: redness after 48 hrs.
Other skin tests:

➢ Mantoux test
➢ Mendel’s test
➢ Vollmer’s patch
➢ Von pirquet test
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
Before the production of sputum, the patient must:

• Gargle with water
• Toothbrush

• Remove the dentures (if any)
Methods of decontamination and digestion:

1. N-acetyl-L-cysteine + NaOH with sodium citrate
*best method for decontamination and digestion
NALC – for digestion (proteolysin; liquefy mucus)
2-4% NaOH – for decontamination
2. Trisodiumphosphate + benzalkonium chloride (zephiran) with 6% oxalic acid
3. Dithiothreitol (sputulysin) + NaOH
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.
1. Lowenstein-Jensen medium – incorporated stain: malachite green

2. Petragnani medium
3. American Thoracic Society medium
4. Dorset Egg medium
5. Agar-based media - for examination of colonies
a. Middle brook 7H10 / Middlebrook 7H11C – for AST; clear Mycobacteria media
b. Duboi’s Oleic Acid Albumin medium
c. Mitchison’s medium
6. Liquid media
a. Bactec 12B
b. Septi-Chek
c. Middle brook 7H9
Culture presented: 8 weeks or 2 months

>Mycobacterium tuberculosis is a slow-grower
Colonies: “cauliflower-like”, dry, wrinkled, non-pigmented o light tan to buff
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
AFB GRADING NATIONAL STANDARD / NATIONAL

STANDARD REPORTING SCALE
No AFB seen in 300 0
viewing fields
1-9/100 viewing fields +n
10-99/100 viewing fields +1
1-10/OIF in at least 50 +2
fields
more than 10/OIF in at +3
least 20 fields
CDC METHOD OF REPORTING AFB

NO AFB SEEN NAFBS

1-2/300 fields doubtful
1-9/100 fields +1
1-9/10 fields +2
1-9/ field +3
>9/ field +4
NATIONAL TUBERCULOSIS ASSOCIATION Mycobacterium leprae

1-2/slide Doubtful - Common name: Hansen’s Bacillus
3-9/slide +1 - AFB, “cigarette-packet or picket-fence”
10 or more/slide +2 appearance
1 or more/OIF +3 - obligate intracellular
- Hydrolyze 3,4-dihydroxy-phenylalanine
(DOPA)
- Causes: Leprosy (Hansen’s disease)
a. Lepromatous – Lepromine (-), many AFB
b. Tuberculoid – Lepromine (+), few AFB
- It has a great tropism to peripheral nerves
- Mode of Transmission: inhalation / direct contact
- Specimen of choice: smear of tissue juice, earlobe secretions or nasal scrapings
- cultured on: Armadillo / rat foot pads (live animals); not cultivable cultivable in agar (in vitro)
- Skin test: Lepromine test
(+) result: redness
Two types of Lepromine test:
• Fernandez test - 24-48 hrs.
• Mitsuda test – 3-4 wks.
• vaccine: BCG (Bacille of Calmette and Guerin) – twice administered
• Stain: Fite Faraco
• Treatment: Sulfone-Dapsone
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
RUNYON CLASSIFICATION OF MOTT (Mycobacterium Other Than Tubercle bacillus)

AKA: Atypical Mycobacteria; NTM (Non-tuberculous Mycobacteria)
1. Based on pigment production
2. Based on growth rate
Group I:
Photochromogens - produces pigment when exposed to light (photoreactive)
• Visible colonies after 10-21 days
Mycobacterium marinum
Mycobacterium asiaticum

Mycobacterium simiae
Mycobacterium kansasii
Group II:
Scotochromogens - produces pigment in the dark and light
Mycobacterium scrofulaceum (scrofula)

Mycobacterium szulgai
Mycobacterium xenopi (both photochromogen and scotochromogen)
Mycobacterium gordonae
Mycobacterium flavescens
Mycobacterium thermoresistible
Group III:
Non-photochromogens - non-pigmenters
Mycobacterium avium-intracellulare complex

Mycobacterium malmoense
Mycobacterium haemophilum
Mycobacterium triviale
Mycobacterium ulcerans
Mycobacterium xenopi
Mycobacterium terrae
Mycobacterium gastri
Group IV:
Rapid growers - visible colonies after 3-7 days
Mycobacterium fortuitum-chelonae complex
Mycobacterium phlei
Mycobacterium smegmatis
Mycobacterium abscessus
Mycobacterium mucogenicum
Biochemical Tests for Mycobacteria:

1. Niacin / Kuno-Runyon test
- most important test for MTB
Reagent: cyanogen bromide + niacin + aniline dye
(+) result: canary yellow
Note: All Mycobacterium produce enzyme to convert niacin to niacin ribonucleotide EXCEPT MTB
Niacin ------> Niacin ribonucleotide
2. Nitrate reduction test

Reagents:
a. HCl
b. sulfanilamide
c. n-napthylethylene diamine
Broth = (+) result: pink/red
Strips = (+) result: blue
(-) result: colorless (broth or strip)
(+) for: Mycobacterium tuberculosis
Rule: If the nitrate reduction test is negative, add powdered zinc / zinc dust. The purpose of the addition
of powdered zinc to negative nitrate reduction test is to detect unreduced nitrate (NO3)
True negative: red (presence of unreduced nitrate)
False negative: colorless (in other words, its positive)

3. Heat Stable Catalase Test
- uses: 68οC for 20 mins.
Medium: Tween 80
Reagent: 30% H2O2
(+) result: >45mm height of gas bubbles (bubbling formation)
(+): M. kansasii
(-): M. tuberculosis
4. Tween 80 hydrolysis test

Substrate: Tween 80 (polyxyethylenesorbitan monooleate)
Detects: Tween 80 lipase
most important identification of: Mycobacterium kansasii (+)
(+) result: pink / red
(-) result: no red (M. avium)
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)
(+) Mycobacterium marinum

(-) Mycobacterium bovis
(-) Mycobacterium kansasii
7. Iron Cyclase test

Reagent: 20% Ferric citrate
(+) M. fortuitum
8. Urease
(+) result: red/pink
M. bovis
M. scrofulaceum
M. gastri
9. Growth on 6.5% NaCl

Mycobacterium triviale
10. TCH (Thiozene-2-carboxylic acid-hydrazide) Susceptibility test

(+) result: Susceptible
(-) result: Resistant
(+) M. bovis; (-) M. tuberculosis
11. Antimicrobial susceptibility test

(+) growth - M. tuberculosis
(-) NO growth - M. bovis
12. Tellurite reduction test

Principle: Tellurite → black metallic tellurium
(+): M. avium
(-): M. kansasii
13. Automated test for Mycobacterium

a. Bactec 460 Middlebrook 7H12 (RIA)
Principle: 14C palmitic acid + organisms = 14 CO2

(+) result: more than 10 growth index
b. Mycobacteria Growth Indicator Tube
• Fluorometric based
c. Bactec 12B + NAP (Inhibition test)
• P-nitro acetylamino beta hydroxypropiophenone (NAP)
Note: To differentiate MTB from M. bovis
M. tuberculosis M. bovis
Niacin test (+) (-)
Nitrate (+) (-)
reduction test
TCH test (-) (+)
Result (+) Result (-

(+) (-)
)
M.
NO color
Niacin test MTB intracellulare, yellow
change
M. bovis
Nitrate M. pink / NO color
MTB
Reduction intracellulare red change
M. NO color
Urease M. avium pink / red
fortuitum change
>45 mm <45 mm
Catalase test M. height of height of
MTB
at 68◦C kansasii gas gas
bubbles bubble
Tween 80 M. avium-
M. NO color
hydrolysis intracellulare pink / red
kansasii change
test complex
Tellurite growth
MTB, M. gray
reduction M. avium (black
kansasii clumps
test ppt)
Arysulfatase Rapid M. pink / NO color
test growers Intracellulare red change
M. NO color
5% NaCl M. gordonae growth
fortuitum change
NO color
TCH NO
change
susceptibility M. bovis MTB growth or
or
test Suscetible
Resistant
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

*The major difference between genera is the number of axial filaments that they have
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
Gram stain: poorly stained
Spp. Treponema pallidum spp. pallidum

Treponema pallidum spp. pertenue
Treponema carateum
Treponema pallidum spp. endemicum
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

• Tertiary Syphilis - Gummas
- tissue destructive stage
- bones, CNS, cardiovascular system
- 10—25 yrs.
*Congenital Syphilis – causes stillbirth; abortion; CREST (Calcinosis cutis, Reynaud’s phenomenon,
Esophageal hypomotility, Sclerodactyly, Telangiectasia)
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
Treponema pallidum subs. pertenue

Causative agent of:
• Yaws
• Prambesia
• Buoba
• Buba
• Parangi
• Parani
Mode of Transmission: traumatized skin contact

*Yaws - NON-venereal infection (sores)
- characteristics: Chronic obstructive sore, if not treated, will progress to crippling eruption
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

Treponema pallidum subs. endemicum
- causative agent of Bejel - NON-venereal infection
- Mode of transmission: direct contact (e.g. fingers)
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)
Stages of Lyme’s disease

1ο - Erythema Chronicum Migrans (ECM)
- RBC
bite site: red ring
2ο – Arthritis
- weeks to months
- arthritis
- neurologic disorders / meningitis
- carditis
3ο - Chronic Arthritis
Alzheimer’s–like (demyelinization)
Multisclerosis
1. Microscopic examination
- specimen of choice: blood, bone marrow

A. Relapsing fever
• PBS (Peripheral Blood Smear) - Giemsa, Wright
B. Lyme’s Disease
• Blood, bone marrow - Giemsa, Wright, Acridine Orange
• CSF - Acridine orange
• Biopsy - Warthin-Starry
• Joint fluid
2. Culture - 2-3wks.
- Nutrionally enriched culture media
A. Relapsing fever
• BAP - microaerophilic
B. Lyme’s Disease
• Keller’s medium
• Chick embryo
• BSK (Barber Stoenner Kelly) – incubated at 33◦C for 6 weeks
3. Serodiagnosis
A. Relapsing fever
• OX-K - (Proteus)
1:80 titer
B. Lyme’s Disease
• ELISA
• IFT (Immunofluroescence Test)
• Western blot
IgM, IgG
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

- fever
- pretibial rash
- serovar automnalis
- Bragg fever
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
Serum + Antigen (Killed Leptospira) = Agglutination (positive result)
b. MIT (Microscopic Agglutination Test) – confirmatory test; gold standard test for Leptospira
Serum + Antigen (Live Leptospira) = Agglutination using Darkfield microscope (positive result)
CHLAMYDIA, RICKETTSIA, MYCOPLASMA AND UREAPLASMA
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”
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
*Urethral swab - the traditional diagnostic specimen to test for Chlamydia

*Transport must be maintained at 4◦C
Chlamydia psittaci (Chlamydophila psittaci)

- causative agent of Parrot Fever / Ornithosis / Psittacosis (causes pneumonia in human)
- infection of birds, parrots, cockatoos, parakeets
- Mode of Transmission: inhalation, fomites, direct contact
- Resistant to sulfonamide
Chlamydia pneumoniae

- causative agent of Taiwan Acute Respiratory Syndrome (TWAR)
- Manifestations: Respiratory infections (pneumonia), Guillain-Barre Syndrome
- Mode of Transmission: inhalation
- Tissue culture: human cell lines and Hep-2 cells
- Lab Diagnosis: Immunofluorescence test, PCR
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
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

Rickettsia Family
- Rickettsia, Erlichia, Coxiella, Rochalimea
- Arthropod vectors: Lice, Ticks, Flea
- ALL CANNOT SURVIVE OUTSIDE THE HOST’S CELL (Obligate intracellular) EXCEPT Coxiella (Mode of
Transmission - inhalation)
- Signs: fever, headache, petechial rashes (ankles, wrists)
- Other signs: conjunctivitis, pharyngitis, mild respiratory distress
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
GROUP SPECIES INFECTION TRANSMISSION

R. rickettsi RMSF (Rocky
Mountain
Ticks
Spotted
Fever)
R. akari Rickettsial pox Mites
R. australis Australian /
Queen’s land Ticks
Fever
Spotted
R. conorii Routonneuce,
fever
Mediterranean
Fever,
Israeli fever,
Indian fever, Ticks
Kenyan fever
Typhus R. prowazekii Epidemic

typhus,
Lice, Fleas
Sporadic
typhus
Brill Zinsser Reactivation
R. typhi Endemic
typhus, Rat Fleas
Murine typhus
Scrub R.
Typhus tsutsugamushi,
Scrub typhus Mites, Chiggers
Orientia
tsutsugamushi
Q Fever Coxiella Q fever
Aerosol, Ticks
burnetti
Ehrlichiosis Human
monocytic
ehrlichiosis,
Erlichia
Morulae, Ticks
chaffensis
Sennetsu fever
(destroy
leukocyte)
Erlichia Human
Ticks
phagocytophila, granulocytic

Erlichia ewingii, ehrlichiosis
Erlichia equi Morulae,
(morulae) Sennetsu fever
(destroy
leukocyte)
Rochalemia Rochalemia
quintana
Formerly
Trench fever Lice
known as:
Bartonella
Quintana
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

OTHER IMPORTANT INFORMATIONS
Key Events in the Development of Medical Microbiology
PERIOD DEVELOPMENTAL NOTES KEY SCIENTIST/S

1665 Publication of the first description of microbes Robert Hooke
1667 Observation of “little animals”; first to describe bacteria; Anton van Leeuwenhoek
invention of microscope; Father of Microbiology
1796 Smallpox Vaccination – first scientific validation Edward Jenner
1850 Advocating Handwashing in the prevention of the spread Ignaz Semmelweis
of disease
1861 Spontaneous Generation disproved Louis Pasteur
1862 Publication of the paper supporting the germ theory of Louis Pasteur
disease
1867 Practice of Antiseptic Surgery Joseph Lister
1876 Discovery of Bacillus anthracis which became the first Robert Koch
proof of germ theory
1881 Utilization of solid culture media for bacterial growth Robert Koch
1882 Outlined Koch’s Postulate Robert Koch
Development of Acid-fast stain Paul Erlich
1884 Gram Stain developed Hans Christian Gram
1885 First Rabies Vaccination Louis Pasteur
1887 Invention of the Petri Dish Richard J. Petri
1892 Discovery of Viruses Dmitri Iosifovich Ivanovski
1893 Zoonosis – first described T. Smith, F.I. Kilbourne
1899 Viral dependence on living host cells for reproduction Martinus Beijerinck
recognized
1900 Proof the mosquitoes carry the agent of yellow fever Walter Reed
1910 Discovered the cure for syphilis Paul Erlich
1928 Discovery of Penicillin Alexander Fleming
1953 Proposed and built the DNA Model J. Watson, F. Crick
1977 Development of the DNA sequencing method W. Gilbert, F. Sanger
1983 Invention of the Polymerase Chain Reaction (PCR) Kary Mulis
1995 Publication of the first microbial genomic sequence The Institute for genomic
Research (TIGR)
Selected Significant Infectious Agents and Dates of Discovery
PERIOD MICROORGANISM DISCOVERER

1876 Bacillus anthracis Robert Koch
1879 Neisseria gonorrohoeae Albert Neisser
1880 Staphylococcus, Streptococcus, Louis Pasteur
Pneumococcus
1882 Mycobacterium tuberculosis Robert Koch
1883 Corynebacterium diphtheria Edward Klebs, Fredrick
Loeffler
1892 Clostridium perfringens William Welch, George Nuttal
1894 Yersinia pestis Emile John Yersin, S. Kitasato
1900 Coccidioides immitis W. Ophuls, H.C. Moffett
1903 Leishmania donovani William Leishman

1905 Treponema pallidum Fritz R. Schandinn, Erich
Hoffman
PERIOD MICROORGANISM DISCOVERER
1918 Brucella abortus Alice Evans
1977 Legionella pneumophila Joseph McDade, Charles
Shepard
1982 Prions Stanley Prusiner
Tissue Culture Media Used in Microbiology Laboratory
TISSUE CULTURE MEDIA SOURCE

Vero Cell Line Kidney cells of an African Green Monkey
Mc Coy Cell Line Mouse Cell line
Chicken Embryo Fertilized chicken egg
A549 Cells Human lung carcinoma
HELA Cell Line Human cervical carcinoma
Hep-2 Cell Line Human epithelial cells of larynx carcinoma
CULTURE MEDIA
CULTURE MEDIA PURPOSE

Alkaline Peptone Broth Vibrio
Bile Esculin Agar Identification of Group D streptococci
Bismuth Sulfite Agar Selective media for Salmonella (stool)
Blood Agar Differentiation for hemolytic patterns
For fastidious organisms
Bordet Gengou Agar Bordetella pertussis
Brilliant Green Agar Selective media for Salmonella
Blood Cystine Dextrose Agar Francisella tularensis
Buffered Charcoal Yeast Extract (BCYE) Legionella spp.
Campy-blood Agar Campylobacter spp.
Cefoperazone Vancomycin Amphotericin Campylobacter spp.
Agar
Cefsulodin-Irgasan-Novobiocin Agar (CIN) Yersinia spp. and Aeromonas
Chick embryo Chlamydia and Rickettsia
Chocolate Agar Neisseria and Haemophilus
Chocolate Agar w/ horse blood Haemophilus spp.
Columbia Colistin Nalidixic Agar (CNA) Selective for g(+) cocci
Cystine Tellurite Blood Agar Corynebacterium diptheriae
Cyclosporine Cefoxitin Fructose Agar Clostridium difficile
(CCFA)
Cetrimide Agar Pseudomonas aeruginosa
Czapek Agar Nocardia asteroids, Aspergillus
Dorset Egg Medium Mycobacterium tuberculosis
Desoxycholate Citrate Agar Salmonella and Shigella (stool)
Dieudonne’s medium Vibrio cholera
Dubois Oleic Agar Mycobacterium tuberculosis
Edward Hayflick Agar Mycoplasma
Eosin Methylene Blue (EMB) – Levine Differential media for LF and NLF
Ellinghausen, McCullough, Johnson and Leptospira interrogans
Harris (EMJH) medium
Fletcher’s semi solid medium Leptospira
Fetal Bovine Serum Agar w/ vancomycin Haemophilus ducreyi
Foot pad of mice Mycobacterium leprae
Gram Negative (GN) Broth For enteric pathogens
Human Blood Bilayer Medium Gardnerella vaginalis

Hektoen Enteric (HE) Agar G(-) enteric pathogens
Kanamycin-Vancomycin Blood Agar (KVBA) Bacteroides
Kelly’s medium Borrelia burgdorferi
Loeffler’s Blood Serum Medium Corynebacterium diptheriae
Lombard Dowell Medium Anaerobic Organisms
Lowenstein Jensen (LJ) Agar Mycobacteria
Lysine Iron Agar Enterobacteriaceae (deamination or
decarboxylation)
Litmus milk media Clostridium spp.
Mac Conkey Agar Differential medium for LF and NLF
Selective for g(-) bacteria
Mac Conkey Agar w/ sorbitol E. coli O157:H7
Mannitol Salt Agar Staphylococcus aureus
McBride Medium Listeria monocytogenes
Middlebrook 7H10 Mycobacteria
Martin Lewis Agar Neisseria gonorrheae
Mueller Hinton Agar Susceptibility test (antimicrobial testing)
Methylene Blue Milk Enterococci
New York City (NYC) Agar Neisseria gonorrheae
Oxidative Fermentative (OF) Medium Differential media for Acinetobacter, Alcaligenes,
Pseudomonas
Petragnani Medium Mycobacteria
Phenyl Ethyl Alcohol (PEA) Medium For Gram (+) cocci
Rabbit Blood Agar w/ yeast extract Haemophilus influenza
Regan Lowe Bordetella pertussis
Salmonella-Shigella Agar (SSA) Salmonella and Shigella
Seller’s Agar Non-fermentative g(-) bacilli
Differential media for Acinetobacter
calcoaceticus, Alcaligenes faecalis and
Pseudomonas aeruginosa
Selenite Broth (enrichment medium) Salmonella spp.
Schaedler Agar For recovery of aerobes and anaerobes
Skirrow Agar Campylobacter spp.
Tetrathionate Broth Salmonella and Shigella spp.
Thayer Martin Agar Neisseria gonorrheae and Neisseria meningitides
Thioglycollate Aerobes, anaerobes, microaerophilic, fastidious
organisms
Thiosulfate Citrate Bile Salt Sucrose Agar Vibrio
(TCBS)
Tinsdale Agar Corynebacterium diptheriae
Todd-Hewitt Broth (selective & enrichment) Streptococcus agalactiae
w/ nalidixic acid and gentamicin/colistin
Trypticase Soy Broth (TSB) Streptococcus pneumoniae, Brucella
Tyrosine Agar Aerobic actinomycetes
Tween 80-albumin Broth Spirochetes
Xylose Lysine Desoxycholate (XLD) Agar Salmonella and Shigella
MYCOLOGY
Mycology - study of fungi
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

7. Monomorphic: fungi with single form only
8. Saprobe: Organism capable of living on decaying organic material
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
 Cell wall: CHITIN (polysaccharide)

 Cell membrane: STEROL (lipid)
 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 (+)
Sexual and Asexual Reproduction

1. Sexual reproduction
a. Requires the formation of specialized fungal structures called spores
b. Fungi that undergo sexual reproduction are termed perfect fungi.
c. Types of spores
1) Ascospores: Spores contained in a saclike structure
2) Basidiospores: Spores contained in a club-shaped structure
3) Oospores: Spores resulting from the fusion of cells from two different hyphae
4) Zygospores: Spores resulting from the fusion of two identical hyphae
2. Asexual reproduction
a. Asexual reproduction only involves division of the nucleus and cytoplasm.
b. Fungi that undergo asexual reproduction are termed imperfect fungi.
c. Imperfect fungi are the only fungal group to produce conidia.
1) Chlamydospore: thicked-walled spores formed by rounding and enlargement within a single hyphal
segment.
 3 types:
a. rounding terminal hyphae – at the end of the hypha
b. intercaly – within hypha
c. Sessile – side of hypha
Ex.: C. albicans
2) Arthrospore/Oidium: barrel-shaped spores/conidia
 Formed by the fragmentation of a septate hyphae into single, slightly thickened cells
Ex.: Geotrichum, Coccidioides, Trichosporon, Aureobasidium
3) Blastospore: formed by budding, daughter cells pinching off (budding off yeasts) from the portions
of the mother cell.
 Typical sporulation of budding yeasts
4) Microconidia and Macroconidia

 Microconidia – small, unicellular conidia
 Macroconidia – large, multicellular conidia
Ex.: Dermatophytes
CULTURE AND ISOLATION

A. Types of Fungal Media
1. Sabouraud dextrose agar (SDA)
• General-purpose culture media, nutritionally poor medium mildly selective for fungi, no longer
commonly used; several different formulations available
2. Sabouraud-brain heart infusion agar (SABHI)
• A nonselective medium for isolation of all fungi
• For systemic fungi
3. Brain heart infusion agar with blood (BHIB)
• Used to grow most fungi, especially those from sterile body sites
• To show dimorphism
• For systemic fungi
4. Selective agars
a. Inhibitory mould agar (IMA)
• IMA is used to grow most fungal pathogens; it is especially formulated to recover the
cyclohexamide-sensitive Cryptococcus.
b. Dermatophye test medium (DTM) or Mycosel agar
• Used to isolate the dermatophytes
5. Differential agars
a. Potato dextrose agar (PDA)
• Used to enhance conidia development
• For pigment production of fungi
b. Bird seed (niger seed) and caffeic acid agars are selective and differential media used to grow C.
neoformans. C. neoformans forms black to brown colonies due to the activity of phenol oxidase.
Chloramphenicol and cycloheximide can be added to make the media selective.
c. Cornmeal agar with Tween 80: Used to differentiate Candida spp.
d. Agars containing rice, casein, and other nutrients are used to differentiate Trichophyton spp.
 Other fungal culture media:

(1) CZAPEK’S AGAR = Aspergillus and Nocardia asteroids
(2) UREA AGAR = Nocardia
(3) COTTON SEED AGAR = Blastomyces dermatitidis
(4) TAP WATER AGAR = Streptomyces
(5) RICE GRAIN MEDIUM = Microsporum canis
(6) SDA WITH OLIVE OIL = Malassezia furfur
(7) SDA WITH CHLOROMYCETIN = Epidermophyton floccosum
(8) THIOGLYCOLLATE MEDIUM = Actinomycetes bovis
BODY SITES AND POSSIBLE FUNGAL PATHOGENS

A. Blood: Candida spp., Blastomyces dermatitidis, Histoplasma capsulatum, and Cryptococcus
neoformans
B. Cerebrospinal Fluid: Cryptococcus neoformans, Candida spp., Histoplasma capsulatum, and
Coccidioides immitis
C. Hair: Microsporum and Trichophyton
D. Nails: Aspergillus, Epidermophyton, and Trichophyton
E. Skin: Candida, Microsporum, Trichophyton, Epidermophyton, and Blastomyces dermatitidis
F. Lungs: Candida albicans, Aspergillus, Rhizopus, Penicillium, Histoplasma capsulatum, Blastomyces
dermatitidis, and Coccidioides immitis
G. Throat: Candida albicans and Geotrichum candidum
H. Urine: Candida albicans and Candida glabrata
I. Genital Tract: Candida albicans
CLASSIFICATION OF FUNGI
A. OPPORTUNISTIC MYCOSES

1. Candida albicans – most common opportunistic pathogen in AIDS patients
a. C. albicans is the most common yeast isolate and is the causative agent of candidiasis/moniliasis, a
general term for Candida infections.
b. C. albicans is normal flora of the mucous membranes lining the respiratory, gastrointestinal, and
female genital tracts. Most adult infections are endogenous, whereas infants acquire infections from
their mothers (exogenous infections).
c. Types of candidiasis
1) Thrush (oral cavity) – characterized by white, creamy patches formation seen most often on the
tongue
- blastoconidia and chlamydospore
2) Vulvovaginitis (vagina)
3) Onychomycosis (nail infections)
4) Paronychomycosis (cuticle infections)
d. C. albicans can also cause systemic infections, including meningitis, UTIs, and heart and lung
infections.
e. Predisposition to Candida infections includes burns, wounds, diabetes mellitus, antimicrobial therapy,
pregnancy, leukemia, and immune problems (#1 opportunistic pathogen in AIDS patients).
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

- causative agent of aspergillosis, aspergilloma, allergy (asthma), ptomycosis (bread mold);
Farmer’s lung disease
- a remarkable clinical form of Aspergillosis is fungus ball appearance on lung cavities
- A particular form of allergic bronchopulmonary disease known as farmer’s lung is associated
with inhalation of large numbers of Aspergillus conidia as well as those of other fungi.
• A. flavus
- Isolated from the vegetation like nuts and grains during either growth or storage
- Produce metabolites that are toxic and carcinogenic in the liver - aflatoxin (hepatotoxin)
• A. niger – brown to black spore
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
Microscopic: Sickle or canoe shaped, multiseptate macroconidia
Macroscopic: white at first, cottony or wooly frequently becoming pink or purple colony
7. Phaeohyphomycosis – Dematiaceous agents

• A – Alternaria
• B - Bipolaris
• C - Curvularia
• D - Dreschlera
• E - Exophiala
8. Pneumocystis jirovecii (carinii)

 Best specimen: Bronchoalveolar lavage
 Protozoan cyst, No ergosterol
 #1 cause of pneumonia in AIDS
 opportunistic infection in AIDS
B. SUPERFICIAL MYCOSES AND CUTANEOUS MYCOSES

1. Superficial mycoses are infections that involve the outer epithelial layers of the skin and top layers of
the hair and nails.
2. Cutaneous mycoses involve deeper layers of the skin and more tissue.
3. Dermatophyte is the term used to group the various fungi that cause infections (dermatophytoses) of
the skin, hair, and nails.
a. The dermatophytes are keratinophilic (i.e., able to metabolize keratin).
b. Dermatophytes contain three genera.
1) Trichophyton: Infects nails, hair, and skin
2) Epidermophyton: Infects skin and nails
3) Microsporum: Infects hair and skin
4. Superficial and cutaneous fungi are rarely invasive to other areas of the body.
5. Dermatophyte skin infection is termed tinea.
6. Types of tinea infections and their causative agents
a. Tinea pedis or athlete's foot: An infection of the spaces between the toes
1) Caused by Trichophyton spp. and Epidermophyton spp.
2) Characterized by itching and scaling
b. Tinea corporis or ringworm: An infection of smooth skin
1) Caused by Microsporum spp. and Trichophyton spp.
2) Characterized by circular patches of scaly skin

c. Tinea unguium or onychomycosis: An infection of the nails
1) Caused by Epidermophyton spp. and Trichophyton spp.
2) Characterized by discoloration, thickening, and progressive destruction of the nails
d. Tinea capitis: An infection of the scalp
2) Characterized by circular bald patches on the scalp
e. Tinea barbae or barber's itch: An infection of beard hair
2) Characterized by skin lesions
f. Tinea cruris or jock itch: An infection of the groin
1) Caused by Trichophyton spp. and Epidermophyton spp.
2) Characterized by itching and scaling of the groin area
7. Identification of the dermatophytes is primarily based on colony morphology and microscopic
appearance. In some cases, it may be necessary to perform an in vitro hair perforation test. Sterile hair
is infected with the isolated fungus and after incubation is examined microscopically for wedge-shaped
perforations
-Non invasive, Person to person contact – garment
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
II. CUTANEOUS MYCOSES

1. TRICHOPHYTON
Trichophyton rubrum
• Microscopically: numerous tear-drop shaped microconidia occurring singly and in clusters along
hypha
• Colonies are fluppy white w/ red colored reverse
Trichophyton mentagrophytes
• Granular to powdery and they usually display abundant grape like clusters and subspherical
microconidia on terminal branches
• Hair penetration test (+)
(+) result: V shape
• Common cause of Tinea pedis (athlete’s foot)
Hair Red Urease

Perforation pigment
(Baiting)
Test
T. mentagrophytes (+) V shape (-) (+)
T. rubrum (-) (+) (-)
Trichophyton tonsuran
• Balloon shape microconidia; “Black dot”, endothrix hair invasion
• Common cause of Tinea capitis

Trichophyton schoenleinii
• cause favus - Favic chandelier (antler)
T. verrucosum - Ectothrix
• Clavate/pyriform microconidia
• Rat tail/ string bean shaped macroconidia
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
III. SUBCUTANEOUS MYCOSES

• All are found in the soil (habitat)
• Acquired thru traumatic implantation of the organism into the subcutaneous tissue (skin
trauma)
• Biopsy, granules (PAS, H and E)
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)
Madura foot (Mycetoma, Maduromycotic) agents

FUNGAL:
Pseudallescheria boydii – most common cause
Madurella mycetomi
Leptosphaeria
Philophora jeanselmei
USUAL BACTERIAL COMPLICATIONS:
Actinomyces (Nocardia, Streptomyces)
• Localized swollen lesion with granules that are compact colonies of the causative agent draining
from sinuses
• In pus, the fungus appears as granulomatous lesions on foot with multiple draining sinus tracts
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)

b. Coccidioides immitis (cocccidioidomycosis)
c. Histoplasma capsulatum (histoplasmosis)
d. Paracoccidioides brasiliensis (paracoccidioidomycosis)
• Exoantigen test - immunodiffusion test; confirmatory test for systemic fungi
a) A Ag - B. dermatitidis
b) 1, 2 & 3 Ag – P. brasiliensis
c) H and M Ag – H. capsulatum
d) HS, HL, F – C. immitis
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
• South American Blastomycosis, Lutz Splendore, Almeida Disease, Paracoccidioidal Granuloma –

lungs, spleen, liver, lymph node, skin
• tissue form: MULTIPLE BUDDING YEAST (mariner’s / navigation wheel)
• mold form: lollipop in appearance
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.
• Reticuloendothelial system parasite – DARLING’S DISEASE

• Blood smear stained with Giemsa
• tissue form: yeast cells intracellular in macrophages
• mold form: tuberculate macroconidia
• Cross reacts with Blastomyces
• H. duboisii – double cell / figure 8, cause African histoplasmois (bone)
4. COCCIDIOIDES IMMITIS – COCCIDIOIDOMYCOSIS

a. Coccidioidomycosis (valley fever) is an infection of the lungs, bones, joints, skin, lymph nodes,
central nervous system, and adrenal glands. Infections can be acute or chronic and self-limiting
or requiring medications.
b. Most infections in the U.S. are in the semiarid southwest desert region (Lower Sonoran Life
Zone). Infections are sometimes called desert or valley fever in the San Joaquin Valley of
California, where many cases are diagnosed.

c. Infections are often acquired through spore inhalation from the environment. Activities that
increase airborne dust, such as plowing and construction, can facilitate transmission.
• San Joaquin Valley Fever, Desert Rheumatism. The Bumps

• tissue form: SPHERULE with endospores
• mold form: barrel-shaped arthroconidia
• Laboratory acquired infection (high risk to laboratory personnel)
LABORATORY DIAGNOSIS OF SYSTEMIC FUNGI: Direct exam of clinical specimens

• Histoplasma – Wright or Giemsa
• Blastomyces, Paracoccidioides, Coccidioides- KOH, PAS, H & E
• Cultures
-SDA – RT
-BHIA + blood- 37◦C
VIROLOGY
General Characteristics of Viruses

1. Viruses are obligate intracellular parasites unable to self-replicate. Once inside living cells, viruses
induce the host cell to synthesize virus particles.
2. The genome is either DNA or RNA (single or double stranded).
3. Viruses do not have a system to produce ATP.
4. Viruses range in size from 25 to 270 nm.
5. The classification of viruses is based on nucleic acid type, size and shape of virion, and presence or
absence of an envelope.
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
Viral morphology (electron microscope)

Capsid symmetry:
• Icosahedral - DNA, (+) sense RNA
• Helical - (-) sense RNA viruses
• Complex (Poxvirus)
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

• Viral Antigen - immunofluorescence
• Serology - ELISA, IFT, Latex
• Viral genome – PCR, SBHT, NBHT
• Virus isolation / culture
• Cell culture - CPE, hemadsorption
• Eggs - pock formation on CAM
• Animals - death of animal
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
Types of Cell Culture

 Cell culture is a lot cheaper and easier to work with (contamination can be a problem however).
 Primary cell lines – have a short lifespan in culture (passed only once); a few generations before
reaching senescence
Ex.: PMKC (Primary Monkey Kidney Cells)
 Diploid cell lines – are derived from embryos and can grow for up to 100x population doublings
before senescence
 Semi continuous cell lines – passed 50X
Ex.: HDF (Human Dermal Fibroblasts)
 Continuous cell lines – are derived from transformed (malignant); passed or grow indefinitely in
culture
Ex.: MDCK (Madine Darby Kidney Cell), HEp-2, Hela cells
Recognition of Viral growth

• CPE (Cytopathic Effect) - changes that happen in a virally infected cell
• Hemadsorption
• Interference - Rubella and Enterovirus
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
RULES FOR DNA AND RNA VIRUSES
DNA Virus (Pa Pa Ad Po2 He He)

Herpesvirus
Hepadnavirus
Adenovirus
Poxvirus
Polyomavirus
Parvovirus

Papovavirus
• All are DS except Parvovirus.
• All are icosahedral except Poxvirus.
• All are enveloped except PAP (Parvovirus, Adenovirus, Papovavirus).
• All multiply in nucleus except Poxvirus.
RNA Virus
• All are SS except Reovirus.
• All are helical except (+) sense RNA virus.
• All are envelope except PCR (PicoRNA virus, Calicivirus, Reovirus)
• All are non-segmented except ROBA (Reovirus, Orthomyxovirus, Bunyavirus,
Arenavirus).
• Positive sense RNA virus – Call Pico and Flo To Come Right.
Call – Calicivirus
Pico – Picornavirus
Flo – Flavivirus
To – Togavirus
Come – Coronavirus
Right – Retrovirus
• Negative sense RNA virus – Pairing of Rats at the Bunny’s Area.
Pairing – Paramyxovirus
Of – Orthomyxovirus
Rats – Rhabdovirus
Bunny’s – Bunyavirus
Area – Arenavirus
DNA VIRUSES
FAMILIES OF DNA VIRUSES
Poxviridae - Variola virus, molluscum contagiosum virus
Herpesviridae - Herpes simplex viruses types 1 and 2, varicella zoster virus, Epstein-Barr virus,
cytomegalovirus, human herpesviruses 6, 7, and 8
Adenoviridae – Adenovirus
Hepadnaviridae - Hepatitis B virus
Papillomaviridae – Papillomavirus
Polyomaviridae - JC and BK viruses
Parvoviridae - Parvovirus B19
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

• Molluscum contagiosum virus - wart like tumors; lesion begins as small papule and gradually
grows into discrete, waxy, smooth, dome-shaped, pearly or flesh colored nodule.
Diagnosis:
• CPE on cell culture or pocks on chorioallantoic membrane (CAM)
PARVOVIRUS – single stranded, icosahedral DNA virus

• Human parvovirus strain B19
• it causes Aplastic crisis or sickle cell anemia because it targets the erythroid progenitor cell
• causative agent of Erythema infectiosum (“fifth disease”, “slapped cheeks syndrome”-
endothelial cells
• Hydrops fetalis – Miscarriages
• Diagnosis: PCR - viral DNA from blood / amniotic fluid
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
Epstein-Barr virus (Human herpesvirus 4) - Heterophile-positive mononucleosis (Infectious

Mononucleosis), Kissing Disease
Cytomegalovirus (Human herpesvirus 5) - Asymptomatic infection, heterophile negative

mononucleosis, fever hepatitis syndrome in neonates and transplant patients, interstitial pneumonia in
immunocompromised patients
• #1 congenital infection
• Culture: HEF (Human Embryonic Fibroblasts)
Human herpesvirus 6 - Roseola infantum (sixth disease)
Human herpesvirus 7 - Roseola and febrile disease in

children
Human herpesvirus 8 - Kaposi's sarcoma
HUMAN PAPILLOMAVIRUS (HPV)

• Icosahedral nucleocapsid
• tropism for squamous epithelial cells causing warts (fingers, sole, face)
• cervical, squamous cell, vulvar, penile cancer
• condylomata acuminata (ano-genital)
ADENOVIRUS - #1 cause of viral conjunctivitis (pink eye)

• Serotypes:
3, 4, 7, 21 - acute respiratory disease
8, 19 – epidemic keratoconjunctivitis
11, 21 - hemorrhagic cystitis
40, 41 - infantile gastroenteritis

• Diagnosis: Cell culture - grape like clusters (CPE)
• 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
FAMILIES OF RNA VIRUSES
Paramyxoviridae - Measles, mumps, respiratory syncytial, parainfluenza, and metapneumo viruses
Orthomyxoviridae - Influenza A, B, and C viruses
Coronaviridae – Coronavirus
Arenaviridae - Lymphocytic choriomeningitis and Lassa fever viruses
Rhabdoviridae - Rabies virus
Filoviridae - Marburg and Ebola viruses
Bunyaviridae - California encephalitis, Hantaan, sin nombre, and Crimean-Congo viruses
Retroviridae - Human T lymphotropic and human immunodeficiency viruses
Reoviridae - Rotavirus and reovirus
Picornaviridae - Rhinovirus, poliovirus, enterovirus, ECHO virus, coxsackievirus, hepatitis A virus
Togaviridae - Rubella virus and western, eastern, and Venezuelan equine encephalitis viruses
Flaviviridae - Yellow fever, dengue, St. Louis encephalitis, hepatitis C, and West Nile viruses
Caliciviridae - Norwalk and Sapporo viruses
PICORNAVIRUS – naked, icosahedral RNA viruses

• ACID RESISTANCE TEST
-Enterovirus – acid-resistant
-Rhinovirus – acid-labile
Site to multiply = Enterovirus - GIT; Rhinovirus - nasal
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

3. Coxsackie B virus – causes internal symptoms (Pleurodynia, Myocarditis,)
- Pleurodynia / Bornholm disease / Devil’s grip – is an uncommon complication of coxsackievirus B
infection and is defined as the sudden occurrence of lancinating pain attacks, commonly associated with
fever, malaise, and headaches.
4. Coxsackie type A16 – Hand, foot and mouth disease
- Hand, foot and mouth disease – symptoms include fever and blisters on the hands, palate and feet
5. ECHO virus (Enteric Cytopathic Human Orphan virus) – aseptic meningitis
6. Enterovirus 72 - Hepatitis A Virus (HAV)
Rhinoviruses - acid labile, 33◦C

• Common cause of common colds (rhinitis)
ORTHOMYXOVIRUS (MYXOVIRUS)
• HELICAL, SEGMENTED, ENVELOPED
• HEMAGGLUTININ (H) and NEURAMINIDASE (N)
• Antigenic changes:
• ANTIGENIC SHIFT - genetic reassortment - pandemic
• ANTIGENIC DRIFT - point mutation - epidemic
Influenza virus (Flu virus)

Question: Can Haemophilus influenzae cause flu? NO
• Type A - Pandemic (Ag shift & drift)
AH1:N1 – Spanish flu, AH2:N2 - Asian flu, AH3:N2 - Hongkong flu, AH5:N1 - Avian flu (Bird flu;
Avian influenza), AH1:N1 - Swine flu
• Type B - Epidemic (Ag drift)
LAB DIAGNOSIS (INFLUENZA)

1. Antigen test - throat washing, nasopharyngeal aspirate → IFT, ELISA
2. Virus isolation - throat swab, nasopharyngeal aspirate - Medium: PMKC, MDCK (Madine Darby Canine
Kidney), embryonated hen’s egg (Hemadsorption)
3. Test: Hemagglutination-Inhibition
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
Measles virus (Rubeola virus)

 Measles
 Koplik spots – a prodromic viral exanthema of measles manifesting on the first day of rash;
pathognomonic for measles; they are described as appearing like “grains of salt on a wet
background”
 2nd Infection – Subacute Sclerosing Panencephalitis (SSPE)
 Nasopharyngeal swab, urine
 Culture on PMK - multinucleated cell

Respiratory Syncytial Virus - RSV
• #1 cause of viral bronchiolitis
• croup, bronchitis, pneumonia and other lower respiratory tract infections
• Nasopharyngeal swab
• DFA, EIA
• Culture: HEP-2 (syncytia), PMK, Human diploid fetal cell
• Treatment: Ribavirin
HENIPAVIRUS - ssRNA
1. NIPAH virus - Encephalitis (pig to man)
2. HENDRA virus - respiratory disease of horses
TOGAVIRUS – single stranded, icosahedral enveloped RNA virus

*zoonotic viruses which are transmissible from animals (arthropods, vertebrates) to man
• Arbovirus
- acquired from mosquitoes (arthropod borne)
- most are RNA genome except for African Swine Fever virus (DNA genome)
• Alphavirus – causes encephalitis
- Chikungunya virus
• Western, Eastern, Venezuelan equine encephalitis virus (WEE, EEE, VEE)
• Rubella virus
- German Measles (3-day rash), “blueberry muffin” baby
- Teratogenic virus; causes fetal defect in the form of CRS (Congenital Rubella Syndrome)
- Direct exam by IF, EIA, HA test (sensitive)
RUBELLA VIRUS
• German Measles (3-day rash), “blueberry muffin” baby
• Teratogenic virus (fetal defect)
• Direct exam by IF, EIA, HA test (sensitive)
RHABDOVIRUS (LYSSA VIRUS)

• Rabies virus - helical, enveloped, BULLET-shaped
• CNS virus that causes encephalitis
• Diagnosis: FAT of rabies Ag (from dog’s brain); Seller’s stain of Negri bodies
• Vesicular stomatitis - horse, cattle, pig
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

• Causes Hemorrhagic fever with renal involvement
REOVIRUS - dsRNA, naked virus

• Rotavirus - segmented, icosahedral wagon wheel-like virus in electron microscope
• Causes viral gastroenteritis in children and infant during winter period
• Orbivirus - Colorado tick fever
RETROVIRUS – icosahedral, RNA virus

 Reverse transcriptase – enzyme that can transcribe ssRNA into dsDNA
 AIDS (Acquired Immunodeficiency Syndrome) – is the late stage of HIV infection that occurs
when the body’s immune system is badly damaged because of the virus
 HIV-1 is also known as HTLV-III (Human T-Lymphotropic Virus type III), ARV (Adenopathy-Related
Virus), and LAV (Lymphadenopathy-Associated Virus)
 Serology – ELISA (screening test), Western Blot (confirmatory test)
 RT-PCR – quantitates HIV-DNA (the circulating virus count or viral load)
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)
ARENAVIRIDAE - HEMORRHAGIC FEVER

• Lymphocytic choriomeningitis virus (LCMV)
• Junin virus - Argentinian Hemorrhagic Fever
• Machupo virus - Bolivia Hemorrhagic Fever
• Lassa fever virus- zoonotic (rats)
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)

• HDV (Viroid like)
a. Co-infection
b. HDV is also called the delta virus. HDV requires but does not encode for HBsAg; therefore, it
only replicates in cells also infected with HBV.
• 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
Milk Bacteriology – Normal Flora

 Pseudomonas syncyanea – blue milk
 Flavobacterium synxanthum – yellow milk
 P. aeruginosa – blue green milk
 S. marcescens – red milk

 Streptococcus lactis – souring of milk
 B. subtilis – hay bacteria; proteolytic action on coagulated milk
 Alcaligenes viscosus – slimy or ropy milk
3. QUALITY CONTROL FREQUENCY

Daily QC – Oxidase, Catalase, GS, Incubator, Refrigerator/Freezer, Water bath
Each use – Gas pak jar, ONPG
Weekly QC – Antibiotic, Autoclave, Biochemical tests
ATCC – Reference strains for QC
-20οC or -70οC – Stock culture storage
2-8οC – Working cultures storage
BIOCHEMICAL PROPERTIES OF ENTEROBACTERIACEAE
TSI LIA Indole Methyl Voges Citrate Urease

Red Proskauer
RAPID
LACTOSE
FERMENTERS
Escherichia A/A + K/K + + - - -
coli gas
Klebsiella A/A + K/K - - + + +
pneumoniae gas
Enterobacter A/A + K/K - - + + -
aerogenes gas
Enterobacter A/A + K/A - - + + -
cloacae gas
LATE
LACTOSE
FERMENTERS
Arizona spp. A/A + K/K + - + - + -
gas + H2S
H2S
Citrobacter A/A + K/A - + - + -
freundii gas +
H2S
Citrobacter A/A + K/A + + - + -
diversus gas
NON-
LACTOSE
FERMENTERS
Proteus K/A + R/A + + - + +
vulgaris gas +
H2S
Proteus K/A + R/A - + - + +
mirabilis gas +
H2S
Providencia K/A + R/A + + - + +
rettgeri gas
Morganella K/A + R/A + + - - +
morganii gas
Salmonella K/A + K/K - + - - -
typhi gas +
H2S

Salmonella K/A + K/K - + - + -
typhimurium gas +
H2S
Shigella K/A K/A - + - - -
dysenteriae
Shigella K/A K/A - + - - -
sonnei
Shigella K/A K/A + + - - -
flexneri
Shigella K/A K/A + + - - -
boydii
Serratia K/A K/A + - + - +
marcesens
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.

28. 90% of cases of Plasmodium infection is caused by Plasmodium vivax and Plasmodium
falciparum.
29. “Crabs” is an ectoparasite.
30. The most predominant species of Plasmodium that is causing Malaria in the world is
Plasmodium vivax.
31. The most predominant species of Plasmodium that is causing Malaria in the
Philippines is Plasmodium falciparum.
32. The causative agent of Gay bowel syndrome is Giardia lamblia.
33. The third Taenia spp. is Taenia asiatica.
34. The common name of Taenia asiatica is Taiwan Taenia.
35. The uterus of Taenia saginata is described as dichotomous or tree-like branches.
36. The uterus of Taenia solium is described as dendritic or finger-like branches.
37. Diphyllobothrium latum adult resembles the adult form of Spirometra.
38. Diphyllobothrium latum egg resembles the egg of Paragonimus westermani.
39. The positive control for the Acetate test is Escherichia coli.
40. Acetate test is incubated at 35◦C for 7 days.
41. The positive result for the Acetate test is blue.
42. The positive control for the Acetamide test is Pseudomonas aeruginosa.
43. The positive control for the Malonate test is Salmonella.
44. Malonate test is incubated at 35◦C for up to 7 days.
45. The positive result for the Malonate test is blue.
46. Acetamide test is incubated at 35◦C for 7 days.
47. The positive result for the Acetamide test is blue.
48. A cellophane paper soaked in mixture of glycerine and malachite green or
methylene blue solution is being used in the kato-thick smear preparation.
49. To detect stipplings, prepare blood films 30 minutes to 1 hour after being drawn.
50. The preferred blood specimen for preparing blood smear for protozoa is obtained
through finger puncture.
51. The definitive host to Plasmodium is female Anopheles mosquito.
52. The color of the chromatoid bodies with trichrome stain is bright red.
53. The appearance of glycogen mass in the protozoan cyst after it is stained with
trichrome stain is colorless.
54. The appearance of nucleus in the trophozoite structures after it is stained with
trichrome stain is red-purple.
55. The appearance of Charcot-Leyden crystals in the trophozoite structures after it is
stained with trichrome stain is bright red.
56. The chromatoidal bodies of protozoan cysts in iodine do not stain.
57. The stains used for Naegleria and Acanthamoeba are H&E and Wright’s stain.
58. Iodine and concentration techniques destroy trophozoites.
59. The hexacanth embryo of Hymenolepis diminuta lacks polar filaments.
60. The hexacanth embryo of Hymenolepis nana has polar filaments.
70. The egg of Taenia have hexacanth embryo in a radially striated shell.
71. The second intermediate host of P. westermani is fresh water crabs or crayfishes.
72. The eggs of Schistosoma mansoni and Schistosoma japonicum are recovered in stool
and through rectal biopsy. S. haematobium eggs are recovered in a concentrated urine
specimen.
73. Flukes and tapeworms are hermaphroditic.
74. An Ascaris egg lacking its mamillated coat is decorticated egg.

75. The nematode parasites with heart to lung migration are Ascaris lumbricoides,
Strongyloides stercoralis and Hookworms.
76. The so-called “Unholy Three” or the “Triad of Infection” composes of Hookworms,
Ascaris lumbricoides and Trichuris trichura.
77. The antigen that is cross-reactive in all adenoviruses is called the hexon.
78. The causative agent of Farmer’s Lungs is Aspergillus.
79. Cornmeal agar plus Tween 80 is used to identify C. albicans through the organism’s
production of chlamydospores.
80. Candida albicans produces blastospores, chlamydospores and pseudohyphae.
81. The stain in Aman medium is LCPB (Lactophenol Cotton Blue). ---the most common
fungal stain
82. Setting of RPMs marked on the face of the rheostat control on the centrifuge
should be checked once monthly.
83 Catalase, oxidase and coagulase reagents must be tested one each day of use and
when vial is first used.
84. Half-life refers to the half a dose of antibiotic to disappear from the blood.
85. Thioglycollate broth is boiled for 10 minutes to drive off oxygen.
86. The positive result for malonate test is blue.
87. The negative result for malonate test is green or yellow.
88. Middlebrook 7H11 is a clear medium for Mycobacteria.
89. Bordetella bronchiseptica is both urease and oxidase positive.
90. The preferred medium for the isolation of Bordetella pertussis is Charcoal-
Cephalexin Agar.
91. The inhibitors in Salmonella-Shigella agar are bile salt and brilliant green.
92. The methods for testing the production of beta-lactamase are chromogenic
cephalosporin method, acidometric method and iodometric method.
93. The meaning of the acronym PPNG is Penicillinase-Producing Gonococci.
94. The zone of inhibition that is considered as sensitive in the Optochin Disk Test or
Taxo P is 14mm or greater.
95. Any zone of inhibition is considered as sensitive in the Bacitracin Disk Test or Taxo
A.
96. Streptolysin O is oxygen labile.
97. Streptolysin S is oxygen stable.
98. The most common pathogen in throat cultures is the Group A Streptococcus
(Streptococcus pyogenes).
99. Staphylococcus aureus is DNAse positive.
100. Staphylococcus epidermidis is DNAse negative.
101. Staphylococcal protein A is the carrier used in coagglutination.
102 Capsule can be used for a serotyping by swelling.
103. The milk is considered as Grade A milk if the bacterial count is 75,000 per mL
when raw, and not to exceed 15,000 bacteria per mL once pasteurized.
104. Schaeffer-Fulton stain is used for the demonstration of spores.
105. Nasopharyngeal swab specimens are recommended for detection of Bordetella
pertussis, Neisseria meningitidis and Haemophilus influenzae.
106. The virus that causes shingles is VZV.
107. Saboraud Dextrose Agar, Potato Dextrose Agar and cornmeal agar are
culture media for fungi.
108. Broad spectrum antibiotics (1) act against gram positive bacteria, (2) act
against gram negative bacteria and (3) act against bacterial and nonbacterial
organisms.

109. Mature cysts of Entamoeba polecki have one nuclei.
110. Trophozoites of Entamoeba histolytica have the following characteristics: (1)
small, delicate nuclear karyosomal chromatin, (2) fine, even peripheral chromatin
and (3) progressive motility with hyaline, fingerlike pseudopods.
111. The enteric cytopathic human orphan virus that causes diarrhea in children is ECHO
virus.
112. Hospital acquired infection is called nosocomial infection.
113. Disease in animals which is spread to humans is called zoonotic disease.
114. A heat-resistant fungi that is commonly associated as a food contaminant is
Aspergillus.
115. A parasite which has characteristic auto-fluorescence is Cyclospora
cayetanensis.
116. An organism which has a characteristic of grayish white, transparent to
translucent, matte or glossy colonies with a large zone of beta hemolysis is Group A
Streptococcus.
117. The types of blood used for detection of microfilaria are capillary and arterial
blood.
118. Gravid proglottid is filled with eggs.
119. Rabies virus can affect CNS.
120. The most common method for detection of toxoplasma is the detection of
antibody specific for toxoplasma.
121. The continent where Chagas Disease was first discovered is America.
122. The C in ToRCHeS stands for Cytomegalovirus. (ToRCHeS = Toxoplasma gondii,
Rubella, CMV, Herpes, Hepatitis, Syphilis)
123. Calcofluor white stains the cell wall of fungi.
124. Campylobacter and Helicobacter are obligate microaerophiles.
125. The coccidia that can be contracted from cats are Toxoplasma gondii.
126. Ova with a large lateral spine belongs to Schistosoma mansoni.
127. The carrier of Ascaris eggs is cockroach. (mechanical or phoretic vector)
128. Calcium alginate swab is toxic to viruses. --- use cotton swab instead
129. Cotton swab is toxic to Neisseria spp.
130. Wood is toxic to Chlamydia spp.
131. The chain of infection composes (1) source, (2) mode of transmission, (3)
susceptible host.
132. The appropriate ratio of blood:broth in preparation of blood culture is 1:5. (ex.
10mL of blood is to 50mL of broth)
133. HTLV-I and HTLV-II can be transmitted via blood, serum, saliva and semen.
134. The best types of water to prepare an agar medium are deionized or distilled
water.
135. Stool specimen is rejected or will be no help in the diagnosis of RSV.
136. The best time to collect blood is before the height of fever.
137. Morphology is a test to determine C. tetani. (tennis racket, lollipop or drumstick
appearance)
138. An AFB found in nasal mucosa is not diagnostic. (normal flora)
139. Diagnostic laboratory media (agar) for bacterial isolation are usually adjusted to
a final pH between 7.0 & 7.5. (Rodriguez)
140. Hemolysis test is one of the tests to differentiate S. pyogenes (β-hemolytic)
and S. pneumoniae (α-hemolytic) from other Streptococci.
141. Bile esculin hydrolysis test is one of the tests to differentiate Listeria (+)
versus Corynebacteria (-)

142. Streptomyces is a filamentous bacteria which is the most common source of
making antibiotics.
143. Too much moisture in disk diffusion in growth in AST leads to smaller zone of
inhibition.
144. Salmonella shows colorless colonies with black centers in SSA (Salmonella
Shigella Agar). --- H2S (+)
145. Shigella shows colorless colonies without black centers in SSA (Salmonella
Shigella Agar). --- H2S (-)
146. SSA, XLD and Tetrathionate are the best culture media for Salmonella culture.
147. Sterilization in cans kills endospores of Clostridium botulinum, endospores of
E. coli and toxins.
148. The concentration of H2O2 used as a disinfectant is 3%.
149. Random urine specimen is the most ideal for GS/CS of ambulatory patients.
150. Catheterized urine specimen is the most ideal for GS/CS of bedridden.
151. Midstream clean-catch urine specimen is the most ideal for GS/CS of women.
152. Vinegar (acetic acid) is the alternative disinfectant if sodium hypochlorite
(chlorox) is unavailable in the laboratory.
153. Resolution refers to the property to see the cellular details that are
microscopically micrometers.
154. Haemophilus can be ruled out in SBAP (Sheep Blood Agar Plate). ---fastidious
organism
155. Staphylococcus aureus is not a cause of venereal disease (STD).
156. Enterococcus is bacitracin-resistant, hippurate hydrolysis test negative, bile
esculin hydrolysis (+), β-hemolytic and 6.5% sodium chloride positive.
157. Motility test can differentiate Listeria (+) and Corynebacterium (-).
158. SIM (Sulfide Indole Motility) medium has two purpose: (1) motility test and (2)
tryptophan medium.
159. Amman medium is a fungal medium.
160. Soap is germicidal.
161. Microsporum canis fluoresce on Wood’s UV lamp.
162. Mycosel agar is for Dermatophytes.
163. Gram staining qualifies sputum for culture.
164 Sugar utilization (Carbohydrate Utilization Test) speciates Neisseria spp.
165 E. coli, S. saprophyticus and Enterococci causes UTI.
166. P. aeruginosa and Burkholderia cepacia complicates Cystic Fibrosis patients.
167 P. aeruginosa causes Swimmer’s ear and burns.
168 Nasal swab is an acceptable specimen for Staphylococcus aureus.
169. MTB (Mycobacterium tuberculosis) is niacin test positive, nitrate reduction test
positive and catalase negative.
170 Clostridium perfringens is lecithinase positive and shows double zone of hemolysis.
171 Gardnerella vaginalis, Neisseria, Streptobacillus moniliformis and
Peptostreptococcus anaerobius are the four bacterias that are inhibited by SPS disk
test.
172. PPM (Proteus, Providencia and Morganella) are PAD (Phenylalanine Deaminase)
positive, urease positive and lysine decarboxylase positive.
173. The positive result of indole test, methyl red test, vogues-proskauer test, urease
test and nitrate reduction test are all the same: red.
174. The positive result of citrate, acetamide, malonate and acetate utilization tests
are all the same: blue --- negative result: green

175. The positive result of LOA test (decarboxylase test), hippurate hydrolysis test,
LIA(Lysine Iron Agar), and oxidase testare all the same: purple
176. The positive result of ONPG (orthonitrophenylgalactopyranoside) test, TSI (Triple
Sugar Iron) and TCBS (Thiosulfate-Citrate-Bile Salt-Sucrose) are all the same: yellow
177. Salmonella paratyphi A is lysine negative and H2S negative.
178. IMViC differentiates Enterobacteriaceae.
179. O-F test differentiates NFO (Non-Fermentative Organisms).
180. Eikenella corrodens, Kingella kingae and Moraxella doesn’t grow on MAC.
181. P. aeruginosa is a pigment-producer (pyocyanin, pyuverdin, pyorubin) and causes
grape-like odor on Cetrimide agar.
182. Haemophilus shows growth in CAP but not in BAP and MAC.
183. F. tularensis is a bacteria known of its requirement cysteine which causes
laboratory acquired infection (BSL-III).
184. Biologic agents which are classified as BSL-I (Biosafety Level-I) post no risk
(Mycobacterium gordonae, Bacillus subtilis).
185. Biologic agents which are classified as BSL-II (Biosafety Level-II) post moderate
risk (Yersinia pestis, Bacillus anthracis).
186. Biologic agents which are classified as BSL-III (Biosafety Level-III) post high
risk but can be treated (Mycobacteria, Brucella, Francisella, molds).
187. Biologic agents which are classified as BSL-IV (Biosafety Level-IV) post high risk
but cannot be treated (viruses).
188. Thionine and fuchsin can speciate Brucella spp.
189. Brucella abortus is CO2 positive, H2S positive and inhibited by thionine.
190. Pasteurella shows “safety pin appearance” once it is stained with Wayson’s stain
(bipolar bodies); it is usually acquired through animal bite wounds.
191. Listeria causes meningitis, sepsis, stillbirth and food poisoning.
192. Erysipelothrix rhusiopathiae is the only Gram positive bacillus that is H2S
producer; Catalase (-); associated with butcher’s cut
193. Legionella is the causative agent of Pontiac fever acquire from air condition;
Culture media: BCYE (Buffered Charcoal Yeast Extract)
194. Leptospira interogans icterohemorrhagica is the causative agent of Weil’s disease
(jaundice); Culture media: Fletcher’s medium
195. Chlamydia trachomatis is the causative agent of LGV (Lymphogranuloma inguinale);
#1 cause of NGU (Non-Gonococcal Urethritis; #1 cause of PID (Pelvic Inflammatory
Disease); Culture media: McCoy cells
196. Borrelia can be demonstrated through Giemsa stained blood smear.
197. The causative agent of Whipple’s disease is Trophyrema whipplei.
198. Ureaplasma urealyticum (Common name: T-strain) causes NGU; Urease (+); Culture
media: Sheperd’s mom A7 medium
199. The vector of Lyme’s disease, RMSF (Rocky Mountain Spotted Fever) and
Ehrlichiosis is the same: ticks.
200. Scrub tyhus (O. tsutsugamushi) is OX-K positive; OX-2 and OX-19 negative
201 RMSF (Rickettsia rickettsi) is OX-2 and OX-19 positive; OX-K negative
202. Rickettsial pox (Rickettsia akari) and Q fever (Coxiella burnetti) are all OX-2,
OX-19 and OX-K negative.
203 BSC (Biologic Safety Cabinet) Class I – air velocity 75 linear feet/min; exhaust air
thru HEPA filter; product (culture) contaminant; biological safety cabinet that allows
room air to pass through and within the cabinet and only sterilizes the air exhausted

204. BSC (Biologic Safety Cabinet) Class II – air velocity 75-100 linear feet/min;
vertical laminar airflow; no product contamination; exhaust and recirculated air through
HEPA filter; must for microbiology laboratory and hospitals
205. BSC (Biologic Safety Cabinet) Class IIa – exhausts air inside the room; biological
safety cabinet that sterilizes the air that flows over the infectious material as well as
air to be exhausted; also, it is the most commonly used biological safety cabinet which
has a fixed opening and 70% of the filtered air is recirculated
206. BSC (Biologic Safety Cabinet) Class IIb – exhausts air outside the building
(filters exhausted air directed outside the building)
207. BSC (Biologic Safety Cabinet) Class III – supply and exhaust air thru HEPA
filter; for BSL-IV (viruses)
208. The basic structural units of fungi are hyphae and spore.
209. Saccharomyces cerevisiae has ascospore.
210. VZV (Variella-Zoster Virus) and influenza virus are the causative agents of Reye’s
syndrome.
211. Tzanck smear is a diagnostic test for HSV and VZV.
212. JC virus (Polyomaviridae) is the causative agent of PML (Progressive Multifocal
Leukoencephalopathy).
213. Prions are the causative agents of Spongiform encephalopathy.
214 The transport virus specimen must be maintained at 4◦C (refrigerator) if there is a
delay of 3 days in testing.
215. The transport virus specimen and sputum must be maintained at -70◦C if there is a
delay of 4 days in testing.
216. Roller drum is used to hold cell culture tube.
217. The test for the presence of bacteria in milk is methylene blue reduction test.
218. When culturing Cryptococcus on Saboraud Dextrose agar, cycloheximide should not
be in the medium.
219. Geotrichum candidum colonies appear as rapid, yeastlike growth.
220. PCR is not a routine instrument in the Microbiology Laboratory. (Example of routine
= Advance Expert System, BacT Alert, Vitek 2)
221. Acridine orange stains the nucleic acids of bacteria.
222. Fungi cannot be recovered by using swab. (Example of suitable recovery for fungi =
biopsy, CSF, aspirate)
223. The color of AFO (Acid Fast Organism) is red.
224. The color of NAFO (Non-Acid Fast Organism) is blue. (Also green if malachite
green is used as the counter-stain)
225. The potassium tellurite color to C. diptheriae is gray black.
226. Corynebacterium amycolatum is a common component of the natural flora found on
human skin and mucous membrane, and as such, is often disregarded by physician as a
contaminant when found in blood cultures. However, C. amycolatum is actually an
opportunistic pathogen capable of causing serious human disease such as endocarditis
and sepsis. (Pleomorphic gram-positive rods with single-cells, V forms, or Chinese
letters)
227. The preferred concentration technique for parasite ova and cyst is formalin-
ethyl-acetate sedimentation.
228. Staphylococcus saprophyticus is resistant to 5 µg novobiocin.
229. Staphylococcus epidermidis is susceptible to 5 µg novobiocin.
230. In the β-lactamase chromogenic cephalosporin method, color change indicates a
positive reaction.
231. DNase positive bacteria: S-M-S (Staphylococcus aureus, Moraxella, Serratia)

232. 0.02-0.04 units of bacitracin (Taxo A) is used for the antibiotic susceptibility
testing of group A beta-hemolytic streptococci.
233. 5 µg of optochin (Taxo P) is used for the antibiotic susceptibility testing of
Streptococcus pneumoniae.
234. Species from the genus Enterococcus are bile esculin positive.
235. A positive Quellung reaction indicates capsular swelling due to an antigen-
antibody reaction.
236. Serratia strains are readily differentiated from Klebsiella on the basis of their:
failure to produce gas from inositol, slowness and reluctance to ferment lactose,
rapid gelatin liquefaction.
237. Cultures of Staphylococcus supplies V factor for culture of Haemophilus.
238. String test is used for the diagnosis of Vibrio cholerae.
239. Corynebacterium ulcerans has the same morphology as Corynebacterium diptheriae
on blood agar plate (BAP).
240. Mycobacterium tuberculosis is best differentiated from Mycobacterium bovis by
niacin test and nitrate reduction test. (MTB – both positive; M. bovis – both negative)
241. Woolsorter’s disease is caused by the pulmonary form of anthrax.
242. Black-Eschar is caused by the cutaneous form of anthrax.
243. In water bacteriology, the following are used as confirmatory test media: Endo
agar, Eosin-methylene blue agar, Brilliant green lactose broth
244. In taxonomic naming of microorganisms: (1) Uses binomial nomenclature in
identifying organisms (2) Genus name can be abbreviated using the first letter (3)
Uses Greek names or words.
245. 35±2 is the preferred incubation temperature (in degree Celsius) for
bacteriological culture.
246. Most pathogenic bacteria grows best in moist environment.
247. Streptococcus pyogenes is the most common pathogenic isolate from the throat.
248. Streptococcus agalactiae is catalase and PYR hydrolysis negative, bacitracin
resistant and CAMP positive.
249. Staphylococcus epidermidis is the most common agent in nosocomial UTI.
250. Pseudomonas, Aeromonas and Vibrio are example of organisms which are oxidase
positive.
251. Salmonella species show colorless colonies without black center in Eosin-
Methylene Blue agar.
252. Bacillus anthracis show flat with irregular, serrated edges and swirling
phenomenon (medusa head colonies) in culture media.
253. Generally, virologists categorize viruses according to the type of genome, then
according to virus families.
254. Serum tube test (Germ tube test) is used for diagnosis of Candida albicans.
255. Schaudinn’s method is used for detecting oocyst.
256. Acanthamoeba castellani exhibits double-walled cysts with an outer wrinkled wall
and an inner polygonal wall.
257. Taenia saginata is the beef tapeworm.
258. Taenia solium is the pork tapeworm.
259. Leishmania donovani is the causative agent of Visceral Leishmaniasis (Kala-azar or
Dum-dum fever).
260. Leishmania tropica is the causative agent of Cutaneous Leishmaniasis (Cutaneous
ulcer, Oriental sore, Classical self-limited ulcer, Jericho boil, Aleppo boil or Delhi boil).
261. Leishmania braziliensis is the causative agent of Mucocutaneous Leishmaniasis
(Espundia or Uta).

262. Thick and thin blood smear is the gold standard method, as well as the DOH
recommended method, to detect and identify malarial species.
263. Haemophilus parainfluenzae is a normal flora of the upper respiratory tract,
medium to large smooth and translucent colonies on chocolate agar, non-hemolytic on
blood agar, requires V factor.
254. Growth around the V disk, no growth around the X disk, and light growth around
the XV disks mean V factor requirement.
255. The intracellular replicative form of the Chlamydia spp. is termed as reticulate
body.
256. An enhanced arrowhead hemolysis at the juncture of a culture and a streak of
beta-lysin-producing strain of Staphylococcus aureaus on a sheep blood agar means the
beta-lysin of Staphylococcus aureus acted synergistically with the culture enhancing
the hemolysis.
257. In the Harada Mori technique, the following take place: Strongyloides filariform
larva float, Hookworm filariform larva settle at the bottom part.
258. Toxoplasma is an important cause of chorioretinitis.
259. Trichuris trichura infection provides a good site for Entamoeba histolytica.
260. Rhizopus is the most common fungal contaminant of commercially prepared food.
261. Nitrite and urease test are the tests used to differentiate Bordetella
bronchiseptica and Alkaligenes faecalis.
262. Staphylococcus aureus is Coagulase (+), PYRase (-).
263. Inactivated catalyst is the most common failure concerning gas pak jar.
264. The following are K/A in TSI: Plesiomonas, Salmonella, Proteus
265. Use 0.1% fuchshin as a replacement for safranin to better visualize Legionella.
266. Ethanol shock test is used to differentiate Clostridium and Bacteroides spp.
267. CSF should be dealt or prioritized first if different specimens (vs. urine, sputum,
wound exudate) arrived in the laboratory at the same time.
268. Neisseria elongata does not ferment glucose and produces gray, translucent,
smooth, glistening colonies and may have dry and clay-like colonies in blood agar.
269. Streptomyces is a filamentous bacteria that grows on Tap Water Agar.
270. No leukocyte but bloody diarrhea: H2S (-), indole (+), motility (+).
271. Four quadrants are streak when using stool specimens.
272. FITC showed no apple green fluorescence: negative.
273. FITC showed faint yet unequivocal apple-green fluorescence: 1+.
274. FITC showed apple-green fluorescence: 2+.
275. FITC showed bright apple-green fluorescence: 3+.
276. FITC showed brilliant apple-green fluorescence: 4+.
277. A dehydrated 25-year-old male patient was admitted to the hospital with
symptoms similar to those of chronic fatigue syndrome. Serological testing proved
negative for recent streptococcal infection, Epstein-Barr virus, and hepatitis. CMV
serological tests should help with a possible diagnosis.
278. Wrap the sample with cheese cloth and submerge it in a funnel filled with water ---
Baermann funnel.
279. Cercaria goes out of snail to go to water.
280. Dracunculus medinensis has a characteristic thick cuticle and a large uterus that
fills the cavity and contains rhabditoid larvae.
281. Mycobacterium bovis is not a non-tuberculoid Mycobacteria.
282. Taenia solium and Diphyllobothrium latum has a lifespan in a human intestine of >25
years.
283. Stool must be transported in the laboratory in room temperature within 1 hour.

284. Hepatitis A vaccine is a best example of inactivated vaccine.
285. Inactivated vaccines: HAV, HBV (recombinant DNA vaccine), influenza, rabies.
286. Live attenuated vaccines: yellow fever, measles, mumps, rubella, smallpox, chicken
pox, rotavirus.
287. Both inactivated and live attenuated: Poliomyelitis (salk and sabin).
288. Humans serves as definitive host to Taenia spp.
289. Clostridium perfringens: lecithinase (+), lipase (-).
290. Capnocytophaga have colonies that are small to medium size, opaque, shiny; non-
hemolytic; pale beige or yellowish color may not be apparent unless growth is scraped
from the surface with a cotton swab; gliding motilityu may be observed as outgrowths
from the colonies or as a haze on the surface of the agar.
291. Streptococcus agalactiae, Listeria monocytogenes, Campylobacter jejuni and
Gardenerella vaginalis can be identified by hippurate hydrolysis test.
292. Vibrio has potential therapeutic resistance against trimethoprim.
293. Hippurate hydrolysis test can be used to differentiate Listeria (+) from
Corynebacterium (-).
294. Sputum cannot transmit Hepatitis B.
295. Modified acid fast stain in Cyclospora has weaker decolorization from the routine
acid fast.
296. Pentatrichomonas hominis has axostyle with full body undulating membrane.
297. Amplification methods: PCR, Ligase Chain Reaction and Branched Chain
Amplification.
298. Staphylococcus aureus is catalase (+), coagulase (+), PYRase (-).
299. Phenol red is the indicator used in XLD.
300. EHEC (Enterohemorrhagic Escherichia coli) is the strain of E. coli associated with
Shiga toxin.
301. M protein is the virulence factor acts with factor H.
302. According to the Kirby-Bauer standard antimicrobial susceptibility testing method,
swarming area should be ignored when interpreting the zone size of a motile, swarming
organism such as a Proteus species.
303. Entamoeba coli exhibits sluggish, non-directional motility and often described as
having a “dirty cytoplasm”.
304. Double-zone of beta hemolysis is the characteristic hemolytic pattern of
Clostridium perfringens.
305. Naegleria fowleri is a free-living ameba which enters mucosal membranes and
causes Primary Amebic Meningoencephalitis.
306. Adult Paragonimus westermani resides on lungs.
307. Giardia lamblia causes crypts in duodenum and subsequently causes diarrhea.
308. Gram stain of a smear taken from the periodontal pockets of a 30-year-old man
with poor dental hygiene showed sulfur granules containing gram-positive rods.
Actinomyces israelli is present.
309. Dermatophytes can be demonstrated using skin scrapings.
310. Ingestion of spores in food or liquid can be responsible for botulism in infants
caused by Clostridium botulinum.
311. Demonstration of spores of aerial mycelium can be done using cellophane tape
method.
312. Hymenolepis nana eggs are characterized by the presence of a thin shell enclosing
an embryo (oncosphere) with six hooklets contained within two layers of membrane.
313. SAF (Sodium Acetate Formalin) is an environment-friendly stool preservative that
is useful for amebic trophozoites.

314. Laboratory aide is the least responsible in specimen collection.
315. Lag phase (period of rejuvinescence) is the phase in the bacterial growth curve
that denotes the increase in size of the bacterial cell BUT not the increase in number.
316. Calcium alginate swabs are acceptable for Neisseria spp. (never for viruses).
317. McCoy cells are used to culture Chlamydia trachomatis.
318. Boiling is a sterilization technique that is non-sporicidal.
319. The following organisms will stain purple using Gram’s stain: Moraxella catarrhalis,
Mycobacterium smegmatis, Listeria monocytogenes.
320. The following tests produce a blue color as a positive result: Citrate test,
Acetamide test, Malonate test, Acetate test.
321. 0.5 McFarland standard is composed of 1.175% barium chloride and 1% sulfuric
acid.
322. A purulent discharge from a necrotic arm wound plated in BAP. Growth of yellow,
creamy pinhead colonies were observed. Biochemical tests yielded the following results:
catalase (+), coagulase (+), novobiocin (S), PYRase (-). The bacterial species in question is
Staphylococcus aureus.
323. Alpha, beta and gamma hemolytic patterns are the bases of the classification
scheme of streptococci developed by Smith and Brown.
324. Bile esculin hydrolysis test can differentiate Group D enterococci from Group A
streptococci.
325. A Neisseria spp. that ferments sucrose in addition to maltose and glucose is
identified as Neisseria secca.
326. A medical technologist observed 1-10 AFB/50 fields. The appropriate grading is
2+.
327. The causative agent of “flat sour spoilage” is Bacillus stearothermophilus.
328. Actinomyces israelli is also known as the “ray fungus”, causing lumpy jaw. The most
characteristic finding in its disease manifestation is the multiple draining sinus tracts
containing characterisitic granules. Colonies are described to be “molar tooth” in culture
medium.
329. Voges-Proskauer test is based on the principle of determination of glucose
fermentation using the butylene glycol pathway.
330. Salmonella enteritidis is the leading cause of diarrhea in the Philippines.
331. Growth on BAP showed gray, spreading, serrated, mucoid, beta-hemolytic colonies.
It was able to grow in 37 and 42 degree Celsius and showed bluish-green colonies on
MHA. The bacteria being described is Pseudomonas aeruginosa.
332. Haemophilus aphrophilus does NOT require X and V factors for growth.
333. Brucella abortus is also known as “Bang’s Bacillus”, this bacteria requires carbon
dioxide for growth. It can grow in the presence of thionine but not in the presence of
basic fuchsin. It causes abortion in cattles.
334. Coxiella burnetti is the only Rickettsiae that is NOT associated with an arthropod
vector transmission.
335. Arthrospore is an asexual fungal spore results from the simple fragmentation of
the mycelium.
336. Czapek’s agar is used to cultivate Aspergillus spp. and Nocardia asteroids.
337. Casein medium is used to cultivate Nocardia spp.
338. Urea agar is used to cultivate T. mentagrophyte and C. neoformans.
339. Cotton seed agar is used to cultivate B. dermatitidis.
340. A patient is suffering from abnormal pigmentation of the skin in his elbow.
Examination with KOH showed intertwining budding yeast cells and hyphae that
resembled “spaghetti and meatballs”. Malassezia furfur is the fungi being described.

341. Balloon-shaped microconidia is a characteristic of Trichophyton tonsurans.
342. 1:8 is the titer for latex agglutination test that would signify an infection with C.
albicans.
343. Virion is a complete viral particle consisting of a nucleic acid genome and a
protective protein coat, regardless of the presence of a lipid-derived envelope.
344. Viroid is an incomplete defective virus (ex. HDV).
345. Poxvirus is the only DNA virus that can also be classified as an RNA virus due to
its complex capsid symmetry.
346. Dorsal root ganglion is the site of latency of the Varicella Zoster Virus (VZV).
347. Direct fluorescence antibody test is the gold standard in the detection of rabies.
348. Coprophilic is a parasite that is able to multiply in fecal matter outside the human
body.
349. Paratenic host is a type of host wherein the parasite does not develop into the
final stage and instead, undergo and arrested stage of development.
350. Rhabditiform larva (L1) is the first stage larva of the nematodes wherein the
buccal cavity is prominent as signified by its active feeding stage.
351. Decorticated egg is an Ascaris egg lacking its outer mammillated albuminous
coating.
352. Hymenolepis nana is the scientific name of the “dwarf tapeworm”.
353. Schistosoma japonicum has a non-operculated egg with small lateral spine.
354. Balantidium coli is the only pathogenic ciliate of man.
355. Hypnozoites may be seen in malarial infections with P. vivax and P. ovale.
356. “Head louse” is the common name associated with Pediculus humanus capitis.
357. The following are rapid grower non-tuberculoid Mycobacteria: Mycobacterium
fortuitum, Mycobacterium chelonei, Mycobacterium phlei, Mycobacterium smegmatis.
358. Skin, hair and nails are dermatophytic specimens.
359. Bacteria are unicellular organisms.
360. Vibrio cholerae is string test positive.
361. Neisseria, Moraxella, Aeromonas, Vibrio and Pseudomonas are oxidase positive.
362. All members of Enterobacteriaceae are oxidase negative EXCEPT Vibrio (oxidase
positive).
363. Bed bug (surot) is a nocturnal ectoparasite.
364. Ancylostoma duodenale (hookworm) may be associated with vertical transmission
and congenital infections; Eosinophilia peaks in approximately 1 month in gastrointestinal
phase.
365. The following are part of the analytical procedure (phase): (1) selecting what kind
of culture media to be used (2) examining the microorganism in the microscope (3)
specimen inoculation.
366. Analysis of the test result is part of the post-analytical phase.
367. Heterophyes heterophyes, Opisthorchis viverrini, Opisthorchis felineus,
Chlonorchis sinensis (HOC) can be acquired through ingestion of undercooked fish.
368. All trematodes that starts with letter “F” (Fasciola hepatica, Fasciola gigantica,
Fasciolopsis buski) can be acquired through ingestion of aquatic plants.
369. Paragonimus westermani can be acquired through ingestion of freshwater crabs or
crayfishes.
370. Echinostoma ilocanum can be acquired through ingestion of snails.
371. The only schistosome with separate sexes is Schistisoma japonicum.
372. The parasite eradicated by ingestion of alcohol is Taenia saginata.
373. Brugia malayi causes upper elephantiasis.
374. Wuchereria bancrofti causes lower elephantiasis.

375. The vector of Leishmania spp. is sandfly (Phlebotomus spp. and Lutzomyia).
376. The unholy three composes of Hookworms, Ascaris lumbricoides and Trichuris
trichuria. (HAT)
377. The soil-transmitted nematodes are Hookworms, Ascaris lumbricoides, Trichuris
trichuria and Strongyloides stercoralis. (HATS).
378. The anticoagulant for blood culture is SPS (Sodium Polyanethol Sulfonate).
379. Taenia asiatica is the third Taenia spp.
380. Slides and photographs serves as quality control in a parasitology laboratory.
381. The mode of transmission of hookworms and Strongyloides stercoralis is skin
penetration.
382. The larva of Ascaris lumbricoides can be found in the sputum.
383. Smears of CSF are prepared from CSF sediment.
384. In a strict sense, Mycoplasma and Ureaplasma are not considered as true bacteria
because they have no cell wall.
385. Acridine orange stains nucleic acids.
386. The best time to collect blood is before the fever spikes.
387. Taenia solium has 7 to 15 uterine branches (finger-like or dendritic appearance).
388. Taenia saginata has 15 to 20 uterine branches (tree-like or dichotomous
appearance).
389. Ascaris lumbricoides is the causative agent of Loeffler’s syndrome and causes
peripheral eosinophilia and appendicitis.
390. Strobila is the scientific name of the body of the tapeworm.
391. Scolex is the scientific name of the head of the tapeworm.
392. Proglottid is the scientific name of the segment of the tapeworm.
393. Blastocystis hominis is often mistaken for cyst of amoeba.
394. The vector of African sleeping sickness is tse tse fly (Glossina spp.).
395. The following migrates through blood, liver, lungs, pharynx and then back to the
small intestine (heart to lung larval migration): ASH (Ascaris lumbricoides,
Strongyloides stercoralis, Hookworm).
396. Venipuncture is NOT recommended for malaria, Babesia, hemoflagellates.
397. India ink (Nigrossin stain) is the stain used to demonstrate uterine arrangement
of Taenia spp. proglottids.
398. Settings of RPM marked on the face of the rheostat control on the centrifuge
should be checked once monthly.
399. The (mechanical) vector for Ascaris lumbricoides is cockroach (Periplaneta
americana).
400. Snail is the first intermediate host of flukes.
401. IL-6 increases during bacterial infection.
402. Infective stage of schistosomes to humans is cercaria.
403. E. coli in MRVP reaction: red
404. The common name of Chlonorchis sinensis is Chinese or Oriental Liver Fluke.
405. The first intermediate host of Diphyllobothrium latum is snail.
406. Entamoeba coli has 1 to 8 nuclei (cyst).
407. Chlamydia, Mycoplasma, Rickettsia and the rest of bacteria have both DNA and
RNA.
408. Acanthamoeba castellani has double-walled wrinkled cyst form.
409. Amastigote (leishmanial form) is the intracellular form of blood and tissue
flagellates.
410. Promastigote (leptomonad form) is the culturable and the infective stage of
Leishmania spp.

411. The infection caused by ectoparasites are called as infestation.
412. Endoparasites are living within the body of the host (infection).
413. Ancylostoma duodenale is an intestinal nematode capable of vertical transmission
and potential cause for congenital infections.
414. Lactobacillus is the predominant microorganism that causes bacon to spoil despite
being stored in vacuum-packed containers.
415. Before collection of hair, nails or skin scrapings for fungal culture, the area must
be first cleansed with 70% alcohol.
416. After determining a strain of Staphylococcus as coagulase negative, novobiocin
resistant would confirm the identification as S. saprophyticus and novobiocin
susceptible would confirm the identification as S. epidermidis.
417. The adult of Fasciola hepatica possesses a cephalic cone and a well-developed
(prominent) shoulders.
418. Streptomyces is characterized by extensive branching aerial hyphae in tap water
agar.
419. Red is the color produced by acid fast bacilli after staining.
420. Blue or green is the color produced by non-acid fast bacilli after staining.
421. ELISA is considered the most common and earliest serological test to detect
antibodies to VZV.
422. M. tuberculosis is best differentiated from M. bovis by niacin and nitrate reduction
tests.
423. The growth rate of photochromogens, scotochromogens and non-photochromogens
is 10-21 days while rapid growers is 3-7 days.
424. Wuchereria bancrofti is considered the most commonly identified species of
filarial worms that infect humans, characterized by a sheath and nocturnal periodicity.
425. Ignaz Semmelweis first demonstrated that handwashing can prevent the spread
of diseases.
426. Negative staining (indirect/relief staining) is utilized to demonstrate the presence
of diffuse capsule surrounding the bacteria. Best example is india ink.
427. In log/exponential phase, microorganisms are most susceptible to antibiotics.
428. The aseptic collection of blood cultures requires that the skin be cleansed with
70% alcohol and then 2% iodine or an iodophore.
429. Opthalmia neonatorum is caused by N. gonorrhoeae as the newborn passes the birth
cannal. AgNO3 drops (Crede’s prophylaxis) were once used. Nowadays, erythromycin is
more commonly used for treatment.
430. Sodium polyanethol sulfonate (SPS) may be used as an anticoagulant in blood
cultures because it (1) prevents phagocytosis and (2) neutralizes the bactericidal
effect of human serum (complement system).
431. Cefsulodin-Irgasan-Novobiocin (CIN) agar is best suited for the recovery of
Yersinia enterocolitica from a patient with gastroenteritis.
432. When testing aminoglycosides and tetracyclines against P. aeruginosa, elevated
concentrations of divalent cations can result to diminished activity of the antibiotics.
433. Dick’s test is used in infection associated with Group A Streptococcus wherein the
positive result is redness of the skin.
434. In the carbohydrate utilization test using CTA medium, Moraxella catarrhalis fails
to produce acid from glucose, maltose, lactose and sucrose. It is catalase, oxidase and
DNase positive.
435. An isolate of E. coli recovered from the stool of a patient with severe bloody
diarrhea should be tested for sorbitol before sending it to a reference laboratory for
serotyping.

436. ONPG test identifies late lactose fermenters.
437. 0.5% sodium desoxycholate is used in string test which differentiates Vibrio (+)
from Aeromonas (-).
438. Haemophilus ducreyi is the only species of Haemophilus which does not require V
factor.
439. In order for a sputum specimen to be acceptable for culture, <10 epithelial
cells/HPF and >25 pus cells/HPF must be seen.
440. The Fite-Faraco acid-fast stain is different from other acid-fast stains because it
uses hematoxylin rather than methylene blue as a counterstain.
441. Stormy fermentation is a characteristic growth pattern of C. perfringens in litmus
milk broth.
442. Coxiella burnetti is associated with Q fever and can be transmitted via inhalation
of aerosols from infected animals.
443. Mycoplasma pneumoniae causes PAP (Primary Atypical Pneumonia) or walking
pneumonia.
444. Phenol oxidase is the yeast enzyme detected using birdseed (niger seed) agar.
445. The mycelia form of Coccidioides immitis (dimorphic mold) produces thick-walled,
rectangular or barrel shaped alternate arthroconidia.
446. Coxsackie B virus is the most common viral syndrome of pericarditis, myocarditis
and pleurodynia (pain upon breathing).
447. When a delay in processing cannot be avoided, specimens for viral isolation must be
held at 4◦C and snap-frozen to -70◦C.
448. The following are correctly matched: (1) Onchocerca volvulus – examination of skin
snips, (2) Cryptosporidium – modified acid-fast stain, (3) Schistosoma haematobium –
examination of urine sediment.
449. The following are considered STAT procedures in diagnostic parasitology: (1) CNS
specimens for examination of free-living amoebae, (2) blood films in a potential
malaria case.
450. The specific gravity of the solution used in the zinc sulfate flotation method is
1.18.
451. An Entamoeba histolytica trophozoite has central karyosome, ingested RBC and
clear pseudopodia.
452. Ventral sucking disk is pathognomonic for Giardia lamblia trophozoite.
453. Undulating membrane is pathognomonic for Trichomonas spp.
454. The decolorizer is weaker in modified acid-fast staining than the acid alcohol in
regular AFB staining.
455. Autofluorescence requires no stain and is recommended for the identification of
Cyclospora cayetanensis oocyst.
456. Eye infections with Acanthamoeba spp. have been implicated in use of
contaminated lens care solutions.
457. Primary Amoebic Meningoencephalitis (PAM) is commonly caused by Naegleria
fowleri.
458. Granulomatous Amoebic Encephalitis (GAE) is commonly caused by Acanthamoeba
castellani.
459. Human Eosinophilic Meningoencephalitis (HEM) is a form of larva migrans
associated with Angistrongylus cantonensis.
460. Amastigote is the hemoflagellate form that can be found in humans in cases of
Leishmaniasis (diagnostic stage).
461. Promastigote is the infective stage of Leishmania spp.
462. Trypomastigote is both diagnostic and infective stage of Trypanosoma spp.

463. Balantidium coli possesses the characteristic “thrown ball” motility.
464. Plasmodium falciparum is capable of exhibiting multiple ring forms and
marginal/applique forms.
465. Plasmodium vivax and Plasmodium ovale are similar because they exhibit
Schuffner’s dots and have a true relapse in the life cycle.
466. The positive result in the Sabin-Feldman dye test is when Toxoplasma loses its
affinity for the methylene blue dye (colorless).
467. The rhabditiform larvae of Strongyloides stercoralis have short buccal cavity and
large (prominent) genital primordium.
468. The rhabditiform larvae of hookworms have long buccal cavity and small genital
primordium.
469. Trichinella spiralis adults are found in small intestine.
470. Trichinella spiralis encysted larva are found in striated muscle.
471. The following are correctly matched: (1) Brugia malayi – nocturnal periodicity,
sheathed; (2) Loa loa – diurnal periodicity, sheathed; (3) Onchocerca volvulus – no
periodicity, unsheathed.
472. Humans acquire infections with Diphyllobothrium latum adult worms by ingestion of
raw freshwater fish.
473. Hymenolepis diminuta is also known as the “rat tapeworm”.
474. Schistosomule is a forked-tailed cercaria minus the tail.
475. Charcot-Leyden crystals in stool may be associated with an immune response and
are thought to be breakdown products of eosinophils.
476. Plerocercoid (sparganum) is the infective stage of the broad fish tapeworm to
humans.
477. The ideal temperature at which to hold fecal specimens for more than 1 hour is
refrigerator temperature.
478. Tuberculin skin test is the screening test for TB recommended by DOTS.
479. Zinc sulfate concentration may NOT allow detection of Infertile ova of
Ascaris lumbricoides.
480. A trematode with male and female adult forms is Schistosoma spp.
481. Dientamoeba fragilis is the protozoa that should be reported to the
clinicians.
482. Nontuberculous colonies that develop pigment in the dark or light, and
take longer than 7 days to appear on solid media are known as scotochromogens.
492. An organism that is (+) in niacin, nitrate reduction, appears to have a
serpentine cording on culture and grows in Tween 80 Hydrolysis after 5 days is
suggestive of M. tuberculosis.
493. Staphylococcus aureus can be used as positive control in Gram staining.
494. E. coli is used for the quality control of positive results in Kovac method
for indole production.
495. Entero test is used to diagnose Giardia lamblia infection from duodenal
specimens.
496. Negative blood smear for malaria is NOT considered a panic value in a
Microbiology laboratory.
497. A lesion from the buccal mucosa of a patient with C. albicans in exudate
shows blastoconidia and chlamydospores.
498. Mineral oil interferes with stool sample testing.
499. A. lumbricoides egg requires a warm humid environment in order for the
embryonated ovum to mature and become infective and is transmitted through
oral fecal route.

500. Nucleic acid amplification test for detection of suspicious Bordetella,
Chlamydia or Neisseria infections require a sample of DNA.
501. An emulsifying agent that stains nuclei of parasites and other structures
to make them easily visible is lugol’s iodine.
502. Anaerobic bacteria that has no definitive virulence factors known and is
associated with inflammatory process in acne is Propionibacterium.
503. The gold standard serologic test for rickettsioses is indirect
immunofluorescent antibody assay.
504. The following are true of virions: (1) contains RNA, (2) reproduce
independently, (3) contains DNA.
505. The order of events should be followed at the conclusions of the shift of
a laboratory worker to prevent the spread of blood borne pathogens: disinfect
area, remove gloves, wash hands remove lab coat.
506. Contact with feline feces in cat litter is a primary source of Toxoplasma.
507. The mode of transmission of Strongyloides stercoralis is direct skin
penetration.
508. Actinobacillus can cause granulomatous disease in animals.
509. In the life cycle of Paragonimus, migration of metacercariae (also the
infective stage) through muscle and tissue causes local pain and immune response
to tissue damage.
510. Impairment of blood supply can make anaerobic indigenous flora become
pathogenic.
511. A single-stranded RNA genome having a helical capsid with envelope is a
characteristic of Filovirus.
512. Test tube dilution / broth dilution method of susceptibility testing
involves subjecting the isolate to a series of concentrations of antimicrobial
agents in a broth environment.
513. Aerobic bacteria can use oxygen as terminal electron acceptor in
respiration.
514. An organism that causes chancroid and which appears as small gram
negative coccobacilli is H. ducreyi.
515. To know if an N95 mask works properly, it must be snug fit.
516. Fastidious organism demand special and enriched nutritional environment.
517. Yersinia pestis is MOST susceptible to changes in temperature.
518. Desiccator preserve moisture-sensitive items.
519. The required preventive maintenance of centrifuges is to check rpm
every 6 months.
520. In thioglycollate broth medium, obligate aerobe can be found at the top
of the tube.
521. Elongated embryonated, operculated ova with terminal spine seen in a
urine specimen is indicative of Schistomosa haematobium.
522. Streptococcus pyogenes is the medically important streptococci that is
characterized by beta-hemolysis, bacitracin sensitive and PYR test positive.
523. The appropriate action if the diameters of the zones of inhibition are too
large or too small with all the antimicrobials being tested: Ensure that plates
are incubated immediately after application of discs.
524. Blood agar is made of nutrients and 5% sheep blood.
525. A device used to incubate, agitate and monitor blood culture bottles for
microbial growth is a blood culture system.

526. An organism appearing as small gram negative coccobacilli that causes
chancroid is H. ducreyi.
527. Onchocerca volvulus is transmitted by a black fly by laying its eggs in
running water and where the larvae attach to the rocks.
528. Considered Personal Protective Equipments: (1) HEPA filter, (2) gloves, (3)
laboratory coat.
529. Sputum sample from a female teenager with cystic fibrosis and diagnosed
with pneumonia shows gram negative bacilli with yellow, smooth columbium.
Further testing gave the following results: oxidase (+), TSI alkaline/alkaline,
glucose oxidized, fluorescence (-), lysine decarboxylase (+). Burkholderia cepacia
is the most likely organism involved.
530. Taenia is the type of tapeworm that is characterized by pollen grain like
appearance of the proglottids.
531. The following are example of strategies in Parasitology Quality Assurance:
(1) set up positive and negative controls, (2) ensure adequate training of all
laboratory staff, (3) standardized reporting of concentration of parasites.
532. Positive cultures of yeast and filamentous fungi are reported with the
organism identification.
533. Hypersensitivity reaction is the cause of Loeffler’s syndrome.
534. Types of growth that an agar slant produce: filiform (straight),
arborescent (treelike), beaded, echinulate, diffuse (spreading), rhizoid.
535. Animals that harbor parasites and serve as an important source of
infection are known as reservoir host.
536. On BA plate, a non lactose fermenter that shows round, smooth, convex,
colonies, appearing as black or very dark purple and has ammonium cyanide odor is
suggestive of Chromobacterium violaceum.
537. Amniotic, peritoneal, synovial, pericardial fluids should be transported to
the laboratory within 30 minutes.
538. A non-lactose fermenter, methyl red positive, motile negative, mannitol
negative, is indicative of S. dysenteriae.
539. Skin scrapings can be transported by itself for virus isolation.
540. Tapeworms anchor themselves to the inside of the intestine of their host
using their scolex.
541. Cutaneous, visceral and mucocutaneous are the forms of Leishmaniasis.
542. Refrigeration of stool specimen is not recommended when amoebic
trophozoites are suspected.
543. Rickettsioses and other related diseases have a/an arthropod vector,
except Coxiella burnetti.
544. S. intercalatum egg resembles S. haematobium and acid fast positive
using the Ziehl-Neelsen test.
545. Automation in a public health laboratory where numerous specimens are
submitted is advantageous to increase productivity.
546. The relationship of the zone of inhibition to the MIC of the test
bacterium is inversely proportional.
547. The following are general guidelines in specimen collection for viral
studies: (1) adequate volume, (2) sterile, chemically clean containers, (3) correct
site.
548. When a laboratory can document a satisfactory performance of daily disk
diffusion susceptibility QC, the frequency of performing this activity can be
reduced to every week.

549. ECM (Erythema chronicum migrans) is the stage of Lyme Disease
infection that produces a characteristic red, ring-shaped skin lesion with a
central clearing that first appears at the site of tick bite.
550. Thayer Martin agar plate is utilized for N. gonorrhoeae and N.
meningitidis cultures.
551. A 10 y/o female was admitted to the emergency room with 2 days of
severe diarrhea after eating cheeseburger sandwich from a local restaurant.
Cultures from three consecutive stool samples contain blood and mucus. Growth
on XLD agar has yellow colonies, HE agar grew yellow colonies, Mac agar has light
pink to dark pink colonies, and MAC with sorbitol agar grew dark pink and many
colorless colonies. The most likely organism identified is Escherichia coli.
552. High concentration of peptone is added to sabouraud dextrose agar
thereby rendering it selective for fungi.
553. A pseudophyllidean cestode is characterized by operculated eggs.
554. The normal stool is brown due to bile pigments.
555. Modified trichome stains are used primarily to demonstrate
microsporidian spores.
556. S. agalactiae is the type of Beta hemolytic streptococci show medium-
sized colony on BAP, hydrolyzes hippurate giving an arrow head streak
intersecting with S. aureus.
557. Citrobacter has the following biochemical reactions: H2S (+), urea (-), LDC
(-).
558. Minimal Inhibitory Concentration gives the most reliable result compared
to gram staining, biochemical tests and 10% KOH preparation.
559. The most commonly used digestion-decontamination reagent for
Mycobacterium suspected organism is 2-4% NaOH.
560. Specimen transport is considered a step during the pre-analytic phase.
561. Escherichia coli can produce an antiphagocytic capsule and has the ability
to adapt for intracellular survival.
562. Scabies is an infestation.
563. To proceed with specimen handling when there is delay in the transport of
specimen, store at 4 degree Celsius for no longer than 24 hours.
564. Bacteria that thrive in extreme heat are called thermophiles.
565. Most bacteria causing food spoilage grow at alkaline pH.
566. The following are characteristics of Enterobacteriaceae: (1) oxidase (-),
(2) able to reduce nitrate to nitrate, (3) all ferment glucose.
567. Test processing will fall within an analytical variable.
568. Testing for antibiotic sensitivity is often done by the Kirby-Bauer
method.
569. Rodents are the intermediate hosts of Echinococcus multilocularis where
the parasite is occasionally transmitted to humans through ingestion of
contaminated food or water.
570. Serologic profile concurs with an individual with chronic infection: HBsAg
(+), IgG anti-HBc (+), IgM anti-HBc (-).
571. Serologic profile concurs with an individual with acute infection: HBsAg
(+), IgG anti-HBc (-), IgM anti-HBc (+).
572. Clostridium perfringens will BEST be identified using the following
laboratory tests: gram stain examination, reverse CAMP test, lecithinase,
hemolysis.

573. The following are considered effective methods of communicating
information to hospital staff: (1) presentation at medical rounds, (2) laboratory
news letter, (3) ward manual prepared by laboratory staff.
574. To isolate Salmonella and Shigella species from other gram negative
enteric bacilli, the medium used is Xylose Lysine Desoxycholate (XLD) agar.
575. There is a need to allow sterilized material to stand for about 10 minutes
in the autoclave chamber before unloading, to allow trapped air to escape from
hot liquids.
576. Spirochetes can be visualized with silver impregnation method.
577. BAP and CAP are considered Nutritive Media.
578. Chlonorchis sinensis is a liver fluke.
579. Wandering larvae in the skin is caused by a hookworm.
580. Infection of this parasite occurs via ingestion of poorly cooked
freshwater fish containing the plerocercoid larval form. The unembryonated
operculated eggs with terminal knob is similar to trematode eggs. The cestode
described is D. latum.
581. Streptococcus agalactiae is a beta-hemolytic Streptococci that gives a
positive hippurate test.
582. The adult female has a distinctive morphology with spiral, winding, “barber
pole” appearing uterus is a characterized of Angiostrongylus cantonensis.
583. The gold standard for laboratory confirmation of malaria is examination
of a Giemsa-stained blood smear.
584. Pour plate method is performed when you need to separate organisms
from a mixed culture of a liquid specimen.
585. All of the following organisms share the same morphologic similarities. All
are small, curved, and motile, gram-negative bacilli: (1) Arcobacter, (2)
Helicobacter, (3) Campylobacter.
586. The gold standard for the confirmation of malarial infection is Giemsa
smear.
587. The inexpensive test to determine foodborne bacteria is culture.
588. Donning of PPE: (1) Gown (2) Mask/Respirator (3) Goggles/Faceshield (4)
Gloves
589. Doffing of PPE (1) Gloves (2) Goggles/Faceshield (3) Gown (4)
Mask/Respirator
590. After removing all PPE, you must wash hands or use an alcohol-based
hand sanitizer.
591. The purpose of mordant in Gram staining is to let gram positive to remain
as purple.
592. Heat is the mordant used in Ziehl-Neelsen acid fast stain.
593. Steam (autoclave) is the best method to achieve sterilization.
594. CAMP test is the best test to identify Group B Streptococcus.
595. Bacitracin susceptibility test is the best test to identify Group A
Streptococcus.
596. S. pyogenes is probably being diagnosed if it involves scraping the back of
the throat.
597. C. neoformans is the most common cause of AIDS meningitis with a CD4+
count of <1000.
598. If someone is infected with Typhoid fever, we have to make sure that the
blood culture is done during the febrile episode. This is part of analytical phase.
599. Nuclear shrinkage (pyknosis) is an example of CPE (Cytopathic Effect).

600. The unit of measurement for bacteria is micrometer.
601. What is the unit of measurement for virus is nanometer.
602. The following are the components of the chain of infection: Source, mode
of transmission, susceptible host
603. The number of viral copies found in the blood and other parts of the
body is being detected in HIV Viral Load.
604. The stain for H. pylori in tissue is toluidine blue.
605. Procalcitonin is the marker to differentiate bacterial from non-bacterial
pneumonia.
606. The C in TORCH stands for cytomegalovirus.
607. Solidifying agent (agar) makes the culture media solidify.
608. 35 +/- 2 C is the temperature of the incubator.
609. Dacron or rayon swab is the type of swab used for the diagnosis of virus
infection.
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

Microbiology - Bacte, Myco, Viro

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* NLE * MIDWIFERY * LET * RAD TECH * CRIMINOLOGY * DENTISTRY * PHARMACY

TABLE OF SPECIFICATION FOR MICROBIOLOGY

4. Equipment and Instrumentation 5%

5. Quality Assurance and Safety 8%

BACTERIOLOGY – study of bacteria

MICROBIOLOGY by CUA Page 1

MICROBIOLOGY by CUA Page 2

1. Strain – is a population of organisms that is differentiated from populations within a particular

2. Biovars – are variant prokaryotic strains characterized by biochemical or physiological differences.

3. Morphovars – are variant prokaryotic strains which differ morphologically.

4. Serovars – are strains with distinctive antigenic properties.

I. Cell Envelope – outermost structure

Gram Stain – General Rule

 All cocci are Gram (+), EXCEPT:

MICROBIOLOGY by CUA Page 3

• Chemicals that can destroy the cell wall:

G (+) Cell wall

G (-) cell wall

Comparison of a Gram positive and Gram negative Bacterial Cell Wall

CELL WALL COMPONENT GRAM POSITIVE GRAM NEGATIVE

MICROBIOLOGY by CUA Page 4

Summary of Gram Staining Technique: Reagent/s and Reactions

Summary of Acid Fast Staining Technique: Reagent/s and Reactions

KEY STEPS ACID NON-ACID

MICROBIOLOGY by CUA Page 5

Summary of Modified Kinyoun Method for Partially/weakly Acid-Fast Organisms

3. Plasma / Inner Membrane

a. Ribosomes (70S) – site of protein synthesis

MICROBIOLOGY by CUA Page 6

Types of Flagellar Arrangements in Bacteria

MICROBIOLOGY by CUA Page 7

c. Pili and fimbriae

Bacteria with Endospores:

Bacteria with Capsule:

Bacteria with Pili:

MICROBIOLOGY by CUA Page 8

Characteristics of Prokaryotes Versus Eukaryotes

CRITERIA OF PROKARYOTE EUKARYOTE

5. MEMBRANE BOUND ABSENT PRESENT

BACTERIAL NUTRITION AND GROWTH

MICROBIOLOGY by CUA Page 9

c. Microaerophilic – organisms that requires 2-10% oxygen for growth

d. Aerotolerant anaerobic – anaerobe that grows well in the presence of oxygen

Enzymes that inhibits the toxic effect of O2:

2. According to CO2 requirement

3. According to nutritional requirement

*Most of G(+) are chemoheteroorganotrophs.

*Saprophytes – require dead organic substances.

MICROBIOLOGY by CUA Page 10

B. Mesophilic – 20-45οC, most of the pathogenic bacteria grow at this temperature

8. As to high salt concentration requirement

9. As to growth factor requirement

MICROBIOLOGY by CUA Page 11

BACTERIAL GROWTH CURVE

A. Lag phase / Period of rejuvinescence / Adjustment phase

B. Log phase / Exponential phase

C. Plateau / Stationary phase

D. Death / Period of Decline

1. Organization and structure of genetic material

2. Replication and expression of genetic material

MICROBIOLOGY by CUA Page 12

Mutation – a process whereby there is a change / exchange of genes

ANABOLISM – there is a synthesis of complex molecules and energy production