MICROBIOLOGY cells , blood cells, parasites

and fungi
- study of microorganisms too small to be seen by Louis Pasteur Credited for his
unaided eyes development of the
- Microbes: bacteria, cyanobacteria. Rickettssiae, principles of vaccination ,
microbial fermentation and
chlamydia, fungi, microscope algae, protozoa,
pasteurization
viruses
Joseph Lister British surgeon and pioneer
of antiseptic surgery
a) Clinical Microbiology
- Study of microbial pathogens that are Introduced the use of
considered health threats to people carbolic acid as a
- Studies the different etiologic agents chemical sterilizing agent
for surgical instruments
Hans Christian Gram Gram staining technique
b) Diagnostic Microbiology
which is used to distinguish
- Involved in examining and identifying organism 2 major groups of bacteria
through laboratory test Alexander Fleming Discovered the powerful
antibiotic, benzylpenicillin (
c) Food Microbiology Penicillim G) from a mold,
- Studies the practical application and use of Penicillium notatum
beneficial microorganisms in food processing Robert Koch Discovered Bacillus
anthracis, Mycobacterium
tuberculosis and Vibrio
Branches of Microbiology
cholera
a) Protozoology
Popularized Koch’s
― study of protozoans , the simplest and postulates
most primitive forms of animal life
b) Mycology
― study of fungi
c) Bacteriology BACTERIOLOGY
― study of bacteria
d) Rickettsiology - A subdivision of microbiology that deals with the
― study of rickettsiae, which are study of bacteria
considered traditional organisms - unicellular organisms that lack a nuclear
between bacteria and viruses membrane and true nucleus
e) Virology -
― study of virus
EUKARYOTIC VS PROKARYOTIC
Pioneers in the History of Microbiology PROKARYOTE EUKARYOTE

Girolamo Fracastoro suggest that disease was Nuclear Nucleoid Nucleus

caused by “invisible living body
creatures” Cell division Binary fission Mitosis

Gave syphilis its name in Cell wall With Without

16th century
Anton Van Leeuwenhoek Cytoplasmic Fluid Phospholipid
Father of Bacteriology and Membrane Phospholipid Bilayer
Protozoology bilayer
Cell Absent Present
His research opened doors organelles
for the discovery of many
Site of Energy Cytoplasmic Mitochondria
microscopic life forms
Production Membrane
which he regarded as
1
animalcules Site of Free Ribosome Rough ER
protein
Discovered the bacteria, synthesis
free living and parasitic
microscopic protest, sperm

MICROBIOLOGY | VILLACERAN, E.
Taxonomy of Bacteria b. Iodine
c. Alcohol
d. Safranin (Appears RED here: Negative)

ACID FAST STAIN

― Used to stain bacteria that have a high

lipid and wax content in their cell walls
and do not stain well with traditional
bacterial stains

BACTERIAL CELL Methods:

a) Ziehl Nelseen (Hot Method)

Parts include the following:
― Carbol Fucshin
― Cell wall ― Steam
― Cytoplasmic membrane ― Acid Alcohol
― Mesosomes ― Methylene Blue/ Malachite Green
― Inclusions b) Kinyoun (Cold Method)
― Endospore ― Carbol Fuchsin
― Capsule ― Tergitol (Cold Reagent)
― Pili ― Acid Alcohol
― Flagella ― Methylene Blue/ Malachite Green

1. CELL WALL In Acid Fast Bacilli testing, when it appears Red it

― Composed mainly of PEPTIDOGLYCAN indicates positive AFB. When it appears Blue or
Green it indicates negative AFB.
Functions:

- Defines the shape of the

2. CELL MEMBRANE
bacteria
― Lipoprotein layer that surrounds the
- protects internal structure
cytoplasm
- Pathogenicity
― Regulates transport of solutes in and
- Determines gram stain
out of the cell
reaction
3. RIBOSOMES
― Sites of protein synthesis
GRAM POSITIVE CELL WALL GRAM NEGATIVE CELL
WALL 4. NUCLEOID
Thick peptidoglycan layer Thin peptidoglycan layer ― Region where DNA is concentrated
5. CYTOPLASMIC MEMBRANE
Consist of glycan Outer membrane: LPS ― selectively permeable membrane
chains of alternating ― Function: Site of energy production
N-acetyl-D 6. MESOSOMES
Lipid A
glucosamine and N –
― point of attachment for
acetyl-D- Muramic
Acid Core polysaccharides chromosomes
7. INCLUSION
Techoic acid (negative Antigenic O ― for nutrient storage
charged) polysaccharide ― Munch granules
i. Babes-Ernst bodies –
Penicillin acts by Corynebacterium diphtheria
preventing
ii. Much granules –
peptidoclycan
synthesis Mycobacterium tuberculosis
2
iii. Bipolar bodies – Yesinia pestis
Steps in Gram Staining (Reagents):

a. Crystal Violet (Appears here: POSITIVE)

MICROBIOLOGY | VILLACERAN, E.
8. ENDOSPORE Bacteria Cell:
― Resting cell, highly resistant
composed mainly of CALCIUM EXOTOXIN ENDOTOXIN
DIPICOLINATE
Source Gram (+) Gram (-)
― Presence of endospore means the
and gram
bacteria is HIGHLY RESISTANT (will not
(-)
die in autoclaving) e.g. is Clostridium
Release Living bacteria Upon lysis
tetani
9. CAPSULE Composition Proteins Lipid A
― increase virulence by preventing
Heat stability Labile Stable
phagocytosis
― medically important encapsulated Toxicity High Low
bacteria:
Lethal dose Small Large
i. Haemophilus influenza
ii. Neisseria meningitides
iii. Streptococcus pneumoniae
iv. Bacillus anthracis
10. PILI
― Ordinary pili
― Sex pili
― Point of sexual attachment for
replication
11. FLAGELLA

For Motility Types:

― Atrichous
 Absence of Flagella
― Monotrichous
 One Flagellum
― Amphitrichous
 Flagella at each end
― Lophotrichous BACTERIAL GROWTH FACTORS
 Many flagella at one end
― Nutrients
― Peritrichous
― Carbon
 Many flagella all over
― Nitrogen
― Minerals
― Salt

Oxygen and Carbon Dioxide

― Obligate aerobe
 cannot live without oxygen
― Obligate anaerobe
 can live without oxygen
― Facultative anaerobe
12. SPORES
 can live with or without oxygen
― Allows bacteria to resist the sterilization
― Aerotolerant aerobe
process, making them viable over time
 can live without oxygen but can
― Composed of calcium dipicolinate
tolerate presence of oxygen
According to position ― Microaerophile
3
 Oxygen is at 5-10%
i. Terminal ― Capnophile
ii. Subterminal  Oxygen is at 15% and Carbon
iii. Central dioxide is as 5-10%

MICROBIOLOGY | VILLACERAN, E.
Temperature

― Psychrophilic
 Also known as cryophilic
 Temperature at 10 °c
― Mesophilic
 Temperature between 30-37°c
― Thermophilic
 Temperature between 50-55°c

pH
Function and use
― Acidophile (pH 3)
i. Simple:
― Alkaliphile (pH 8-10)
ii. Enriched
iii. Enrichment
iv. Selective
CULTURE MEDIA v. Differential
vi. Selective differential
According to physical state/consistency:

― Liquid
― Semisolid GRAM POSITIVE COCCI
― Solid
a) STAPHYLOCOCCUS
Composition o Gram (+) cocci arranged in tetrads or
clusters
― Synthetic/ chemically defined o Facultative anaerobes
― Complex/ non synthetic o Catalase positive
― Tissue o Oxidase Negative
o Non-motile
Dispensing
o Grows in 7.5-10% NaCl
― Plated
a. Weighing (Agar)
b. Dissolving (Water)
c. Sterilization (Autoclave)
d. Dispensing (Plate)

Staphylococcus aureus

― Tubed ― Most clinically significant species of

a) Weighing Staphylococci
b) Dissolving ― Present in various skin surfaces
c) Dispensing ― Cause of nosocomial or hospital- 4
d) Sterilization acquired infections

MICROBIOLOGY | VILLACERAN, E.
 Virulence factor: giving boils
b) STREPTOCOCCUS
i. Enterotoxin
ii. Toxic Shock Syndrome Toxin 1 o Gram (+) cocci arranged in pairs or
iii. Exfoliative Toxin chains
iv. Cytolytic toxins o Aerotolerant anaerobes
v. Enzyme o Catalase negative
vi. Protein A o Some are capnophilic
o Non-motile
Colonial Characteristics

― Medium to large; round smooth, entire,

Streptococcus pneumoniae
translucent, creamy; mostly pigmented
yellow and Beta hemolytic ― Gram-positive diplococci
― Capnophilic
― Neufeld Quellung positive

Virulence Factor:

― Capsular polysaccharide

Clinical Significance:

― Pneumonia
― Sinusitis
― Otitis Media
CATALASE TEST ― Bacteremia
― Meninigitis
― Ability of organism to breakdown H202
into O2 & H20 Laboratory Diagnosis

MICRODASE TEST ― Small, round, glistening, dome-shaped, mucoid

colonies.
― “Modified oxidase test” ― Colonies tend umbilicated as they age; alpha
― Reagent: hemolytic
Tetramethylparaphenylenediaminedihyd
rochloride with dimethyl sulfoxide GRAM POSITIVE BACILLI

a) Corynebacterium diphtheriae
Blue to Purple = Positive o Pleomorphic Gram (+) Bacilli
No Color Change = Negative o Non motile
o Virulence factor:
o Diphtheria toxin
o Clinical significance:
COAGULASE TEST o Respiratory diphtheria
o Cutaneous diphtheria
― Determines the ability of bacteria to convert
fibrinogen into fibrin.
Colonial Characteristics
― Clot formation
― Cystine- tellurite blood agar and
Types: Tinsdale’s agar
― Black colonies with brown halo
i. Bound coagulase
 Slide Method 5
 Clumping means positive
b) Listeria monocytogenes
ii. Free coagulase
o Motile
 Tube Method
 Formation of Clot is positive
MICROBIOLOGY | VILLACERAN, E.
 Virulence factor: ― MacConkey Positive
― Glucose Fermenters
― Hemolysin ― Reduces Nitrate to Nitrite
― Protein p60 ― All motile at body temperature (37°)

Clinical significance:
a) Escherichia coli
― Stillbirth and spontaneous abortion
Clinical significance:
― Bacteremia
― UTI, Septicimia, CNS and
c) Mycobacterium tuberculosis diarrheal diseases
o Slender, slightly curved rod shaped
microorganism measuring 0.2 – 0.6 µ in Disease syndromes
diameter and 1-4 µ in length
i. Enterotoxigenic E.coli (ETEC)
Clinical significance: a. Montezuma’s Disease
b. Turista’s Disease
― Pulmonary Tuberculosis c. Traveller’s Disease
ii. Enteroinvasive E. coli (EIEC)
Colonial Characteristics
a. Inflammatory Diarrhea (slimy
and with RBC)
― LJ MEDIUM
iii. Enteropathogenic E. coli (EPEC)
― tan to buff in color, rough, dry, warty,
granular “CAULIFLOWER a. Infantile Diarrhea (Mucous
APPEARANCE” without RBC but with WBC)
iv. Enterohemorrhagic E. coli (EHEC)
― 2 months to grow
v. Enteroadherent E. coli (EAEC)

Colonial Characteristics

― EMB(Eosinophil Methylene Blue):

selective differential for G- bacilli
― Greenish Metallic Sheen
GRAM NEGATIVE COCCI Appearance

a) Neisseria gonorrhoeae Klebsiella

o Gram (-) diplococci
o Obligate aerobes ― K. pneumoniae, K. ozaenae, K.
o Capnophilic rhinoscleromatis
o Nonmotile ― Exhibits large mucoid growth
o Catalase positive
Clinical significance:
o Oxidase positive
― RTI
Clinical significance:
― Wound infections
― Urethritis & cervicitis ― UTI
― conjunctivitis ― Blood infections
― CSF infections
Colonial Characteristics
Colonial Characteristics
― CAP: Small, tan, translucent and raised
― EMB: pink mucoid colonies
GRAM NEGATIVE BACILI ― capsulated slimy colony
6
ENTEROBACTERIACEAE Antimicrobial Susceptibility Testing

― Gram negative bacilli ― a test that provides a clinician with therapeutic

― Facultative anaerobes guidelines. It indicates which antibiotic is
― Oxidase Negative
MICROBIOLOGY | VILLACERAN, E.
 effective in killing the bacteria that has caused
the infection or disease.

Types of Antimicrobial Assays

a) Disk Diffusion susceptibility test (Kirby bauer

method)
― Rapid and cost effective procedure in
determining the susceptibility of bacterial
pathogens to antimicrobial agents
― Based on growth inhibition surrounding
antibiotic impregnated disks
― Yields qualitative results

Susceptible Intermediate Resistant

Susceptible
Large zone of Inhibition is larger Small zone of
inhibition when given a inhibition
larger dose

― Usually used in microbiology

Must know:

Standard medium: 4mm thick Mueller – Hinton agar

(MHA) Standard inoculum size:1.5 x 108 CFU/mL

Standard turbidity: 0.5 McFarland standard: 99.5 mL 1%

H2SO4 + -.5mL 1.175% BaCl2

b) Broth Dilution Susceptibility test

― Used to determine broth the minimal
inhibitory concentration (MIC) and
minimal bactericidal concentration
(MBC)
― Yields quantitative results

Antimicrobials: Agents against bacteria

Antibiotics

― Drugs that are administered to etither kill bacteria

or inhibit growth by opreventing reproduction.

Types of antibiotics

a) Bacteriostatic
― agents that only inhibit microorganism
growth in vitro and that can kill
standardized inoculate of bacteria at
high concentration
― only inhibits the growth
b) Bactericidal 7
― lethal to standardized inoculate at
clinically achievable concentration
― more reliable

MICROBIOLOGY | VILLACERAN, E.