MICROBIOLOGY

LECTURE \ FIRST SEMESTER

(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

HISTORY OF BACTERIOLOGY ○ A selective media because it only wants to grow

ANTON VAN LEEUWENHOEK M. tuberculosis in the culture media
● First true microbiologist ○ Inhibits other bacteria because selective media
● Father of Protozoology and Bacteriology (Microbiology) wants to select a particular bacteria
● Discovered “animalcules”
○ which are tiny & moving organisms now called as SELMAN WAKSMAN
“microorganisms” ● “Father of Antibiotics”
● Self made microscope (50x to 300x) magni cation to ● Discovered Streptomycin & Neomycin
study bacteria and protozoa ○ Source of antibiotics are the microorganisms
‣ ○ Streptomycin- from the bacteria “Streptomyces”
LOUIS PASTEUR
● “Aseptic technique” ALEXANDER FLEMING
○ prevention of contamination from unwanted ● Discovered Penicillin
microorganisms ○ Penicillium notatum
● Developed vaccines against: ○ MOA: Inhibits Cell wall synthesis
○ Anthrax (1881)- Bacillus anthracis ● Also discovered Lysozyme
○ Rabies (1885) - Rabies virus ○ an enzyme with antibacterial property which are
● IMPROVED: Fermentation & Pasteurization richly seen in our tears
○ Fermentation: important/signi cant in wine
industry EDWARD ABRAHAM
● First to correct biochemical structure of Penicillin
IGNAZ SEMMELWEIS
● Handwashing: PAUL EHRLICH
○ most e ective means of preventing diseases ● Discovered Salvarsan/606 Drug/ Arsphenamine
○ Happy Birthday Song (2x) or 20 seconds ○ 1st modern antimicrobial agent
○ An arsenic compound
ROBERT KOCH ○ In 1910, it is used for the treatment of 3 diseases:
● Discovered • Syphilis (Treponema pallidum)
○ Bacillus anthracis (Anthrax Bacillus) • American Trypanosomiasis/ Chaga’s Dx
○ Mycobacterium tuberculosis (Koch’s Bacillus) (Trypanosoma cruzi)
● First to cultivate bacteria on boiled potatoes, gelatin, meat • Relapsing fever (Borrelia)
extracts and protein • Malaria-like illness
○ Upon cultivating these bacterias, he developed
di erent culture media HIERARCHICAL CLASSIFICATION SYSTEM
KOCH’S POSTULATE Species speci c epithet, lower case
1. The microorganisms must be present in every case of disease Latin adjective or noun
but absent in a healthy host Genus
2. Suspected microorganisms is isolated from a diseased host &
contains similar species
grown in pure culture Family contains similar genera
3. Grown microorganism in a culture medium, when inoculated in
a healthy individual, same disease must be present from the Order contains similar families
rst host
4. Same organism must be isolated again from the diseased host Class contains similar orders

‣ Points out the process of transmission of disease contains similar classes;

‣ Once an organism is considered pathogenic, it can invade Phylum equivalent to the Division
our immune system and can cause a disease taxa in botany
EXAMPLE OF CLASSIFICATION
FANNY HESSE Family: Micrococcaceae
● “Agar” - Genus: Staphylococcus
○ solidifying agent in the use of culture media Species: aureus
Accepted abbreviation: S. aureus
JULIUS RICHARD PETRI Informal: staphylococci
● Discovered “petri dish” -
○ transparent glass where we place our culture GENERAL PROPERTIES OF MICROORGANISMS
media Microorganisms- belong to the Protista Biologic Kingdom
1. Prokaryotes
MARTINUS BEIJERINCH 2. Eukaryotes
● “Enrichment-culture technique” 3. Virus
○ Kapag kokonti ang tubo ng bacteria, enrich or 4. Prions
propagate growth of certain bacteria 5. Viroids
● Selective media
○ Ex: Lowenstein gensen agar
• a selective media for M. tuberculosis

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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

PROKARYOTIC VS EUKARYOTIC CELLS

Characteristics Prokaryotic cells Eukaryotic cells

Major group Bacteria Fungi

Archaea (blue green algae) Protozoa
Algae
Plants
Animals

Size of cell Generally smaller Typically 10-100 um in diameter

Typically 0.2-2.0 um in diameter

Motility (Locomotory organelle) Simple Flagella Complex Flagella

“Motile/gumagalaw” Some have pseudopodia

Cell Wall Present Absent (Generally no cell wall)

• Gives shape to bacteria Except: (Miss Universe) Except: (have CW)
• Mycoplasma • Fungi (chitin)
• Ureaplasma • Plants (cellulose)
Composition of Bacterial C.W: • Complex carbohydrate
‣ Peptidoglycan or Mycoplasma and Ureaplasma
‣ Murein or ‣ Cell wallless/de cient
‣ Mucopeptide layer • No cell wall = no shape
‣ Pleomorphic bacteria
• Vary in size and shape
• No shape

Cell Membrane (Plasma Present Present

Membrane) • Lack sterols except: • With sterols
• Site of energy/ATP • Mycoplasma • ergosterol
production of bacteria • Ureaplasma • cholesterol
Mycoplasma and Ureaplasma
‣ Cell membrane contains
STEROLS
‣ Require sterol/cholesterol for
GROWTH
‣ Can grow on culture media
with Increased/High Lipid
Content

NUCLEUS

Characteristics Prokaryotes Eukaryotes

Nucleus “Nucleoid” “True nucleus”

• Contains genetic materials
Stain:
• Feulgen Stain
• stains nucleoid and has
increase affinity with
nucleic acids specifically
DNA

Nuclear membrane Absent Present

Chromosomes Present Present

Single, close, circular, & double Multiple & linear

stranded

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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

CYTOPLASM

Characteristics Prokaryotes Eukaryotes

Cytoskeleton Absent Present

Ribosomes Present Present

smaller larger
70S (50S + 30S) 80S (60S + 40S)
smaller size 70S in organelles

Mitochondria Absent Present

ATP Production takes place in the
cell membrane

Golgi complex Absent Present

Endoplasmic reticulum Absent Present

Triglyceride fats Absent Present

Svedberg unit BFM FLUORESCENCE DARK FIELD ELECTRON

‣ Non-SI unit for sedimentation rate Organism
Group Requirement :
(Spirochete (BLT)
Borrelia Parasites
‣ Rate at which particles of a given size and shape travel to Fluorescent stain/ Leptospira Viruses only
Fluorochromes Trepanoma
the bottom of the tube under centrifuge force
Bacteria + +/- + -
VIRUS
● Obligate intracellular microorganisms Fungi + +/- - -
○ Only survive once they are attached to a cell
Parasite
○ Strictly grow inside the cell (intracellular) + +/- - +/-
● Composition
-
○ Has PROTEIN COAT Viruses *useful +/- - +/-
• Coats the nucleic acid in EM
○ Either DNA or RNA (never both)
BRIGHT FIELD MICROSCOPE
VIROIDS ● Most common microscope for
● Composition ○ Gram Staining
○ No Protein Coat ○ Acid Fast Staining
○ Has NUCLEIC ACID ○ Potassium Hydroxide
• Both DNA and RNA ● NOTE! If questioned what are the three methods are
● Cause plant diseases but have not been proven to cause usually employed under BFM? What techniques or
human disease although the RNA of the hepatitis D virus procedures in microbiology are usually used when we use
(HDV) is viroid like BFM?
● Used for stained and Unstained specimens.
PRIONS ● Color illumination
● Composition ○ this is the sign to provide maximum illumination
○ Has PROTEIN COAT and resolution when observing images using a
○ No Nucleic Acid microscope
● Very abundant in the brain QUICK RECALL
○ Infects the CNS mainly the brain Two lens system used in BFM
○ Causes subacute progressive degenerative ● Ocular lens (10X magni cation)
diseases of the CNS (TSK (si)C Mad) ● Objective
• Transmissible Spongiform Encephalopathy ○ Scanner (Red) (4x magni cation)
• Scrapie ○ LPO ( Yellow) (10X magni cation)
• Kuru ○ HPO (Blue) ( 40x Magni cation)
○ Oil Immersion Objection (White or Black) (1000X
• Creutzfeldt Jakob Disease
magni cation
• Mad Cow Disease To get the total magni cation Just multiply the Objective lens to
MICROSCOPY FOR DIAGNOSTIC MICROBIOLOGY the Ocular lens
● Microorganisms are microscopic TOTAL MAGNIFICATION = OBJ x OCU
● 4 main microscopes used in diagnostic microbiology

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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

DARK-FIELD MICROSCOPE ● Resolution: 0.5nm

● Visualize/ motility of SPIROCHETES ● Before viewing we 1st nee to:
● Also used for uorescent stains ○ Fixative (for spx preservation)
○ SUBSTITUTE for uorescent microscope • Glutaraldehyde
● Higher resolving power than BFM • Osmium tetroxide
○ Dehydrating Agent (to remove water)
PHASE-CONTRAST MICROSCOPE • Alcohol
● Inclusion bodies seen on virus and Chlamydia, living ○ Stain (Heavy metal stains)
cells/ natural state • Lead citrate
• Uranyl acetate
INCLUSION BODIES ● Transmission EM — internal structure, organelles
** the inclusion bodies in virology and bacteriology are di erent
‣ VIRUSES ● Scanning EM — external structure/ surface
• Considered as obligate/intracellular microorganism
TOOLS MAGNIFICATION (X) APPLICATION
• For them to grow, they will be cultured in tissue
culture media Eyes 0 Gross Examination
• Hindi sila tumutubo sa commercially available na
culture media Magnifying glass 5 Gross Examination
• CYTOPATHIC EFFECT (CPE)
• When they grow in tissue culture media (e ect) Dissecting Gross detailed examination
2.5 — 3.0
• Ex: Cytomegalovirus microscope and manipulation
• Tissue culture media
Bright eld
• Para malaman na tumubo ito makikita ka ng compound 10 — 2000 Cells stained
OWL’S EYE —> which is an inclusion body microscope
‣ CHLAMYDIA
• Aggregates of speci c types of proteins/nutrients Dark- eld
Cells not readily stained for
compound 10 — 400
microscope bright- eld microscopy
Chlamydia Halberstadler
trachomatis Prowazek body Phase contrast 10 — 400 Living or unstained cells

Preparations using
Chlamydia Levinthal Cole Lillie uorochrome stains, which
Psittaci body Fluorescence 10 — 400 can directly stain cells or be
connected to antibodies that
attach to cells
● Microlymphocytotoxicity test for HLA (Human
Leukocyte Antigen)
○ Inverted Phase Contrast Microscope
FLUORESCENT MICROSCOPE
● Requires uorescent stain/ uorochrome

Calcofluor white Cell wall of Fungi

(Chitin) — high affinity
to chitin

Acridrine Orange High affinity with

NUCLEIC ACID

Auramine Rhodamine AKA Truan’s method

High affinity with
Mycolic acid
(Mycobacterium spp.)

Fluorescein For
Isothiocyanate (FITC) immunofluorescent
techniques
‣ Bacteria
• Bordetella
• Chlamydia
CONFOCAL MICROSCOPY
• Legionella
● This is useful to obtain high resolution images and for 3
‣ Parasites
dimensional reconstruction of biological models
‣ Viruses
SCANNING PROBE MICROSCOPY
ELECTRON MICROSCOPE ● This measures surface features by moving a sharp probe
● Used for VIRAL morphology over the object’s surface.
● There are two types of scanning probe microscope
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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

○ Scanning tunneling microscope

○ Atomic force microscope

FUNDAMENTALS OF BACTERIOLOGY
BACTERIAL MORPHOLOGY
SHAPE
● along with other properties, shaped is used to identify
bacteria. It is determined by the mechanism of cell wall
assembly

Bacilli rods

Cocci spheres

1. Vibrio - straight rod or with single rigid curve

2. Spirillum - rigid helical cord
3. Spirochete - exuous helical rod (BLT)
Spiral shapes a. Borrelia burgdorferi
b. Leptospira interrogans
c. Trepenoma pallidum

vary in size and shape

Pleomorphic Mycoplasma
Ureaplasma

ARRANGEMENT

diplococci, diplobacilli

Gram negative (-) Diplococci

Pairs Neisseria (kidney/co ee bean shape)
Gram positive (+) Diplococci
Streptococcus pneumoniae ( ame/bullet shape)

Chains streptococci, staphylococci

Grape-like clusters staphylococci processes ( respiration, photosynthesis), detection of

environmental cues for chemotaxis.
Groups of four tetrads ○ Sa cell membrane sakanya dumideretso yung
(e.g. Peptococcus) signal when there is a chemical agent approaching,
cuboidal
magkakaroon ng chemotaxis (lalapit sa agent na
Packets of eight
(e.g. Sarcina) yun)
● Lipid bilayers with oating proteins (internal membrane
Also known as chinese character/XYVL proteins
(e.g. Corynrbacterium)
Palisades
○ Amphipathic lipids BACTERIAL APPENDAGES
- Mga nakaattach externally
• Polar ends and non polar tails - Flagella
○ Membrane proteins
CAPSULE
- outer layer
- Not all bacteria possesses a capsule, there are speci c
SIZE bacteria that have capsules
● Haemophilus ducreyi
○ smallest pathogenic bacillus (0.2 x 0.5 um)
○ Caused a disease called chancroid
• painful ulcer
○ Chancre
• painless ulcer that can be seen in Syphilis
(T. pallidum)

● Bacillus anthracis
○ largest pathogenic bacillus (1 x 3-10 um)
○ Disease caused: anthrax

BACTERIA STRUCTURES
CELL MEMBRANE/PLASMA MEMBRANE
● Site of energy production
● Selective permeable barrier, mechanical boundary cell,
nutrient and waste transport, location of many metabolic
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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

CELL WALL ● Consists of glycan chains of alternating N-acetyl-D-

● Also known as the peptidoglycan/murein layer/ glucosamine(NAG) and N-acetyl-D-muramic acid (NAM)
mucopeptide layer ● Teichoic acid
● Rigid structure that lies just outside the cell membrane ○ contributes to the negatively charged of the cell
● Stain: DYAR stain (congo red) - old stain wall
● Functions
○ Protects bacteria against osmotic pressure
• Hindi sila mabilis masira kasi may cw
○ Gives shape to the bacteria
○ Confer the gram’s reaction of the bacteria
○ A usual target of anti-microbial drugs (e.g.
penicillins, cephalosporins)

CASE STUDY
Penicillins - MOA: inhibits the cell wall synthesis
Scenario:
Patient X had a cough for a long time and it is already chronic. He
decided to undergo self medication. He took penicillin as a
treatment for a week but he is still sick. Because of that, he go for
a check up and undergo in a lab test. Based on the lab result,
Mycoplasma pneumoniae is the cause of the infection. What is the
rationale??

Rationale
Mycoplasma has no cell wall and Patient X took penicillin as a
treatment without knowing that it inhibits the cell wall synthesis
so, it is nonsense. Therefore, the rationale is that Patient X must
not do self medication because antibiotics has di erent mode of GRAM-NEGATIVE CELL WALL
action.
● Composed of a thin layer of peptidoglycan.
● No teichoic acids
● Structure: ● Periplasmic space
○ Meshlike polymer or identical subunits forming ○ space between outer membrane and gram
long strands negative cell wall
a. Two alternating sugars ○ involved in the peptidoglycan synthesis
N-acetylglucosamine (NAG), ● Outer membrane
N-acetylmuramic acid (NAM) ○ proteins
b. Alternating D- and L-amino acid ○ phospholipids
○ LPS (lipopolyssacharide)
• AKA as endotoxin

ACID-FAST CELL WALL

● Has a gram-positive reaction
○ When we gram stain a bacteria w/ acid fast cell
wall= they stain gram positive
○ For example, Mycobacteria= gram positive w/
acid fast cell wall
● Concurs resistance to drying and chemicals
○ Bacteria with acid fast cell wall (also known as
AFB= Acid fast bacteria) cannot be destroy easily
the cell wall because they are resistant to drying
and chemicals
● Contain a waxy layer of glycolipids and fatty acid
○ (hydroxymethoxy acid or mycolic acid) bound to
the exterior of the cell wall.

● Important for identi cation

○ Gram positive cell wall
○ Gram negative cell wall
○ Acid fast cell wall
GRAM POSITIVE CELL WALL
● THICK = TEIC
● Composed of a very thick protective peptidoglycan
(murein layer)

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 MICROBIOLOGY
LECTURE \ FIRST SEMESTER
(BACTE) MIDTERMS
INTRODUCTION TO MICROBIOLOGY

ACID FAST ORGANISM (MILCN) 3. SPECIAL — we stain the speci c structure of bacteria
● Bacteria and parasite ● Ex: NUCLEOID
1. Mycobacteria ○ Feulgen stain
2. Isospora ● Flagella
3. Legionella micdadei ● Capsule
4. Cryptosporidium ● Inclusion body (etc)
5. Nocardia
STAINS FOR ACID FAST ORGANISMS
OTHER CLINICALLY RELEVANT ORGANISMS W/ CW
Most common AFS
CONTAINING MYCOLIC ACID AFS (ZN AND
‣ Ziehl Neelsen = hot method
● Not acid fast cell wall bc kokonti lang ang mycolic acid KINYOUN)
‣ Kinyoun = cold method
● NoGoRhoTsuCO
1. Nocardia — partically acid fast bacilli (PAFB) May binago sa original
2. Gordonia MODIFIED ZN AND ‣ Recommended for:
KINYOUN
3. Rhodococcus • Nocardia
4. Tsukamurella • Cryptosporidium

5. Corynebacteria Most SENSITIVE and SPECIFIC acid fast stain

● All are gram positive bacilli AKA Truant’s stain
‣ A uorescent stain
STAINING ‣ A di erential stain
AURAMINE
● We do staining to observe the appearance of the PHODAMINE Primary = auramine rhodamine
microorganism Mordant = none
● To di erentiate microorganisms Decolorizer = 0.5% acid alcohol
Secondary = (Potassium) K permanganate/
● Gives a preliminary result/clue
quenching agent
○ Ex: gram neg/gram pos
Di erentiates
PAPPENHEIM
SALT = most commonly used stain old method ‣ M. tuberculosis (red)
CLASSIFICATIONS OF STAIN ‣ M. smegmatis (blue)

STAIN CATION ANION Di erentiate

BAUMGARTEN ‣ M. Tuberculosis (blue)
BASIC Colored Colorless ‣ M. Leprae (red)

ACIDIC Colorless Colored For M. leprae

FITE-FARACO Cause leprosy “ketong” = Hansen’s disease
CHROMOPHORE Hansen’s bacillus
● Colored ion in the salt
● Nagbibigay ng kulay sa stain

MORDANT
● Intensify yung pagkapit ng stain sa spx
● Increases a nity/attachment of stain with the spx.
● Pampakapit

2 PREPARATORY METHOD BEFORE STAINING

1. Smear preparation (glass slide)
2. Fixation
• For preservation
• Para hindi mawash o yung spx
• Para dumikit if magsstain
• Alcohol
• Heat (common)

STAINS
1. SIMPLE — 1 stain only is being used
○ Methylene blue
○ Basic fuchsin
○ Crystal violet
2. DIFFERENTIAL — not only 1 stain is being used
○ Gram stain (gram pos/gram neg)
○ Acid Fast stain (AFB/ nonAFB)
● 4 components
○ Primary stain
○ Secondary stain
○ Mordant
○ Decolorizer

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