MICROBIOLOGY! 5.

Biotechnology
BY: ANNA CASTRO
6. Industrial Microbiology
LECTURE
7. Genetic Engineering and Recombinant DNA
I. INTRODUCTION TO MICROBIOLOGY
Technology
A. Scope of Microbiology
D. Branches of Microbiology
1. specialised area of biology that focuses on
1. Bacteriology - bacteria
microscopic organisms = microorganisms
2. Mycology - fungi
a) examples are bacteria, viruses, fungi,
3. Protozoology - protozoans
protozoa, algae, helminths (parasitic
4. Virology - viruses
worms)
5. Parasitology - parasites
B. Coverage
6. Phycology and Algology - seaweeds and
1. Natural history of microbes
algae
a) primordial soup > coacervates >
7. Microbial morphology, physiology, taxonomy,
amino acids > proteins
genetics, molecular biology, ecology
2. Microbe-human and microbe-environmental
a) morphology - form
interactions
b) physiology - function
3. Genetics
c) taxonomy - identification, classification,
a) Mutations
and naming
4. Metabolism
E. Energy seen in the nutrient flow
a) Krebs cycle
1. Autotrophs
b) Glycolysis
a) Photoautotrophs - light
5. Infection & Disease
b) Chemoautotrophs - chemical
a) I: localised condition
2. Heterotrophs
b) D: pathological disorder of organs
3. Saprophytes
6. Drug Therapy
F. Microbes are Versatile Chemical Machines
a) Macrolytes, Beta lactase, prozantia
1. Microbes can create many products
7. Immunology
2. They improve life and can mould civilisations
a) Immune System - attack/defense system
- can be a make or break situations.
of body > watch immune system in YT
Outbreaks of disease on the human
b) Serology & Allergy
population or on an organism that causes an
8. Genetic Engineering
incurable disease.
a) modify, alter, etc genes
3. Biotechnology
9. Industry
a) genetic engineering - deliberate
a) Food production
modification of an organism through the
b) Products like jeans
manipulation of its genetic material
10. Agriculture
b) recombinant DNA - DNA molecules
a) Na and K channels > proteins
formed through laboratory methods of
11. Ecology
genetic recombination to bring together
a) Examples: BT Eggplant
genetic material from different sources
C. Practical Uses
c) unlimited potential
1. Immunology
d) bioengineers
a) Serology - serum
(1) use microbes as a natural pesticide,
b) Allergy
viruses as vaccines, pigs producing
2. Public health microbiology and epidemiology
human hemoglobin (protein in RBCs)
a) PH microbiology - prevention of disease
or insulin
b) Epidemiology - statistics of disease
e) Bioremediation
(1) morbidity, mortality, etc
(1) water sewage systems
3. Food/Dairy/Aquatic Microbiology, Water
(2) to use naturally occurring or
Testing
deliberately introduced microbes (or
4. Agricultural Microbiology

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 other forms of life) to consume and
break down environmental pollutants
G. Microbes and Infectious Diseases
1. Pathogens
2. Plague
3. Emerging and reemerging diseases
H. CHECKPOINT
1. Microorganisms - too small to be seen with
the naked eye
2. Ubiquitous - live everywhere
3. Relationship with man - beneficial or harmful
4. Microbiology has a very diverse scope
a) research, diseases (prevention and
treatment), environmental and industrial
uses
II. HISTORICAL DEVELOPMENT IN MICROBIOLOGY
A. Anton van Leeuwenhoek
1. dutch merchant > lens grinder (1670)
2. animalcules > protozoans
3. 40 years wrote about what he studied
4. Sept. 7, 1683 - first described bacteria
5. 1723 - died at the age of 90
B. Robert Hooke
1. fascinated with the microscope
2. English man
3. cells from observed corks
4. elongated stalks > threadlike fungi
C. Louis Pasteur
1. French scientist, 1850s
2. Microbes can be possible agents of
infectious disease
3. Studied why wines turn sour
a) Saccharomyces cerevisiae: yeast >
wine
4. Set down the foundations for Germ Theory
of Diseases as well as Control of Bacterial
Contamination by Heat!
a) Sterilisation - kills all microbes
b) Pasteurisation - kills certain bacteria;
those that cannot survive at 50 - 55
degrees
c) Germ Theory was proved through 2
swan-necked flasks with sterilised broth;
one has its neck broken and microbial
growth occurs while the other doesnt
have its neck broken which prevented
the entrance of microbes and its growth
> disproved the theory of
Spontaneous Growth > changed the
treatment of diseases
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(1) 1 germ = 1 disease
5. Discovered the cause of the disease of
silkworms > pebrine
a) caused by microsporidian parasites;
leaves silkworms unable to spin silkworm
thread
b) Frances economy was dependent upon
the production of silk by silkworms
6. Anti-rabies Vaccine
a) Vaccines are attenuated pathogens -
alive but very weak which allows the
body to easily overcome it the body is
able to generate antibodies and store the
memory of that
D. Robert Koch
1. East Prussia
2. RKs Postulate
a) microorganism must be found in the
diseases organism and not in healthy
ones
b) Microorganism is isolated and grown in
pure culture
c) Cultured microbe is introduced into a
healthy organism.
d) Once the healthy organism is diseased,
the microbe is again involuted from the
the disease organism and recaptured
e) Terms:
(1) Mixed culture: multiple microbes
(2) Pure culture: only one kind of
microbe
(3) Inoculate: to transfer microbes
(4) Inoculum: microbe to be transferred
(5) Control
(a) -: nothing
(b) +: one independent variable
E. Golden Age of Microbiology
1. 1856 to 20th Century
2. Competition between France and Germany
was fierce
3. Kochs Camp (Germany)
a) von Behring - 1st Nobel Prize for a
Vaccine
b) Koch - TB, Typhoid fever, Diphtheria
4. Pasteurs Campl (France)
a) Pasteur - discovered the toxin of
diphtheria
b) Ronald Ross - vectors for malaria
c) David Bruce - tsetse flies is the carrier for
sleeping sickness
 d) Masaki Ogata - bubonic plague
5. Nobel Prize
a) Alfred Bernhard Nobel
(1) discovered dynamites > became
very wealthy because of it
(2) became a pacifist all his wealth,
and properties was used to finance
the awards B. Prokaryotic Profiles
b) For: Chemistry, Physics, Physiology or 1. Prokaryotic Cell
Medicine, Literature and Peace, a) External
Economics (1) Appendages
c) Occurs every December 10 in Oslo or (a) Flagella (lum)
Stockholm i) locomotion organelles of
(1) Winner gets a medallion, scroll, and some bacteria; propellers
$1 Million ii) has different movements, #,
F. Discoveries in the Environment and direction
1. Sergius Winogradsky - certain bacteria use iii) composed of microtubles
carbon dioxide to synthesise sugars with a basal nuclei
2. Martinus Beijerinck - soil bacteria trap
nitrogen and make it available to the plants
G. Into the Twentieth Century
1. Blood products and vaccines became
prevalent during and after WW1
2. Microbes were used in the industrial
processes
3. Virology grew due to the advent of Electron
Microscopes
4. Biochemical research was prevalent
a) synthesis of proteins
b) genetic engineering and biotechnology
5. Molecular Genetics started
III. MICROBIAL CELL BIOLOGY (b) Arrangement
A. Size, Shape, and Arrangement of Bacterial Cells
1. Shape and Arrangement

(c) Cilia
(d) Pili
i) shorter than flagella
ii) longer and thicker than cilia
iii) bristles
iv) can be used for sexual
reproduction - allows for
adherence
v) present singly or in pairs
(e) Fimbriae
! i) bristle-like
2. Size ii) present in multiple numbers

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 iii) used to adhere to host tissue (b) for protection and increased
(f) General Purpose virulence
i) Biofilm/Attachment i) virulence - increased
ii) Conjugation/Reproduction capacity to withstand
(2) Bacterial Surface Coating pressure, drugs, etc
(a) Glycocalyx (c) present in both gram + and - but
i) glycoprotein polysaccharide are different for each
covering (d) Used by scientists to classify
ii) increases pathogenicity bacteria
iii) prevents phagocytosis (e) determines bacterial shape
iv) forms biofilms (f) made of peptidoglycan
i) Types of Peptidoglycan
(1) NAG: N-Acetyl
Glucosamine
(2) NAM: N-Acetyl Muramic
Acid
(3) Cell Membrane
(a) bilipid layer = proteins +
(b) Slime Layer phospholipids
i) composed of (b) fluid mosaic model
polysaccharides, (c) for protection
glycoproteins, and (d) controls the transport of
glycolipids molecules into and out of the cell
(c) Capsule (e) selectively permeable
i) is a more solid bacterial (f) mesosomes - infolding of cell
coating membrane; endosymbiosis
ii) composed of (g) Functions
polysaccharides i) transport, enzymatic activity,
b) Cell Envelope signal transduction,
(1) Basic Types: intercellular joining, cell-cell
recognition, attachment
c) Internal
(1) Cytoplasm
(a) !
(2) Ribosomes
(3) Inclusions
(4) Nucleoid
(5) Cytoskeleton
(6) Endospore
C. Gram Staining

(2) Cell Well

(a) Outermost layer of the cell
envelope

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 1. Nontypical Cell Walls
a) several bacterial groups lack even a cell
wall
(1) Ex: Mycobacterium and Nocardia
b) have special long chain fatty acid
mycolic acid or cord factor
c) stained by ACID-FAST stain
2. L-Forms/L-Phase Bacteria
a) bacteria lose their cell wall due to the
mutation of the cell wall forming gene
b) these are induced artificially by lysozyme
or penicillin
c) Gram + > Protoplast (lost
peptidoglycan layer which is the cell
wall)
d) Gram - > Spheroplast (lost f) Structure
peptidoglycan
D. Bacterial Endospores
1. Endospores
a) enables bacteria to withstand extreme
environments
(1) high temperature, drying, freezing,
radiation, and chemicals
b) dormant bodies
c) caused by changes in nutrient sources
of environmental requirements of the cell
d) Produced by Gram + bacteria
e) Life Cycle
(1) Vegetative Cell > Sporulation >
Endospore (if extreme condition)

(1) Exosporium - outermost thin layer

(2) Spore coats - layers of spore-
specific proteins
(3) Cortex - below spore coat, loosely
linked peptidoglycan
(4) Core - inside cortex and consists of
(a) Core wall, CM, Cytoplasm,
Nucleoid, Ribosomes, and
Cellular essentials
(b) Gell like core - 10 to 15% water
(c) Contains dipicolinic acid which
is absent in vegetative cells
(d) Enriched in Ca 2+ which is in
complex with the dipicolinic acid
(calcium dipicolinate)
(e) 10% of dry weight
i) reduces water availability
and helps to dehydrate
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 (f) Intercalates in DNA and (3) Main Functions:
stabilises DNA (a) storage material that is
(g) Contains small-acid soluble hydrolysed as needed
proteins (SASP) (b) building material
(h) Binds to DNA and confers 3. Function:
resistance from UV, Dessication, a) backbones of nucleon acids
and heat - Changes DNA b) call wall polymer
structure from B form to A form c) source of energy
i) A forms resists pyrimidine (1) cellular respiration allow cells to
dimmer formation extract energy from glucose
(i) Carbon and energy source molecules by breaking them down in
during germination a series of reactions
IV. Macromolecules d) Carbon skeleton source of raw metrical
A. Carbohydrates for synthesis of other small organic
1. Sugars and its polymers molecules
2. Types: B. Proteins
a) Monosaccharides 1. Monomeric units: Amino Acids
(1) aka as simple sugars a) Amino Acid structure
(2) monomers of more complex sugars (1) Carboxylic group
(3) General Molecular Formula: CH2O (2) amine group
(4) Usual Structure: (3) R group which differs giving rise to
(a) carbonyl group whose location different amino acids
varies b) Cysteine: R group is SH which allows for
i) Aldose or aldehyde sugar disulphide bonds which are important
(1) if carbonyl is at the end 2. Levels of Organization
ii) Ketose or ketone sugar a) Primary
(1) if carbonyl is somewhere (1) focuses on the sequence and %
in the middle composition of the polypeptide
(b) multiple hydroxyl groups b) Secondary
(5) Classification according to: (1) Structures formed due to interaction
(a) Location of Carbonyl Group of the amino acids in a polypeptide
(b) Size of the Carbon Skeleton (3-7 (2) Types
C) (a) Alpha helix
i) Trioses - 3 C sugar (b) Beta sheets
ii) Pentoses - 5 C sugar (c) Random
iii) Hexoses - 6 C sugar c) Tertiary
b) Disaccharides (1) 3D structure of a polypeptide
(1) two monosaccharides joined by a d) Quaternary
glycosidic bond (1) structure of proteins which are
(a) covalent bone formed by composed of more than one
dehydration > produces water polypeptide
(b) maltose = 2 glucose 3. Functions
(c) sucrose = glucose + fructose a) Enzymatic - selective acceleration of
(d) lactose = glucose + galactose chemical reactions
(e) ! b) Storage - storage of amino acids
c) Polysaccharides c) Hormonal - coordination of an organisms
(1) the legit macromolecule activities
(2) hundred to a few thousand of d) Contractile and motor proteins -
monosaccharides connected by movements
glycosidic bonds e) Defensive - protection against disease

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 f) Transport - of substance (2) 2 FA + 1 Phosphate group +
g) Receptor - response of cell to chemical Glycerol
stimuli (a) Phosphate group has a small
h) Structure - support charged molecule attached to it
C. Lipids that varies in turn different
1. diverse group of hydrophobic molecules phospholipids can be produced
2. Kinds: (b) creates a hydrophobic and
a) Fatty Acids hydrophilic end
(1) Structure: has hydrophobic and (c) structure themselves into
hydrophilic regions bilayers when in aqueous
(2) Kinds solutions
(a) Saturated c) Steroids
i) no double bonds; as mont (1) carbon skeleton composed of four
as possible H are bounded fused rings + different chemical
to each C groups
(b) Unsaturated D. Nucleic acids
i) has one or more double 1. Monomeric units: Nucleotides
bonds a) five carbon sugar
ii) usually cis bonds (1) Deoxyribose (DNA)
b) Glycerides (2) Ribose (RNA)
(1) Neutral glycerides b) nitrogen congaing (nitrogenous) base
(2) Phosphoglycerides (1) Pyrimidines - one ring 6 C
c) Non-glyceride Lipids (a) Cytosine
(1) Sphinholipids (b) Thymine (DNA) or Uracil (RNA)
(2) Sphingomyelins (2) Purines - 2 rings - 1 6 C and 1 5 C
(3) Glycolipids (a) Adenine
(4) Steroids (b) Guanine
d) Complex lipids c) phosphate group
(1) Lipoproteins 2. Two types:
3. Biological Important Lipids a) DNA
a) Fats (1) blueprint of life
(1) assembled from smaller molecules b) RNA
by dehydration (1) intermediary molecule
(a) glycerol (2) kinds:
i) an alcohol; each of its three (a) mRNA
carbons contain a hydroxyl (b) tRNA
group (c) rRNA
(b) Fatty acid (FA) 3. Other nucleotides:
i) long carbon skeleton with a a) ATP
carboxyl group at one end b) NADP
(c) hydrocarbon chain c) FAD
(2) hydrophobic due to the large 4. Function
number of C-H bonds a) store, transmit, and help express
(3) 3 FA + Glycerol bonded by an Ester hereditary information
linkage by dehydration reaction E. Location of Macromolecules in a Cell
between a hydroxyl and carboxyl 1. DNA
group = triacylglycerol = tryglyceride 2. Ribosome
(4) Major Function 3. Cytoplasm
b) Phospholipids 4. Cell Membrane
(1) Major constituent of cell membranes 5. Cell Wall

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 6. Capsule a) unicellular or multicellular
F. Importance of Water b) two important groups:
1. Properties (1) Diatoms
a) Polar (2) Dinoflagellates
b) Cohesive c) Do not infect but can be poisonous
c) High surface tension (1) Paralytic > affect muscles
d) High Specific Heat (2) Neurotoxin > affects brain
e) Capable of H-bonding 4. Fungi
V. Microbial Taxonomy a) decomposers of organic matter
A. Nomenclature b) saprophytic
B. Taxonomy 5. Yeast cells
1. Organisation, Classification, and a) anaerobic process > fermentation
Nomenclature > Identification b) catabolic in nature
C. Carl vin Linne (Linnaeous) (1) sugar to alcohol
D. 8 Taxa (2) faster than glycolysis
1. Domain 6. Viruses
2. Kingdom a) non-living
3. Phylum (Division for plants) b) cannot reproduce on its own >
4. Class requires a host
5. Order VI. Reports
6. Family !
7. Genus LABORATORY
8. Species I. Exercise 1: Microbiology Equipment and Instruments
E. Development from Two Kingdoms to Three A. Sterilising Equipment
Domain System 1. Autoclave - specialised pressure cooler
1. Taxonomists classified all species as Plant or a) 15 psi at 121 degrees Celsius for 15
Animal minutes
2. Five Kingdoms: Monera (prokaryotes), b) distilled water to a required level
Protista, Plantae, Fungi, Animalia 2. Oven
3. Recently, Three Domain System a) similar to incubator but max temp is 200
a) Bacteria degrees Celsius
b) Archaea 3. Bunsen burner or Alcohol Lamp
c) Eukarya a) dry heat sterilisation of transfer
d) Supported by data from sequenced instruments
genomes b) denatured alcohol or 90% ethanol
F. Assigning Specific Names B. Growth and Incubation Equipment
1. Binomial nomenclature 1. Incubator
G. Microbes a) Cell culture - control of gas
1. Bacteria b) Microbiology - temperature control
a) best known of all microbes c) 5-70 degrees Celsius
b) important in research, clinical medicine d) for Bacterial growth 37-40 degrees
c) existed 3.5 B years ago and just evolves e) for Fungi
to continuously survive 2. Water bath
d) occupy all niches on Earth a) maintenance of temperature of broth and
2. Protozoa media for a short period of time
a) Eukaryotes 3. Refrigerator
b) diverse cellular bodies a) control growth or prevent overgrowth
c) free living and parasitic b) 1 - 12 degrees Celsius - inactive
d) decomposers or photosynthetic enzymes and low metabolic activity for
3. Algae cells

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 C. Culture Equipment ii) not ideal for colourless
1. Culture tubes/test tube organisms > needs stains
a) for slants and deeps (2) Dark field
b) for broth (a) improves contain of unstained,
2. Culture Plates/Petri dishes transparent specimens
a) hold media for cultivation of microbes (b) excludes the unscattered beam
D. Transfer Instruments from the image
1. Inoculating wire loop/needle (c) dark black background with the
a) loop - streaking specimen as a bright image
b) needle - stabbing into deeps and used (d) disadvantage
for Dalmau i) sample must be strongly
2. Pipets, pipettors, and tips illuminated which can
a) assure delivery of accurate volumes of damage the sample
piques (3) Phase contrast
b) for glass pipettes use aspirators (a) difference in refractive index as
E. Hoods and Biosafety cabinets difference in contrast
1. UPM - Biosafety Level 3 (b) difference in density and
2. Prevents escape of microbes and noxious composition of the imaged object
chemicals into the environment give rise to phase change in the
3. helps maintain asepsis light as it passes through them
F. Glasswares and Other Tools > phase objects
1. Beakers, Erlenmeyer Flask (c) cell structures are visible
a) mixing and heating during media prep (d) allows study of live specimens
2. Graduated Cylinder
a) measuring liquids
3. Stirring Rod
a) mixing during prep of media, reagents,
and stains
4. Glass Slides
5. Amber bottles and dropper
a) storage and dispensing of chemicals and
stains
6. Wash bottle
a) wash off stains or dispense water onto
slides
7. Staining Tray
8. Top Loading Balance
a) accurate weighing of solid reagents
9. Stove/Hot Plate/Microwave
II. Exercise 2: Microscopy
(4) Fluorescence
A. Microscopy
(a) makes use of fluorescent dyes
1. technical field using a microscope
which different affinities for
2. Main Type of Microscopes
different molecules
a) Optical
(b) Makes use of high energy light
(1) Bright field
(ex. UV) since fluorochromes
(a) simplest and most common
emit lower frequency lights
(b) uses white light to illuminate
b) Electron
specimen
(1) Makes use of electron beam instead
(c) disadvantages
of light beam
i) limited resolving power

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 (2) needs prep of samples (2) Magnifying Parts
(3) Two kinds (a) Ocular eyepiece
(a) Scanning (b) Objective lenses
i) surface of the specimen - 3D i) Scanning - 4x
image ii) LPO - 10x
(b) Transmission iii) HPO - 40-44x
i) internal structure iv) OIO - 97-100x
ii) need sample prep - slicing III. Exercise 3: Aseptic Techniques
1. Asepsis - absence of bacteria, viruses, and
other microbes
a) without infection
b) Kinds of Culture
(1) Pure - one kind of organism
(2) Mixed - two or more organisms with
distinct characteristics
c) Goal of Microbiologist - maintain a PURE
culture
2. Aseptic techniques
a) prevent contamination and protect the
investigator
3. Aseptic Transfers
a) Terms
(1) Broth
(a) liquid culture media
3. Structure of a Compound Light Microscope (2) Agar
a) Mechanical (a) sold or semi-solid culture
(1) Base - support of the whole (3) Agar Slant
microscope (a) agar solidified at an angle
(2) Arm - connects upper part to the (4) Agar Deep
base (a) solidify straight up with the agar
(3) Body tube - contains ocular or at the bottom
eyepiece IV. Exercise 4: Culture Media
(4) Draw tube - contains lenses A. Classification According to:
(5) Revolving nosepiece - holds 1. Culture type
objective lenses a) Cell Culture Media
(6) Dust shield - found on top of b) Microbiological Culture Media
revolving nosepiece 2. Composition
(7) Stage and stage clips a) Synthetic/defined
(8) Mechnical stage - includes knobs (1) contains specific trace of element
which allows for slide movement; and and compound constituents
rulers for plotting b) Non-synthetic/complex
(9) Adjustment Knobs (1) contains uncharacterised mixture of
(a) Coarse - LPO compound that are inconsistent per
(b) Fine - HPO and OIO sample
b) Optical Parts 3. Use/Functional Class
(1) Illuminating Part a) Selective
(a) Mirror (1) promotes the growth of a select
(b) Lamp group of organisms
(c) Iris Diaphragm (a) e.g.
(d) Condenser

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 i) PDA with tartaric acid - only (1) +
fungi (a) metallic green sheen - for intense
(1) replacement - acid product due to lactose
Saborauds Dextrose fermentation
Agar i) e.g. Escherichia coli
ii) eosin methylene blue agar (b) pinkish purple - for weak lactose
(1) only for gram negative fermenter
bacteria (2) -
b) Differential (a) no colour change
(1) contains specific indicators in the 2. MacConkey Agar
solution which react with the a) Selective and Differential
biochemical products produced by b) selective for Gram -
certain microbes c) composition: Lactose, bile salts, neutral
(a) e.g. red and crystal violet, peptone
i) MSA - Mannitol Salt Agar d) pink in colour
ii) MacConkey Agar e) bile salts & crystal violet inhibits Gram +
iii) Salmonella-Shigella Agat f) neutral pH red indicator
c) General Purpose (1) hot pink in acidic condition
(1) contains most of the requirements for (2) rose in neutral
a wide spectrum of bacteria (3) tan in alkaline
(a) e.g. nutrient broth g) Tests if Lactose fermenter (differential)
d) Enrichment (1) +
(1) promotes fastidious growth of (a) pink since acid is produced
microbes lowering the pH of the media
(a) e.g. which reacts with the neutral red
i) BAP (2) -
ii) Chocolate Agar (a) colourless
e) Minimal 3. Blood Agar Plates
(1) specifically used for wild-type a) composition: Trypticase Soya Agar + 5%
microbes Sheeps blood
(2) contains a carbon source but without (1) Trypticase is a source of protein
amino acids b) pH 7.3
(3) e.g. M9 minimal medium c) inhibits Neisseria and Haemophilus
4. Physical State/Fluidity (1) If heated to become chocolate agar
a) Solid State these genus are not inhibited
(1) 1.5 - 2.0 % agar (a) since it releases the V factor
b) Semi-solid which is an essential growth
(1) 0.5 % agar factor
(2) for motility of microbes d) Kinds of hemolysis
c) Liquid (1) Beta
(1) no agar (a) complete lysis of RBC
B. Specific Agars (b) blood clears up
1. EMB - Eosin Methylene Blue Agar (c) e.g.
a) Selective and Differential i) Streptococcus pyogenes
b) Gram - ii) Streptoccocus agalactiase
c) composition: Eosin and Methylene Blue (2) Alpha
dye (a) partial hemolysis
d) dark red in colour (b) greenish-gray or brownish
e) Aniline inhibits the growth of Gram + discolouration of BAP
f) Tests if Lactose Fermenter

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 (c) haemoglobin is reduced to b) Phenylethyl alcohol - disrupts lipid
methemoglobin structure of gram -
(d) e.g. 7. PDA with 10% Tartaric Acid
i) Escherichia coli a) ensures that only fungi will grow
ii) Streptococcus pneumonia b) lowers pH to 3.4-3.6
(3) Gamma (1) inhibits bacterial growth due to low
(a) no hemolysis occurs pH
(b) e.g. actual
Agar + - Stops
i) Staphylocossus epidermidis color
4. Mannitol Salt Agar dark red metallic green no colour Gram -
a) isolation of pathogenic Staphylcocci from EMB
(strong)! change
pinkish purple
mixed culture (weak)
b) bright red
light pink pink (since colourless/ Gram +
c) composition: MacConkey
acidic) no change
(1) beef extract
blood red beta - clears! no colour Neisseria
(2) peptone alpha - change and
BAP
(3) agar greenish gray/ Haemop
brown hilus
(4) 7.5% NaCl - too high for other
bacteria and inhibits their growth Mannitol bright red yellow no colour
Salt change
(5) Mannitol - alcohol of carbohydrate
mannose Hektoen black ppt no colour Gram +
Enteric change
(a) fermentation of mannitol changes
media colour to yellow Phenylethyl clear Gram -

(6) Phenol red indicator

PDA w/ bacteria
(a) yellow in acidic Tartaric
(7) Results Acid

(a) +
i) yellow in colour due to the
fermentation of mannitol
which lowers the pH and
causes phenol red to
become yellow
5. Hektoen Enteric Agar
a) or Salmonella Shigella Agar
b) moderately selective
c) composition
(1) bile salts - inhibit Gram +
(2) Bromothymol blue and acid fuchsin
dye - lower toxicity
(3) Lactose, sucrose, and salicin -
fermentable carbohydrates which
promote growth
(4) Sodium thiosulfate - provides sulfur
(5) Ferric ammonium citrate - provides a
source of iron for production of
hydrogen sulphide and sulfur from
sodium thiosulfate > black ppt
6. Phenylethyl alcohol agar
a) inhibits gram -
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https://quizlet.com/4675318/microbio-225-lab-test-3-
selective-and-differential-media-imvic-biochem-tests-
with-some-bacterial-samples-flash-cards/ > for positive
and negative results of different tests
V. Exercise 5: Sterilisation
A. Sterilisation
1. total elimination of all forms of microbes
B. 2 Types
1. Chemical Sterilisation
a) chemicals like phenols, iodine derivative,
etc
2. Physical Sterilisation
a) agents like heat or radiation or filtration
C. Heating
1. most common type of physical sterilisation
2. Dry Heat
a) uses hot air without water vapour
b) coagulates proteins in organism >
oxidative free radical damage > drying
of cells > cell death
c) e.g.
(1) oven
(2) incineration for wire loops and
needles
 d) kinds b) plate is divided into several parts for a
(1) Red heat dilution series
(2) Flaming B. Characterisation
(3) Hot air 1. Colony Morphology
3. Moist Heat a) For Bacteria and Yeast
a) for heat labile supplies
b) coagulates proteins and denatures it with
aid of water vapour > cell death
(1) water vapour has high penetrating
property
c) Kinds
(1) Below 100 degrees Celsius
(a) Pasteurization
i) Flash method - high temp,
short time
(1) 75 degrees for 15 secs
ii) Holding - 63-55 degrees for
30 minutes
(b) Inspissation - stiffening of protein
without coagulation
i) 75 - 80 degrees
(2) At 100 degrees Celsius
(a) Boiling - water bath
(b) Steaming
(3) Above 100
(a) Autoclaving - steam under
pressure b) Molds
i) 121 degrees Celsius at 15 (1) colour and size
psi (2) hyphae
VI. Exercise 6: Pure Culture Techniques - Isolation, (a) septate (septum divides)
Characterisation, & Preservation (b) ceonocytic (so septum)
A. Isolation (3) spores
1. Capture Plate (a) sporangiospores (round)
a) from air (b) condidiospores (branches)
b) microbes are everywhere (4) sexual reproduction
2. Swab Inoculation 2. General Characteristics
a) from skin a) Bacteria
b) human bodies harbour normal flora (1) white, cream, yellow
3. Streak (2) circular
4. Surface (3) mucoid
a) quantify the number of bacteria b) Yeast
b) diluted sample spread over plate surface (1) white and opaque
5. Pour (2) looks similar to bacteria
a) broth culture to petri plate then added (3) how as patches with a glossy surface
with more broth culture c) Molds
b) subsurface colonies are present (1) filamentous (hyphae) and fuzzy
6. Miles and Misra (2) spores are powdery
a) determine the number of colony forming (3) bright to dull colours
units C. Preservation
1. Mineral Oil

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 a) does not solidify (1) increase in size of cell
b) submerge microbes with mineral oil d) For multicellular organism growth is an
c) incubate for 24-48 hours then refrigerate increase in size of organism
at 4 degrees Celsius e) For unicellular organisms growth is an
d) can last for months or years increase in number
2. Subsequent Transfer 2. Generation Time (or Doubling Time)
a) prevent microbes from dying due to dried a) time require for a group of cell or colony
up medium to double its population
b) microbes is restreaked every 1-2 weeks b) varies depending on species and growth
c) use nutrient broth for growing conditions
d) incubate for 24-48 hours then refrigerate c) constant until nutrients are deplete or
at 4 degrees Celsium there is an accumulation of toxic
e) label species and date of streaking metabolic products
3. Saline Suspension d) at low favourable conditions: doubling
a) suspend in 1% salt solution (NaCl) time increase and growth slows
b) bacteriostatic: inhibits bacterial growth e) G = t/n
when stored at room temp (1) G is generation time
4. Refrigeration (2) t is time interval between initial
a) 0-4 degrees Celsius population Po and population after a
b) 2-3 weeks (bacteria) period of time, t, Pt
c) 3-4 months (fungi) (3) n number of generations
5. Very Low Temperature (a) n = (logPt - logPo)/(log2)
a) suspend in Nutrient Broth _ 15% glycerol f) Growth curve describes the change in
b) freeze at -15 to -30 degrees Celsius cell number against time
6. Crypreseration: Liquid Nitrogen (1) Lag Phase
a) Frozen culture (-196 degrees Celsius)
b) protecting agent: glycerol or DMSO to
prevent cell damage
c) 10-30 years storage
d) expensive
7. Drying in Vaccum
a) dry over Calcium Chloride in vacuum
and store in the refrigerator
8. Lyophilization: Freeze-Drying
a) sublimation
(1) remove water and other solvents to
stop metabolic activity
(2) bacteria become dormant
b) can last up to 30 years
(1) preserve in vials in the dark and
refrigerate at 4 degrees Celsius
VII. Exercise 7: Enumeration of Microbial Growth
(a) microbes are acclimatising to
A. Definition
environment
1. Growth
(b) factors:
a) increase in the cellular constituents of an
i) status of cells
organism
ii) difference in environment
b) Population Growth
iii) number of viable organisms
(1) increase in the number of cells in a
in inoculum
population
(2) Log or Exponential growth phase
c) Cell Growth

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 (a) microbes growth (number of (3) Calculation
cells) at an geometric rate due to (a) cell/mL = (Caverage/Af) x (100
the presence of nutrients that are mm2/0.01 mL) x DF
in excess i) Cave - average number of
(b) biomass increase linearly with cells
time ii) Af - area of microscopic field
(3) Stationary growth phase iii) DF - dilution factor
(a) microbes growth rate = microbes b) Viable Cell Count
death rate (1) live cells
(b) substrate may be exhausted (2) advantage: only live cells
(c) inhibitory products of metabolic (3) disadvantage: underestimation
may accumulate in medium (4) Procedures
(d) 109 cells/mL (a) Serial Dilution
(e) total number of viable organisms i) Step-wise dilution of
= constant microbes in solution
i) some die and the nutrients ii) usually, dilution factor =
they release are used by constant which allows for a
other cells that continue to geometric progression
divide iii) ten-fold dilution at a 10 mL
(4) Decline or death phase scale
(a) too much waste are present and (1) each tube = + 9 mL of
microbes die 1% peptone water
B. Methods of Growth Estimation (2) + colony = 1 mL of stock
1. Indirect Methods microbial solution
a) Biomass Determination (3) and vortex mixed
(1) Cell Mass iv) to be plated on media in
(a) wet weight = weight of packed different methods
centrifuged cells (1) pour plating, surface
(b) dry weight = weigh after heating plating, and miles and
or using desiccator misra
b) Turbidity Measurements (b) Pour Plating
(1) cell mass i) colony range = 30 - 300
(2) spectrophotometer ii) pour 1.0 or .1 mL and add
(3) blank = uninoculated broth or melted nutrient agar
distilled water iii) allows for surface and
(4) 540 nm subsurface colonies
2. Direct Methods iv) < 30 statistically unreliable
a) Total Cell Count or Direct Microscopic v) >300 too crowded and
Counts colonies become indistinct;
(1) total cell count: alive and dead TNTC
(2) use Hemocytometer grid (c) Surface or Spread Plating
(a) 9 major squares = 1mm x 1mm i) inoculate 0.1 mL and use L
(b) count: 4 corners and center rod to spread
(c) chamber hight with cover = 0.1 (1) dry sterilise with 70%
mm ethanol before and after
(d) major square volume (a) 2 different beakers =
i) 1mm x 1mm x 0.1 mm = .1 before beaker and
mm3 after beaker
(e) count systematically (d) Miles and Misra
(f) total count >= 100 cells i) colony range = 10 - 30

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 ii) inoculum = 20 micrometers (1) 1% Barium Chloride + 1% Sulfuric
iii) Plate dilutions of 1/10, 1/100, acid = Barium sulfate (fine ppt)
1/1000, ad 1/10,000 (2) usually 0.5 McFarland
iv) Calculations (a) 0.05 mL of barium chloride and
9.95 mL or sulphuric acid = 1.5 x
108 suspension/mL

(1) Colave - average number

of colonies
(2) DF - dilution factor
(3) n1 - # of plate in lower
dilution that is valid
(4) n2 - # of plates in next
highest dilution
(5) DF - dilution factor of
first count - lowest
dilution?
v) Other Results
(1) Case: No valid dilution
(all TNTC)
(a) EST >= 1.0 x 106
CFU/mL
(2) Case: No valid dilutions
(no colonies observed)
(a) EST <= 1.0 x 102
CFU/mL
(5) Units of results: CFU - colony forming
units
(6) unknown if colony came from a cell
or group of cells
3. Other Methods of Estimations
a) MPN
(1) pattern of positive test + tables
b) Methylene Blue Reduction Test
c) McFarland Standard

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