microb -

Shapes

Cubical/spherical/cylindrical

Cytoplasmic membrane

Encases an internal matrix (cytoplasm) from outside

Selective permeability

Chromosomes

One or more chromosomes containing DNA

Ribosomes

Protein synthesis/translation

Prokaryotic cells Eukaryotic cells

• Animals, plants, fungi & protists

• Contain membrane enclosed organelles that perform useful

functions (metabolisms, nutrition, synthesis)

• Nucleus – most visible organelle

• Few internal structures

• Lack nucleus & organelles

Cytoplasm

Aqueous mixture of macromolecules (e.g. proteins,

lipids, nucleic acids & polysaccharides), maintain turgidity

Cell wall*

Outer layer surrounding cell that located outside the cell membrane

Provide strength & structural strength to a cell

Cell

What is unicellular?

Consisting only single

cell

What is multicellular?

Consisting of

numerous cell

Characteristic Prokaryotic Eukaryotic

Cell size 0.2-2.0 μm in diameter 10-100 μm in diameter

Nucleus No nuclear membrane/nucleoli True nucleus, consisting of nuclear membrane and nucleoli

Membrane enclosed organelles Absent Present (e.g: lysosomes, golgi complex, endoplasmic

reticulum, mitochondria, chloroplasts)

Flagella Consist of two protein building blocks Complex; consist of multiple microtubules

Cell type Usually unicellular (some cyanobacteria maybe

multicellular)

Usually multicellular

Cell wall Usually present, chemically complex

(typical bacterial cell wall includes peptidoglycan)

When present, chemically simple

Plasma membrane No carbohydrates and generally lacks sterols Sterols and carbohydrates that serve as receptors present

Cytoplasm No cytoskeleton/cytoplasmic streaming Cytoskeleton; cytoplasmic streaming

Ribosomes (Large subunit) (Small

subunit)

70S (50S + 30S) (23S + 5S rRNA) (16S rRNA) 80S (60S + 40S) (25S/28S + 5S + 5.8S rRNA) (18S rRNA)

Chromosome (DNA) Single circular chromosome; lacks histones Multiple linear chromosomes with histones arrangement

Cell division Binary fission Mitosis

Sexual reproduction No meiosis; transfer of DNA fragments only Involves meiosis

Example Bacteria & Archaea Animal, Plant, Fungi & Protist cell

MacroscopicMicroscopicUltramicroscopicAtomic

Microbial dimension Energy & Nutrient flow

Roles???

• constitute a major fraction of global biomass (2 x 1030 microbial cells) & key

reservoirs of nutrients essential for life

• Vital components of structure & function of ecosystems

• Flow of energy & food through the earth’s ecosystems

Photosynthesis

• Phototrophs : light fueled conversion of CO2 to O2 & organic materials

• Photosynthetic microorganisms contributing majority O2 to the atmosphere (>50%

earth’s photosythesis )

Decomposition & nutrient cycling

• Breakdown of dead matter into simple compounds & direct back into natural cycles

• Microorganisms>>main forces that drive the structure & content of soil, water, &

atmosphere

Majority free existence

Relatively harmless often beneficial

Close associations with other organisms

(Parasites, hosts)

Usually in cm & m

Range of μm, nm, mm

Lived & evolved for billions of years……….

Bacteria

• Unicellular microorganisms

• Three major shapes:

o Bacillus (rod like)

o Coccus (spherical/ovoid)

o Spiral (corkscrew/curved)

• Mostly have peptidoglycan cell wall

• Binary fission

• May possess flagella

• Use wide range of chemical substances

for nutrition

Archaea

• Prokaryotic cell

• Common freshwater plankton

• Single celled organisms- cell bounded by a single

lipid membrane

• Lack nucleus & organelles

• Cell wall lack of peptidoglycan

• Reproduce asexually (fission/budding)

• Including:

o Methanogens

o Halophiles

o Extreme thermophile

• Mostly chemoautotrophs (grow on simple

inorganic chemical), others

heterotrophs

Fungi

• Eukaryotic cell

• Heterotrophic

• Unicellular/multicellular/ dimorphic

• Thick cell wall (Chitin & glucan)

• Mostly produce spores (haploid, mitosis)

• Growth as hyphae

• No chlorophyll

• Decomposer known as saprotrophs

Protozoa

• Eukaryotic microorganisms

• Microscopic unicellular & microscopic

• Complex internal structure

• Variety of shapes (2-200 μm)

• Mostly free living or parasites

• Motile

• Binary fission (most common), sexual

& asexual reproduction

• Nucleus enclosed with in membrane

• Obtain nourishment by absorption or

ingestion

Algae

• Eukaryotic cell

• Unicellular/multicellular

• Lack defined structure, no roots,

stems/leaves

• Photosynthetic organisms (O2 & CHO)

some chemoheterotrophic, others

saprobes & parasites

• Reproduction in vegetative

(fragmentation) and both asexual (spore

formation, binary fission) & sexual forms

(fusion)

• Free living some symbiotic

Virus

• Infectious agent

(bacteriophages,

mycophages,

virophages)

• Reproduce in living hosts

(obligate intracellular

parasites)

• No metabolism using host cell’s metabolic

machinery

• Structurally simple, a core made of DNA/RNA &

surrounding by protein coat (encased by

envelope)

• Cannot growth in synthetic culture media

• Acellular (no cytoplasm/cellular organelles)

Historical Foundations of Microbiology

Prominent discoveries in

the past 300 years……

Microscopy

Scientific

method

Development of

medical microbe

Microbiology

techniques

some forms of life could arise from

vital forces present in non

living/decomposition matter

Abiogenesis

Inedible Enhance

flavor

Spoil food

Human sickness/ disease

Black plague

Smallpox

Spontaneous generation

From the very earliest, humans noticed that when

certain food spoiled

Unclear of transmission source & path

Simply organisms can arise from non living matter

Redi experiment (1665)

Francesco Redi, Italy

Test on spontaneous generation theory

“life is necessary to produce life”

Maggots are not spontaneously

produced in rotten meat

Jablot experiment (1710)

Louis Jablot, French

Microbes are present in dust particles

Only the open vessel developed

microorganisms

cells cells

Needham experiment (1745)

John Needham, England

Both heated & unheated test containers

teemed with microbes

life had been created from nonlife

Supports spontaneous generation of organisms

Pasteur experiment (1859) • Disproved spontaneous generation of microbes

• microorganisms are everywhere - even in the air

• Microbes caused fermentation & spoilage

Louis Pasteur, France

Test whether sterile nutrient broth could

spontaneously generate microbial life

Air and dust were the source of microbes

True awareness of widespread

distribution of microorganisms and

their characteristics was made possible

by the development of first microscopes

Antonie van Leeuwenhoek

(1632-1723)

• Dutch microscopist, first observer

for bacteria & protozoa

• Refuted the doctrine of

spontaneous generation

• Lay foundations for the sciences

of bacteriology and protozoology

Robert Hooke

(1655)

• first to use a microscope to

observe microbes/living things

• first used the term “cells” (1665) to

describe the small chambers

within cork

• Laid the groundwork for

development of “cell theory”

• Deductive approach taken by scientists to explain

a certain natural phenomenon

• General observations of phenomenon to develop

a set of facts to explain

• Form a hypothesis-a tentative explanation that

can be supported/refuted (scientific thought)

A valid hypothesis will allow for experimentation & testing & can

be shown to be false

• A lengthy process of experimentation analysis, and

testing eventually leads to conclusions

>>>support/refute hypothesis

explanatory

predictive

Information How do scientists apply scientific method???

Developing a hypothesis

based on existing theory

and then designing a

research strategy to test

the hypothesis

To predict what is expected to happen.....under

known condition

Does not mean

results are invalid

require

• reworking

• additional tests

Discard/modify to

fit the results of

experiment

Should not be

immediately

accepted

Test & retest

• Results must be published & repeated by other

investigators

• If hypothesis is supported by evidence & survives

rigorous scrutiny, it moves to next level of

confidence-it becomes a theory

• If evidence of a theory is so compelling that next

level of confidence is reached, it becomes a

Law/principle

Theory is a collection of statements.

propositions/concepts that

explains/accounts for a natural event

Is not the result of a single experiment repeated

over & over again but is an entire body of ideas

that expresses/interprets many aspects of a

phenomenon

Technologies

Edward Jenner and the introduction of smallpox vaccine

Early experiments on the sources of microorganism led to the

profound realization that MICROBES are EVERYWHERE

Edward Jenner (1749-1823)

• Father of Immunology

• Pioneer of vaccination

• 1796:Inoculation with cowpox gave immunity to smallpox,

was an immerse medical breakthrough & saved countless

lives

• 1967: World Health Organization (WHO) began a global

vaccination program

• 1980: The disease was officially declared eradicated

Discovery of Spores & Sterilization

John Tyndall

(1820-1893)

• Irish scientist

• Discovered some bacteria existed in

o Heat sensitive form (vegetative cell)

o Heat stable form (endospore)

• Need prolonged/intermittent heating to

destroy

• Resulted a method of sterilizing liquid by

heating it to boiling point (tyndallization) on

successive days

Ferdinand Cohn

(1828-1898)

• German botanist

• Discovered heat resistant forms of

bacteria (Endospore) Bacillus & Clostridium

spp.

• Sterilization technique requires the

elimination of all lifeforms including

endospores & virus

• Laid ground work for bacterial

classification

Development of Aseptic Techniques

Dr. Oliver Wendell Holmes Sr (1809 - 1894)

• Mothers of home births had fewer

infections (puerperal fever) than those who

gave birth in hospitals (US)

Dr. Ignaz Semmelweis (1818 – 1865)

• correlated infections with physicians

coming directly from autopsy room to

maternity ward

Joseph Lister (1827 – 1912)

• British surgeon and scientist

• introduced aseptic techniques reducing

microbes in medical settings and preventing

wound infections

• Involved disinfection of hands using

chemicals prior to surgery

• Use of heat for sterilization

The human body is a source of infection

Robert Koch

(1843-1910)

Founder of Bacteriology

Many diseases are caused by the growth of microbes

in the body and not by sins, bad character, or

poverty, etc.

Two major contributors:

• Louis Pasteur

• Robert Koch

• German scientist

• Demonstrated role of bacteria in causing disease

• Established Koch’s postulates :a sequence of

experimental steps that verified the germ theory

• Identified cause of anthrax, TB, and cholera

• Developed pure culture methods

Discovery of Pathogens & Germ Theory of Disease

Pure cultures and Microbial Taxonomy

Pure culture

“Suspected pathogen must be

isolated and grown away from

other microorganisms in

laboratory culture”

Walther Hesse & Koch

When a solid surface

incubated in air, masses of

microbial cells developed,

each having a characteristic

shape and colour. Solid

media provided an easy way

to obtain pure cultures.

Developed the transparent

double-sided “Petri dish” in

1887, standard tool for

obtaining pure cultures

Richard Petri

• Taxonomy: organizing, classifying, and naming living

things

o Formal system originated by Carl von Linné

• Concerned with:

o Classification: orderly arrangement of organisms into

groups (taxa) on the basis of similarities or

relationships

-May applied to existing named taxa or newly described taxa

o Nomenclature: assigning names to taxonomic groups

o Identification: determining and recording traits of

organisms for placement into taxonomic schemes

• Require knowledge of their morphologic, biochemical,

physiological, and genetic characteristics

• Importance of microbial taxonomy

v Allows scientists to organize huge amounts of knowledge

v Allows scientists to make predictions and frame hypotheses

about organisms

v Places organisms in meaningful, useful groups with precise

names, thus facilitating scientific communication

v Essential for accurate identification of microorganisms

Taxonomy

Domain - Archaea, Bacteria, & Eukarya

• Kingdom

• Phylum or Division

• Class

• Order

• Family

• Genus

• Species

Levels of classification

• Binomial (scientific) nomenclature

• Gives each microbe 2 names:

o Genus - capitalized

o species - lowercase

• Both italicized or underlined

o Staphylococcus aureus (S. aureus)

• Inspiration for names is extremely varied and

often imaginative

Assigning Scientific Names

• Cell shape

Ø Spherical/ovoid- coccus (cocci)

Ø Cylindrically shaped- rod/bacillus

Ø Curved/loose spiral shapes – spirilla

Ø Long, thin cells/chains of cells-filamentous

Ø Tightly coiled/ extensions of cells as long tubes/stalks-spirochete

• Some Bacteria & Archaea remain together in groups/clusters after

cell division

-----------------------------------------------------------------------------------------------------------------

Can be oval/elongated/fattened on one side

Diplococci: A pair of attached cocci. Remain attached after dividing

some cocci form long chains (Streptococcus)

Tetrads: Groups of four. Divide in two planes

Three-planes & attached in cubelike groups of eight (Sarcinae)

Multiple planes & form grapelike clusters/ broad sheets (Staphylococcus)

-

------------------------------------------------------------------------------------------------------------------------------------

Varied rod shapes (blocky, spindle shaped, round ended, long & threadlike

(filamentous), clubbed/ drumstick shapes)

Diplobacilli: A pair of attached bacilli. Remain attached after dividing

Streptobacilli: Chain like arrangement

Coccobacillus: Intermediate shape between coccus & bacillus. Oval rods

Have one or more twists-- Vibrio: comma shaped cell. Look like curved rods

-----------------------------------------------------------------------------------------------------------------------------------

Cell morphology

Cocci

Bacilli

Spirilla: Helical, corkscrew shaped bacteria with rigid bodies, use whip like external flagella to move

Spirochetes: Helical bacteria with flexible bodies, use axial filaments (internal flagella) to move

Spiral/curvi form shaped bacteria

Cell size

• Cells of Bacteria & Archaea vary in size (0.2 μm - >700 μm)

-rod-shaped species 0.5 & 4 μm wide & < 15 μm long

-Epulopiscium fishelsoni (0.6 mm in length)

-Thiomargarita namibiensis (750 μm in diameter)

• Small cells - more surface area relative to cell volume than

large cells >>higher surface-to-volume (s/v) ratio

-higher S/V ratio of small cells: faster rate of nutrient & waste

exchange per unit of cell volume

- Free-living smaller cells tend to grow faster than larger cells &

for a given amount of resources (nutrients for growth)

• Prokaryotic cells grow faster & evolve more rapidly than

eukaryotic cells

Halophilic archaean, Haloquadratum walsbyi, discovered in 1980

by AE Walsby, in a coastal hypersaline pool on the Sinai Peninsula

in Egypt. Cultured only in 2004. Thin (0.15 um) square-shaped

structure.

Cell membrane

cytoplasmic membrane (plasma membrane/inner membrane)

• Thin structure lying inside cell wall & enclosing cytoplasm of cell

• “gatekeeper”- for entrance and exit of dissolved substances

• 8–10 nm wide, physically weak [prokaryotes by phospholipids & protein, some

bacteria strengthened by hopanoids, eukaryotes by carbohydrates & sterols

(cholesterol)]

• Phospholipid [both hydrophobic (water-repelling) & hydrophilic (water attracting)

components] bilayer containing embedded proteins

• Bacteria and Eukarya: hydrophobic component-fatty acids & hydrophilic component

of a glycerol molecule containing phosphate & one of several other functional groups

(e.g. sugars, ethanolamine/choline) bonded to the phosphate

• Archaea: Constructed from either phosphoglycerol diethers [C20 side chains (phytanyl

group)/ or diphosphoglycerol tetraethers (C40 side chains (biphytanyl group)]

• Three major functions:

Ø cell’s permeability barrier, preventing the passive leakage of solutes into or out

of the cell

Ø cytoplasmic membrane anchors several proteins that catalyze a suite of key

cell functions

Ø cytoplasmic membrane of Bacteria and Archaea plays a major role in energy

conservation and consumption

Peripheral membrane proteins are loosely attached & some are lipoproteins

[proteins that contain a hydrophobic lipid tail that anchors the protein into the

membrane]

Peripheral membrane proteins

typically interact with integral membrane proteins that involved in important

cellular processes (e.g. energy metabolism and transport)

Fatty acids point inward toward each other to

form hydrophobic region

Hydrophilic portion exposed to either

environment/cytoplasm & interacts with

cytoplasmic milieu

This membrane structure is called a lipid

bilayer/a unit membrane because each

phospholipid “leaf” forms half of the unit

Proteins embedded in the membrane

>>integral membrane proteins

Peripheral membrane proteins are more

loosely attached

• Other functions:

Ø Breakdown of nutrients & production of energy

Ø Synthesis of cell wall components

Ø Assists with DNA replication

Ø Site of photosynthesis: Photosynthetic bacteria have membrane

extensions called thylakoids, where photosynthesis occurs.

Ø Secretes proteins

Ø Contains bases of flagella

Ø Responds to chemical substances in the environment

Impermeable

large proteins, ions, and most polar molecules–larger than pores in integral

proteins that function as channels

Permeable

smaller molecules (water, O2, CO2, simple sugar) easily pass through

Substances that dissolve easily in lipids (O2, CO2, non-polar organic

molecules) enter & exit easily than other substances>>>membrane consist

mostly phospholipids

Movement of Materials Across Membranes

Passive Active

• Substances cross

membrane from

high

concentration>>low

concentration

• Move with

Concentration

gradient/different

• Without any

expenditure of

energy (ATP)

• Move substances

from low

concentration

>>high

concentration

• Use energy (ATP)

Passive Processes

Simple diffusion

• Net movement of molecules/ions from high

concentration>>>low concentration.

• Equilibrium: Net movement stops when molecules are evenly

distributed

• Used by cells to transport small molecules (O2, CO2) across

their membranes

Facilitated diffusion

• Net movement of molecules/ ions from high concentration to

low concentration

• Substance to be transported combines with a carrier protein

in plasma membrane

• Extracellular enzymes may be used to break down large

substances before they can be moved into the cell by

facilitated diffusion

Osmosis

• Net movement of water (solvent) molecules across a semipermeable membrane from high

concentration to low concentration

• Osmotic Pressure: Pressure required to prevent the movement of pure water into a solution

• Bacterial cells can be subjected to three different types of osmotic solutions:

Isotonic (equal)

Ø Concentration of solutes equals that found inside a cell

Ø Waters leaves & enters cell at the same rate (no net change)

Ø Cell’s contents are in equilibrium with solution outside cell wall

Hypotonic (hypoosmotic, under/less)

Ø Concentration of solutes outside the cell is lower than that inside the cell

Ø Net movement of water into the cell

Ø Most bacteria live in hypotonic environments, swelling is contained by cell wall, G-

may burst/osmotic lysis>>excessive water intake

Hypertonic (above/more)

Ø Solute concentration is higher outside the cell

Ø Net movement of water out of the cell

Ø Most bacterial cells shrink & collapse/plasmolyze >>water leaves the cells by

osmosis

Active Processes

Active Transport

• Requires carrier proteins or pumps in plasma membrane

Group Translocation

• Similar to active transport, but substance transported is chemically altered during

process

• After modification, the substance remains inside cell

• Important for cells to accumulate various substances even though they in low

concentrations outside the cell

• Require energy supplied by high energy phosphate compounds

[phosphoenolpyruvic acid (PEP)]

• E.g. glucose is phosphorylated during group translocation in bacterial cells.

• Endocytosis (phagocytosis, pinocytosis, etc.) does not occur in procaryotic cells

Cell wall

Semirigid structure that lies outside the cell membrane in almost all bacteria

confers structural strength on the cell to keep it from bursting due to osmotic pressure

• Cell wall thin (8-12 nm) , wavy

• Two layer lipid membrane

• With outer membrane (lipopolysaccharide, LPS)

• Periplasmic space present in all

• Peptidoglycan, lipopolysaccharide, lipoproteins

• Porins proteins, more lipid

• Less peptidoglycan & less penetrable

• More resistance to molecules

• Gram reaction: pink/red

Negative Positive

• Cell wall thick (20-80 nm), smooth

• One layer lipid membrane

• No outer membrane

• Periplasmic space present in some

• Peptidoglycan, teichoic acid & lipotechoic acid

• No porins proteins, less lipid

• More peptidoglycan & penetrable

• Less resistance to molecules

• Gram reaction: blue/purple

N-acetylglucosamine (NAG)

N-acetylmuramic acid (NAM)

Gram Stain Mechanism

Based on differences in structure of cell wall (G+ & G-)

& reaction to various reagents

• Crystal violet (primary stain)

Dye enters cytoplasm

• Iodine (mordant)

Forms large crystals with dye that difficult to escape

through cell wall

• Alcohol

G+:Dehydrate peptidoglycan>> make it more

impermeable to crystal violet iodine

G-: colourless, dissolve outer membrane & leaves small

holes in thin peptidoglycan layer through crystal violet

iodine diffuse

• Safranin (counterstain)

G->>pink

*G+ cells----->G- response>>>>cells dead

Bacillus, Clostridium, Mycobacterium-gram variable

LPS has two components:

• O polysaccharides: Antigens, used to identify

bacteria

• Lipid A: Endotoxin causes fever and shock

Porins: Membrane proteins that allow the passage of

nucleotides, disaccharides, peptides, amino acids

Cell walls containing:

• Polysaccharides

• Proteins/glycoproteins or

• Some mixture of these macromolecules

Certain methane-producing Archaea (methanogens) contain

pseudomurein (similar to peptidoglycan)

Archaea lack pseudomurein contain other polysaccharides

• Methanosarcina sp. have thick polysaccharide walls composed of

polymers of glucose, glucuronic acid, galactosamine uronic acid, and

acetate

• Extremely halophilic (salt-loving) Archaea e.g. Halococcus, contain

large amounts of sulfate in cell wall

• Paracrystalline surface layer (S-layer) S-consist of interlocking

molecules of protein/glycoprotein

-Methanocaldococcus jannaschii

• Functions:

Ø serving as protection from osmotic lysis

Ø as the interface between the cell and its environment (selective

sieve) allowing the passage of low-molecular-weight solutes,

excluding large molecules/structures (viruses/lytic enzymes)

Ø Retain proteins near the cell surface that must function outside the

cytoplasmic membrane

Archaea

Atypical cell walls

No walls/very little wall material

Mycoplasmas

• No cell wall, smallest known bacteria that can grow and reproduce

outside of host cells

• Pass through most bacterial filters

• Unique plasma membrane contains lipids (sterols) >>protect them

from osmotic lysis

• Mycoplasma pneumoniae is the cause of primary atypical bacterial

pneumonia (walking pneumonia)

Archaebacteria

• May lack cell walls/have cell walls without peptidoglycan

• Composed of protein, polysaccharides/peptidoglycan-like molecules,

but never do they contain murein (pseudomurein) * Unique feature

distinguishes the bacteria from the Archaea

Cytoplasm

• Substance of cell inside plasma membrane

• Thick, aqueous, semitransparent, elastic

• Contains:

Ø 80% water

Ø Proteins

Ø Carbohydrates

Ø Lipids

Ø Inorganic ions (much higher concentration)

Ø Low molecular weight compounds

• Prokaryotes: major structures

Ø Nuclear area (containing DNA)

Ø Ribosomes

Ø Inclusions

• Prokaryotic lacks certain features of eukaryotic cytoplasm

Ø Cytoskeleton

Ø Cytoplasmic streaming

The Nuclear Area (nucleoid)

• Contains single chromosome, a long circular molecule of

double stranded DNA (bacterial chromosome- not

surrounded by nuclear envelope [membrane] & not include

histones)

• Can be spherical/elongated/dumbbell shaped

• Chromosome is attached to the plasma membrane, protein

in plasma membrane>>replication of DNA &segregation of

new chromosome to daughter cells in cell division

• Occupy 20% of the intracellular volume of active cells

Plasmids

• Small, circular, double stranded DNA molecules

• Extrachromosomal genetic elements that not connected to

bacterial chromosome, replicate independently of

chromosomal DNA

• Associated with plasma membrane proteins

• Contain from 5 -100 genes that are usually not essential for

survival of bacterium under normal environment conditions

• Found in many bacterial cells in addition to chromosomal

DNA

• Carried genes for:

Ø Antibiotic resistance

Ø Tolerance to toxic metals

Ø Production of toxins

Ø Synthesis of enzymes

• Transferable, plasmid DNA is used for gene manipulation in

biotechnology

Ribosomes

• Site of protein synthesis (translation)

• Present in all eucaryotic and procaryotic cells.

• Made up of protein and ribosomal RNA (rRNA).

• Procaryotic ribosomes (70S) are smaller and less dense than

eucaryotic ribosomes (80S)

Ø Prokaryotic:small subunit (30S) & large subunit (50S)

Ø Eukaryotic:small subunit (40S) & large subunit (60S)

• S is Svedberg units indicate relative rate of sedimentation

during ultra high speed centrifugation [sedimentation rate:

function of size, weight & shape of a particle]

• Several antibiotics (streptomycin, gentamycin, erythromycin,

chloramphenicol) work by inhibiting protein synthesis by

procaryotic ribosomes, without affecting eukaryotic ribosomes

Inclusions

• Reserve deposits in the cytoplasm of cells

• Some are common in bacteria, others are limited (serve as a

basis for identification)

Metachromatic Granules (volutin)

• Large inclusions

• Stain red with blue dyes (e.g. methylene blue)

• Contain inorganic phosphate (polyphosphate) that can be used

in the synthesis of ATP

• Found in bacteria, algae, protozoa, and fungi

• Characteristic of Corynebacterium diphtheriae, causative agent

of diphtheria

• Useful for identification purposes

Polysaccharide Granules

• Consist of glycogen & starch

• Carbon & energy reserves

• Glycogen granules stain reddish brown & starch granules

appear blue with iodine

Lipid inclusions

• Common lipid storage material

• Polymer-β-hydroxybutyric acid-unique to bacteria

• Revealed by staining cells with fat soluble dyes (Sudan dyes)

• E.g. Mycobacteria, Bacillus, Azotobacter, Spirillum

Sulfur Granules

• Contain sulfur and sulfur containing compounds.

• “Sulfur bacteria” (Thiobacillus) obtain energy by oxidizing

sulfur and its compounds

Carboxysomes

• Contain enzyme ribulose 1,5-diphosphate carboxylase,

necessary for carbon fixation during photosynthesis

• Found in nitrifying bacteria, cyanobacteria, and thiobacilli

Gas vacuoles

• Hollow cavities found in many aquatic bacteria.

• Each vacuole consists of row of several individual gas

vesicles that are hollow cylinders covered by protein

• Used to regulate buoyancy so cells can remain at

appropriate water depth>>> to receive sufficient O2, light,

nutrients

Magnetosomes

• Contain iron oxide (Fe3O4 ), which acts like a magnet

• Formed by several G- bacteria to move downward until reach a suitable

attachment site

• May protect cells against hydrogen peroxide accumulation

• Magnetite from bacteria can be used in the production of magnetic tapes

for sound & data recording

Endospores

• “resting” cells formed by certain G+ bacteria

Ø Bacillus, Anoxybacillus, Paenibacillus, Clostridium

Ø Sporomusa ovata, Coxiella spp., Acetonema spp. (G-ve)

• Highly durable dehydrated cells with thick cell walls and additional layers

• Formed internal to bacterial cell membranes

• Can survive extreme temperatures, disinfectants, acids, bases, lack of water, toxic chemicals,

and radiation

• Sporulation/sporogenesis: Process of endospore formation within a vegetative (parent) cell

take several hours

• Important for survival during adverse environmental conditions

Ø Type of dormant cell, intracellular structures

Ø Formed by vegetative cells in response ~ limiting factor

Ø Highly resistant to environmental stresses

• The endospore might be located terminally (at the end), subterminally (near one end),

centrally inside the vegetative cell

• The water present in forespore cytoplasm is eliminated by the time sporulation is complete &

endospore do not carry out metabolic reactions

• Contains only DNA, small amount of RNA, ribosomes, enzymes, few important small

molecules, dipicolinic acid, calcium ions

• Retain viability under appropriate environmental condition

• Germination: The return of an endospore to its vegetative state, triggered by

physical/chemical damage to the endospore’s coat

• Important in clinical & food industry because they are resistant to processes that kill

vegetative cells (heating, freezing, desiccation, chemicals, radiation)

• Can survive in boiling water for several hours or more, thermophilic bacteria surviving in

boiling water for 19 hours

Endospores

• One cell produces one spore

1. Spore septum: Newly replicated DNA is

isolated by an ingrowth of the

plasma membrane

2. Spore septum becomes a double-layered

membrane that surrounds

chromosome and cytoplasm (forespore)

3. Thick layers of peptidoglycan are laid down

between the two membrane

layers of forespore

4. Spore coat forms: Thick layer of protein

around the outer membrane. This

coat makes endospore resistant to many

harsh chemicals

5. Maturation: Cell wall ruptures, endospore is

released

• Bacterial motility is typically

provided by structures known

as flagella.

• The eukaryotic flagellum, which

operates as a flexible whip-like tail

utilizing microtubules that are

powered by ATP.

• The bacterial flagellum is rigid in

nature, operates more like the

propeller on a boat, and is powered

by energy from the proton motive

force.

Flagella

There are three main components to the

bacterial flagellum:

the filament – a long thin appendage that

extends from the cell surface. The filament

is composed of the protein flagellin and is

hollow. Flagellin proteins are transcribed in

the cell cytoplasm and then transported

across the cell membrane and cell wall. A

bacterial flagellar filament grows from its

tip (unlike the hair on your head), adding

more and more flagellin units to extend the

length until the correct size is reached. The

flagellin units are guided into place by a

protein cap.

the hook – this is a curved coupler that

attaches the filament to the flagellar motor.

the motor – a rotary motor that spans both

the cell membrane and the cell wall, with

additional components for the gram

negative outer membrane.

The motor has two components: the basal

body, which provides the rotation, and

the stator, which provides the torque

necessary for rotation to occur.

The basal body consists of a central shaft

surrounded by protein rings, two in the

gram positive bacteria and four in the gram

negative bacteria.

The stator consists of Mot proteins that

surround the ring(s) embedded within the

cell membrane.

Rotation of the flagellar basal body occurs

due to the proton motive force, where

protons that accumulate on the outside of

the cell membrane are driven through pores

in the Mot proteins, interacting with

charges in the ring proteins as they pass

across the membrane. The interaction

causes the basal body to rotate and turns

the filament extending from the cell.

Rotation can occur at 200-1000 rpm and

result in speeds of 60 cell lengths/second

(for comparison, a cheetah moves at a

maximum rate of 25 body lengths/second).

Flagella

• long, thin appendages (15–20 nm wide) free at

one end & anchored into the cell at the other

end

• Tiny rotating machines that function to push/pull

the cell through a liquid

• Present in many bacteria [flagellum (plural,

flagella)] and Archaea [archaellum (plural,

archaella)]

• A bacterium may have one/several flagella Filament

o Outermost region

o Contains globular protein

(flagellin)

o Not covered by a sheath like

eucaryotic filaments

Hook

o Wider segment that anchors

filament to basal body

Basal Body

o Complex structure with a central

rod surrounded

by a set of rings

o Fli proteins as the motor switch,

reversing the direction of rotation of

the flagella in response to intracellular

signals

Gram negative Gram positive

Gram-negative bacteria

Ø L ring> anchored in outer membrane

Ø P ring>anchored in peptidoglycan layer

Ø MS & C rings> located within cytoplasmic

membrane & cytoplasm

Gram-positive bacteria

Ø Only inner pair of rings present

Ø Surrounding inner ring & anchored in the

cytoplasmic membrane & peptidoglycan

are a series of proteins called Mot proteins

Bacterial flagella move by rotation from basal body

Flagellar movement may be either clockwise

(CW)/counterclockwise (CCW)

Bacteria may be capable of several patterns of

motility

Ø Runs/swims: bacterium moves in one direction

Ø Tumbles: bacterium changes direction. Caused by

reversal of flagellar rotation

Some bacteria are motile but lack flagella they move by gliding

-motility is a slower & smoother form of movement & typically

occurs along the long axis of the cell

Taxis

Movement of a cell toward or away from various stimuli

Chemotaxis: Movement in response to a chemical stimulus

Phototaxis: Movement in response to a light stimulus

enhance a cell’s access to resources/allow it to avoid harmful

substances that could damage or kill it

The occurrence of tactic behavior provides evidence for the

ecological (survival) advantage of flagella in bacteria and other

prokaryotes

Rotation can occur in a clockwise (CW) or

a counterclockwise (CCW) direction, with different

results to the cell. A bacterium will move forward,

called a “run,” when there is a CCW rotation, and

reorient randomly, called a “tumble,” when there is a

CW rotation.

Fimbriae

Bristle like short fibres (0.03-0.14 μm) occurs on

the surface of a cell, shorter in length than pili

Made up of fimbrillin protein

Present on both Gram positive & negative

bacteria

§ Salmonella typhimurium

§ Shigella dysenteriae

Enable cells to stick to surfaces including

• animal tissues in the case of pathogenic

bacteria

• form pellicles (thin sheets of cells on a liquid

surface)

• biofilms on solid surfaces

Not for motility, not receptor for viruses

Similar to fimbriae but are typically longer (0.5-2 μm) only

one/few pili are present on surface of a cell

Made up of pilin protein

Found in all gram-negative bacteria & many gram-positive

bacteria

§ Escherichia coli

§ Pseudomanas

§ Neisseria gonorrhoeae

Function:

o facilitating genetic exchange between cells in conjugation

(conjugative/sex pili)

o Enabling adhesion of pathogens to specific host tissues that

they subsequently invade (type IV & other pili)

Can be receptors for certain types of viruses

Important virulence factor & pathogenicity

Pili

Eukaryotic flagella and cilia

Used for cellular locomotion/moving

substances along the surface of cell

Contain cytoplasm and are enclosed by

plasma membrane

Flagella :projections are few and long in

relation to the size of cell

Cilia : projections are numerous & short

resembling hair

Anchored to plasma membrane by a basal

body, and both consist of nine pairs of

microtubules (doublets) arranged in ring,

plus another two microtubules in the center

of the ring (9+2 array or 9+0 array)

Microtubules: long hollow tubes made up of

a protein called tubulin

Eukaryotic flagellum moves in wavelike

manner

Capsule and Slime Layer

Many bacteria & Archaea secrete sticky/slimy materials

(polysaccharide/protein) on their cell surface

§ not considered part of the cell wall because not confer

significant structural strength on the cell

These layers referred as “capsule” & “slime layer”

Capsule: layer that organized in a tight matrix that excludes small

particles and is tightly attached

Slime layer: polysaccharide substance that is loosely attached to the cell

wall

Function:

o assist in the attachment of microorganisms to solid surfaces

o development and maintenance of biofilms

o acting as virulence factors & preventing dehydration

§ Bacillus anthracis (anthrax)-thick capsule of protein

§ Streptococcus pneumoniae (pneumonia)-thick capsule of

polysaccharide

*Encapsulated cells>>avoid destruction by the host’s

immune system

o outer surface layers of virtually any type bind water and likely protect

the cell from desiccation in periods of dryness

Eukaryotic cell wall

Most eukaryotic cells have cell wall, much simpler than prokaryotic

Ø Algae,plants and some fungi: Cellulose

Ø Fungi: Chitin (polysaccharide)

Ø Yeasts: Glucan and mannan (polysaccharides)

Glycocalyx

A layer of material containing substantial amounts of sticky

carbohydrates

Covalently bonded to proteins & lipids in plasma membrane, forming

glycoproteins and glycolipids that anchor glycocalyx to the cell

Strengthens the cell surface, help attach cells together and contribute

to cell cell recognition

Plasma (cytoplasmic) membrane

• Very similar in function & basic structure with procaryotic

• Have different in the types of membrane proteins

• Contain carbohydrates that are important for cell-cell recognition and serve

as sites for bacterial attachment

• Contain sterols (not found in prokaryotic plasma membranes) which

associated with the ability of membranes to resist lysis resulting from

increased osmotic pressure

• Movement across eucaryotic cell membranes:

Ø Simple diffusion

Ø facilitated diffusion

Ø Osmosis

Ø active transport

• Group translocation does not occur in eukaryotic cells

• Can use endocytosis: Process in which plasma membrane encircles particles

outside of cell and bring it into the cell

• Two important types of endocytosis:

Ø Phagocytosis

-Cellular projections called pseudopods engulf particle and bring them into the

cell

-Used by white blood cells to destroy bacteria & foreign substances

Ø Pinocytosis

- Plasma membrane fold inward, bringing extracellular fluid into the cell along

with whatever substances are dissolved in the fluid

Cytoplasm

• Encompasses substance inside the plasma

membrane and outside the nucleus

• Has a complex internal structure,

consisting microfilament, intermediate

filaments & microtubules >>>cytoskeleton:

a complex network of thread and tube-like

structures, which provides support, shape,

and movement.

• Helps distribute nutrients and move the

cell over surface (cytoplasmic streaming)

• Many important enzymes found in

cytoplasmic fluid of prokaryotes are

sequestered in organelles of eukaryotes

Nucleus

• Usually spherical/oval frequently the largest structure

& contain DNA

• Surrounded by nuclear envelope

• Nuclear pores-tinny channels in the membrane

Ø allow nucleus to communicate with cytoplasm

Ø Control movement of substances between

nucleus & cytoplasm

• Nucleolus: Dense region where ribosomes are made

• DNA (genetic material) is combined with histones and

exists in two forms:

Ø Chromatin (Loose, threadlike DNA)

Ø Chromosomes (Tightly packaged DNA, found

during cell division)

• Functions

Ø House and protect cell’s genetic information

(DNA)

Ø Ribosome synthesis

Endoplasmic Reticulum (ER)

• An extensive network of flattened membranous sacs/tubules

(cisterns)

• Two types of ER:

Ø Rough Endoplasmic Reticulum (RER)

o continuous with nucleus membrane & usually unfolds into a

series of flattened sacs

o Outer surface is studded with ribosomes

o Protein synthesized by ribosomes attached to rough ER enter

cisterns within ER for processing & sorting

o Functions:

v Synthesis and modification of proteins

v Synthesis of cell and organelle membranes

v Packaging, and transport of proteins that are secreted

from the cell e.g.: Antibodies

Ø Smooth Endoplasmic Reticulum (SER)

o Extends from rough ER to form a network of membrane

tubules

o Without ribosomes on the outer surface of its membrane

o Functions:

v Lipid Synthesis: Phospholipids, fatty acids, and steroids

(sex hormones)

v Breakdown harmful substances (alcohol, antibiotics,

etc.)

v Helps develop tolerance to drugs and alcohol

v Regulates sugar release from liver into the blood

v Calcium storage for cell and muscle contraction

Ribosomes

• Site of protein synthesis (translation), present in all

eucaryotic & procaryotic cells

• Made up of protein and ribosomal RNA (rRNA)

• May be found free in the cytoplasm or associated

with the rough endoplasmic reticulum (RER)

• Eucaryotic ribosomes (80S):larger & more dense

than procaryotic ribosomes (70S)

• Eucaryotic ribosomes have two subunits:

Ø Large subunit: 60S

Ø Small subunit: 40S

• Free ribosomes unattached to any structure in

cytoplasm>>synthesize proteins

• Membrane bound ribosomes attached to nuclear

membrane & endoplasmic reticulum>>> synthesize

proteins destined for insertion in plasma

membrane/export from the cell

• Located within mitochondria>>synthesize

mitochondrial proteins

Golgi complex

• Consists of 3-20 cisterns that resemble a stack of pita break,

cisterns are often curved, giving a cuplike shape

• Works closely with the ER to secrete proteins

• Functions:

Ø Receiving side receives proteins in transport vesicles from

ER

Ø Modifies proteins into final shape, sorts, and labels them

for proper transport

Ø Shipping side packages and sends proteins to cell

membrane for export or to other parts of the cell

Ø Packages digestive enzymes in lysosomes

Lysosomes

• Formed from Golgi complexes & look like membrane enclosed

sphere

• Have only a single membrane & lack internal structure

• containing at least 40 different digestive enzymes, which can

break down carbohydrates, proteins, lipids, and nucleic acids

• Optimal pH for lysosomal enzymes is ~5

• Found mainly in animal cells

• Functions:

Ø Molecular garbage dump and recycler of macromolecules

(e.g.: proteins)

Ø Destruction of foreign material, bacteria, viruses, and old/

damaged cell components. Important in immunity

Ø Digestion of food particles taken in by cell

Vacuoles

• Membrane bound sac

• Different types, sizes, shapes, and functions:

• Central vacuole: In plant cells. Store starch, water, pigments, poisons, &

wastes. May occupy up to 90% of plant cell volume

• Contractile vacuole: Regulate water balance, by removing excess water

from cell. Found in many aquatic protists

• Food or Digestion Vacuole: Engulf nutrients in many protozoa (protists).

Fuse with lysosomes to digest food particles

Mitochondria (Singular: mitochondrion)

• Spherical/rod shaped organelles appear throughout the

cytoplasm of most eukaryotic cells

• Structure:

Ø Inner/outer membrane

Ø Intermembrane space

Ø Cristae (inner membrane extensions)

Ø Matrix (inner liquid)

• Number of mitochondria per cell varies among cell types

• Powerhouses of the cell-Site of cellular respiration:

Food (sugar) + O2-----> CO2 + H2O + ATP

• Change chemical energy of molecules into the useable

energy of the ATP molecule

• Contain their own DNA, 70S ribosomes, & machinery

necessary to replicate, transcribe & translate the information

encoded by their DNA

• Can reproduce more/less on their own by growing & dividing

in two

Chloroplasts

• A membrane enclosed structure that contains both the

pigment chlorophyll & the enzymes required for the light

gathering phases of photosynthesis

• Disc shaped with three membrane systems:

Ø Outer membrane: Covers chloroplast surface.

Ø Inner membrane: Contains enzymes needed to make

glucose during photosynthesis. Encloses stroma

(liquid) and thylakoid membranes.

Ø Thylakoid membranes: Contain chlorophyll, green

pigment that traps solar energy, stacks of thylakoids

are called grana (singular: granum)

• Capable of multiplying on their own within the cell-by

increasing in size and then dividing in two

Bacterial Growth

• Growth is defined as an increase in the number of cells ,

not an increase in the size of the individual cells

• Bacteria normally reproduce by binary fission (“binary”-

two cells have arisen from one)

cells elongate to

~twice their

original length

then form a

partition that

constricts the cell

into two daughter

cells

• Some variations in binary fission

Ø Bacillus subtilis>>septum forms without cell wall

constriction

• A few bacterial species reproduce by budding

• Form a small initial outgrowth (bud) that enlarge until its size approaches

that of the parent cell and then it separate that yields totally new daughter

cell, with the mother cell retaining its original identity

Generation time

• Time required for a cell to divide and its population to

double

• Under ideal circumstances, a growing bacterial population

doubles at regular intervals

• In bacteria, each new fission cycle/generation increases the

population by a factor of 2/double of it

Growth is by geometric progression:

n=the number of generations

Assumption:

Individual generation time is the same for all cells in the

population

• Varies among organisms & with environmental conditions

(e.g. temperature)

• Most bacteria generation time: 1-3 hrs

Generation time

Mean growth rate constant (k) = n/t

Generation time (g) or doubing time = t/n

Bacterial Growth Curve

• Growth of cells over time

• Four basic phases

Ø Lag

Ø Log

Ø Stationary

Ø Death

Lag phase

• Immediately after inoculation of the cells into fresh medium,

population remains temporarily unchanged (adaptation to

new environment)

• No apparent cell division occurring, cells may be growing in:

Ø Volume/mass

Ø Synthesizing enzymes

Ø Proteins

Ø RNA

Ø Increasing in metabolic activity

• Length of the lag phase is dependent on a wide variety of

factors:

Ø Size of inoculum

Ø Time necessary to recover from physical damage/shock

in the transfer

Ø Time required for synthesis of essential

coenzymes/division factors

Ø Time required for synthesis of new (inducible) enzymes

that are necessary to metabolize the substrates present

in the medium

• Even through population of cells is not increasing , individual

cells are metabolically active

Exponential (log) phase

• Best condition for growth

• All cells are diving regularly by binary fission & are growing by geometric

progression

• Cells divide at a constant rate depend on :

Ø Composition of the growth medium

Ø Conditions of incubation

• Rate of exponential growth of a bacterial culture is expressed as generation

time, also the doubling time of the bacterial population

• Growth is balanced and genetically coordinated

Stationary phase

• Population growth (size) is limited

Ø Exhaustion of essential nutrients

Ø Accumulation of inhibitory metabolites/end product

Ø Depletion of oxygen

Ø Development of an unfavourable pH

Ø Exhaustion of space, lack of “biological space”

• Number of cells able to divide (viable cells)= number that are unable

to divide (non viable cells)

• Like lag phase, is not necessarily a period of quiescence

• Bacteria produce secondary metabolites (e.g. antibiotics) during

stationary phase of the growth

• Secondary metabolites are defined as metabolites produced after

active stage of growth

• During stationary phase, spore forming bacteria have to

induce/unmask the activity of dozens of genes that may be involved

in sporulation process

Death phase

• Very short phase,

• Viable cell population declines (turbidimetric measurements/ microscopic

counts cannot observe death phase)

• Number of viable cells decreases geometrically (exponentially), essentially

the reverse of growth during log phase

• Factors contribute to cell death:

Ø Cell lysis by autolytic enzymes

Ø Effects of toxic metabolites

Direct measurement of Microbial Growth

Plate counts

• Measure number of viable cells (≥24 hrs to form)

• A colony is from short segments of a chain/from a

bacteria clump

• Reported as colony forming units (CFU)

• The Food & Drug administration convention count

only plates with 25-250 colonies/30-300 colonies

• To obtain countable colony counts, serial dilution

needed on the original inoculum

• Time consuming & tedious to perform, used only

viability/in some statutory tests of food/drinking

water

Guidelines for calculating the cfu Guidelines for calculating the colony forming unit per g or mL

ü 25 – 250 or 30 – 300 (excluded by spreaders or lab accidents) colonies &

average the counts

v If …..

Ø Only one plate of a duplicate pair yields 25 – 250 or 30 – 300 colonies

count both plates, unless excluded by spreader or lab accidents

Ø If count ratio > 2

take lowest value

Ø Spreaders

count area that has well distributed cfus and estimate counts by multiplying

total area

Ø No colonies

check for inhibitory substances, if none, report estimated count as less than

the lowest dilution

Ø No plate with 25 – 250 or 30 – 300 colonies & ≥ 1 plates have more than 25 – 250

or 30 – 300 colonies

Select plate(s) having nearest to 250 or 300 colonies & report count as est. CFU

per ml/g

Ø All plates have fewer than 25 – 250 or 30 – 300 colonies

Record the actual number of colonies on the lower dilution & report count as

est. CFU per ml/g

TNTC: Too numerous to count

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

* 1 ml was added in each plate

Ø Crowded plates (>250 or 300 colonies)

Count colonies in portion of the plate that are representative of colony

distribution

Total area of Petri plates 56 cm2, if colony < 10 colonies/cm2, count 12 boxes (6

vertical and 6 horizontal across the plates= 12cm2)

v If colony > 10 colonies/cm2, count 4 boxes or squares with each 1 cm2

v When > 100 colonies/cm2, report as greater than (>) the plate area 100 x

56 x highest dilution factor (not as TNTC)

Ø Spreader or spreading colony > 50% of plate area

label as Spr. If not, count area that has well distributed cfus and estimate

counts by multiplying total area

Filtration

• Rapid method to estimate bacterial populations in water

• useful when evaluating large sample volumes or performing coliform

tests

• Volume of sample depends on types of sample

Ø drinking water/bottled water: 100 ml

Ø Polluted water: 0.001-10

• If sample volume <10 ml, mix the sample with 10-100 ml of sterile

distilled water/deionized water before filtration (ensure an even

distribution of bacteria across the entire filter surface)

• Non-potable water samples must be diluted to a level at which the

bacteria can be measured

• Sample volume should be adjusted to obtain plates with 20 to 200

CFU/filter

Ø Total coliform:~ 20-80 coliform colonies /filter

Ø Fecal coliform:~ 20 to 60 coliform colonies/filter.

Ø *Analyze 3 different sample volumes when coliform number is

uncertain

• Filtration set preferably autoclavable

• Membrane (usually modified cellulose e.g. nitrocellulose, nylon or

PVDF) must be sterile, pore size 0.45 um or 0.2 um

• After filtration the filter is place on suitable media either with or

without absorbent pad

Sample volume by sample type—total coliform test

Sample volume by sample type—fecal coliform test

• Report test results as the number of colonies per 100 mL of

sample

mL sample: actual sample volume and not the diluted volume

• Indistinct colonies—If growth covers the entire filtration area of

the membrane or a portion of it, and colonies are not discrete, report

the test results as “Confluent growth with or without coliforms.”

• High colony density—If the total number of colonies exceeds 200

per membrane or the

colonies are too indistinct for accurate counting, report the results as

“Too numerous to count, (TNTC)”

When testing non-potable water, if no filter meets the desired

minimum colony count

Reporting results Direct Microscopic Count

• Direct counting the cells in a population

• Viable, dead, viable but nonculturable (VBNC) & viable but difficult to

culture (VBDC) organisms

• Do not distinguish between living & dead cells

Ø Direct microscopic observation on specially etched slides

(Petroff-Hausser chambers/hemacytometers)

o a very small sample (e.g. 10 μL of a cell suspension) of the

population is placed into a counting chamber with known

volume

o The number of bacterial cells visible in the chamber is

counted

Ø Electronic counters

(Coulter counters, count microorganisms as they flow through a small

hole/orifice)

o A sample from the population is placed in the machine

o Sample is passed between two electrodes, every time a cell

passes between the electrodes it causes a disturbance in the

electrical field, and the cell is counted

• Population count is determined immediately, no incubation time is required

Epifluorescence microscopy

• Black polycarbonate membrane filter with 0.2 um

pore size is used

• Sample can be stained in the funnel itself with

fluorochrome

Ø Acridine orange (AO)-acridine orange direct

counts (AODC) ->green (high amounts of

RNA)/ orange (high amounts of DNA)

Ø 4',6-diamidino-2-phenylindole (DAPI)

Ø Fluorescein isothiocyanate (FITC)

Ø LIVE/DEAD® BacLight™ stain from molecular

probe-live bacteria appear green (SYTO 9)

and dead bacteria appear red (propidium

iodide)

Cells per ml =

(Total filterable area on membrane filter / Cells in number

of microscope fields counted)

Volume of sample filtered

The Most Probable Number Method

• tube-dilution method (mostly 3 or 5-tube series) where multiple tube

dilution is carried out to extinction

• It uses a medium that is less sensitive to toxicity and supports the

growth of stressed organisms

• Applicable to examination of total coliforms in chlorinated primary

effluents and under other stressed conditions

• Suitable to examine of turbid samples, muds, sediments, sludge

• Does not provide a direct measure of the bacterial count more variable

and tends to yield higher result

• Can produce estimates of bacterial concentration that are below

detectability of most other methods e.g. plating

• Three stages

1. Presumptive

2. Confirmed

3. Completed

Presumptive test:

• A series of lauryl tryptose broth containing Durham

tubes are inoculated with decimal dilutions of sample

• Formation gas at 35˚C, within 48 hr constitutes a ‘+’

(acid & gas) for members of total coliform group

Confirmed test:

• ‘+’ presumptive test are streaked onto Eosine

Methylene Blue agar (EMB)

• transferred to brilliant green lactose bile broth (BGLB)

• EMB: green metallic sheen, BGLB: gas production,

after 24/48 hr at 35˚C

Completed test:

• Streaking inoculum from the BGLB tubes onto EMB

plates for 24 hr at 35˚C

• Typical & atypical colonies are transferred into lauryl

tryptose broth fermentation tubes & onto nutrient

agar slants

• Gas formation in the fermentation tubes & present of

gas negative rods

Basic assumptions:

I. organisms are randomly and evenly distributed

throughout the sample

II. organisms exist as single entities, not as chains, pairs

or clusters and they do not repel

one another

Disadvantages:

• tedious and laborious

• Certain non coliform bacteria may suppress coliforms

or act synergistically to ferment

lauryl tryptose broth è false positive results

• Brilliant green bile broth when chlorinated primary

effluents are tested (storm water mixed with

sewage) è false positive results

III. proper growth medium, temperature and incubation

conditions have been selected to allow even a single

viable cell in an inoculum to produce detectable growth

IV. population does not contain viable, sub-lethally injured

organisms that are incapable of growth in the culture

medium used

Methods for Measurement of Cell Mass

• Direct physical measurement

Ø Dry weight (Volume of cells after centrifugation)

Ø Wet weight

• Direct chemical measurement of some chemical

component of cells

Ø Total N

Ø Total protein

Ø Total DNA content

• Indirect measurement of chemical activity

Ø Rate of O2 production/consumption

Ø CO2 production/consumption

• Turbidity measurement

Employ a variety of instruments to determine amount of

light scattered by suspension of cells (bacteria scatter

light in proportion to their numbers)

Turbidity/optical density is directly related to cell

mass/cell number, after construction & calibration of

standard curve.

Lack sensitivity limited to ~107 cells ml-1

The requirements for Growth

Chemicalphysical

• Temperature

• pH

• Osmotic

Source of

• carbon

• nitrogen

• sulfur

• phosphorus

• trace elements

• O2

• Organic growth factors

• Environmental factors that affect bacterial

growth and how this relate to isolation and

culture of bacteria

Ø Psychrophiles (cold living microbes) - grow at

0° C but optimum ~15° C

Ø Psychrotrophs - grow at 0° C also but optimum

20 - 30° C – important in food spoilage

Ø Mesophiles (moderate- temperature-loving

microbes) - grow best at moderate around 37°C

– many pathogens fall in this category

Ø Thermophiles (heat loving bacteria) - have a

growth optimum at 50-60° C

Ø Hyperthermophiles - have growth optima of

80° C or higher (archaea)

Temperature Minimum growth temperature

Lowest temperature at which the species

will grow

Optimum growth temperature

Temperature at which the species growth

best

Maximum growth temperature

The highest temperature at which grow is

possible

pH • Acidophiles: Organisms capable of

living at low pHs

• Alkaliphiles: Organisms capable of

living at very high pHs

• Most bacteria have optimum ~pH 6.5-

7.5, <5 & >8.5 do not grow well (except

acetic bacteria, sulfur oxidizing

bacteria)

• Optimum pH for Molds & yeasts is

usually 5-6

• Acidity inhibits most microbial growth

and is used frequently for food

preservation (e.g.: pickling)

• Alkalinity inhibits microbial growth, but

not commonly used for food

preservation

• Culturable bacteria often produce acids

that affect interfere with their own

growth

o To neutralize acids & maintain

proper pH>>>chemicals buffers

(peptones, amino acids,

phosphate salts) are included in

growth media

• Miroorganisms are made up 80- 90% water.

Hypertonic solutions:

Ø High osmotic pressure removes water from cell,

causing shrinkage of cell membrane (plasmolysis)

Ø Used to control spoilage and microbial growth

Ø Salted meat, honey, sweetened condensed milk

Hypotonic solutions:

Ø Low osmotic pressure causes water to enter the

cell

Ø In most cases cell wall prevents excessive entry of

water microbe may lyse or burst if cell wall is weak

Halophiles: Require moderate to large salt concentrations.

(Ocean water contains 3.5% salt)

Most marine bacteria

Ø Extreme /Obligate Halophiles: require very high

salt concentrations (20 to 30%)

Ø Facultative Halophiles: Do not require high salt

concentrations for growth, but tolerate 2% salt or

more

Osmotic pressure

Oxygen

Microbes use O2 produce more energy from nutrients than

anaerobes

Aerobes/Obligate aerobes

microorganisms that require O2 for their energy yielding processes

Anaerobes/ Obligate anaerobes

microorganisms that do not require O2 for their energy yielding

processes

E.g. Clostridium

Facultative anaerobes

• capable using either respiratory/fermentation processes,

depending on availability of O2 in cultural environment

• Efficiency in producing energy decreases in absence of O2

• E.g. E. coli & yeast

Aerotolerant anaerobes

• use fermentation to produce ATP

• do not use oxygen, but can protect from reactive oxygen

molecules

• Characteristic: ferment carbohydratesàlactic acid

• E.g. Lactobacilli

Microaerophiles

can grow in presence of O2 but only at concentration less than 20%

v/v found in atmosphere

• Limited toleranceàsensitive to superoxide free radicals &

peroxides, produce in lethal concentration under O2 rich

condition

Chemical requirements

• Most important requirements for microbial growth

• Makes up 50% of dry weight of cell

e.g. D-glucose, D-ribose or L-arginine

• Structural backbone of living matter , needed for all

all organic compounds that make up living cell

• Chemoheterotrophs:

Ø Obtain carbon from their energy source:

lipids, proteins, and carbohydrates

• Chemoautotrophs and Photoautotrophs:

Ø Obtain carbon from carbon dioxide

• Makes up 14% of dry cell weight

• Used to form amino acids, DNA, RNA

• Sources of nitrogen:

• Protein: Most bacteria

• Ammonium: found in organic matter

• Nitrogen gas (N2): Obtain N directly from atmosphere

(nitrogen fixation) Important nitrogen fixing bacteria,

live free in soil or associated with legumes (peas, beans,

alfalfa, clover, etc.)

• Legume cultivation is used to fertilize soil naturally

• Nitrates: Salts that dissociate to give NO3

• Used to form synthesis sulfur containing amino acids

and vitamins (thiamin & biotin)

• Sources of sulfur:

Ø Protein: Most bacteria

Ø Hydrogen sulfide

Ø Sulfates: Salts that dissociate to give SO42-

• Used to form DNA, RNA, ATP, and phospholipids.

• Sources: Mainly inorganic phosphate salts & buffers

C

N

S

P

Trace elements (micronutrient)

• Mineral elements that required by living organisms

in very small amounts

• E.g: Iron, Copper, Molybdenum, Zinc

• Essential for functions of certain enzymes

(cofactors)

• Naturally present in tap water & other compounds

of media

Organic growth factors

• Essential organic compounds an organisms is unable

to synthesize

• function as coenzymes and are required for essential

metabolic reactions

• Many bacteria can synthesize all their own vitamins

(humans-only a few),but some do require that certain

vitamins be supplied

• Other organic growth factors for bacteria include

amino acids, purines, and pyrimidines

H2O present in agar. Usually prepared either in 1.5% or 2%

Light

Especially for autotrophs or phototrophs-generate ATP

Function optimally at specific light intensities and utilise specific wavelengths

High light levels may cause photo inhibition and retard photosynthesis rates

Pressure

• Especially when working with deep water microbiology (> 1000 m deep)

• Most organisms on land or on the surface of water is always subjected to a pressure

of 1 atm

• Deep sea the hydrostatic pressure can reach 600 to 1100 atm mainly barotolerant

• Some bacteria in the gut of deep sea invertebrates (amphipods and holothurians)

can grow more rapidly at high pressures (Ex. Photobacterium, Shewanella, Colwellia )

Moisture

Culture media

• A nutrient materials prepared for the growth of microorganisms in a laboratory/

mixture of nutrients on which microorganisms can grow outside their nature

habitat

• composition of these media is calculated to supply the organism with sources of

energy and elements allowing its optimum growth

• can be liquid or gel

• Inoculum: Microbes that introduced into a culture medium to initiate growth

• Culture: microbes that grow & multiply in/on a culture medium

• Criteria needed for culture medium

ü right nutrients for specific microorganism

ü Sufficient moisture

ü Proper adjusted pH

ü Suitable level of oxygen

ü Sterile

• Most media are available from commercial sources, have premixed components

& require only the addition of water

• Solidifying agent –agar (thickener)

• Agar media are usually contained in test tubes (slants)/Petri dishes

• Unique Properties of Agar:

v Melts above 95 ˚C

v Once melted, does not solidify until it reaches 40˚C

v Cannot be degraded by most bacteria

v Polysaccharide made by red algae

Classification of Culture Media Based on Consistency

Solid media

• Contains 1.5-2.0% agar per

weight/solidifying agent to solidify

the liquid, nutrient part of the

media

v Agar melts above 95oC

v Agar once melted, does not

solidify until it reaches 40oC

v Cannot be degraded by most

bacteria

• Useful for isolation,

characterization of bacterial

isolates, purification

• Ideal for culture storage &

biochemical reaction observation

• E.g.,: Nutrient agar, MacConkey

agar, Blood agar, Chocolate agar

• bacteria appear as smooth, rough,

mucoid, round, irregular,

filamentous, punctiform

Semi solid media

• Contains 0.5% or less agar per

weight

• Bacteria motility observation,

fermentation study & preservation

• Cultivation of microaerophilic

bacteria

• E.g.: Stuart’s and Amies media,

Hugh and Leifson’s oxidation

fermentation medium, and

Mannitol motility media.

• Bacteria appears as a thick line in

the medium

Liquids (broth)

• Without addition of agar

• Allows bacteria to grow uniformly

with turbidity

• Growth of a large number of

bacteria

• For cultivation, maintain

microorganisms, fermentation

studies, biochemical tests

• E.g.: Nutrient broth, Tryptic soy

broth, MR-VP broth, phenol red

carbohydrate broth

Classification of Culture Media Based on Basic of Composition

Synthetic/chemically defined media

• exact chemical composition are known

• entirely free of animal/plant-derived components

(including microbial derived components e.g. yeast

extract)

• Minimal medium that provides only the exact

(including any growth factors) needed by the

organism for growth

• Must contain organic growth factors that serve

as a source of carbon & energy

• Use for studying the minimal nutritional

requirements of microorganisms, for enrichment

cultures, and for a wide variety of physiological

studies

• Not wisely use, costly

• exact chemical constitution of the medium is not known

• usually contain complex materials of biological origin E.g. blood/milk/

yeast extract/beef extract, the exact chemical composition of which

is obviously undetermined

• usually provide the full range of growth factors that may be required

by an organism so they may be more handily used to cultivate

unknown bacteria/bacteria whose nutritional requirement are

complex (i.e., organisms that require a lot of growth factors, known

or unknown)

• usually used for cultivation of bacterial pathogens, other fastidious

bacteria, fungi

• Composition may vary slightly from batch to batch

• Energy, carbon, nitrogen, and sulfur requirements are primarily met

by protein fragments (peptones)

• Vitamins and organic growth factors provided by meat and yeast

extracts.

• Two forms of complex media:

Ø Nutrient broth: Liquid media

Ø Nutrient agar: Solid media

Non-synthetic/chemically undefined/complex media

Classification of Culture Media Based on Oxygen

Aerobic media

• Common media for most of the aerobic microbes that

grow in ambient 20% O2

Simple media • Basal media that used for

culture non-fastidious

bacteria without any

enrichment source

• a non-selective medium

• E.g. Peptone, meat extract,

sodium chloride & water

Complex media (undefined medium)

• Media other than basal media

• Additional of complex

ingredients in unknown

proportions e.g: yeast

extract/carbon hdrolysate

• Provide special nutrients

Anaerobic media

• For cultivation of anaerobic bacteria at low oxygen,

reducing oxidation-reduction potential

• contains extra nutrients (Vitamin K, Hemin),

oxygen that get reduced by a physical/chemical

process. The addition of glucose (1%),

thioglycollate(0.1%), ascorbic acid (0.1%), cysteine

(0.05%), or iron fillings added to cause the medium

to reduce O2

• PRAS (PreReduced Anaerobically Sterilized)

media made specifically for anaerobes containing

Oxyrase (an enzyme system that offers a way to

remove dissolved oxygen from liquid, gas or

semisolid products)

• medium is boiled in a water bath to force out

dissolved O2 and packed with sterile paraffin

• Plates must be grown in oxygen free containers

(anaerobic chambers)

• Examples:

Ø RCM (Robertson cooked meat) isolation for

Clostridium sp.

Ø Thioglycolate broth– It has sodium glycolate

that maintains low oxygen.

Classification of Culture Media Based on Chemical composition/Application/Purpose

• Special purpose media are needed to facilitate recognition, enumeration and isolation of certain types of bacteria

General purpose media/basic media

• Simple media that supports most non-

fastidious bacteria without any enrichment

source

• a non-selective medium

• E.g. Peptone water, nutrient broth/agar

• Routinely used medium in the lab for

primary isolation & sub-culturing of

microorganisms

• Staphylococcus and Enterobacteriaceae grow

in this media

• requires addition of other substances e.g.:

blood, serum, egg yolk

• E.g.: Blood agar, Chocolate agar, Loeffler

medium (LSS), Monsor’s taurocholate,

Lowenstein Jensen media.

• Blood agar identifies hemolytic bacteria,

chocolate media for N. gonorrhea

Enriched media

Selective media

• growth of selective/desired bacteria & suppress the growth of unwanted

microbes

• are agar based

• Any agar media can be selective by addition of

Ø bile salts

Ø Antibiotics

Ø Dyes

• adjust the physical conditions of a culture medium, e.g. pH & temperature

to render it selective for organisms

• Examples:

Ø Saboraud’s Dextrose Agar: pH of 5.6 discourages bacterial growth.

Used to isolate fungi

Ø Brilliant Green Agar: Green dye selectively inhibits gram-positive

bacteria. Used to isolate gram-negative Salmonella

Ø Bismuth Sulfite Agar: Used to isolate Salmonella typhi. Inhibits growth

of most other bacteria

Ø MacConkey Agar: It has bile salts that inhibit the growth of gram-

positive bacteria. Selective isolation for Enterobacteriaceae

Ø TCBS Agar: Light green translucent media Bile salt inhibits the growth of

unwanted bacteria. Selective for Vibrio cholera. V. cholera produces

acid by fermentation of sucrose that acts as indicator called

bromothymol blue and yellow colonies appears

Differential/indicator media

• Easier to distinguish colonies of the desired

organisms from other colonies growing on the

same plate based on the basis of their colony

colour

• Incorporation of

Ø Dyes (neutral red, phenol red, methylene

blue)

Ø Metabolic substrates

Ø pH indicators

• Examples:

Ø Mannitol salts agar (mannitol fermentation

= yellow)

Ø Blood agar (various kinds of hemolysis i.e.

α, β and γ hemolysis)

Ø Mac Conkey agar (lactose fermenters, pink

colonies whereas non- lactose fermenter

produces pale or colorless colonies.

Ø TCBS (Vibrio cholerae produces yellow

colonies due to fermentation of sucrose)

Selective & differential characteristics can combined in a

single medium

• Used both to distinguish colonies of a desired organisms,

Inhibit unwanted commensal/ unwanted microbes & help

to recover pathogen from a mixture of bacteria

• Examples:

Ø Mannitol Salt Agar

• Used to distinguish and select for S. aureus

• High salt (7.5% NaCl) discourages growth of

other organisms

• pH indicator changes color when mannitol is

fermented to acid

Ø MacConkey Agar

• Used to distinguish and select for Salmonella

• Bile salts and crystal violet discourage growth

of gram-positive bacteria

• Lactose plus pH indicator: Lactose fermenters

produce pink/red colonies, nonfermenters are

colorless

Enrichment culture

• Used to favor the growth of a microbe that may be found

in very small numbers

• Unlike selective medium, does not necessarily suppress

the growth of other microbes

• Typically used as a broth medium

• Used mainly for fecal and soil samples

• After incubation in enrichment medium, greater numbers

of the organisms, increase the likelihood of positive

identification

• Selenite F broth, tetrathionate broth, and alkaline peptone

water (APW) are used to recover pathogens from fecal

specimens

Transport media

• Transport specimens after collection to control the overgrowth of

contaminating organisms/commensals

• Act as temporary storage, maintains the viability of pathogens in the

specimen and prevents them from drying (desiccation)

• Some of these media (Stuart’s & Amie’s) are semi solid in consistency

• Addition of charcoal serves to neutralize inhibitory factors

• Examples:

Ø Cary Blair transport medium & Venkatraman Ramakrishnan

(VR) medium-used for transport faeces from suspected cholera

patients

Ø Sach’s buffered glycerol saline used to transport faeces from

patients suspected to be suffering bacillary dysentery

Ø Pike’s medium used to transport streptococci from throat

specimen

Special Culture Techniques

• Used to grow bacteria with unusual

growth requirements

• Bacteria that do not grow on artificial

media:

Ø Mycobacterium leprae (leprosy):

Grown in armadillos

Ø Treponema pallidum (syphilis):

Grown in rabbit testicles

Ø Obligate intracellular bacteria

(rickettsias & chlamydias): Only

grow in host cells

• Bacteria that require high or low CO2

levels:

Ø Capnophiles: Grow better at high

CO2 levels & low O2 levels. Similar

to environment of intestinal

tract, respiratory tract, and other

tissues

• Media is usually also supplemented

with reducing agents to reduce

oxygen stress

• Sodium bicarbonate and sodium

borohydride are mixed with a small

amount of H2O to produce CO2 and

H+

• A palladium catalyst in the jar

combines with the O2 in the jar and

the H+ to remove O2

• Oxyrase is a bacterial respiratory

enzyme that can be put in media; it

combines O2 with H+ to remove O2

by forming H2O and turns the petri

dish into a mini-anaerobic chamber

Anaerobic Jar

Candle light jar

• 3-5% CO2 and 8-10% O2 (0.3% and 21% in the

atmosphere, respectively)

• method increases CO2 levels (up to 6%) and

concurrently reduces O2 (to < 15%). Suitable for

Neisseria and Campylobacter

• Some microaerophiles are actually capnophilic

(requiring elevated CO2 levels to grow)

• Strict aerobes may not grow well in a candle jar,

depending on the species

CO2 generating packet

• The gas generator is crushed to mix the chemicals it

contains and start reaction that produce CO2

• Reduce O2 to ~5% and CO2 ~ 10%

air lock

• filled with inert gas- nitrogen gas (N2), are used before filling the

interchange with gas mix (5% CO2, 10% H2, 85% N2) to reduce the

amount of oxygen introduced into the chamber

Anaerobic chambers

Maintenance & Preservation

• Necessary to maintain the viability and purity of the

microorganism

• Aseptic conditions to avoid contamination

Periodic Transfer to Fresh Media

• periodically preparing a fresh culture from the previous stock

culture

• culture medium, storage temperature, and time interval at which

the transfers are made vary with the species and must be

ascertained beforehand

• temperature and type of medium chosen should support a slow

rather than a rapid rate of growth so that the time interval

between transfers can be long

• Heterotrophs commonly remain viable for several weeks/months

on a medium (NA)

• Disadvantage: failing to prevent changes in the characteristics of

a strain due to the development of variants and mutants.

Refrigeration

• stored at 0-4°C either in refrigerators/cold-rooms

• applied for short duration (2-3 weeks, metabolic activities are

greatly slowed down)

Paraffin Method/ preservation by overlaying cultures with

mineral oil

• simple and most economical method

• sterile liquid paraffin in poured over the slant (slope) of culture

and stored upright at room temperature--ensures anaerobic

conditions and prevents dehydration of the medium

• Helps pure culture to remain in a dormant state thus can be

preserved form months to years (varies with species)

• Advantage--remove some of the growth under the oil with a

transfer needle, inoculate a fresh medium, and still preserve the

original culture

• changes in the characteristics of a strain can still occur

Cryopreservation

• freezing in liquid nitrogen at -196°C or in the gas phase above the

liquid nitrogen at -150°C)

• in the presence of stabilizing agents e.g. glycerol/Dimethyl

Sulfoxide (DMSO) –prevent cell damage due to formation of ice

crystals and promote cell survival for long storage times

• Expensive method

Lyophilization (Freeze-Drying)

• process where water and other solvents are removed from a

frozen product via sublimation

• microbial cells are dehydrated and their metabolic activities are

stopped; microbes go into dormant state and retain viability for

years

• stored in the dark at 4°C in refrigerators, freeze-dried products are

hygroscopic and must be protected from moisture during storage

• most frequently used technique by culture collection centers

• Many species of bacteria remained viable and unchanged in their

characteristics for >30 years

Success in growing and maintaining strains

• As soon as you receive a culture streak it on an agar plate, refrigerate

the plate as soon as it grows well

• Plates and slants keep better than broth cultures

• Start seed cultures from a single colony

• Grow strains in an appropriate growth medium near the optimum

temperature.

• Grow strains that require oxygen in flasks with a large surface: volume

ratio and shake it by putting on a shaker

• If you want to maintain cultures in broth, subculture broth cultures

once a week.

• Grow cultures only until there is good visible growth and then

refrigerate the culture to maintain cell viability. Do not store cultures in

the incubator

• Store all cultures in the refrigerator

• Cell growth on a plate or in a broth culture does not mean the culture

is alive. Dead cells generally look the same as live cells

• Maintain strains on streak plates and check carefully for contaminants

• Restreak cultures on slants or plates about once a month

Control of Microbial Growth

Terminology of Microbial Growth

• Killing or removing all forms of

microbial life (including

endospores) in a material/object

an object

• Canned food sterilization

• Sufficient heat treatment to kill

microorganisms and endospores

• Destruction of vegetative

pathogens

Sterilization

Commercial sterilization More resistant endospore of thermophilic

bacteria may survive, but they will not

geminate & growth under storage conditions

Disinfection

May use physical/chemical methods

o Disinfectant: Applied to inanimate objects

o Antiseptic: Applied to living tissue (antisepsis)

o Degerming: Mechanical removal of most microbes in

a limited area ( E.g.: Alcohol swab on skin)

o Sanitization: Use of chemical agent on food-handling

equipment to meet public health standards & minimize

chances of disease transmission (E.g.: hot soap & water.

Antisepsis• Destruction of vegetative

pathogens on living tissue Treatment is almost always by chemical antimicrobials

Degerming• Removal of microbes from a limited

area (skin around an injection site) Mostly a mechanical removal by an alcohol soaked swab

Usually done by heat (steam) under

pressure or sterilizing gas (e.g.

ethylene oxide)

Biocide/germicide

bacteriostasis

Sepsis

Asepsis

Sanitization

• Treatment intended to lower

microbial counts on eating and

drinking utensils to safe public

health levels

May be done with high

temperature washing /dipping

into a chemical disinfectant

Kill microorganisms

Capable of inhibiting the

growth or multiplication of

bacteria

from Greek for decay/putrid

Indicates bacterial contamination

Absence of contamination

Rate of Microbial Death

• When bacterial population are heated/treated with antimicrobial chemical,

bacteria usually die at constant rate

• Factors influence the effectiveness of antimicrobial treatments

Number of Microbes

More microbes present, more time needed to eliminate the entire population

Type of Microbes

Endospores are very difficult to destroy. Vegetative pathogens vary widely in

susceptibility to different methods of microbial control

Environmental influences

Presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials,

temperature, pH etc.

Time of Exposure

Chemical antimicrobials and radiation treatments are

more effective at longer times. In heat treatments,

longer exposure compensates for lower temperatures

Mode of action of the agent

How does it kill/inhibit microorganisms

o Act on microbial cells to dissolve them (Antiseptic: e.g.

alcohols & quaternary ammonium compounds)

o Penetrate cells & cause the release of amino acids,

nuclear material, & other important chemical

constituents

o Penetrate microbial cell walls & inactivate essential

membrane transport systems>>cells can no longer

obtain the nutrients to survive & reproduce

o Coagulate certain vital materials in cells, thereby

destroying the microorganisms

o Disrupt the metabolism of the cells so that they can no

longer assimilate nutrients>>starve & die

Mode of Action

• An antimicrobial agent’s adverse effect on cells is known as its mode/

mechanism of action

• Cellular targets of physical and chemical agents fall into four categories:

Cell walls Cell

membranes

Protein &

nucleic acid

synthesis

Protein

structure &

function

Effects of Agents on cell wall

chemical agents damage the cell wall by

o blocking cell wall synthesis

o digesting it

o breaking down surface

Cell wall becomes fragile & lysed easily

Penicillins: interfere with the synthesis of cell wall in bacteria

Detergents & alcohol disrupt cell walls especially in gram-negative bacteria

Effect on Cell Membrane

• If membrane is disrupted, a cell

loses its selective permeability

>>>usually leads to cell death

• Surfactants (detergents): lower the

surface tension of cell membranes

• Surfactants are polar molecules

with hydrophilic and hydrophobic

regions that can bind to the lipid

layer & penetrate the internal

hydrophobic regions of

membranes

• This process “opens up” the once

tight interface, leaving leaky spots

that allow harmful chemicals to

seep into the cell and important

ions to seep out

• Alcohols: dissolving membrane

lipids & stripping membranes away

from cells

Drugs bind to the ribosomes of bacteria, stops peptide bonds from

forming

Bacterial cells inhibited from forming proteins required in growth &

metabolism>> inhibited from multiplying

Drugs used to treat infections are chemicals that block protein synthesis

in microbes without adversely affecting human cells

DNA must be regularly replicated & transcribed in growing cells, any agent

that either impedes these processes/changes the genetic code is

potentially antimicrobial

Some agents bind irreversibly to DNA, preventing both

transcription & translation, whereas others are mutagenic agents

Gamma, ultraviolet/X radiation causes mutations>>

permanent inactivation of DNA

Chemicals (e.g. formaldehyde, ethylene oxide) interfere with DNA and

RNA function

Effect on Protein & Nucleic Acid Synthesis

The antimicrobial properties of some

agents arise from their capacity to

disrupt the structure of, or denature,

proteins

Denaturation occurs when the bonds

maintain the secondary and tertiary

structure of the protein are broken

Breaking these bonds will cause the

protein to unfold/create random,

irregular loops and coils

Denature proteins is through

coagulation by moist heat

Chemicals e.g. strong organic

solvents (alcohols, acids) can

coagulate proteins

Antimicrobial agents (e.g.: metallic

ions) attach to active site of the

protein & prevent it from interacting

with its correct substrate

Alter Protein Function Physical Methods of Microbial Control

Heat

Kills microorganisms by denaturing their enzymes and other proteins

Heat resistance varies widely among microbes

Thermal Death Point (TDP)

Lowest temperature at which all of the microbes in a liquid

suspension will be killed in 10 min

Thermal Death Time (TDT)

Minimal length of time in which all bacteria will be killed at a given

temperature

Decimal Reduction Time (DRT, D value)

Time in minutes, in which 90% of a population of bacteria at a given

temperature will be killed (Used in canning industry)

Half-life == 30% of D-value

Two physical states of heat used in microbial control:

• moist

Ø occurs in the form of hot water, boiling water, or steam (vaporized

water)

Ø usually ranges from 60˚- 135˚C, more effective than dry heat

Ø Kills microorganisms by coagulation & denaturation of proteins

• Dry

Ø air with a low moisture content that has been heated by a flame/

electric heating coil

Ø moderate temperature dehydrates the cell, removing the water

necessary for metabolic reactions, and alters protein structure

• Bacterial endospores exhibit the greatest resistance

• Vegetative states of bacteria & fungi are the least resistant to both

moist & dry heat

• Destruction of spores requires temperatures above boiling, as

some can survive ≥20 hours of boiling

Four ways of moist heat

i) Sterilization with Steam Under Pressure

• High temperatures are commonly achieved by steam under

pressure in an autoclave for -method of sterilization

Ø Steam heated to 121–134 °C (250–273 °F) with a holding time

of at least 15 minutes at 121 °C/3 minutes at 134 °C

Ø Duration of the process is adjusted according to the

bulkiness of the items in the load (thick bundles of material

or large flasks of liquid) & how full the chamber is

Ø Range of holding times varies from 10 min for light loads to

40 min for heavy or bulky ones; the average time is 20 min

• Most effective when organisms contact steam directly, killed

within 15 minutes

• Are contained in a small volume of liquid

ii) Nonpressurized Steam (tyndallization)

• Suitable for substances that cannot withstand high temperature of

the autoclave

• Items are exposed to free-flowing steam for 24 hours, and then

again subjected to steam treatment which repeated for 3 days in a

row

• Temperature never gets above 100˚C, highly resistant spores could

survive even after 3 days of this treatment

iii) Boiling Water

• Temperature at 100˚C-desinfection

• Exposing materials to boiling water for 30 minutes will kill most non-spore-

forming pathogens

• items can be easily recontaminated when removed from the water

iv) Pasteurization

• Technique of applying heat to consumable liquids to kill potential agents of

infection and spoilage, while at the same time retaining the liquid’s flavor

and food value

• Flash method: exposes liquid to heat exchangers at 71.6˚C for 15 seconds,

effective against certain resistant pathogens e.g. Coxiella, Mycobacterium.

but do not kill endospores/thermoduric microbes

• High Temperature Short Time (HTST) pasteurization: exposes milk to a

temperature of about 63˚C for 30 min-lowers total bacterial counts so milk

keeps well under refrigeration

• Ultra High Temperature Treatments (UHT) —exposes milk to a higher

temperature 134˚C for 2-5 secs

Dry heat sterilization

• Kills by oxidation effects

Direct flaming

• Sterilize inoculating loop by heating the wire to a red glow

Incineration

• at very high temperatures destroys all microorganisms

• Sterilize & dispose of contaminated paper cups, bags and

dressings

Hot air sterilization

• Temperature of about 170˚C maintained for ~2 Hrs ensures

sterilization

• Oven

Low temperature

• The effect depends on the particulate microbe & the intensity of the

application

• Most microorganisms do not reproduce at ordinary refrigerator

temperatures (0˚-7˚C), slowing the growth of microorganisms

significantly and helping preserve refrigerated products e.g. foods/

medical supplies

• Many microbes survive ( but do not grow) at the subzero

temperatures used for store foods

• Freezing below −2 °C may stop microbial growth and even kill

susceptible organisms

High pressure

• Denatures proteins in vegetative cells but endospores may survive these

pressures

• High-pressure processing (pascalization) is used to kill bacteria, yeast,

molds, parasites, and viruses in foods while maintaining food quality and

extending shelf life

• The application of high pressure between 100 and 800 Mpa

• This is not commonly used for disinfection or sterilization of fomites.

• The application of pressure and steam in an autoclave is effective for killing

endospores, it is the high temperature achieved, and not the pressure

directly, that results in endospore death

Desiccation

• Drying or dehydration

• Microorganisms cannot grow or reproduce but can remain viable for

years

• May regrow when conditions are more favorable and water content is

restored

• Freeze-drying, or lyophilization-item is rapidly frozen (“snap-frozen”) &

placed under vacuum

• Resistance of vegetative cells to desiccation varies with the species

and the organism’s environment

Osmostic Pressure

• Water activity: water content of foods and materials

• can be lowered by the addition of solutes e.g.: salts

or sugars

• High concentrations of solutes create a hypertonic

environment that cause water to leave the microbial

cell

• Used in preservation of foods

• E.g.: Concentrated salt solutions used to cure meats

and thick sugar solutions used to preserve fruits

Radiation

• X-rays and gamma rays can be used to sterilize many packaged materials

Ø Laboratory: sterilize materials that cannot be autoclaved e.g. plastic Petri

dishes and disposable plastic inoculating loops

Ø Clinical: sterilize gloves, intravenous tubing,& other latex and plastic items

used for patient care, heat-sensitive materials including tissues for

transplantation, pharmaceutical drugs, and medical equipment

• Low level ionizing radiation used for food preservation-processing spices and

certain meats and vegetables

Nonionizing radiation

• Wavelength ≥ 1 nm

• uses less energy than ionizing radiation

• Cannot penetrate cells or packaging

• Ultraviolet (UV) light-causes thymine dimers that inhibit correct replication of the

DNA during reproduction of the cell

• energy emitted from atomic activities and dispersed at high

velocity through matter or space

• characterized by a range of wavelengths (electromagnetic

spectrum)

• Various effects on cells, depending on its:

Ø Wavelength

Ø Intensity

Ø Duration

• Sterilizing radiation: radiation that kills microorganisms

Ø Ionizing

Ø Nonionizing

Ionizing radiation

• gamma rays, X rays or high energy electron beams

• wavelength shorter than nonionizing radiation, < 1 nm

• Carry more energy

• Can pass into the cell, where it alters molecular structures and

damages cell components

• UV wavelengths ~260 nm- most effective for killing microorganisms

• UV radiation used to control microbes in the air

• UV light can be used effectively by both consumers and laboratory

personnel to control microbial growth

Ø Small portable UV lamps incorporated into water purification

systems for use in homes.

Ø Germicidal lamps used in surgical suites, biological safety

cabinets, and transfer hoods

Ø UV light used to disinfect vaccines and medical products

• Disadvantages of UV light:

Ø Not very penetrating and will not pass through plastics/glass,

cells must be exposed directly to the light source

Ø Can damage human eyes, prolonged exposure can cause burns

and skin cancer

Filtration

• effective method to remove microbes from air and liquids

• Used to sterilize heat sensitive materials e.g. culture media, enzymes,

vaccines, antibiotic solutions

• Using thin membranes of cellulose acetate, polycarbonate, and a

variety of plastic materials (Teflon, nylon)

• Pore size can be controlled

Ø 0.22 μm & 0.45 μm-bacteria

Ø 0.01 μm virus & large protein molecules

• Applications:

Ø Alternative method for sterilizing milk & beer without altering

their flavor

Ø Water purification but not removing soluble molecules (toxins)

Ø High-efficiency particulate air (HEPA) filters are widely used to

remove microorganisms > 3 μm in diameter to provide a flow of

sterile air to hospital rooms and sterile rooms

Chemical Methods of Microbial Control

• Used to control the growth of microbes both living tissue and inanimate objects

• Antimicrobial chemicals occur in liquid, gaseous, or solid state

• Serve as disinfectants, antiseptics, sterilants (chemicals that sterilize), degermers/

preservatives

• Agents that actually kill are called -cidal agents, with a prefix indicating the type of

microorganism killed (bactericidal, fungicidal, and viricidal)

• Agents that do not kill but only inhibit growth are called -static agents

(bacteriostatic, fungistatic, and viristatic compounds)

• Desirable qualities in a germicide including:

Ø rapid action in low concentrations

Ø solubility in water or alcohol and long-term stability

Ø broad-spectrum microbicidal action without being toxic to human and

animal tissues

Ø penetration of inanimate surfaces to sustain a cumulative or persistent

action

Ø resistance to becoming inactivated by organic matter

Ø noncorrosive or nonstaining properties

Ø sanitizing and deodorizing properties

Ø affordability and ready availability

• Germicides are evaluated in terms of their effectiveness in destroying microbes in

medical and dental settings

• Three levels of chemical decontamination procedures:

Ø Low levels of disinfection:

o eliminate only vegetative bacteria, vegetative fungal cells, & some

viruses

o Used to clean materials e.g.: electrodes, straps, and fixtures that touch

the skin surfaces but not the mucous membranes

Ø Intermediate

o level germicides kill fungal (but not bacterial) spores, resistant

pathogens and viruses

o used to disinfect items (respiratory equipment, thermometers)

that come into intimate contact with mucous membranes but

are noninvasive

Ø High

o kill endospores and, if properly used are sterilants

o Used for medical devices that are not heat-sterilizable e.g.:

catheters, endoscopes, & implants

The effectiveness of the agents depends on:

• numbers and kinds of microbes that are present

• kinds of materials that are being treated

• time of exposure required

• strength and mode of action of the agent

Effect of Antimicrobial Agents on Growth

• Antibacterial agents are classified as:

Ø -static

Ø -cidal

Ø -lytic (cell lysing)

by observing their effects on cultures using viable and turbidimetric

growth assays

• Bacteriostatic agents (antibiotics) are typically inhibitors of some important

biochemical process, e.g.: protein, synthesis, and bind relatively weakly; if the agent

is removed, the cells can resume growing.

• Bactericidal agents (formaldehyde) bind tightly to their cellular targets and kill the

cell. The dead cells are not lysed, and total cell numbers, reflected in the turbidity of

the culture, remain constant.

• Bacteriolytic agents (detergent) kill cells by lysing them, and this affects both viable

and total cell numbers An example of a bacteriolytic agent

Assaying Antimicrobial Activity

• Minimum inhibitory concentration (MIC): determining the smallest

amount of the agent needed to inhibit the growth of a test organism

Dilution method

• inoculate a series of tubes of liquid growth medium containing a test

organism and dilutions of the agent.

• Following incubation, the tubes are scored for growth

(turbidimetrically) and the MIC is revealed as the lowest concentration

of antimicrobial agent that completely inhibits growth.

Metabolism: all chemical reactions that occur

within a living organism

Microbial Metabolism

Catabolic

Anabolic

• Degradative reactions that release energy

by breaking down large, complex

molecules into smaller ones

• Often involve hydrolysis, breaking bonds

with water

• Catabolic reactions are coupled to ATP

synthesis:

ADP + Pi + Energy -----------> ATP Provide building blocks for

anabolic reactions & furnish energy

needed for anabolic reactions

• Adenosine triphosphate (ATP)

• stores energy from catabolic

reactions & releases it to drive

anabolic reactions

• Biosynthetic reactions that build large

complex molecules from simpler ones

• Require energy and often involve

dehydration synthesis

• Anabolic reactions are coupled to ATP

hydrolysis (breakdown):

ATP -----------> ADP + Pi + Energy