Introduction and Historical Development in Microbiology

Organism - object / structures invisible to the naked eye

are called microorganism = human eye cannot see object smaller than

30u (1/1000 of an inch in dia.)

Microbiology – science that deals with the study of microorganisms and their activities

Medical Microbiology – deals with the study of disease

producing organism affecting human = also concerned with prevention and

control of disease.

Scope of study (order of decreasing size)

a) Algae (Phycology)

b) Fungi (Mycology)

c) Protozoa (Protozoology / Parasitology)

d) Bacteria (Bacteriology)

e) Virus (Virology)

Features of Microorganism:

* All microorganisms are not alike

* Some of them are very small while others are

relatively larger in size

* Some of them possess plant-like

characteristic,

while others have animal-like and few of

them

have neither plant-like nor animal-like

characters

BRIEF HISTORYBRIEF HISTORY

before microorganism were discovered, ancient before microorganism were discovered, ancient people regarded diseases as a form people regarded diseases as a form

of punishment sent by GOD for the sins of menof punishment sent by GOD for the sins of men disease were not new to man disease were not new to man Biblical disease - Leprosy Biblical disease - Leprosy

- Tuberculosis - Tuberculosis

- Syphilis - Syphilis

- Plague - Plague treatment and prevention of these diseases treatment and prevention of these diseases

were sought by sacrifices to appease the anger were sought by sacrifices to appease the anger of Godof God

Varro = during the 2nd century B.C.– postulated the concept of contagion

diseases are transmitted

by invisible creature

Roger Bacon (13th century) = postulated that invisible

living things called “germs” produce disease

Fracastorius (1546) = postulated that disease is caused

by invisible living things and can be transmitted by direct contact (person to person contact)

Spontaneous Generation TheorySpontaneous Generation Theory

Aristotle – proposed the Aristotle – proposed the

““Spontaneous Generation Theory”Spontaneous Generation Theory” also called Abiogenesisalso called Abiogenesis state that living things could develop spontaneously state that living things could develop spontaneously

from non-living materials from non-living materials supported by appearance of living creatures in supported by appearance of living creatures in

decaying meat, stagnating ponds, fermenting grain, decaying meat, stagnating ponds, fermenting grain, and infected woundsand infected wounds

Experiments to disprove Experiments to disprove Spontaneous Generation TheorySpontaneous Generation Theory

Francesco Redi 1668Francesco Redi 1668 did an experiment with flies and wide-mouth did an experiment with flies and wide-mouth

jars containing meatjars containing meat he demonstrated the appearance of maggots he demonstrated the appearance of maggots

in decomposing meat as a result on the in decomposing meat as a result on the deposition of eggs by fliedeposition of eggs by flie

Rudolph Virchow Rudolph Virchow 18581858 11stst person to propose the Theory of person to propose the Theory of

BiogenesisBiogenesis

‘ ‘Cells can only arise from preexisting cells’Cells can only arise from preexisting cells’

John Tyndall John Tyndall proved that dust carried germs proved that dust carried germs also demonstrated the great resistance of spore to also demonstrated the great resistance of spore to

heatheat introduced (Tyndallization/Fractional Sterilization) a introduced (Tyndallization/Fractional Sterilization) a

method by which spore can be destroyed by heating method by which spore can be destroyed by heating using a free-flowing steam for 30-60 minutes at 100using a free-flowing steam for 30-60 minutes at 100°°C C for 3 consecutive days. for 3 consecutive days.

Anton Van LeeuwenhoekAnton Van Leeuwenhoek 11stst to describe bacteria in 1677 with the use of to describe bacteria in 1677 with the use of

a simple microscope utilizing crude a simple microscope utilizing crude lenseslenses

considered as the Father of Bacteriology for being the considered as the Father of Bacteriology for being the first person described the 3 morphologic forms of first person described the 3 morphologic forms of bacteria: rod, spherical, spiralbacteria: rod, spherical, spiral

Robert Hooke 1678Robert Hooke 1678 developed the compound microscope and was able to developed the compound microscope and was able to

confirm Leeuwenhoek’s discoveriesconfirm Leeuwenhoek’s discoveries first person to coin the word “cell” to describe the first person to coin the word “cell” to describe the

tiniest components of the living systemtiniest components of the living system

Agostino Bassi 1800 Agostino Bassi 1800 proved that a fungus cause a disease in “silkworm” proved that a fungus cause a disease in “silkworm”

called Muscardine called Muscardine

Rayer and Davaine 1850Rayer and Davaine 1850 were able to observed the presence of microorganism were able to observed the presence of microorganism

in blood of animals dying from a disease called in blood of animals dying from a disease called Anthrax. Anthrax.

Edward JennerEdward Jenner - developed the concept of vaccination - developed the concept of vaccination

Paul ErlichPaul Erlich - developed the used of staining to - developed the used of staining to demonstrate bacterial cell morphology for better demonstrate bacterial cell morphology for better visualization visualization

Louis Pasteur Louis Pasteur published the results of an experiment he did to published the results of an experiment he did to

disprove spontaneous generation in microscopic disprove spontaneous generation in microscopic organismsorganisms

introduced Pasteurization a method of killing introduced Pasteurization a method of killing organisms found in dairy products organisms found in dairy products

Joseph Lister 1860sJoseph Lister 1860s demonstrated the importance of using antiseptic to demonstrated the importance of using antiseptic to

control spread of disease-producing organismcontrol spread of disease-producing organism uses a chemical disinfectant (aqueous phenol) to uses a chemical disinfectant (aqueous phenol) to

prevent surgical wound infections.prevent surgical wound infections. also developed the first pure culture technique also developed the first pure culture technique

using liquid medium which was the key to using liquid medium which was the key to identification of bacteria identification of bacteria

Robert Koch 1876Robert Koch 1876

establish proof of microbial etiology of 3 establish proof of microbial etiology of 3 important diseases: important diseases:

Cholera (Cholera (Vibrio cholerae)Vibrio cholerae) Tuberculosis (Tuberculosis (Mycobacterium tuberculosis)Mycobacterium tuberculosis) Anthrax (Anthrax (Bacillus anthracis)Bacillus anthracis)

formulated the Koch’s postulates which provides formulated the Koch’s postulates which provides proof that a specific bacterium caused a proof that a specific bacterium caused a particular disease. particular disease.

also developed a solid culture medium for also developed a solid culture medium for isolation of bacteria in pure culture. isolation of bacteria in pure culture.

Koch’s postulatesKoch’s postulates::

1. The organism must always be found in diseased

animals and not in healthy one2. Organisms isolated from the diseased

animals must be grown in pure culture away from the

diseased animals 3. Organisms grown in pure culture must initiate and

reproduce the disease when re-inoculated into

susceptible animal4. Organisms must be re-isolated from

experimentally infected animals

Exceptions to the Kock’s Postulate:

#1) Carrier - some individual harbors the organism but do not manifest the disease and can transmit the disease to others

#2) Some or certain organisms/bacteria cannot be cultured in

vitro (cannot grow in artificial culture media) Ex. Mycobacterium leprae / Treponema pallidum

#3) Certain animals not susceptible to certain microorganism some animals are by nature immune

- cannot replicate even with the same specie

some bacterial specie are host specific Ex. Vibrio cholerae cannot cause

chicken cholera or vice versa

Classification and Identification of Bacteria

Protist = group of single–celled microorganism which

do not form highly differentiated tissue and organ

system.

In the early history living organism were classified into 2 kingdoms: 1. Plant

2. Animal = after the discovery of microorganism, it was found that many microorganism possess both plant and animal characteristic or neither plant or animal characteristic. So a new kingdom was proposed for microorganism

(Kingdom Protista) = all bacteria, fungi, algae and protozoa are group in kingdom protista = viruses are not included because they do not have cellular

organization.

= Kingdom Protista is divided into 2 groups based on cellular organization:

1. Procaryotes – lower protista 2. Eucaryotes – higher protista

A.Eucaryotes – Higher protista Characteristics

1) Size greater than 5um2) Possess a well developed true nucleus enclosed

in a nuclear membrane 3) Contain sets of chromosomes 4) Cytoplasm contains mitochondria endoplasmic

reticulum and vacuoles 5) Cell membrane continous with well-defined endoplasmic reticulum and nuclear membrane 6) Ribosome (sedimentation coefficient) 80s 7) Motility organelle multistranded Eg: Algae (Red – Brown), fungi, protozoa, slime

mold

B) Procaryotes – Lower protista

Characteristic:

1) Size less than 4um

2) Nucleus primitive and is homogenous with the

cytoplasm of cell and not enclosed in a

nuclear

membrane (naked)

2) Posses single chromosome (haploid)

3) Do not have mitochondria and endoplasmic

reticulum

4) Cell wall made up of complex rigid layer

5) Ribosome (sedimentation coefficient) 70s

6) Motility organelle unstranded

Eg: Bacteria, Blue – Green algae

Laboratory Procedure Employed in the Identification

of Bacteria

1. Isolation of bacteria in pure (axenic) culture.

= depends on source of clinical specimen

= blood, spinal fluid and closed abscesses yield pure

bacterial culture of microorganism

= sputum, skin and body fluids, stool usually contains mixed organism.

2. Bacterial colony morphology (macroscopic)

= colony is composed of descendant of a single cell,

a clone or lump of cell

= usually the result of rapid multiplication of cell

when inoculated in a medium containing 2%

agar incubated for 18 – 24 hours in favorable

atmosphere.

= charac. microbial growth pattern on artificial media as observe when inspected with the

unaided eye

= determine size, shape, texture, presence/absence

of pigmentation, odor of the colonies

4 types of bacterial colony :

1) Smooth (S-colonies) - gives the appearance of homogeneity and uniform texture

without appearing as liquid or mucoid colonies.

= characteristic of freshly isolated wild – type organism.

Ex. Shigella, Salmonella, Proteus and E. coli

2) Rough (R-colonies) granulated and rough in appearance.

= produced by mutant strain of organism

3) Mucoid (M-colonies) - exhibits a water-like glistening

confluent appearance.

= seen among organism that forms well-defined capsule or slime layer.

4) L colonies – seen among cell-walless bacteria. Ex. Mycoplasma

3. Microscopic morphology and staining reaction.

= determines shape, arrangement and staining reaction = light microscopic examination of gram-stained

preparation with the oil immersion lens and without

coverslip is routinely used to determine bacterial morphology.

= staining artificially coloring the organism with dyes

= advantages: 1) organism are made more clearly visible

2) differences between all can be demonstrated

3 types of staining procedure :

1. Simple – use of 1 dye only.2. Differential – used of 2 or more dyes.

A)gram – differentiates gram positive from gram

negative bacteria.B)acidfast – differentiates acidfast from non-acidfast organism.

3. Special – used to detect special bacterial structure (capsule, flagella, spores, and intracellular)

inclusion granuleA) Negative B) Positive

4. Biochemical

= detect different metabolic products and sugar

fermentation reaction of various species

of microorganisms.

= 60% of common pathogens are identified by metabolic test.

= commercial kits now available especially for identification of enterobacteria.

5. Serological – detect presence of antibodies in patient

serum against specific antigens. Ex. Widal test, TPI, VDRL

6. Animal pathogenicity

7. Antibiotic susceptibility test – important use for the

purpose of treatment.

8. Epidemiologic test – useful in tracing source of outbreak of certain bacterial

diseases.

Specimen

smear cultivation (pure culture)

microscopic colonial morph. exam. biochemical

serologic animal path. antimicrobial susceptibility

Bacterial Morphology

Bacterial cell is a complete unit of any living organism. All its functions are genetically controlled and performed by that particular cell structure whether it be physiologic or biochemical.

Bacterial morphology includes:

Form, Arrangement, Size and Staining reaction

I. Form= determine by heredity= genetically most bacteria are monomorphic

(maintain a single shaped) = however some environmental conditions can

alter shape = some are genetically pleomorphic

(can have many form) = determined by the configuration of the cell wall

= detected by brightfield microscopy of stained smear

A. Coccus (spherical-shaped) = round-like a ball, perfect sphere, globe = can be oval, elongated, or flattened on one side

Variations : 1. Ovoid shape- both sides rounded ends are pointed.

Ex. Streptococcus

2. Lancet-shape - one end is pointed, other end is flat.Ex. Pneumococcus

3. Coffee-bean shape- flat on one side, opposite side convex or appear as letter “D” form.Ex. Neisseria

B) Bacillus (rod-shaped) = cell appears longer than wide or cylindrical form

= both sides parallel and ends are convex = varies in actual form depending on the species

Variations:1. Clubbed/drumstick shaped–swollen on one end.

Ex. C. diphtheriae / C. tetani

2. Corset-shaped – both sides swollen, end flat or

concave. Ex. Bacillus anthracis

3.Fusiform - both sides parallel ends pointed (thick at the center and tapered)

towards the end

C) Spiral = bacteria w/ more than one somatic curved

= may be regarded as bacillary form twisted to

form a helix.

2 types: 1. Flexible – spirals that can contract and relax

whose long axis bends when in motion.

Ex. Spirochetes

Genus Treponema - tightly coiled spiral

with corkscrew appearanceEx. T. pallidum

Genus Borrelia- much less tightly coiled having

the appearance of an extremely long

undulating pole. Ex. B. recurrentis

Genes Leptospira- tightly coiled spiral with

sharp-hooked like bends.

Ex. L. interrogans

2. Rigid – spirals that cannot contract and relax and

move by rotation in corkscrew-like (long axis remains) rigid when in

motion

Ex. Spirillum minor

D) Intermediate forms

A. Coccobacillary–when a rod is short thick, wide/plump.

= these form is intermediate between a

spherical and rod.

Ex. Haemophilus, Brucella

B. Vibrio - a gently curved bacteria (comma-shaped)

Ex. V. cholerae

II. Arrangement = is the result of the number of planes in which

the cell divides and how they remain attached afterwards.

A) Sphericals

1. Singly – occurs as a single spherical cell

2. Chain – common among ovoid-form resulting

in one plane division with daughter cells

remained attached to one another (Streptococci)Ex. Streptococcus pyogenes

3. Pair–common with lancet-shaped and coffee-

bean shaped form resulting from one

plane division with daughter remain in

pair after dividing (Diplococci)Ex. Streptococcus

pneumoniae Neisseria gonorrheae

4. Cluster – common with sphericals resulting from many plane division with daughter cell in

grape-like agglomeration bunch.

(Staphylococci) Ex. Staphylococcus aureus

5. Tetrads – (Packets of 4) – result from 2 plane division with daughter cell

separating from one another to form group of

4 cells. Ex. Graffya tetragena

6. Sarcinae – (Packets of 8) – results in 3 planes division with cells remain

attached in cube-like group of 8.Ex. Sarcina lutea

B) Rods = divides only across their short axis

= fewer arrangement of bacilli than cocci

= most bacilli appears as single rods

1. Chain – result in one plane division with daughter

cell remain attached to one another.

Ex. Bacillus anthracis

2. Palisade – arrangement like fence due to slipping

movement of daughter cells. Ex. Diptheroids

3. Chinese-letter arrangement – common with clubbed-

shaped rods resulting from a snapping post

division movement of the daughter cells.

Ex. Corynebacterium diptheriae

4. Packets of cigarette – arrangement like bundles.

Ex. Mycobacterium leprae

5. Serpentine – chain arrangement commonly seen among virulent strain of Myco. tuberculosis

C) Spirals = no characteristic cell arrangement, most occur

singly

= different species vary in size, length, rigidity and

amplitude of their coils

III. Bacterial size

1. Micrometer 2. Ultrafiltration 3. Ultracentrifugation

•Cocci – 0.5 – 3.0um dia.

•Bacilli – 0.2 – 2.0um dia. X 0.5 – 20um in length

•Vibrio and Spirilla – 0.2-2.0um dia. X 0.5-100um L

•Spirochetes – 0.1 – 3.0um dia. X 0.5 – 250um L

IV. Staining Reaction

= putting a color to facilitate identification

= unstained bacteria are colorless and transparent

= can be determined through the used of

basic aniline dyes

Types: 1. Simple = employs one dye only = sufficient to det. shaped and

arrangement

2. Differential = employs the use of more than one dye

= enables microbiologist to differentiate one

group from the other by the color as well

as shape = it is based on relative affinity of diff.

bacterial cell for the stained used

2.a) Gram = diff. gram (+) from gram (-) bacteria

= gram (+) those that retain the primary stain and deep violet or purple color

= gram (-) those decolorize with ethyl

alcohol and stained by the counter

stain appearing pink or red in color

2.b) Acidfast = differentiate acidfast from

non-acidfast bacteria

3. Special = use to color and determined bacterial

structures like capsule, flagella, inclusion granule, endospore and

etc. 3.a) Negative staining

3.b) Positive staining

BACTERIAL CELL

ULTRASTRUCTURES

General Division:

I. External - Capsule - Fimbriae - Flagellum

II. Internal - Cell Wall - Cytoplasmic Membrane - Cytoplasm- Mesosome - Nuclear Body- Ribosome - Plasmid - Inclusion Granule - Endospore

BACTERIAL ULTRASTRUCTURE

A. Bacterial Surface Coating: = are extracellular polysaccharide polymers which

surrounds the bacterial cell

Capsule = well-formed thick viscous jelly-like structure

firmly attached to the cell wall surrounding the cell

= not readily removed. = easily visualized by negative staining using

India ink method

Slime layer

= loose and irregularly arranged meshwork of

fibrils totally detached from the cell but

still surrounds the cell

= when present are more easily washed off

Chemical Comp.: = water – main component 98-99%= 1-2% of bacterial capsule are

chemically polysaccharide, Except capsule of:

B. anthracis-polypeptide (D-glutamic acid)

S. pyogenes-Hyaluronic acidFunction:

= protection from phagocytosis = correlates with virulence

= act as antigen (used in the identification)

and typing of capsulated bacteria

= allows bacteria to adhere/attach to various

surfaces in its natural environment in order to survive

Detection: 1. Negative staining (India

Ink)2. Special staining (Capsular

method) 3. Serological (Quellung

reaction)

B. Appendages Flagellum & Axial filaments Fimbriae

FLAGELLUM: = long, thick, helical protein filament of uniform

length and diameter

= commonly seen among free-swimming bacteria

= originates in cytoplasmic membrane = composed of 3 parts:

1. Basal body – anchors the flagellum to the

cell wall and plasma membrane

2. Hook – attached to the basal body and

connects to the filament

3. Filament – external to the cell and contains

the flagellar protein

Chemical Comp.: protein monomer - Flagellin

Function - Motility organelle of many pathogenic bacteria

Detection: 1. Darkfield / Phase contrast microscopy

(Wet mount / Unstained smear)2. Brightfield/Electron Microscopy – using stained specimen 3. Serological – use of specific antisera against

flagellar (H) antigen 4. Swarming phenomenon 5. Motility medium 6. Hanging drop preparation

Types and Arrangement of flagella in relation to the distribution and number:

I. Monotrichous – single polar flagellum at one end

II. Multitrichous – more than one flagella

Types:A) Lophotrichous – tuft of flagella at one

polar end

B) Amphitrichous – single flagellum or tulf of

flagella at both polar end

C) Peritrichous – flagella distributed all over the

body of the bacteria

Axial Filaments = flagella-like sheathed filaments

located in the periplasmic space (between

the inner) and outer membrane of

the cell= move by traveling helical wave on

opposite direction

= motility organelle of spirochete

Fimbriae / Pili / Microfibrils = short, straight, thin hair-like filaments usually

distributed around the body of bacteria.= originates in the cytoplasmic membrane = found virtually among all gram (-) bacteria but

not in gram (+) bacteria

2 types according to function:

1. Common/ordinary – for attachment or adherence

to mucosal surfaces of host cell during colonization and infection.

2. Sex pili – responsible for attachment of donor and

recipient cell during bacterial conjugation whereby genetic material (DNA) from one

cell is transferred to another (reproduction).

Chem. Comp. - Protein (Pilin)

Detection: Electron Microscopy

Cell wall (Peptidoglycan, Murien, Mucopeptide / Glycopeptide)

= complex, rigid, multilayered structure that protects the

protoplast and the underlying fragile plasma membrane

= found in all pathogenic free-living bacteria except Mycoplasma (cell-walless bacteria)

Chemical Comp.:1. Protein (Mesodiaminopimelic acid,Isomers of D-glutamic)

acid and D-alanine

2. Polysaccharide (N-acetylmuramic acid and N-

acetylglucosamine)= responsible for rigidity of cell wall

3. Lipids

Function :1. Responsible for the characteristic shape of bacterium2. Provides strong structural support necessary to keep

bacterial cell from rupturing due to changes in

the environmental osmotic pressure 3. Contain somatic O antigen that can serologically

identify particular bacteria 4. Site of action of some antibiotics 5. Determines differences in gram staining reaction

Detection :1. Microscopy of smear prepared from special

staining method.2. Electron microscopy. 3. By chemical methods using lysozyme.

COMPARISON OF GRAM POSITIVE AND GRAM NEGATIVE CELL WALL

Gram PositiveGram Positive Gram NegativeGram Negative

Peptidoglycan Peptidoglycan Very thick homogenous single Very thick homogenous single layerlayer extensively crossed-linked. extensively crossed-linked. Constitute 50-90% of the cell Constitute 50-90% of the cell wall wall material. material. (NAM, NAG, D-alanine)(NAM, NAG, D-alanine) Diaminophemilic acidDiaminophemilic acid

More complex layer More complex layer composed composed of thin peptidoglycan with of thin peptidoglycan with few few crossed-linked and an outer crossed-linked and an outer membrane layer. membrane layer. Constitute 5-10% of the cell Constitute 5-10% of the cell wall material.wall material.

Auxiliary Auxiliary compounds compounds

Teichoic acid (Polymer of ribitol Teichoic acid (Polymer of ribitol and) and) glycerol phosphateglycerol phosphateLipoteichoic acidLipoteichoic acidSurface protein Surface protein

No teichoic acid No teichoic acid Outer membrane contains: Outer membrane contains: Phospholipids-located in Phospholipids-located in the the inner membrane inner membrane Lipoprotien – connects OM Lipoprotien – connects OM toto the peptidoglycan the peptidoglycan LipopolysaccharideLipopolysaccharide (LPS/Endotoxin layer) (LPS/Endotoxin layer) – – located in the outerlocated in the outer layer of the OMlayer of the OM - contains the lipid A - contains the lipid A

Penicillin Penicillin sensitivity sensitivity

Sensitive Sensitive ResistantResistant

Response to Response to lysozyme lysozyme

Digest/ DegradeDigest/ Degrade Resistant Resistant

Gram Positive

Gram Negative

Damage to the cell wall may result to:

= when a gram positive bacteria is exposed to lysosyme will degrade the

peptidoglycan layer resulting to complete

removal of the cell wall producing a osmotically fragile

wall-less spherical body called Protoplast

= since all cell wall components is removed, therefore incapable of regeneration

Spheroplast= when a gram negative bacteria is exposed to

lysozyme it looses the peptidoglycan layer

but retain the outer membrane leaving a

less fragile spherical body capable of regenerating the cell wall.

Protoplast: comprises the naked cytoplasmic membrane and

its content

PERIPLASM = space between the plasma inner membrane

and the outer membrane layer = readily observe among gram negative

bacteria = consist of a gell-like substances which

help secure nutrients from the

environment

CYTOPLASMIC/PLASMA/CELL MEMBRANE: = thin elastic bilayered semi-permeable

membrane lying underneath the cell wall enclosing

the cytoplasm of the cell.

Chemical Composition - Lipoprotein - (composed of phospholipids and protein

molecules)

Functions: 1. Serve as selective permeability barrier

(transport of nutrient material in & out of cell)

2. Site of electron transport and oxidative phosphorylation of substances

involve in the generation of chemical energy (ATP)

3. Helps in the excretion of metabolic waste product

4. Site for excretion of enzyme involved in OM synthesis, CW synthesis and in the

secretion of extracytoplasmic & extracellular

substances:A. Permease for active uptake of

nutrientB. RespiratoryC. Polymerase that manufacture

substancesof the cell wall

D. Hydrolytic

Detection - Electron microscopy

MESOSOME: = usually seen as an invagination of the

membrane associated cytoplasmic sac seen in Gram

(+) cell = contains lamellar, tubular and vesicular

structure = often associated with division septa Chemical Composition - Lipoprotein

Functions: 1. Site for the synthesis of

cytochrome oxidase and reductase enzyme

2. Provide support for enzymes present in the cell

membrane 3. Responsible for compartmenting

DNA during cell division and sporulation

Detection- Electron microscopy

Cytoplasm = refers to everything that is enclosed by the

cytoplasmic membrane = site for most bacterial metabolism = 80% is composed of water

NUCLEAR BODY: (Nucleus / Nucleoid)= genetic material of bacteria = contains a single circular molecule of double

stranded DNA network which runs parallel to the

long axis of the cell = found homogenous within the cytoplasm not

enclosed in a nuclear membrane. = constitute 2-3% of the cell weight. = no definite form and seen at all stages of

growth cycle of the bacteria.

Chemical Composition – DNAFunction:

1. Controls the growth and metabolic activity of

the bacteria 2. Responsible for hereditary

characteristics of the cell. Detection- Electron microscopy, Feulgin staining

(+)

PLASMID/EPISOME: = extrachromosomal genetic material capable of

autonomous replication usually located near

the chromatin body.

Chemical Composition – DNA

Function:1. Transfer of genetic material from one cell to another

by conjugation (Transmissible Plasmid)2. Carry genes for activities like:

a) Antibiotic resistance b) Toxin production c) Synthesis of enzyme d) Tolerance to toxic

metals

Detection– Feulgin staining (+), Electron microscopy

RIBOSOME: = histone-like particles composed of ribosomal

RNA (rRNA) and protein molecules found in the cytoplasm of the cell.

= site of action for many antibiotics that inhibit protein

synthesis.= have a sedimentation coefficient of 70S and are composed of 50 S and 30 S subunits

containing 16 S, 23 S and 5 S RNA respectively

= procaryotic ribosome - 70s= eucaryotic ribosome - 80s

Function – Site for protein synthesis

Detection– Feulgin staining (-), Electron microscopy

INCLUSION / CYTOPLASMIC GRANULE: = large granules found anywhere in the

cytoplasm= are accumulation of organic and inorganic

substances which serves as source of energy and

nutrient supply to the organism

Kinds:1. Babes Ernst / Volutin / Metachromatic granules

Chemical Composition – polymerized metaphosphate which can be

used in the synthesis of ATP

Function – reserve energy supply

Demonstration – simple staining / special staining (Albert’s stain)

2. Lipid granules

Chemical Composition – polymer of beta-

hydroxybutyric acidFunction – reserve food supply.

3. Sulfur granules – derived energy by oxidizing

sulfur and sulfur-containing

compounds.

Function – reserve energy supply for sulfur-containing bacteria.

4. Glycogen granule – polymer of glucose.

Function – reserve food supply.

ENDOSPORES:

= highly refractile body formed within vegetative

bacterial cells in response to adverse

environmental

condition and due to deficient nutrient supply

= known as endospore, because it is formed inside

the

bacteria cell and is genetically controlled

= are metabolically inactive bacterial cell that are

highly

resistant to desiccation, heat and various

chemical

agents

= found in the cytoplasm of rod-shaped

sporeforming

bacteria of the genus Bacillus & Clostridium

= Composed of five parts: 1. Core – located at the center which is the

spore cytoplasm/protoplast.

- contains the nucleus and enzyme dipicolinic acid

2. Spore wall – enclosing the core.3. Cortex – laminated structure surrounding the

spore wall

4. Spore coat – multilayered membrane enclosing the

cortex 5. Exosporium – the outermost covering of the

spores which give the spore a

rigid appearance.

= contains Calcium Dipicolinate which aids in heat resistance within the core by making the

protein and nucleic acid more resistant to denaturation

= germinate under favorable nutritional condition after

an activation process that involves damage to the spore coat

= helpful in identifying some specie of bacteria especially those sporeforming bacilli (Clostridium and Bacillus)

Classification accdg. to:

A. Location - Central, Subterminal, Terminal

B. Shape - a) Ovoid b) Circular

C. Swollen/not swollen (swollen when the diameter is more than the of

the) bacterial cell, not swollen diameter the same as bacterial cellDetection:

1. Microscopy of stained smearA) Negative staining B) Positive staining

2. Phase contrast microscopy

PHYSIOLOGY OF BACTERIAL CELL

Nutrition = process by which chemical

substances (nutrients) either organic or inorganic are use

in cellular activities of microorganism for

metabolism and growth

Growth = orderly increase of all chemical constituents

of the cell including size and number

= process entails replication of all cellular structures,

organelles and protoplasmic components of

the cell Generation time

= measure of the growth rate of microbial species. = varies in length of time accdg. to environmental

conditions.

Bacterial growth requirement:

1) Water – most important requirement = vehicle for the entry of all

nutrients into the cell and elimination of their

waste product = form an integral part of the

cell protoplasm

2) Nutrient requirement: = source of Carbon and Nitrogen

a) Carbon = major building block for constructing cell material

Types of bacteria acdg. to carbon requirement

1. Autotroph (Lithotroph)= req. only H2O, CO2 & inorganic

substances = utilize CO2 as source of carbon= Photoautotroph- light as energy

source = Chemoautotroph - chemical reaction as

energy source 2. Organotroph

= requires organic substances for growth

= unable to utilize CO2 as energy source Photoorganotroph - light as energy

source Chemoorganotroph - chemical

reaction as energy source

3. Heterotroph = requires both organic and inorganic

substances for growth

b) Nitrogen Requirement

= main reservoir of nitrogen is nitrogen gas

(N2)

which make up 79% of earth atmosphere

= must be degraded into their basic building

block

(Protein Amino acid; Nucleic acid

Nucleotides)

3) Inorganic Ions = small amount needed ex. Sulfur,

Phosphorous, Magnesium, Calcium, Manganese, Zinc,

Cobalt, Copper

4) Growth Substances = organic nutrient essential to an organism

metabolism that cannot be synthesized and must be provided in the culture medium

Ex. yeast extract, whole blood, serum, B-complex vitamins, amino acids, purins and pyrimidines

Prototrophic = bacteria that do not require exogenous source of growth

factor because they are capable of synthesizing their own

Auxotrophic = bacteria that requires additional

growth factor in the culture medium

for growth to occur

II. Physical Requirement

A)Temperature 1. Phychrophile/Cryophile

= 5-300C opt. 10-200C= grow at refrigeration

temp. of 40C - 80C= responsible spoilage food

refrigeration = cold –loving bacteria = found normally in cold

water

2. Mesophiles = 10 – 450C opt. 20-400C= saprophytic – 26-350C= parasitic – 37-450C= bacteria pathogenic for human

3. Thermophiles = 25-800C opt. 50-600C= hotspring, tropical soil, hot water heater

4. Thermoduric = 80-1000C= resist high temperature but cannot grow

and multiply

B) Oxygen = required by particular bacterium to satisfy its energy needs 5 groups of bacteria on the basis of their O2

requirement

1) Obligate anaerobes = grows only under

condition of high reducing intensity and for which

oxygen is toxic (complete absence of

oxygen)= requires oxygen - free

environment to survive

2) Aerotolerant anaerobes = organism that are not

killed by exposure to oxygen

3) Facultative anaerobes = capable of growth under both aerobic

and anaerobic condition

4) Obligate aerobes = requires oxygen for growth

5) Microaerophilic = organism that grows best at low

oxygen tension

6) Capnophiles = requires 5-10% carbon dioxide and

oxygen on primary isolation

C) Hydrogen Ion Concentration (PH) = optimum ph for pathogenic

bacteria 7.2 – 7.6

1. Acidophilic – below ph 6.5 – 7.6 2. Basophilic – (alkalophilic) – 8.4 – 9.0 3. Normophilic (neutrophilic) – ph 7.5 – 8.0

D) Osmotic Pressure = direct pressure – resistant = osmotic pressure – sensitive = plasmolysis – shrinkage = osmophile – bacteria that can

grow on high osmotic pressure

= Ex. Halophile – seawater bacteria grow best at

high concentration of salt

Measurement of Bacterial Growth

1. Cell Concentration (cell number) number of cell per unit volume of culture. Can be counted directly by a

microscope counting chamber.

DETERMINATION:

1) Total direct plate count A) Bacterial Counting

Chamber (Petroff-Hauser

Counter)

B) Coulter Counter (Electron Particle Counter)

= measures both distribution of size

and number of bacteria in

bacterial suspension

2) Indirect Viable Count = plate sample of culture

and make dilutions of microbial

population inoculate suitable solid medium

incubate formation of viable

colony

USES:A) Solving problems on bacterial

cell division B) Genetic C) Infection D) Microbial Inactivation

2) Cell Density (Cell Mass)= total protoplasm/dry weight of the cell per

unit volume of culture

= include living and dead cell

DETERMINATION:1. Absorbance Spectrophotometer

= measuring optical density of broth culture of

microorganism 2. Turbidimetric Technique

= useful in determining mass of cells during bacterial growth

3. Nitrogen Determination 4. Centrifugation

USES: 1. Study of bacterial nutrition 2. Biochemistry

Bacterial Growth Curve (5 PHASES)

1. Lag phase (Phase of Rejuvenescence/Phase of)Physiologic Growth

= after inoculation bacteria requires a period of

adjusting and adapting to new environment

marked increase macromolecular

component of the cell increase in cell size

but no detectable increase in cell number

= no cell division occur

2. Exponential phase (Logarithmic phase) = cell in state of balanced growth = characterized sharp rise in growth

curve indicating rapid growth and multiplication

= bacterial cell double its growth per unit of time

3. Stationary phase (phase of equilibrium/”plateau”)

= manifestation of unbalanced growth growth rate zero = viable count remains constant for a short period eventually gives way to decreasing population

= number of living bacteria equals number of bacteria dying

4. Phase of Decline (Death phase)= growth rate decreases complete cessation of multiplication = result in the decline in total viable count= most bacteria dies due to :

1. lack of nutrient material in the medium 2. accumulation of waste product excreted by

the bacteria 3. change in ph of the environment

Consequence of growth & multiplication is reproduction

3 methods of bacterial reproduction 1. Binary fission 2. Branching 3. Sporulation

2 methods of bacterial multiplication 1. Binary fission 2. Branching

STERILIZATION STERILIZATION

ANDAND

DISINFECTIONDISINFECTION

STERILIZATION STERILIZATION - the process of destroying all forms of microbial - the process of destroying all forms of microbial lifelife

in terms of their ability to reproduce and in terms of their ability to reproduce and multiplymultiply

including spores including spores

PHYSICAL AGENTS FOR STERILIZATIONPHYSICAL AGENTS FOR STERILIZATIONA. HEAT:A. HEAT:

= most reliable method of sterilization= most reliable method of sterilization= method of choice for material which are not = method of choice for material which are not

damage damage by heat by heat

= denature proteins and damage membrane= denature proteins and damage membrane

2 types of heat:2 types of heat:

1. DRY HEAT:1. DRY HEAT:= preferred method for sterilization of = preferred method for sterilization of

glasswares, such glasswares, such as glass syringes, test tube, petri plates as glass syringes, test tube, petri plates

and and material such as oil, jellies and powdersmaterial such as oil, jellies and powders

= less effective than moist heat as it requires = less effective than moist heat as it requires longer longer

time of heating and higher temperature time of heating and higher temperature = kills the organism by oxidizing their chemical = kills the organism by oxidizing their chemical

constituent constituent

a) Hot air oven: a) Hot air oven: = most widely used method of sterilization by = most widely used method of sterilization by

dry heat dry heat = used for sterilization of glasswares such as = used for sterilization of glasswares such as

flask,flask, pipettes, test tube, swab pipettes, test tube, swab = principle of killin= principle of killing: heating at 160g: heating at 160OOC or 180C or 180OOC C

forfor 30 minutes 30 minutes b) Direct Flamming/Heating b) Direct Flamming/Heating

= direct heating of instrument or object in a = direct heating of instrument or object in a flame till itflame till it

becomes red hotbecomes red hot= easy way of sterilization but has limited = easy way of sterilization but has limited

applicationapplication= useful method of sterilization for non-= useful method of sterilization for non-

inflamable inflamable material such as bacteriological loop or material such as bacteriological loop or

needle,needle, forceps, scissors forceps, scissors

c) Incineration c) Incineration = direct burning of contaminated materials= direct burning of contaminated materials= efficient method for sterilization and disposal = efficient method for sterilization and disposal

of of soiled dressing, bedding of patient and soiled dressing, bedding of patient and

animal animal carcassescarcasses

2. Moist Heat = kills bacteria faster than dry heat 1. Boiling

= 30 minutes boiling at 1000C is adequate to kill

vegetative cell of bacteria, fungi, protozoa

= sporeformer requires 5½ hours boiling to kill the organism

2. Free – Flowing stream = live stream Arnold sterilizer = same sterilizing action with boiling

3. Steam Under Pressure = heating material at 15 lbs/square inch

pressure for 15 – 20 minutes at 1210C = most efficient and reliable method of

sterilization = commonly used for sterilizing of:a. Hospitals surgical & medical

instrumentb. Bacteriological media not

destroyed by heat c. Hospital supplies

4. Tyndallization (Fractional/Intermittent sterilization) = for sterilization of liquid & semi-solid material

easily destroyed by heat . Ex. culture medium contg. egg, serum,

carbohydrate= heating material at 80-1000C 30 minutes 3

conc. days = kills both sporeformer & non-sporeformer

bacteria

5. Pasteurization = consist of heating material at 620C for 30

minutes followed by rapid cooling

= use to kill only disease – producing organism found in milk, beverages, milk products and in preparation bacterial vaccine

Mechanism of moist heat: Denetuaration and coagulation of

protein inside bacterial cell

B) Radiation = produce damage to DNA resulting to killing of the organism = 2 types :

A. Non-ionizing radiation Ex. Ultraviolet rays – have low

penetrating power = used to destroy bacteria, fungi,

viruses that are sensitive to U-V

treatment= primarily used to control airborne

infections in nurseries, hospital wards & OR

B. Ionizing radiation Ex: X-ray, alpha, beta and gamma rays

= have greater penetrating power = use extensively for sterilization of cutgut , plastic syringe, catheter, prosthesis,

swab and dressing pack C.) Filtration = process of separating microorganism from

contaminated solution = employs the use of filters for removal of

microorganism that are sensitive to heat

= for sterilization of heat – labile C.M containing serum,

plasma, urea

D) Ultrasomic vibration = are sound waves with high frequency

causes desruption & disintegration of the cell

= uses 1. research laboratories 2. treating sewage H2O

E) Lyophilization ( Freeze-drying )= not reliable = causes protein denaturation & damage of the

membrane w/ leakage of intracellular organic compounds

F) Laser’s = sterilization of medical instrument and

operating room

Chemical agents :Disinfection = process destroying / eliminating

potentially hazardous & pathogenic organism

= aims to destroy the disease-= aims to destroy the disease-producing producing

organismsorganismsDisinfectant = agents that kills growing (vegetative)

form of organism but not necessarily

sporeformer = applied to non-living/inanimate object

Bactericidal / Germicidal = implies the killing action of

chemical substancesBacteriostatic = substances that inhibit / prevent

growth & multiplication of bacteria

Antiseptics = subs. that either kill / prevent growth = applied to living tissue (animate

object)

found in the surface of skin found in the surface of skin and and

mucous membranemucous membrane

Mech. of action:1. damage cell membrane function 2. denatures protein 3. induce extensive nucleic acid damage

Agents that damage cell membrane function

I – Surface active agents : = destroys structural integrity of cell membrane

(protein & lipids)= interferes w/ normal membrane function = widely used as detergent, wetting agent and

emulsifier 3 types:

cationic = most useful = includes Quarternary ammonium

cpd., Benzalkonium chloride ( Zephiran )

anionic = soap, surfactant, sanitizer = help remove microbe by rubbing

nonionic = not effective, non-toxic

II – Phenolic compound = disrupt cell membrane function leaking cell

content

A) Carbolic acid (phenol)= excellent for disinfecting feces, blood, pus,

sputum and other proteinous material = primarily use for testing new bactericidal

agent

B) Cresol / Creolin Lysol - alkyl derivative phenol = disinfect inanimate object esp.

contaminated with TB bacilli Creolin - septic tank disinfectant

C) Diphenyl compound (Hexachlorophene)= effective against gram + bacteria (staph &

strep)

III – Alcohol = disorganize lipids cell membrane = denature protein = widely used antiseptics = has ability to remove lipids from skin

surfaces

Ethanol / Ethyl alcohol= skin disinfectant = disinfection clinical thermometer = effective against gram (+) , gram (-) , AF

bacteria

Isopropyl alcohol= most effective, less volatile = bactericidal property greater than ethanol = used as skin antiseptic = has a better fat solvent property and more

bactericidal = effective at a concentration of 50-70%

Agents that denatures protein 1. Acid & alkalies 2. Alcohol 3. Acetone

Agents that modify functional group of protein & nucleic acid

1. Salts of heavy metals – mercury, silver , arsenic

Silver cpd. widely used as antiseptic

AgNO3 (silver nitrate) – routinely used as

prophylaxis against opthalmia neonatorum

2. Oxidizing agents (Halogen Compound) iodine – wound/skin disinfectant

- active against tubercle bacilli chlorine – H2O disinfectant Hydrogen peroxide – use for cleaning

wound

3. Dyes Triphenyl methane dyes (aniline acid)Acidine dyes (Flavines) – yellow color

4. Alkalyting agent (Formaldehyde, Glutaraldehyde,)

Ethylene oxide = exert lethal effect on protein substances

Formaldehyde – in aqueous solution is bactericidal, sporocidal and also effective against

viruses = use for inactivating

vaccine and preservation of fabrics

formalin = in aqueous solution of 37%

formaldehyde is used for preserving

fresh tissue specimen = as gas, used for

fumigating operating room, hospital ward,

laboratories

Glutaraldehyde = used as cold sterilant for sterilizing

medical and surgical instruments

Ethylene Oxide = used for sterilization of

polyethylene tubing, heart lung machine, electronic hospital devices and sensitive medical instrument

Chemotherapeutic Agent

= one of the most valuable method of treating infection

Antibiotics : are naturally occurring metabolic products of primarily soil bacteria & fungi

Desirable Properties :

1. Selective Toxicity 2. Bactericidal 3. Should not be allergenic and with no adverse reaction

on large doses or prolonged use 4. Active in the presence of plasma, body fluids &exudates 5. Should have broad-spectrum activity 6. Water soluble & stable 7. Susceptible organism should not become resistant 8. Cheap

Antibiotic producers 1. Penicillium 2. Bacillus 3. Streptomyces 4. Cephalosporium

Mech. of action :1. Interfere with cell wall synthesis 2. Interfere with cell membrane function 3. Interfere with Protein synthesis 4. Interfere with nucleic acid metabolism

Cell Wall Inhibitors :1. Beta – lactam antibiotics

A. PenicillinB. Cephalosphorins

2. Fosfomycin 3. Cycloserine 4. Vancomycin 5. Bacitracin

Penicillins 1. Natural PCN – Benzyl PCN G

- Phenoxymethyl PCN V 2. Semi – synthetic PCN

A) Penicillinase – resistant PCN’s Methicillin Nefcillin Isoxazolyl PCN – Cloxacillin

Drug of choice - Dicloxacillin For PCN resistant - Oxacillin

S. aureus & S. epidermides B) Extended spectrum PCN’s

Ampicillin Amoxicillin

C) Antipseudomonas Carbenicillin Ticarcillin Piperacillin

Cephalosporins = fermentation product fungus

Cephalosporium

First Generation – bactericidal against most gram (+) and many gram (-) bacteria , Except :

Enterobacter Proteus ( Indole + ) Pseudomonas Cephalothin Cephapirin Cefazolin Cephradine Cephalexin Cefadroxil

Second Generation – Gram (-) bacilli – Enterobacter - Proteus

Cefamandole - Hemophilus Cefoxitin – N. gonorrhea Cefaclor Serratia Cefuroxime Bacteroides

Third Generation – less active against gram (+) but more active against gram (-) bacteria

Cefoperazone Ceftazidine more active against Cefpiramide Pseudomonas Cefpirome

II : Cell Membrane Inhibitors 1. Polymixins – reserved for serious pseudomonas infection 2. Polyenes

Amphotericin B – for deep seated mycoses Nystatin – seperficial & cutaneous fungus infection

3. Azoles – Imidazole antifungal agent – Ketaconazole - Miconazole - Clotrimozole

4. Triazoles – Itraconazole - Fluoconazole

III : Inhibitors of DNA function 1. Mitomycin 2. Nalidixic acid & Quinolones 3. Norfloxacine & Ciprofloxacin 4. Metronidazole – for anaerobic infection 5. Novobiocin – gram (-) 6. Griseofulvin

IV : Inhibitors Protein Synthesis

A. Actinomycin – active against gram (+) & gram ( -)

B. Rifampicin – major drug for TB & Leprosy

C. Streptomycin

Gentamycin Aminoglycosides

Tobramycin

Amikacin

D. Chloramphenicol

E. Nitrofurans

F. Erythromycin

G. Clindamycin / Lincomycin

V : Metabolite Analogue

1. Sulfonamide

= gram (+) gram, (-) Nocardia, Chlamydia,

Pneumococcus

2. Sulfone

= Myco. leprae

3. Para-aminosalicyclic acid

(PAS) – tuberculosis