BACTERIAL CULTIVATION - PowerPoint Presentation

Slide1

BACTERIAL CULTIVATION

SELMA ABDUL SAMAD

BCH-10-05-02

S1-MSc.BIOCHEMISTRYSlide2

NUTRITIONAL REQUIREMENTS OF BACTERIA

Nutrients

are substances used in biosynthesis and energy production. Hence required for

bacterial growth

Macronutrients (

constitute 95 % of dry cell weight

)

C,O,H,N,S,P –

Components of

carbohydrates,lipids,proteins,nucleic

acids

K,

Ca,Mg,Fe

– Exist as

cations

with different roles –

eg.,in

enzymes,heat

resistance to

endospores

, cofactors,

cytochromes

etc

.Slide3

Micronutrients – Mn,Zn,Co,Mb,Ni,Cu

– As part of enzymes and cofactors

Special requirements –

eg

.- Diatoms

need

silicic

acid

(H

4

SiO

4

)

to construct their silica cell wall

eg

.- Halophytes

need more sodium

All nutrients should be present balancedSlide4

REQUIREMENTS FOR C , H , O

Usually satisfied from same source

Usual sources for carbon are

CO

2

for

autotrophs

(

from atmosphere or cellular metabolism

)

Glucose for

heterotrophs

Most bacteria – flexible in case of carbon source Slide5

Based on oxygen requirement – aerobic and non-aerobic

Aerobes – require O

2

for growth

Obligate aerobes-

eg.vibrio

cholerae

Facultative aerobes – most medically important bacteria

Anaerobes-

eg

. Clostridia

Obligate- die on oxygen exposure

Facultative- grow well in anaerobic conditions

Microaerophilic

- grow well in low oxygen tensionSlide6

NUTRITIONAL TYPES OF BACTERIASlide7
Slide8

REQUIREMENTS FOR N , P , S

Nitrogen –

for synthesis of amino

acids,nitrogenous

bases,some

carbohydrates and

lipids,enzyme

cofactors etc

SOURCE –

aminoacids,ammonia,nitrates

(to NH3 by

phototrophs

),nitrogen(N2 fixation by

rhizobium

)

Phosphorous –

nucleic

acids,phospholipids,nucleotides,cofactors,proteins,cell

components

SOURCE-inorganic

phosphate,organic

P(eg.Hexose.6.P)

Sulfur –

aminoacids

(

cys,met

),some

carbohydrates,biotin,thiamine

SOURCE- sulfatesSlide9

GROWTH FACTORS

Organic components required because they are essential cell components or precursors of such components but cannot be

synthesised

by the organism are called

growth factors

For bacteria they are

Amino acids

Purines

and

pyrimidines

vitaminsSlide10

ENVIRONMENTAL FACTORS - GROWTHSlide11

CULTURE MEDIA

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions.

A culture of

bacillus

anthacisSlide12

A culture medium must supply suitable carbon and energy sources and other nutrients, sometimes including "growth factors" (defined below). It is important to note that

no one medium will support the growth of all microorganisms

Culture medium – a liquid/gel designed to support the growth of micro-organisms

2 common types – nutrient broth and nutrient agar

Basically differentiated to

Defined

(known quantity of all ingredients)

Undefined

(has some complex ingredients)Slide13

SYNTHETIC/DEFINED MEDIA

C source – CO2(as Na2CO3 / bicarbonate)

N source – nitrate / ammonia

Also sulfate and phosphate

Used for –

photolithoautotrophs

like

cyanobacteriaSlide14

COMPLEX/UNDEFINED MEDIA

Undefined components like peptones, meat extract, yeast extract

(each one serves as C, N and energy source)

Eg

. Nutrient broth,

tryptic

soy broth,

MacConkey

agar etc.Slide15

OTHER TYPES OF MEDIA

SIMPLE/BASAL MEDIA

Eg.Nutrient

broth –

peptone,meat

extract,NaCl

and water.

Nutrient agar is 2% agar added to broth

SELECTIVE MEDIA

0 Favor growth of a particular micro organism

0 bile salts/dyes(basic

fuchsin

/crystal violet) – favor growth of gram

negative bacteria and inhibit gram positive bacteria

0

endo

agar, EMB agar,

MacConkey

agar – contain dyes that

suppress gram positive bacteria

0 Media with only cellulose as C and energy source – selects

cellulose digesting bacteriaSlide16

ENRICHED MEDIA

0

substances like blood ,serum or egg are added to basal media

0 Used to grow bacteria which are more exacting in their

nutritional needs.

0

eg

. Blood agar, chocolate agar , egg media

ENRICHMENT MEDIA

0 Substances which have a stimulating effect on the bacteria to

be grown or an inhibitory effect on those to be suppressed are

incorporated in the media(usually liquid media)

0 An absolute increase in the number of the wanted bacterium

relative to the other

0

eg

.

Tetrathionate

broth (inhibits

coliforms

, allows typhoid –

paratyphoid bacilli to grow freely

0

Selenite

F broth – favors dysentery bacilliSlide17

DIFFERENTIAL MEDIA

0 Distinguishes between different groups of bacteria

0 Blood agar (also an enriched media)

distinguish between hemolytic and non-hemolytic bacteria

Hemolytic bacteria (

eg

. Many streptococci and staphylococci

isolated from throat ) produce clear zones around their colonies

0

MacConkey

agar (also a selective media)

since it contains lactose and neutral red dye , lactose fermenting

colonies appear pink to red in color , thus getting

distinguished.

Slide18

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus cultureSlide19

ISOLATION OF PURE CULTURE

Pure culture – population of cells arising from a single cell, to characterize an individual species

Different methods are used

SPREAD PLATE –

A mixture of cells spread out on an agar surface – each cell grows into a colony – each colony represents a pure culture ,and the number of viable organisms

.

STREAK PLATE –

A mixture of cells transferred to the edge of an agar plate with an inoculating loop and streaked out in a patternSlide20
Slide21
Slide22

POUR PLATE

The original sample is diluted several times – small volumes of diluted sample is mixed with liquid agar that has been cooled to about 45 degree , poured immediately into sterile culture dishes – each cell forms individual colonySlide23

STERILISATION and DISINFECTION

STERILIZATION – The process by which an article, surface or medium is freed of all living microorganisms either in vegetative or spore state.

DISINFECTION – The destruction or removal of all pathogenic organismsSlide24

STERILISING AGENTS

PHYSICAL AGENTS

Sunlight

Drying

Dry heat : flaming, incineration, hot air

Moist heat :

pasteurisation

, boiling, steam under pressure, steam under normal pressure

Filtration : candles, asbestos pads, membranes.

Radiation

Ultrasonic and sonic vibrationsSlide25

CHEMICAL AGENTS

Alcohols : ethyl, isopropyl,

trichlorobutanol

Aldehydes

: formaldehyde,

glutaraldehyde

Dyes

Halogens

Phenols

Surface active agents

Metallic salts

Gases : ethylene oxide, formaldehyde, beta

propiolactoneSlide26

SUNLIGHT

Has UV rays that are sterilizing along with heat rays

Bacteria suspended in water are readily destroyed by exposure to sunlight

DRYING

Moisture is essential for bacterial growth(4/5 of bacterial cell weight is of water)

Spores are unaffected by drying

HEAT

Most reliable method of sterilization

Influenced by nature (dry or moist) , temperature and time, type and characteristics of microorganisms present and which material to be

sterilised

Heating denatures

protein,causes

oxidative

damage,toxicity

due to elevated electrolytes etc.Slide27

DRY HEAT

FLAMING – Inoculating loop or wire, the tip of forceps and searing spatulas are held in a

bunsen

flame till they become red hot.

INCINERATION – Used for safely destroying contaminated materials – like cloth , animal carcasses and pathological materials

HOT AIR OVEN – Most widely used method – used for instruments and

glasswares

, forceps, scissors ,scalpels etc. Also

paraffin,dusting

powder, fats and grease.

The spores of a

nontoxigenic

strain of Clostridium

tetani

are used as a

microiological

test of dry heat efficiency.Slide28

MOIST HEAT

PASTEURISATION – for milk – heated either at 63` for 30 min or 72` for 15-20 min

All

nonsporing

pathogens are destroyed

BOILING – vegetative bacteria are killed almost immediately at 90-100` C ,but

sporing

bacteria require prolonged periods of boiling.

It is only a means for disinfection and not

sterilisation

STEAM UNDER PRESSURE – The principle of autoclave or steam

steriliser

is that water boils when its vapor pressure equals that of the surrounding

atmosphere.Hence

when pressure inside a closed vessel increases, the temperature at which water boils also increases. The condensed water ensures moist conditions for killing the microbes presentSlide29

FILTRATION

Helps to remove bacteria from heat labile liquids such as sera and solutions of sugars or antibiotics used for preparation of culture media

RADIATION

NON IONISING LOW ENERGY TYPE - electromagnetic rays with wavelengths longer than those of visible light are used.

Also

uv

radiations are used

IONISING HIGH ENERGY TYPE - x rays, gamma rays and cosmic rays are highly lethal to DNA and other vital constituents.

VIBRATIONS

Has no practical value in

sterilisation

and disinfection since micro organisms vary in their sensitivity to themSlide30

CHEMICAL AGENT

S

ALCOHOLS – ethyl alcohol and isopropyl alcohol are most frequently used.

They have no action on spores

Methyl alcohol is effective against fungal spores(hence used for treating cabinets and incubators)

ALDEHYDES – Formaldehyde is active against the amino group in the protein molecule.

It is bactericidal and

sporicidal,also

has lethal effect on virus

Glutaraldehyde

is also used and is less toxic than the former

DYES – aniline dyes and

acridine

dyes are used as skin and wound antiseptics

Both are just

bacteriostatic

and has low bactericidal activity.

Aniline dyes(brilliant

green,malachite

green,crystal

violet) against gram positive

organisms.Same

with

acridine

dyes too.Slide31

HALOGENS – iodine in aqueous and alcoholic solution has been used widely as a skin disinfectant.

Actively bactericidal with moderate action against spores

Chlorine and its compounds used as

dinsinfectants

in water supplies, swimming pools etc.

PHENOLS – cause cell membrane damage ,releasing cell contents and causing

lysis

.

Low

conc

ppt

proteins.

Membran

bound

oxidases

and

dehydrogenases

are inactivated by

conc

of phenol that are rapidly bactericidal for microbes

GASES – it

alkylates

the amino , carboxyl ,hydroxyl and

sulfhydryl

groups in protein molecules. In addition , it reacts with DNA and RNA.

Its use as a disinfectant presents a potential toxicity to human beings, including

mutagenicity

and carcinogenicity.

It’s effective against viruses and spores.Slide32

SURFACE ACTIVE AGENTS – Substances which alter energy relationship at interfaces, producing a reduction of surface or interfacial tension are referred to as surface active agents.

They usually act on bacterial membranes

Cationic agents act on the phosphate groups of the cell membrane and also enter the cell

METALLIC SALTS – salts of heavy metals have a greater action.

Salts of silver, copper and mercury are used as disinfectants

They are protein coagulants and have the capacity to combine with free

sulfhydryl

groups of cell enzymes, when used at appropriate conc.

Eg

. Copper salts , phenyl mercury nitrate,

and mercurochrome.Slide33

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