BACTE CULTURE MEDIA

Bacterial Cultivation

vivo environment

vitro environment

– process of growing microorganisms in culture by taking bacteria from the infection site (in ________________)

by some means of specimen collection and

growing them in the artificial environment of

the laboratory (in _________________)

• To grow and isolate all bacteria present in a

clinical specimen.

• To determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers (normal microbiota).

• To obtain sufficient growth of clinically relevant bacteria to allow identification, characterization,and susceptibility testing.

Main Purpose of Bacterial Cultivation

CARBON

NITROGEN

NON-METALLIC ELEMENTS

METALLIC ELEMENTS

VITAMINS

WATER

ENERGY

NUTRITIONALREQUIREMENTS FOR GROWTH:

Carbon

: the most essential and central atom

common to all cellular structures and functions.

AUTOTROPHS

HETEROTROPHS

2 CARBON

Autotrophs

: These organisms can be cultivated in a medium consisting solely of inorganic compounds.

Heterotrophs

: These organisms cannot be cultivated in a medium consisting solely of inorganic compounds; they must be supplied with organic nutrients.

Nitrogen

proteins

nucleic acids

: essential atom in many

cellular macromolecules, particularly

_________ and ________

SULFUR

PHOSPHORUS

2 Non-metallic elements

Sulfur

– integral to some amino acids and is

therefore a component of proteins.

organic compounds (sulfur-containing amino acids)

inorganic compounds (sulfates).

Sources OF SULFUR:

Phosphorus

– necessary for the formation of the

nucleic acids DNA and RNA and also for

synthesis of the high-energy organic compound

adenosine triphosphate (ATP).

Metallic Elements

• Ca2+, Zn2+, Na+, K+, Cu2+, Mn2+, Mg2+, Fe2+,&

Fe3+ - some of the metallic ions necessary for

continued efficient performance of varied

cellular activities

osmoregulation

regulation of enzyme activity

electron transport

ACTIVITIES OF METALLIC ELEMENTS

Vitamins

sources of coenzymes, which are required for the formation of active enzyme systems.

Water

: all cells require distilled water in the medium so that the low-molecular-weight nutrients can cross the cell membrane.

Energy

: active transport, biosynthesis, and biodegradation of macromolecules.

PHOTOTROPHS

CHEMOTROPHS

2 ENERGY

Phototrophs

– use radiant energy.

Chemotrophs

glucose

H2S or NaNO2

– dependent on oxidation of chemical compounds. Organic molecules (_________); Inorganic compounds (___________)

CULTURE

CULTURE MEDIUM

COLONY

CULTURE AND CULTURE MEDIA

CULTURES

– are growth of microorganism on a culture medium

CULTURE MEDIUM

– is a liquid, semi-solid or solid medium utilized to observe growth patterns of microorganism as well as for transport and storage (cultivate or we propagate or

microorganisms for testing)

COLONY

- is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike

peptone

meat extract

yeast ectract

mineral salt

carbohydrates

agar

water

COMMON INGREDIENTS OF CULTURE MEDIA

Peptone

free amino acid

peptides

proteoses

carbohydrates

nucleic acid fractions

minerals

vitamins

Hydrolyzed product of animal and plant proteins:

___________, _________ and _______ (large sized peptides).

•Provides nitrogen; as well __________, _____________, _________ and _______.

Meat extract

: supply amino acids, vitamins and mineral salts.

Yeast extract

: is a bacterial growth stimulants.

Mineral salts

•Phosphates as a source of phosphorus.

•Sodium chloride

•Sulfates as a source of sulfur.

Carbohydrates

Sucrose in TCBS agar (Thiosulfate–Citrate–Bile Salts–Sucrose agar)

Lactose in MacConkey agar

: Simple and complex sugars are a source of carbon and energy.

Assist in the differentiation of bacteria.

•eg. _______________ differentiates vibro species.

_______________________differentiates enterobacteria.

AGAR

micro-organism

: an inert polysaccharide of seaweed.

Not metabolized by _________.

(80-90oC)

(40-50oC)

1.5% W/V concentration.

0.4-0.5% W/V concentration.

Property of AGAR:

• It has high gelling strength

• High melting temperature_________

• Low gelling temperature__________

• It forms firm gel at _____________

• It forms semisolid gel at _______________

• Solidify culture media

• May provide calcium and organic ions to inoculated bacteria.

Uses: OF AGAR

Water

Deionized or distilled water must be used in the preparation of culture media.

• pH buffers

• Inhibitory Agents (dyes, salts, antibicrobials)

Other Ingredients: FOR CULTURE MEDIA

• specimen submitted

• infection process

• arranged

• most enriched

• most selective

Selection of Medium

Based on:

• type of ____________ for culture

• characteristic of organisms involved in the ____________

NOTE:

• If several plates will be inoculated for a given specimen = media should be ________ from _____________to ____________

PURE CULTURE

MIXED CULTURE

STOCK CULTURE

TYPES OF CULTURE MEDIA

PURE CULTURE

– It is composed of only one species

MIXED CULTURE

– It is composed of more than one species.

STOCK CULTURE

- Is composed of several species contained in a separate culture medium (one species per culture medium)

- cultivated in a large volume of broth and aliquoted in small vials

- It is used for academic and industrial purposes.

• I. ACCORDING TO CONSISTENCY

• II. ACCORDING TO COMPOSITION

• III. ACCORDING TO HOW THEY ARE DISPENSED

• IV. ACCORDING TO USE

CLASSIFICATION OF CULTURE MEDIUM

LIQUID MEDIUM

SEMI-SOLID MEDIUM

SOLID MEDIUM

ACCORDING TO CONSISTENCY

0% agar

aerobes

anaerobes

facultative anaerobes

tubed media

Trypticase Soy broth (TSB)

Brain heart infusion (BHI)

thioglycolate

LIQUID MEDIUM

• - Contains _________ (solidifying agent)

• - Allows growth of _______, ________, and __________

• - Commonly referred to as ___________

• o Examples: __________________, ______________, and __________

0.2 -0.5% agar

screw cap

Sulfide indole motility medium (SIM)

SEMI-SOLID MEDIUM

• - Contains ______________

• - Usually place in _________

tubes/plain test tube plugged with a

cotton ball

• - Examples: _________________

1.5-2% agar

TSI

Mac Conkey

BAP

Chocolate agar

SOLID MEDIUM (most common)

• - Contains _________

• - Examples: _____, _______, ______, __________

SYNTHETIC OR DEFINED MEDIUM

NON-SYNTHETIC OR COMPLEX MEDIUM

TISSUE CULTURE MEDIUM

ACCORDING TO COMPOSITION

medium

user

Davis

Mingioli Medium

BG-11 medium

ACCORDING TO COMPOSITION

A. SYNTHETIC OR DEFINED MEDIUM

• - Components of the _______ is known by the _____

• - Made at the laboratory by using adding certain grams of various components (for research purposes)

• Ex: ______ and ___________, __________

peptones

meat

yeast extracts

digested

ground up

Nutrient Broth(NB) medium

TSB

MacConkey(MAC) agar

NON-SYNTHETIC OR COMPLEX MEDIUM

• - Exact contents are not known (_______, ________, _________)

• - Use of _______ or _______ animal organs (liver/heart)

• - Useful in nurturing and isolation of medically significant bacteria

• Ex: ________________, ______, __________________

obligate intracellular bacteria

Rickettsia

Chlamydia

W138

HeLa 229 cells

McCoy cells

TISSUE CULTURE MEDIUM

• - It used for __________________ (__________, _________).

• Examples: _______, ____________ and ___________

HeLa 229 cells

McCoy & W 138 cells

For Chlamydia:

HeLa 229 cells

= human cervical tissue cells

McCoy & W 138 cells

= human fibroblasts

Embryonated eggs (avian)

For Rickettsia:

PLATED MEDIUM

TUBE MEDIUM

ACCORDING TO DISPENSING/DISTRIBUTION

PLATED MEDIUM (solid)

- Dispensed in petri dishes

1. Glass typed (must be autoclaved before using)

2. Disposable

- 2 types of Petri dishes

• Butt

• Slant

• Butt-slant

TUBE MEDIUM (liquid, semi-solid, solid)

A. SIMPLE/NUTRITIVE/SUPPORTIVE MEDIA/GENERAL

PURPOSE MEDIA

B. ENRICHMENT MEDIA (liquid-type media)

C. ENRICHED MEDIA (solid/non-selective media)

D. SELECTIVE MEDIA

E. DIFFERENTIAL MEDIA

F. SPECIAL MEDIA

ACCORDING TO USE

nutrients

growth

non-fastidious organisms

Nutrient Agar

Broth

TSB

A. SIMPLE/NUTRITIVE/SUPPORTIVE MEDIA/GENERAL

PURPOSE MEDIA

- Contains _______ that supports _______ of ____________

- Routinely used in the laboratory (w/o supplements)

• Examples: __________/________, ________

nutrients required

growth

specific nutrients

desired orgs.

supplement to plates

gas requirements

ENRICHMENT MEDIA (liquid-type media)

• Contains ____________ for the ______ of particular bacteria

• Contains ____________ (w/o supplements)

• Incubated and sub-cultured to isolate the _________

• Can be used as a ___________ to detect______________

Vibrio spp.

8.4

ENRICHMENT MEDIA

• Alkaline Peptone Water (APW) = best for _____ spp. (@pH _____)

SALMONELLA SPP.

feces

urine

water samples

Selenite F = _____ spp. (from ______, _____, _________)

Thioglycolate

___________ (for anaerobes)

SALMONELLA

PROTEUS SPP.

Tetrathionate (for _____ & _____ spp.)

Group B streptococci

LIM broth (for _____________)

Streptococcus agalactiae

EXAMPLE OF LIM BROTH

GN Broth

__________ (for Gram-negative organisms)

fastidious orgs

C. ENRICHED MEDIA (solid/non-selective media)

Contains specific nutrients (w/ supplements) for __________

blood

vitamins

yeast extracts

ENRICHED MEDIA (solid/non-selective media)

Supplements (________, ________, _________)

BAP (5% SHEEP’S OR HORSE BLOOD)

CAP

ENRICHED MEDIA (solid/non-selective media)

example:

BAP (5% sheep’s or horse’ blood)

enriched & differential (hemolytic patterns)

Haemophilus sp.

CAP = for _____ spp

enrichment media (liquid-type media) is isolate the desired orgs.

enrichment media (solid/non-selective media) for fastidious orgs

ano pinag kaiba ng enrichment media (liquid-type media) and enrichment media (solid/non-selective media)

SELECTIVE MEDIA

Contains one or more inhibitory agents that kills other organism

specific growth condition/chemical

other than the “selected” by ___________________

• (inhibits growth of another and supports another)

CEFSULODIN-IRGASAN-NOVOBIOCIN (CIN)

CEFOPERAZONE, VANCOMYCIN, AMPHOTERICIN (CVA)

PHENYLETHYL ALCOHOL AGAR (PEA)

AGAR/CNA AGAR (Columbia Nalidixic Acid Agar)

THAYER MARTIN AGAR

SELECTIVE MEDIA EXAMPLE AGAR

Cefsulodin-Irgasan-Novobiocin (CIN) Agar

selective media

Examples: ________________________ (for Yersinia and Aeromonas)

CVA

Cefoperazone

Vancomycin

Amphotericin

(for Campylobacter)

Phenylethyl Alcohol Agar (PEA)

Agar/CNA AGAR (Columbia Nalidixic Acid Agar)

(inhibits growth of gram negative and supports gram positive)

THAYER MARTIN AGAR

AND ITS MODIFICATION

Gentamycin Blood Agar

Bacitracin chocolate agar

BAP w/ Ampicillin

Phenylethyl Alcohol agar (PEA)

Columbia colistin-nalidixic acid agar (CNA)

Bacteroides Bile-Esculin Agar (BEA)

SELECTIVE MEDIA

• Other Examples:

Gentamycin Blood Agar

1. –Streptococcus

Bacitracin chocolate agar

2. – Haemophilus

BAP w/ Ampicillin

3. – Aeromonas

4.Phenylethyl Alcohol agar (PEA)

5. Columbia colistin-nalidixic acid agar

– Gram-pos bacteria

6.Bacteroides Bile-Esculin Agar (BEA)

– anaerobes like B. fragilis

Crystal/gentian violet, basic/carbol fuchsin, bile salt

Potassium tellurite, sodium azide

Alcohol, chloral hydrate

Inhibitory substances: selective media

Crystal/gentian violet

basic/carbol fuchsin

bile salt

X gram+ve (gram positive)

Potassium tellurite

sodium azide

= X gram-ve (gram neg)

Alcohol

chloral hydrate

= X swarming phenomenon (swarming bacteria)

E. DIFFERENTIAL MEDIA

These are media that allow visualization of metabolic differences

• between groups of bacteria

Mac Conkey

BAP

EMB

HEA

E. DIFFERENTIAL MEDIA

Examples: ____________, ______, _____ and

______

Mac Conkey

pink colonies

colorless colonies

selective

differential media

– differentiates lactose fermenters (_________) from non lactose fermenters (___________); acts as both _______ and ______________

BAP

differentiates hemolytic pattern (streptococci)

F. SPECIAL MEDIA

high protein

high fats

Use for bacteria with special growth requirements (_________, ________)

Löwenstein-Jensen (LJ) medium

Thiosulfate-citrate-bile salt-sucrose (TCBS) agar

F. SPECIAL MEDIA

Examples:

Löwenstein-Jensen (LJ) medium

whole eggs

malachite green

inspissation

_____________________ = for mycobacteria (________, _____________) *not autoclaved = ____________

Thiosulfate-citrate-bile salt-sucrose (TCBS) agar

thru boiling

= selective for Vibrio *not autoclaved = _________

MAC CONKEY AGAR

BLOOD AGAR

CHOCOLATE AGAR

HEKTOEN ENTERIC AGAR

XLYOSE-LYSINE-DEOXYCHOLATE AGAR

MANNITOL SALT AGAR

EOSIN METHYLENE BLUE AGAR

THIOSULFATE CITRATE BILE SALT AGAR

COMMONLY USED AGARS

MAC CONKEY AGAR

Contains neutral red that provides a differential test