Culture media - nutrient material prepared for the growth of microorganisms in the laboratory Inoculum - microbes introduced into a culture medium to initiate growth Inoculation - process of transferring or isolating microorganisms from clinical specimen or from pure culture to a culture medium

Culture

- microbes that grow and multiply in or on

a culture medium

-

Types of culture:

1.Pure culture

- has only one species

or one strain of

microorganism

2.Mixed culture

- has two or more

species or strain of

microorganism

3. Contaminated

- unwanted microorganisms are accidentally

grown

ex. fungi in bacterial medium

4.Stock

- for research and schools

Classification of Culture

Media

A. According to Nutritional Composition

1. Simple or Basic Media

contain basic requirement for growing microorganism

beef extract, peptone and water Peptone proteins with shorter chains of amino acid (easily digested by bacteria)

Beef extract provides vits, other organic growth factors, organic

nitrogen and carbon compounds

Use: to grow non-fastidious microorganisms

A. Liquid Media

Ex. Nutrient broth (1% peptone, 0.5%NaCl

and water)

Peptone water(peptone water and 1%

meat extract)

B. Solid Media Ex. Nutrient agar (nutrient broth and 2% agar)

2. Complex Media

chemical composition is not specifically defined or difficult to estimate

with added ingredients

provide special nutrients

Ex. BAP, CAP

3. Synthetic Media or Defined Media

specially prepared media for research purposes

exact composition is known

Ex. Peptone water -1%peptone +0.5%NaCl in

water

B. According to Physical State/Consistency

1. Solid

1.5 to3% agar (seaweeds)

Colony visible growth of microorganism on solid media

2.Liquid

No agar

evidenced by turbidity, pellicle formation and precipitation

Peptone water, NB

3.Semi- solid

with 0.5% agar

used to demonstrate swarming growth of Proteus and other motile organisms

motility

transport media

SIM (Sulfide Indole Motility Medium)

C. According to manner of Dispensing or

Formation

1.Tubed

1.1 Liquid

1.2 Semi- solid

1.3 Solid

1.3.1 Butt (use stab)

1.3.2 Slope or Slant (use streak)

1.3.3 Butt-slant (stab-streak)

2.Plated

solid media in Petri dishes

MSA, EMB, Mac Conkey, SSA.

D.According to Function or

Application

1. Basal or Base

basic medium from which other culture media are prepared

NB, NA, Trypticase Soy Agar

2. Enriched

with additional requirements or additives for

fastidious organisms to grow

blood, serum, ascitic fluid

best source of blood is sheep because only

desired organisms will produce hemolysis

horse, rabbit

BAP, CAP, LSS (Loefflers Serum Slant)

Ex. BAP (Blood Agar plate)

Use: cultivation of fastidious microorganisms

hemolytic reactions

Components: Trypticase Soy Agar or NA+ 5%

defibrinated blood

Gamma hemolysis (nonhemolysis)

Streptococcal species do not lyse(hemolyse) sheep rbc

no discrete zones are formed around the colony

Alpha hemolysis

Streptococci modify hemoglobin to green pigment (biliverdin and other heme compounds)

zone of partially lysed red cells surround the colonies greenish discoloration Beta hemolysis

Streptococci create clear zone (complete lysis of rbc)

CAP (Chocolate Agar plate)

Use: cultivation of Haemophilus influenza

Neisseria gonorrhoeae

Components: Peptone base + 5%

defibrinated blood added while

heating at 750C

LSS (Loefflers Serum Slant)

Use: to cultivate Corynebacterium diptheriae

Component: NA with horse serum

3.Selective Media and Differential Media

with inhibitors to prevent growth of unwanted

organisms and favor desired organisms

A. Inhibit growth of Gram positive

organisms

Eosin Y, methylene blue (EMB)

Bile salts, brilliant green (SSA)

bile salts, crytal violet (MacConkey)

B.Inhibit growth of Gram negative organisms

1.K tellurite

2.Na azide

C. Inhibit swarming growth of Proteus

1.Chloral hydrate

2.Alcohol ex. PEA

D. Inhibit contaminants or invaders

1. Penicillin

2. Streptomycin antibiotics

3. Malachite green

Indicators are added to demonstrate Hydrogen Sulfide production, CHO fermentation, pH

Hydrogen sulfide indicator:

sodium thiosulfate ( SSA, XLD)

Ferric ammonium citrate (HEA)

Carbohydrate fermentation:

Lactose (SSA)

Sucrose, lactose, salisin (HEA)

.

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of Colony

Phenyl ethy Alcohol

Agar(PEA)

selective medium used to cultivate

Gram positive

organisms

Inhibits E. coli, Salmonella sp.,

Shigella.

Enterobacter,

Pseudomonas

Allows Gram positive

organisms

Staphylococcus,

Streptococcus,

Bacillus,

Clostridium

phenylethyl alcohol,

inhibits or

reduces

growth of

Gram

negative

organisms by

interfering

with DNA

synthesis

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of Colony

TCBS (Thiosulfate Citrate Bile Salts

Sucrose)

differential and selective

plating medium

for Vibrio

Ox bile Sodium

citrate

Sucrose pH indicator Thymol blue

Bromothymol

blue

H2S indicator

Sodium

thiosulfate

Ferric citrate

SF-

Yellow

Ex. V. cholera

NSF-

Green

ex. V. parahemol-

yticus

Vibrio parahemolyticus

Vibrio cholera

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of

Colony

EMB (Eosin

Methylene Blue)

used to isolate fecal coliforms

inhibits Gram positive

organisms

Eosin Y Methylene

Blue

Lactose

pH indicator

Eosin Y

Methylene

blue

LF- purple with

green metallic

sheen

(E. coli)

NLF- colorless

(Shigella and

Salmonella)

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of

Colony

MacConkey

selective medium for

Gram

negative

organisms

Bile Salt Crystal

violet

Lactose pH indicator Neutral red

(red at a pH

below 6.8

colourless at

pH greater

than 6.8)

LF- pink to red

(E.coli,

Klebsiella,

Enterobacter)

NLF colorless

(Salmonella,

Shigella,

Proteus)

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of

Colony

SSA

(Salmonella

Shigella Agar)

selective and differential

media for

isolation of

pathogenic

enteric bacilli

Salmonella

and Shigella

Brilliant green

Bile Salts

Citrate

Lactose pH indicator

Neutral red

H2S

indicator

Sodium

thiosulfate

Ferric

citrate

LF- red

(E. coli,

Klebsiella,

Enterobacter)

NLF white with or without

black center

Salmonella-

colorless with

black center

Shigella-

colorless

Medium Inhibitor Fermentable

Carbohydrate

Indicator Color of

Colony

MSA (Mannitol Salt

Agar)

selective isolation of

Staphylococci

High concentration

of salt (7.5%)

inhibits

bacteria

Mannitol Phenol red

The organism (Staphylococcus aureus) indicated

by the orange arrow is capable of mannitol

fermentation, signified by the color change in

the surrounding media. The organism

(Staphylococcus epidermidis) indicated by the

blue arrow is not capable of mannitol

fermentation.

Medium Inhibitor

Fermentable

Carbohydrate

Indicator

Color of

Colony

Bismuth Sulfite Agar

(BSA)

isolation and cultivation of

Salmonella

species

Salmonella

typhi

Brilliant green Glucose Bismuth sulfite

H2S indicator

Ferric

Sulfate

Metallic colonies

with black

ring

Medium Inhibitor Fermentable

Carbohydrates

Indicator Color of

Colony

Hektoen Enteric Agar

(HEA)

used to isolate

species of

Salmonella,

Shigella

Bile salts Sucrose Lactose Salicin

pH indicator

Bromothymol

blue

H2S indicator

Ferric

ammonium

citrate

Sodium

thiosulfate

LF- carrot

orange/salmon

colored

colonies with

(E.coli)

NF- green

colonies

(Shigella)

colonies with

black center

Salmonella

typhimiurium

E. Coli

Enterococci

fecalis

Medium Bacteriostatic

Agent

Fermentable

Carbohydrates

Indicator Color of

Colony

Xylose Lysine

Deoxycholate

Bile Salt Xylose Lactose Sucrose

pH indicator Phenol red

H2S indicator Ferric ammonium

citrate

Sodium

Thiosulfate

LF- yellow

colonies(E,c

oli)

NLF-

Red(Shigella

red colonies

with black

center(Salm

onella)

NLF yellow (E.coli)

selective and differential medium for isolation of

enteric pathogens

Shigella and Salmonella

4. Differential Media

dyes or indicators are added to differentiate 2

groups of organisms growing together

MSA

phenol red

differentiates Staphylococcus sp. based on Mannitol fermentation

organisms that ferment mannitol --- acidic product is

formed and cause phenol red in the agar to turn

yellow

pathogenic staphylococci (Staphylococcus aureus)

ferment mannitol

non pathogenic staph will not ferment mannitol

MacConkey

neutral red differentiates lactose from non-lactose fermenters

SSA

neutral red differentiates Salmonella from Shigella sp.

EMB

Eosin Y and Methylene blue differentiates E. coli from other enterics

5. Enrichment Medium

liquid medium to which certain substances are added to enhance the growth of pathogenic

organisms and suppress the unwanted

Selenite broth

enrichment medium for Salmonella typhi

APW (Alkaline Peptone Water)

solution enrichment medium for Vibrio cholerae

6. Transport

used when there is anticipated delay in bringing

the specimen from source

to laboratory

Cary-Blair

Vibrio sp.

Stuart medium

V. cholerae

7. Assay Media

for research

used for assay of vitamins, a. acids and antibiotics

8. Special Media

A. Special Media for Biochemical Test

1. Tryptone Broth

- identifying Gram- based on Indole

production

2. MR-VP or Peptone Glucose Broth