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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

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

Fermentable
Carbohydrate

Indicator

Color of Colony

Medium

Inhibitor

TCBS (Thiosulfate Ox bile


Citrate Bile Salts
Sodium
Sucrose)
citrate
differential and
selective
plating medium
for Vibrio

Vibrio parahemolyticus

Fermentable
Carbohydrate
Sucrose

Indicator

pH indicator
Thymol blue
Bromothymol
blue
H2S indicator
Sodium
thiosulfate
Ferric citrate

Vibrio cholera

Color of Colony

SFYellow
Ex. V. cholera
NSFGreen
ex. V. parahemolyticus

Medium
EMB (Eosin
Methylene Blue)
used to isolate
fecal coliforms
inhibits Gram
positive
organisms

Inhibitor
Eosin Y
Methylene
Blue

Fermentable
Carbohydrate
Lactose

Indicator

Color of
Colony

pH indicator
Eosin Y
Methylene
blue

LF- purple with


green metallic
sheen
(E. coli)
NLF- colorless
(Shigella and
Salmonella)

Medium
MacConkey

selective
medium for
Gram
negative
organisms

Inhibitor
Bile Salt
Crystal
violet

Fermentable
Carbohydrate
Lactose

Indicator
pH indicator
Neutral red
(red at a pH
below 6.8
colourless at
pH greater
than 6.8)

Color of
Colony
LF- pink to red
(E.coli,
Klebsiella,
Enterobacter)
NLF
colorless
(Salmonella,
Shigella,
Proteus)

Medium

SSA
(Salmonella
Shigella Agar)
selective and
differential
media for
isolation of
pathogenic
enteric bacilli
Salmonella
and Shigella

Inhibitor

Brilliant green
Bile Salts
Citrate

Fermentable
Carbohydrate

Lactose

Indicator

Color of
Colony

pH indicator LF- red


Neutral red (E. coli,
Klebsiella,
H2S
Enterobacter)
indicator
NLF white
Sodium
with or without
thiosulfate
black center
Ferric
Salmonellacitrate
colorless with
black center
Shigellacolorless

Medium

MSA
(Mannitol Salt
Agar)
selective
isolation of
Staphylococci

Inhibitor

Fermentable
Carbohydrate

High
Mannitol
concentration
of salt (7.5%)
inhibits
bacteria

Indicator

Color of
Colony

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

Bismuth
Sulfite Agar
(BSA)
isolation and
cultivation of
Salmonella
species
Salmonella
typhi

Inhibitor

Brilliant green

Fermentable
Carbohydrate

Glucose

Indicator

Bismuth
sulfite
H2S
indicator
Ferric
Sulfate

Color of
Colony

Metallic
colonies
with black
ring

Medium

Inhibitor

Hektoen
Bile salts
Enteric Agar
(HEA)
used to
isolate
species of
Salmonella,
Shigella

Fermentable
Carbohydrates

Sucrose
Lactose
Salicin

Indicator

Color of
Colony

pH
indicator
Bromothymol
blue

LF- carrot
orange/salmon
colored
colonies with
(E.coli)

H2S
indicator
Ferric
ammonium
citrate
Sodium
thiosulfate

NF- green
colonies
(Shigella)
colonies with
black center
Salmonella
typhimiurium

E. Coli
Enterococci
fecalis

Medium

Xylose Lysine
Deoxycholate

Bacteriostatic
Agent

Fermentable
Carbohydrates

Bile Salt

Xylose
Lactose
Sucrose

Indicator

pH indicator
Phenol red

H2S indicator
Ferric ammonium
citrate
Sodium
Thiosulfate

selective and differential


medium for isolation of
enteric pathogens
Shigella and Salmonella

Color of
Colony
LF- yellow
colonies(E,c
oli)
NLFRed(Shigella
red colonies
with black
center(Salm
onella)
NLF yellow
(E.coli)

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