CULTURE MEDIA

(Principle, Purpose & Interpretation)

Ma.Elizabeth P. Bonghanoy
 MacConkey Agar

•Purpose
- It is both selective and differential
medium designed to isolate and
differentiate enteric based on their
ability to ferment lactose.

- It differentiates microorganisms in the

colon-typhoid-dysentery group.
MacConkey Agar

Inhibitor: crystal violet

and bile salts

CHO: Lactose

pH indicator: Neutral Red

MacConkey Agar
 MacConkey Agar

• Interpretation
✓Lactose fermenting strains – They
grow red/pink and surrounded by
acid precipitated bile.

✓Non-fermenting lactose strains –

The results are colorless and
transparent.
 Eosin Methylene Blue Agar

• Purpose
- Both a selective and
differential medium used to
isolate fecal coliforms.
Eosin Methylene Blue Agar

Inhibitor: Eosin Y and

methylene blue
CHO: Lactose/Sucrose
pH Indicator: Eosin Y and
Methylene blue
Eosin Methylene Blue Agar
 Eosin Methylene Blue Agar

• Interpretation
✓Lactose Fermenting: pink (slow
fermenters) – purple complex

✓Non Lactose Fermenting: Colorless

 Eosin Methylene Blue Agar

Rapid fermentation of lactose & production of

strong acids.
- The metallic green sheen is due to the acid
production from the amide bonding between
the eosin and methylene blue

E. coli: Green Metallic Sheen

 Eosin Methylene Blue Agar

Klebsiella has capsular antigens (K antigens),

leading to different serogroups12. These well
developed polysaccharide capsules give the
colonies their characteristic mucoid appearance.
The pink color is attributed to its slow
fermenting property.

Klebsiella: Pink mucoid colonies

 Eosin Methylene Blue Agar

Some strains with a K antigen

possess a capsule which
attributes to the mucoid shape.
The dark center is due to
precipitation of methylene blue.

Enterobacter: Pink colonies

with dark center.
“FISH EYE” colony
 Thiosulfate Citrate Bile Salts
Sucrose Agar

• Purpose
- Used for the selective isolation of Vibrio
cholerae and other enteropathogenic vibrios.

- Used to control outbreaks of the crown-of-

thorns seastar (Acanthaster planci).
Thiosulfate Citrate Bile Salts
Sucrose Agar

Inhibitor: Ox bile and sodium

cholate
CHO: Sucrose
pH Indicator: Bromthymol blue and
Thymol blue
Thiosulfate Citrate Bile Salts
Sucrose Agar
 Thiosulfate Citrate Bile Salts
Sucrose Agar

• Interpretation
 Salmonella and Shigella Agar

• Purpose
- Used as a selective and differential medium
for the isolation of Salmonella and
some Shigella species from clinical and
non-clinical specimens.

- It was also developed to aid in the

differentiation of lactose and non-lactose-
fermenters from clinical specimens, suspected
foods, and other such samples
Salmonella and Shigella Agar

Inhibitor: bile salts, brilliant green

and sodium citrate
CHO: Lactose
pH Indicator: neutral red
H2S indicator: (Fe+NH4 Citrate)
 Salmonella and Shigella Agar

or Sodium
Thiosulfate (not
metabolized)
 Salmonella and Shigella Agar

• Interpretation

Non Fermenting
✓Salmonella: H2S (+), colorless with
black center
✓Shigella: H2S (-), Colorless, without
black center
 Salmonella and Shigella Agar

• Interpretation

Fermenting
✓Coliform bacteria: H2S (-), pink color
✓Enterobacter and Klebsiella: larger
than E. coli, mucoid, pale, opaque
cream to pink
 Hektoen Enteric Agar

• Purpose
• is a selective as well as
differential media for the
isolation and differentiation of
enteric pathogens from
clinical specimens.
Hektoen Enteric Agar

Inhibitor: bile
salts and acid
fuchsin
CHO:
Lactose/Sucrose/
Salicin
pH Indicator:
Bromthymol blue
H2S indicator:
(Fe+NH4 Citrate)
 Hektoen Enteric Agar

or Sodium Thiosulfate
(not metabolized)
 Hektoen Enteric Agar

• Interpretation
✓Salmonella colonies: blue-green, with
hydrogen sulfide gas.

✓Shigella appear greener

than Salmonella, with the color
fading to the periphery of the colony.
 Hektoen Enteric Agar

• Interpretation
✓Rapid lactose fermenters (such
as E. coli): moderately inhibited,
produce bright-orange to salmon
pink colonies.

✓Proteus strains: small transparent

and more glistening or watery in
appearance
Hektoen Enteric Agar
 Thioglycollate Broth

• Purpose
- Used to find out the growth characteristics
of various bacteria based on their oxygen
requirements.

- Differentiation of obligate
aerobes, obligate anaerobes, facultative
anaerobes, microaerophiles,
and aerotolerant organisms.
 Thioglycollate Broth

• Purpose
- Recommended for the cultivation of
aerobic, microaerophilic, and anaerobic
microorganisms.

- Recommended for sterility testing of

antibiotics, biologicals, and foods and for
determining the phenol coefficient and
sporicidal effect of disinfectants.
 Thioglycollate Broth

PRINCIPLE
CHO: Glucose
Inhibitor: Bile Salts
Growth factors: Dextrose, pancreatic digest of
casein, yeast extract, L-cystine
Reducing agents: Thioglycolate, cystine, and
sodium sulfite
 Thioglycollate Broth

PRINCIPLE
- Low concentration of agar prevents downward
diffusion of oxygen, allowing for the growth of
anaerobic organisms toward the bottom of the tube

- L-cystine and sodium thioglycollate

allows Clostridium and other strict anaerobes to grow
in this medium even under aerobic conditions.
 Thioglycollate broth

• Interpretation
✓Gram-negative, facultatively
anaerobic bacilli generally produce
diffuse, even growth throughout the
broth.

✓Gram positive cocci frequently grow

as discrete “puffballs”.
 Thioglycollate broth

• Interpretation
✓Strict aerobic bacteria such
as Pseudomonas spp., tend to grow
toward the surface of the broth

✓Strictly anaerobic bacteria grow at

the bottom of the broth.
Thioglycollate broth
 Tryptone Soy broth

• Purpose
- For use as a general growth medium
for the isolation and cultivation of
microorganisms.

- Supports the growth of non-fastidious

as well as moderately fastidious
microorganisms
 Tryptone Soy broth

• Purpose
- Recommended for use in the cultivation, storage,
maintenance, and transportation of pure cultures of
microorganisms

- Not used for the isolation of pathogens from clinical

specimens but may be used for maintaining or
subculturing bacterial strains (e.g.,
Enterobacteriaceae and Staphylococci). After
supplementation with blood (e.g. 5% sheep blood)
 Tryptone Soy broth

• Principle
CHO: Glucose
Amino acids and other complex nitrogenous
substances: Enzymatic digests of casein and
soybean
Osmotic balance: Sodium chloride
Buffering agent: Dipotassium hydrogen
phosphate
 Tryptone Soy broth

• Principle
- The addition of a small amount of
agar renders the broth suitable for
the cultivation of obligatory
anaerobes, such as Clostridium
species.
 Tryptone Soy Broth

• Interpretation
✓After the incubation period, the
broth should appear turbid, an
indication of organism growth.

✓In the case of TSB with phenol red,

a red to yellow color change also
accompanies turbidity, an
indication of acid production from
dextrose utilization.