2. Index
Introduction
Definition
History
Classification
Preparation, sterilization and testing of media
Simple media, Enriched media and Enrichment media
Selective and Differential media
Transport media
Biochemical media
Fungal, Parasitic and Viral media
Anaerobic media
Disposal of media
Non culturable microorganisms
3. Introduction
Microorganisms have to be grown (cultured)for them to be
identified.
Cultivation is growing microorganisms in culture by taking
bacteria from infection site by specimen collection and
growing them in artificial environment.
Culture is the most common diagnostic method used for
detection of bacterial infections.
6. A.Based on consistency
Liquid media or broth:no agar
Semi solid media:0.5%agar
Solid media:2%agar
7. B.Based on the method of growth detection
Conventional culture media
- Simple or basal media
- Enriched media
- Enrichment broth
- Selective media
- Differential media
- Transport media
- Anaerobic media
Automated culture media
8. Preparation of culture media
Weigh the dehydrated media in required quantity.
Dissolve in purified/distilled water as per the manufacturer instruction
Heat to boiling to dissolve the medium completely
Check the pH of the media by using narrow range pH paper
Compare the colour of the pH against the chart provided.
9. Sterilization of culture media
• The following methods are used to sterilize culture media:
>Autoclaving
>Steaming at 100⁰C
>Inspissation
>Filtration
10. Dispensing sterile media into petri dishes
Pour plates under sterile conditions within the hood
Lay out the sterile petri dishes on clean surfaces
Name of the medium and date of preparation should be mentioned on the plate.
Media should not be warmer than 60⁰C when poured
Mix the medium gently by rotating the flask,avoid air bubbles
Hold the plug of flask in such a position that it does not get contaminated
Flame sterilize the neck of the flask
Pour 15-20ml of medium into each disc(90-100mm diameter)
11. Storage of culture media
>Stored at dark place
>At 2-8⁰C
Testing of culture media
>Sterility testing-
• Done for routine media to which sheep blood or other
substances are added after autoclaving.
• 5% batch incubated at 35-37⁰C overnight.
• All media to be checked visually before using.
12. Name of the medium Controls
Sheep blood agar Alpha hemolysis -Streptococcus viridans
Beta hemolysis -Streptococcus pyogens
Gamma hemolysis -Enterococcus spp
Chocolate agar Alpha hemolysis -Streptococcus
pneumoniae
Nutrient agar Pigmented strain of Pseudomonas
MacConkey agar LF –Escherichia coli
NLF-Pseudomonas
TCBS(Thiosulfate-citrate-bile
salts-sucrose agar)
Sucrose fermenting-Vibrio cholerae
Wilson Blair H2S producing-Salmonella typhi
H2S non producing-Salmonella para typhi A
Alkaline peptone water Positive control-Vibrio cholerae
Negative control-Escherichia coli
>Performance testing
13. Name of the medium Controls
Selenite F broth Positive control-Salmonella
Negative control-Escherichia coli
Potassium tellurite agar Black coloured colonies-Corynebacterium
diphtheriae
CLED(Cystine-Lactose-
Electrolyte-Deficient agar)
Staphylococcus aureus-yellow colonies
Escherichia coli-yellow colonies
Pseudomonas-green colonies
Triple sugar iron agar Acid/acid , with/without gas-Escherichia coli
Acid/acid,H2S-Proteus
Alkaline/no change-Pseudomonas
Simmon’s citrate medium Positive control-Klebsiella Pneumoniae
Negative control-Escherichia coli
Christensen’s urea medium Positive control-Proteus spp
Negative control-Escherichia coli
Bile esculin agar Positive control-Enterococcus species
Negative control-Streptococcus viridans
Cetrimide agar Positive control-Pseudomonas aeruginosa
Negative control-Escherichia coli
14. Simple/basal media:
They contain minimum ingredients that support the growth of non
fastidious bacteria.
Name of media Components Primary purpose
Peptone water Peptone(1%)NACI,(0.5%)water >Used as growth medium
>Base for carbohydrate
media
>Used for indole test
Nutrient broth Peptone water , meat
extract(1%)
Isolation of non-fastidious
organisms
Nutrient agar Nutrient broth,2%agar Isolation of non-fastidious
organisms
Semi-solid agar Nutrient broth,0.2 to 0.5%agar Determination of bacterial
motility
16. Enriched media:
Substances like blood , serum , egg are added to the simple/basal
medium to support growth of fastidious bacteria.
Enriched media components Primary purpose
Blood agar 5-10%sheep blood added to
molten NA at 45⁰C- 55⁰C
Isolation of Staphylococcus ,
Streptococcus.
Chocolate agar 5-10%sheep blood added to
molten NA at 72⁰C
Hemoglobin , hemin(X factor)
and Co-enzyme nicotinamide
adenine dinucleotide(NAD or V
factor)
Isolation of Streptococcus
pneumonae , Neisseria gonorrhoeae
, Hemophilus spp.
20. Name of the media Components Primary purpose
Serum containing enriched
medium
• Loeffler serum slope
Nutrient broth, glucose,
ox/sheep/horse serum
Isolation of
Corynebacterium
diphtheriae.
Egg containing enriched
medium
• Lowenstein Jensen
medium
• Dorset egg medium
>Potato flour, L-Asparagine ,
Monopotassium phosphate ,
Magnesium citrate ,
Malachite green , glycerol ,
Egg suspension , distilled
water
>Nutrient broth , whole
fresh eggs.
>Isolation of Mycobacterium
tuberculosis and Atypical
mycobacteria .
>Isolation of
Corynebacterium
diphtheriae.
Buffered charcoal yeast
extract agar
Yeast extract, agar, charcoal
and salts supplemented with
L-cysteine , HCL , ferric
pyrophosphate and alpha-
ketoglutarate.
>Enrichment media for
Legionella spp.
>Supports growth of
Nocardia.
21. Loefflers serum slope Lowenstein-Jensen medium Dorset egg agar Buffered charcoal yeast
extract agar
22. Enrichment media:
They are liquid media added with some inhibitory
agents which selectively allow certain organism to
grow and inhibit others.
23. Enrichment media components Primary purpose
Selenite F broth Peptone-base broth ; sodium
selenite toxic for most
Enterobacteriaceae
Isolation of Samonella species
Tetrathionate broth Peptone-base broth;iodine and
potassium iodide,bile salts and
sodium thiosulfate inhibit Gram
positive organisms and
Enterobacteriaceae
Isolation of Salmonella and
Shigella spp.
Alkaline peptone water Peptone ,sodium
chloride,distilled water.
Isolation of Vibrio cholerae
Gram negative broth Peptone-base broth with glucose
and mannitol;sodium citrate and
sodium deoxycholate act as
inhibitory agents
Isolation of Enteric pathogens
26. NAME OF THE
MEDIUM
CONSTITUENTS USE
MacConkey
Agar
Peptone base with lactose
Bile salts (Sodium
taurocholate)
Indicator: Neutral Red
SELECTIVE - Gram
Negative bacilli
DIFFERENTIAL - Lactose
fermentation
CLED
(Cystine lysine
electrolyte deficient
agar)
Peptone base with lactose,
tryptone, L-cystine
Indicator: Bromothymol
blue
DIFFERENTIAL - Lactose
fermentation
(mostly for bacteria from
the urinary tract)
EMB AGAR
(Eosin Methylene
Blue)
Peptone base with lactose
Indicators: Eosin Y and
Methylene blue
DIFFERENTIAL - Lactose
fermentation
(for Enteric bacilli)
28. NAME OF THE
MEDIUM
CONSTITUENTS USE
BEA
(Bile Esculin Agar)
Nutrient agar base with ferric
citrate
Brown colour of the medium is
due to hydrolysis of esculin
Indicator: Sodium
Desoxycholate (Bile salt)
DIFFERENTIAL - Isolation and
presumptive identification of
group D streptococci and
enterococci
POTASSIUM
TELLURITE
AGAR
Sheep blood agar base with
potassium tellurite
SELECTIVE: Corynebacterium
diphtheriae
(Black coloured colonies)
TCBS AGAR
(Thiosulfate citrate
bile sucrose)
Peptone base agar with yeast
extract, bile salts, citrate,
sucrose, ferric citrate and
sodium thiosulfate
Indicator: Bromothymol blue
SELECTIVE: Vibrio spp
(Yellow coloured colonies)
DIFFERENTIAL: Sucrose
fermentation
30. NAME OF THE MEDIUM CONSTITUENTS USE
SS AGAR
(Salmonella Shigella Agar)
Peptone base with lactose, ferric citrate and
sodium citrate
Inhibitors: Brilliant green and bile salts
Indicator: Neutral red
SELECTIVE - Salmonella and some Shigella spp
WILSON BLAIR
AGAR
Peptone agar base with beef extract,
dextrose, disodium phosphate and Bismuth
sulphite
Inhibitor: Brilliant green dye
Indicators: Ferrous sulphate
SELECTIVE – Isolation and preliminary
identification of Salmonella spp from clinical
specimens
DIFFERENTIAL – Dextrose fermentation
DCA
(Deoxycholate citrate agar)
Peptone base agar with lactose, sodium
citrate, sodium thiosulfate, ferric citrate
Inhibitor: Sodium deoxycholate (Inhibits Gram
positive organisms)
Indicator: Neutral Red
DIFFERENTIAL - Salmonella and Shigella spp from
other Gram negative enteric bacilli
XLD AGAR
(Xylose Lysine
Deoxycholate)
Yeast extract agar with lysine, xylose, lactose,
sucrose and ferric ammonium citrate
Inhibitor: Sodium deoxycholate (Inhibits Gram
positive organisms)
Indicator: Phenol Red
DIFFERENTIAL - Salmonella and Shigella spp from
other Gram negative enteric bacilli
HE AGAR
(Hektoen Enteric Agar)
Peptone base agar with bile salts, lactose,
sucrose, salicin and ferric ammonium citrate
Indicators: Bromothymol blue and acid
fuschin
SELECTIVE - Salmonella and some Shigella spp
DIFFERENTIAL - Salmonella and Shigella spp from
other Gram negative enteric bacilli
34. NAME OF THE
MEDIUM
CONSTITUENTS USE
BORDET-
GENGOU AGAR
Potato-glycerol-based medium enriched with 15 to
20% defibrinated blood
Inhibitor: Methicillin (2.5 microgram/mL)
SELECTIVE – Bordetella pertussis and
Bordetella parapertussis
REGAN LOWE
AGAR
Charcoal agar with horse blood, cephalexin and
amphotericin B
ENRICHMENT and SELECTIVE –
Isolation of Bordetella pertussis
THAYER MARTIN
AGAR
Blood agar base enriched with haemoglobin and
supplement B
Inhibitors: Colistin, Nystatin, Vancomycin,
Trimethoprim
SELECTIVE – N. gonorrhoeae and N.
meningitidis
NYC AGAR
Peptone agar base with cornstarch supplemented with
yeast dialysate, 3% hemoglobin and horse plasma
Antibiotic supplements include:
1. Vancomycin (2 microgram/mL)
2. Colistin (5.5 microgram/mL)
3. Amphoterecin B (1.2 microgram/mL)
4. Trimethoprim (3 microgram/mL)
SELECTIVE – Neisseria gonorrhoeae
Also supports growth of Ureaplasma
urealyticum and some Mycoplasma
spp.
37. NAME OF THE
MEDIUM
CONSTITUENTS USE
CAMPY
BLOOD
AGAR
Brucella agar base with sheep blood and the
following:
Vancomycin(10 mg/L)
Trimethoprim(5 mg/L)
Polymyxin B(2500 U/L)
Amphoterecin B(2 mg/L)
Cephalothin(15 mg/L)
SELECTIVE – Campylobacter
spp.
CVA AGAR
(Cefoperazone,
Vancomycin,
Amphoterecin meduim)
Blood supplemented with the 3 drugs to inhibit
growth of most Gram-negative bacteria, Gram-
positive bacteria and yeast, respectively.
SELECTIVE – Campylobacter
spp
SKIRROW AGAR
Peptone and soy protein base agar with lysed horse
blood
Inhibitors:
1. Vancomycin – Gram positive organisms
2. Polymyxin B and Trimethoprim – most Gram
negative organisms
SELECTIVE – Campylobacter
spp
39. NAME OF THE
MEDIUM
CONSTITUENTS USE
BURKHOLDERIA
CEPACIA SELECTIVE
AGAR
Bile salts
Gentamicin
Ticarcillin
Polymyxin B
Peptone
Yeast extract
SELECTIVE - Burkholderia cepacia
(Cystic fibrosis patients)
CNA AGAR
(Columbia Colistin-
Nalidixic acid agar)
Columbia agar base with
10 mg/L colistin
15 mg/L Nalidixic acid
5% sheep blood
SELECTIVE - Isolation of Gram-
positive cocci
CIN AGAR
(Cefsulodin-Irgasan-
Novobiocin agar)
Peptone base with yeast extract, mannitol
and bile salts
Inhibitors: drugs
Indicator: Neutral red and Crystal violet
SELECTIVE - Isolation of Yersinia spp
and Aeromonas spp
DNase agar
Tryptose, Deoxyribonucleic acid, Sodium
Chloride, Agar
DIFFERENTIAL – Deoxyribonuclease
enzyme producing property
Eg. Serratia marcescens
41. NAME OF THE
MEDIUM
CONSTITUENTS USE
MANNITOL SALT
AGAR
Peptone base, mannitol
Inhibitor: 7.5% concentrated salt
Indicator: Phenol red
SELECTIVE DIFFERENTIATION -
Staphylococci
PEA
(Phenylethyl alcohol
agar)
Nutrient agar base
Inhibitor: Phenylmethanol
(inhibits Gram negative
organisms)
SELECTIVE - Isolation of:
Aerobic Gram positive
cocci and bacilli
Anaerobic Gram positive
cocci and negative bacilli
SSA
(Streptococcal selective
agar)
5% sheep blood agar base with
colistin and trimethoprim-
sulfamethoxazole
Indicator: Crystal violet
SELECTIVE - Streptococcus
pyogenes and Streptococcus
agalactiae
45. MILK AGAR: NON-SELECTIVE SOLID MEDIUM
CONSTITUENTS:
Peptone agar base with yeast extract and milk solids
USE:
Identification of organisms that can hydrolyse casein eg. Streptomyces,
Pseudomonas and Actinomadura.
For differentiation of aerobic actinomycetes based on casein proteolysis.
It is used to demonstrate the pigment producing properties of certain
organisms.
46. MUELLER HINTON AGAR
Non-selective, non-differential medium
Constituents: Beef Extract, Acid Hydrolysate of Casein, Starch and Agar.
Uses
1. Routine susceptibility testing of non-fastidious microorganisms by the Kirby-Bauer disk
diffusion technique.
2. It can be used to cultivate Neisseria
3. It is specified in FDA Bacteriological Analytical Manual for food testing, and procedures
commonly performed on aerobic and facultative anaerobic bacteria.
48. NAME OF THE
MEDIUM
CONSTITUENTS USE
CARY-BLAIR
Soft agar semisolid medium with
sodium thioglycolate, disodium
hydrogen phosphate, NaCl and
CaCl2
To transport enteric pathogens
Eg. Shigella, Salmonella, Vibrio
cholerae and E. coli
For detection of Campylobacter spp
from faeces (when transport time <
2 hours)
STUART’S
Soft agar medium with sodium
thioglycolate (mercaptoacetate),
sodium glycerophosphate, CaCl2,
Distilled water and methylene blue
To maintain viability of gonococci on
swabs during their transmission.
Also for Shigella and E. coli
AMIES
Soft agar medium with sodium
thioglycolate (mercaptoacetate),
NaCl, KCl, CaCl2, MgCl2, Na2PO4,
Potassium dihydrogen phosphate,
CHARCOAL (finely powdered),
distilled water
To maintain viability of gonococci on
swabs during their transmission.
Also for Shigella and E. coli
50. NAME OF THE
MEDIUM
CONSTITUENTS USE
PIKE’S
Blood agar containing
1 in 10 lakh Crystal violet
1 in 16000 Sodium azide
Distributed as for stab cultures
in tubes or bottles
Preservation of Streptococcus
pyogenes, pneumococci and
Haemophilus influenzae in
nose and throat swabs
SACH’S
BUFFERED
GLYCEROL
SALINE
Distilled water and glycerol with
NaCl, Disodium hydrogen
phosphate, potassium
dihydrogen phosphate and
phenol red
Preservation and transport
of Salmonella and Shigella
species and Escherichia coli in
facial specimens. It is not
suitable for Vibrio cholera,
Campylobacter species
or Yersinia enterocolitica.
53. FERMENTATION TEST MEDIA
Constituents:
1. Nutrient medium base: Peptone water, serum peptone water and serum agar
2. Carbohydrate or related compound under test (SUGARS):
MONOSACCHARIDES Pentoses (Arabinose/ Xylose/ Rhamnose)
Hexoses (Glucose/ Fructose/ Mannose/ Sorbose/ Galactose)
DISACCHARIDES Sucrose/ inulin/ Dextrin/ Glycogen
POLYHYDRIC
ALCOHOLS
Glycerol/ Erythritol/ Adonitol/ Mannitol/ Dulcitol/ Sorbitol/ Inositol
GLYCOSIDES Salicin/ Coniferin/ Aesculin
ORGANIC ACIDS Tartrate(Dextro/ Laevo/ Meso) / Citrate/ Mucate/ Gluconate/ Malonate
3. Suitable Indicator:
ANDRADE’S INDICATOR (NaOH with acid fuchsin till colour becomes yellow)
BROMOCRESOL PURPLE
PHENOL RED
BROMOTHYMOL BLUE
4. A small inverted tube (DURHAM TUBE) completely filled with liquid and
containing no air bubbles is usually included in each culture tube or bottle to
detect gas.
54. TSI (Triple Sugar Iron): DIFFERENTIAL MEDIUM
CONSTITUENTS: Agar slant of special medium with Phenol Red (pH
sensitive dye), 1% lactose, 1% sucrose, 0.1% glucose, sodium
thiosulfate and ferrous sulfate.
Carbohydrate fermentation is indicated by the production of gas and a
change in the color of the pH indicator from red to yellow.
USES:
•Differentiate members of the Enterobacteriaceae family from other
gram-negative rods
•Differentiation among Enterobacteriaceae on the basis of their sugar
fermentation patterns
55. INTERPRETATION OF TSI:
An alkaline/acid (red slant/yellow butt) reaction: Indicative of dextrose fermentation only
An acid/acid (yellow slant/yellow butt) reaction: Fermentation of dextrose, lactose and/or
sucrose
An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation
Blackening of the medium: production of hydrogen sulphide
Gas production: Bubbles or cracks (formation of CO2 and H2)
56. OXIDATIVE FERMENTIVE MEDIA
A semi-solid tubed medium containing the carbohydrate along with a pH
indicator.
Constituents: Peptone base agar with NaCl, Dipotassium hydrogen phosphate,
distilled water and bromothymol blue.
Fermenting organisms: Enterobacteriaceae, Aeromonas, Vibrio
Oxidizing organisms: Pseudomonas
Non-fermenting organisms: Alcaligenes faecalis
57. DECAROXYLASE MEDIUM
Constituents: Decarboxylase test medium base (Peptone, meat extract,
glucose, pyridoxal, bromocresol blue, cresol red and distilled water)
The media is divided into four equal parts. One part is tubed without the
addition of any amino acid, and the tube is labeled as ‘Control’. The remaining
three parts are dispensed onto three tubes, to which L-lysine hydrochloride,
L-arginine hydrochloride, and L-ornithine hydrochloride are added separately.
Uses:
•Decarboxylase test is used to differentiate the members of the
Enterobacteriaceae family with closely related physiological characteristics on
the basis of their ability to produce the enzyme decarboxylase
59. NAME OF THE
MEDIUM
CONSTITUENTS USE
MR
(METHYL RED)
Ingredients per liter of
deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate
Positive Reaction: A distinct red color
Examples: E. coli, Yersinia sps, etc.
Negative Reaction: A yellow color
Examples: Enterobacter aerogenes, Klebsiella
pneumoniae, etc
INDOLE
KOVACS
MEDIUM
P-dimethylaminobenzaldehyde
Hydrochloric acid (37%)
Amyl alcohol
Positive: Formation of a pink to red color (“cherry-
red ring”) in the reagent layer on top of the medium
within seconds of adding the reagent.
Examples: Escherichia coli, Haemophilus
influenzae, Klebsiella oxytoca, Proteus sp., Enterococcus
faecalis, and Vibrio sp.
Negative: No color change
Eg most Haemophilus sp.,
most Klebsiella sp., Neisseria sp., Proteus mirabilis, P.
penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yer
sinia sp
60.
61. NAME OF THE
MEDIUM
CONSTITUENTS USE
VP
(VOGES-
PROSKAUER)
VP REAGENT A: BARRITT’S
REAGENT A
Alpha naphthol 5%
Absolute alcohol
VP REAGENT B: BARITT’S
REAGENT B
Potassium hydroxide
Deionized water
Positive Reaction: A pink-red color at the
surface
Examples: Viridans group streptococci,
Enterobacter, Klebsiella, Serratia
marcescens, Vibrio eltor.
Negative Reaction: A lack of a pink-red
color
Examples: Streptococcus mitis, Citrobacter
sp., Shigella, Yersinia, Salmonella, Vibrio
parahaemolyticus
62.
63. NAME OF THE MEDIUM CONSTITUENTS USE
SIMMONS’
CITRATE
MEDIUM
Koser’s medium with agar and
Bromothymol blue as the indicator.
Koser’s medium includes:
NaCl, MgSO4, Ammonium dihydrogen
phosphate, potassium dihydrogen
phosphate and sodium citrate
Positive Reaction: Growth with color change
from green to intense blue along the slant.
Examples: Salmonella, Edwardsiella,
Citrobacter, Klebsiella, Enterobacter,
Serratia, Providencia, etc.
Negative Reaction: No growth and No color
change; Slant remains green.
Examples: Escherichia, Shigella,
Morganella, Yersinia etc.
CHRISTENSEN’S
UREASE
MEDIUM
Peptone agar base with NaCl, dipotassium
hydrogen phosphate, Glucose (10%), Urea
(20%)
Indicator: Phenol red
to distinguish urease-positive
Enterobacteriaceae.
can also be used to differentiate between
yeasts : Candida albicans (negative urease)
and Cryptococcus neoformans (rapid
production of urease).
detect the presence of Helicobacter pylori
65. NAME OF THE
MEDIUM
CONSTITUENTS USE
MALONATE
AGAR
Distilled water with yeast extract,
ammonium sulphate,
dipotassium hydrogen phosphate,
potassium dihydrogen phosphate,
NaCl and sodium malonate
Indicator: Bromothyol Blue
Differentiate among Enterobacteriaceae:
Klebsiella pneumoniae is positive (blue at
24 hours), Escherichia coli is negative(cult
ure remains green).
Also used to separate Salmonella arizonae
(positive) from other Salmonella spp(neg
ative).
Differentiate Enterobacteriaceae in food
and dairy products.
CETRIMIDE
AGAR
Distilled water, agar, glycerine,
MgCl2, Potassium sulfate,
pancreatic digest of gelatin,
cetyltrimethylammonium
bromide
selective medium for the isolation
of Pseudomonas aeruginosa
used for determining the ability of an
organism to produce fluorescein and
pyocyanin (Antibiotica)
66.
67. NAME OF THE
MEDIUM
CONSTITUENTS USE
HYDROLYSIS OF
TRIBUTYRIN
Peptone agar base with
water, yeast extract and
Tributyrin
Lipolytic organisms
remove the opacity by
converting the fat to
water soluble butyric
acid
