1. Nutrient Agar
Nutrient agar is a solid medium used in microbiology to grow a wide range of non-fastidious organisms, including bacteria and fungi.
Composition of Nutrient Agar:
| Component | Amount |
|---|---|
| Peptone | 0.5% |
| Beef Extract | 0.3% |
| Sodium Chloride | 0.85% |
| Agar | 1.5% |
| Water | 1000 ml |
2. Blood Agar
It is a general purpose, enriched and differential solid medium, which supports the growth of most ordinary bacteria. It is also useful to detect and differentiate hemolytic bacteria, especially Streptococcus species.
Blood supplies a number of required nutrients for the growth of fastidious organisms.
Composition of Blood Agar:
| Component | Amount |
|---|---|
| Nutrient agar | 500 ml |
| Sterile Defibrinated blood | 25 ml |
Procedure:
Transfer sterilized nutrient agar to 50°C water bath.
When it is cooled to 50°C, add aseptically sterile defibrinated sheep or horse blood.
Mix gently and dispense aseptically in sterile petri dishes.
pH of the medium should be adjusted to 7.3.
Blood should be free from hemolysis.
Sheep or rabbit blood can be used for most of the pathogens.
Use of human blood should be avoided, since certain substances in human blood may be inhibitory to the growth of certain pathogens such as hemolytic Streptococci.
Sheep blood inhibits the growth of Haemophilus haemolyticus (human throat commensal). Note:
3. Chocolate (heated blood) agar
It is used to grow Haemophilus influenzae and other pathogens which require highly nutritious medium, such as Neisseria meningitidis and Streptococcus pneumoniae.
Formula and preparation:
It is prepared by using the same procedure used for the preparation of blood agar except after adding blood, the medium is heated at 70°C in a water bath until it becomes brown in color. The medium should be mixed gently for about 10–15 minutes.
Allow it to cool to about 45°C.
Dispense aseptically in sterilized petri dishes.
Note:
The medium should not be overheated.
The duration of heating should not be prolonged.
4. Crystal violet blood agar
This is used as a selective medium for Streptococcus pyogenes.
• Principle:- At a concentration of 1:500,000 crystal violet inhibits the growth of Staphylococcus aureus and retards the growth of commensals (throat specimen).
• Formula and preparation
1. Sterile blood agar: 100 ml.
2. 0.02% (w/v) crystal violet: 1.0 ml.
Mix well and dispense aseptically in sterile petri dishes.
5. Neomycin Blood Agar
This is used as a selective medium for obligate anaerobes.
Principle:
Facultative anaerobic Gram-negative rods are inhibited at a concentration of 70 µg/ml neomycin sulfate.
Formula and Preparation
a) Prepare stock neomycin sulfate (70,000 µg/ml)
Neomycin sulfate → 0.5 g
Sterile water → 5 ml
(This contains 0.35% neomycin base)
b) Preparation of working neomycin (37500 µg/ml)
Add 2 ml of 70,000 µg/ml stock neomycin solution to 8 ml sterile water.
Blood agar → 250 ml
Working neomycin → 1.0 ml
Mix well and pour aseptically in sterile Petri dishes.
Concentration of solution C = 700 µg/ml neomycin/ml.
6. Brain Heart Infusion Broth and Agar
It is an enriched medium used for the growth of fastidious bacteria, yeasts, and molds.
Preparation of BHI (Liquid Medium)
I. Beef heart infusion → 250.0 g
II. Calf brain infusion → 200.0 g
III. Proteose peptone → 10.0 g
IV. Dextrose → 2.0 g
V. Sodium chloride → 5.0 g
VI. Disodium phosphate → 2.0 g
VII. Distilled water → 1000 ml
Dissolve the contents in 800 ml of distilled water and make the final volume to 1000 ml.
Sterilize the medium by autoclaving at 121°C for 15 minutes.
Dispense aseptically after cooling the agar medium to 45–50°C.
Note:
With the addition of 1.5% agar, BHI agar can be used as a solid medium.
7. Bordet Gengou’s Medium
It is a selective medium used for the isolation of the causative agent of whooping cough, Bordetella pertussis.
Formula and Preparation
I. Potato infusion — 125.0 g
II. Peptone — 10.0 g
III. Sodium chloride — 5.0 g
IV. Glycerol — 20.0 g
V. Agar — 30.0 g
Procedure
Mix potato with glycerol and about 100 ml of distilled water; boil and strain through gauze.
Add the filtrate to the mixture of other ingredients and keep for 5 minutes.
Boil the solution to dissolve the agar.
Make the final volume 1000 ml.
pH of the medium should be adjusted to 6.4–6.6.
Sterilize the medium in 100 ml volumes in bottles or flasks.
Sterilize the containers by autoclaving at 121°C for 15 minutes.
Store at 2–8°C.
8. Brilliant Green Agar
This medium is used for the selective isolation of Salmonella.
Formula and Preparation
I. Proteose peptone — 10.0 g
II. Yeast extract — 3.0 g
III. Sodium chloride — 5.0 g
IV. Lactose — 10.0 g
V. Sucrose — 10.0 g
VI. Phenol red — 0.08 g
VII. Brilliant green — 0.0125 g
VIII. Agar — 20.0 g
These contents are dissolved in 800 ml of distilled water and final volume is made to 1 liter.
pH of the medium should be adjusted to 6.9.
Dispense into flasks, bottles, and tubes and sterilize by autoclaving at 121°C for 15 minutes.
Store at 2–8°C.
9. Cetrimide Agar
It is a selective plate medium used to isolate Pseudomonas species from a mixed bacterial flora.
It inhibits the growth of bacteria such as Staphylococcus aureus and coliforms.
Formula and Preparation
-
Peptone 20.0 g
-
Magnesium chloride 1.4 g
-
Potassium sulphate 10.0 g
-
Cetrimide (cetyltrimethylammonium bromide) – 0.3 g
-
Agar 15.0 g
-
Distilled water 1000 mL
Preparation Steps:
-
Add ingredients except cetrimide; heat to dissolve.
-
Add cetrimide.
-
Adjust the final pH to 7.2 ± 0.2.
-
Dispense in flasks and sterilize at 121°C for 15 minutes.
10. Dorset Egg Medium
It is a semisolid slope medium used to culture Corynebacterium diphtheriae to detect volutin granules.
It can also be used for maintaining stock cultures.
Formula and Preparation
Nutrient broth – 20 mL
Peeled whole egg – 1
Preparation Steps:
Wash egg carefully using soap and water, then place in 70% alcohol for 30 minutes.
Break egg aseptically; mix yolk and white with nutrient broth.
Strain the mixture through sterile gauze into a sterile bottle.
Dispense into tubes, keeping the mixture as a long slope.
Sterilize inspissator at 85°C for 1 hour daily for 3 consecutive days or keep tubes at 85°C for 30 minutes until solidified.
Incubate at 37°C in a slanted position until medium is solidified.
11. Deoxycholate Citrate Agar (DCA)
It is a selective and differential medium used to isolate Salmonella and Shigella species.
Addition of extra bile salts inhibits coliforms but allows Salmonella and Shigella to grow.
Helps differentiate lactose fermenters (pink colonies) and non-lactose fermenters (colourless colonies).
Formula and Preparation
-
Beef extract – 5.0 g
-
Peptone – 5.0 g
-
Lactose – 10.0 g
-
Sodium citrate – 5.0 g
-
Sodium thiosulphate – 5.0 g
-
Sodium deoxycholate – 5.0 g
-
Ferric ammonium citrate – 1.0 g
-
Neutral red – 0.02 g
-
Agar – 15.0 g
-
Distilled water – 1000 mL
Preparation Steps:
-
Dissolve ingredients in distilled water.
-
Heat with agitation until completely dissolved.
-
Cool to 50–55°C and adjust pH to 7.3.
-
Pour into Petri dishes.
12. Eosin Methylene Blue Agar (EMB)
It is a selective and differential medium used for growing Enterobacteriaceae and lactose fermenting organisms.
Eosin and methylene blue inhibit Gram-positive bacteria.
Lactose fermenters produce dark colonies with metallic sheen (e.g., E. coli) while non-lactose fermenters remain colourless.
Formula and Preparation
-
Peptone – 10.0 g
-
Lactose – 10.0 g
-
Dipotassium hydrogen phosphate – 2.0 g
-
Eosin Y – 0.4 g
-
Methylene blue – 0.065 g
-
Agar – 15.0 g
-
Distilled water – 1000 mL
Preparation Steps:
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Dissolve all ingredients by heating.
-
Avoid overheating to prevent dye precipitation.
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Adjust pH to 7.2 ± 0.2.
-
Autoclave at 121°C for 15 minutes.
-
Cool and pour into plates.
13. Fether’s Medium
It is used as a selective medium for the growth of Legionella.
Formula and Preparation
-
Peptone — 10.0 g
-
Beef extract — 0.3 g
-
Sodium chloride — 5.0 g
-
Agar — 15.0 g
-
Distilled water — 1000 mL
Preparation Steps:
-
Dissolve the ingredients in water by heating and sterilize at 121°C for 15 minutes.
-
Cool to 50–55°C and add 0.1% ferric salt solution.
-
Mix well and dispense aseptically in sterile culture tubes (15 × 225 mm).
-
Sterilize the medium by inspissation at 80–85°C for one hour for three consecutive days.
14. Lowenstein Jensen (LJ) Acid Medium
It is used to isolate Mycobacterium tuberculosis and other mycobacteria. Malachite green prevents contamination by other bacteria.
Formula and Preparation
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Homogenized whole egg (without shell) — 7–8 mL
-
L-asparagine — 0.6 g
-
Magnesium sulfate — 0.6 g
-
Potassium dihydrogen phosphate — 1.0 g
-
Magnesium citrate — 0.6 g
-
Malachite green (2% aqueous solution) — 20 mL
-
Glycerol — 12 mL
-
Distilled water — 600 mL
Preparation Steps:
-
Wash the shells and immerse in 70% alcohol.
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Mix yolk and white thoroughly; strain to remove fibers.
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Add weighed ingredients, glycerol, and malachite green.
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Adjust pH to neutrality (6.2–6.4) and mix well.
-
Dispense into tubes and inspissate at 85°C for 45 minutes on 3 consecutive days.
-
Store at 2–8°C.
15. Loeffler Serum Medium
It is a slope medium used to culture Corynebacterium diphtheriae for metachromatic granule demonstration.
Formula and Preparation
Sterile sheep serum — 3 parts
Sterile nutrient broth — 1 part
Preparation Steps:
Dissolve 0.5% glucose and 1% litmus in nutrient broth.
Mix with serum and adjust reaction to pH 7.2–7.4.
Dispense in tubes.
Solidify by inspissation at 75–80°C for 2 hours.
16. MacConkey Agar
It is a differential and selective medium used to differentiate lactose fermenting from non-lactose fermenting enteric bacteria.
Some Lactose Fermenting Bacteria
-
Escherichia coli
-
Enterobacter cloacae
Some Non-lactose Fermenting Bacteria
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Shigella spp.
-
Proteus spp.
-
Pseudomonas aeruginosa
Also used to detect organisms like Streptococcus pyogenes, Streptococcus pneumoniae and Bordetella pertussis species.
Formula and Preparation
-
Peptone — 20.0 g
-
Lactose — 10.0 g
-
Bile salts — 1.5 g
-
Sodium chloride — 5.0 g
-
Neutral red — 0.03 g
-
Agar — 20.0 g
-
Distilled water — 1000 mL
Preparation Steps:
-
Dissolve the ingredients in distilled water.
-
pH of the medium should be adjusted to 7.0–7.2.
-
Sterilize in autoclave at 121°C for 15 minutes.
-
Cool the medium to 45–50°C and pour into sterile plates.
17. Mannitol Salt Agar
It is a differential and selective agar medium used to isolate Staphylococcus aureus and detect mannitol fermenters.
Formula and Preparation
-
Beef extract — 10 g
-
Peptone — 10 g
-
Sodium chloride — 75 g
-
Mannitol — 10 g
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Agar — 25 g
-
Phenol red — 0.025 g
-
Distilled water — 1000 mL
Procedure:
-
Dissolve ingredients in distilled water by heating.
-
Adjust pH to 7.3–7.5.
-
Autoclave at 121°C for 15 minutes.
-
Cool the medium to 45–50°C and dispense in sterile plates.
18. Motility Indole Urea (MIU) Medium
Used to differentiate enterobacteria species.
Formula and Preparation
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Peptone — 20 g
-
Tryptone — 3.0 g
-
Sodium chloride — 5.0 g
-
Potassium dihydrogen phosphate — 3.0 g
-
Agar — 5.0 g
-
Distilled water — 1000 mL
19. Mueller Hinton Agar
This medium is used for the disc diffusion method of antimicrobial susceptibility testing of bacteria (Kirby–Bauer method).
It is also used for testing Neisseria and Haemophilus species, with the addition of blood into the medium.
Formula and Preparation
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Beef extract – 300.0 g
-
Peptone – 17.5 g
-
Starch – 1.5 g
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Agar – 17.0 g
-
Distilled water – 1000 mL
Preparation Steps:
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Boil all ingredients in 1000 mL distilled water in a conical flask.
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Adjust pH of the medium to 7.4 ± 0.2.
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Sterilize by autoclaving at 121°C for 15 minutes.
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Cool to 50–55°C and pour into sterile Petri dishes.
20. Phenylalanine Agar
This medium is used in the phenylalanine test, mainly to assist in identification of enterobacteria.
Formula and Preparation
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Peptone – 3.0 g
-
Yeast extract – 3.0 g
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Sodium chloride – 5.0 g
-
DL-phenylalanine – 2.0 g
-
Dipotassium hydrogen phosphate – 1.0 g
-
Agar – 15.0 g
-
Distilled water – 1000 mL
Preparation Steps:
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Dissolve ingredients in distilled water and adjust pH to 7.3.
-
Dispense in 1 mL amounts into screw-cap tubes.
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Sterilize in autoclave at 121°C for 15 minutes.
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Slant tubes before solidification.
21. Sabouraud Dextrose Agar
Used to culture Candida albicans and other fungi.
Formula and Preparation
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Peptone – 10.0 g
-
Glucose – 40.0 g
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Agar – 15.0 g
-
Distilled water – 1000 mL
Preparation Steps:
-
Dissolve ingredients in distilled water; pH should be adjusted to 5.6 ± 0.2.
-
Sterilize at 121°C for 15 minutes.
-
Cool to 50–55°C, mix well, and dispense 15–20 mL amounts into sterile Petri dishes or 7–10 mL amounts into sterile tubes.
22. Salmonella–Shigella (SS) Agar
It is a selective medium used to isolate Salmonella and Shigella species from fecal specimens.
This medium inhibits Gram-positive organisms because it contains bile salts and brilliant green.
Lactose fermenters produce red colonies, while non-lactose fermenters appear colourless.
Salmonella may produce black-centered colonies due to H₂S formation (Ferric citrate).
Formula and Preparation
Peptone – 5.0 g
Beef extract – 5.0 g
Lactose – 10.0 g
Bile salts – 8.5 g
Sodium citrate – 8.5 g
Sodium thiosulphate – 8.5 g
Brilliant green – 0.00033 g
Ferric citrate – 1.0 g
Neutral red – 0.025 g
Agar – 15.0 g
Distilled water – 1000 mL
Preparation Steps:
Dissolve ingredients in distilled water by heating; boil for 1 minute.
Adjust pH to 7.0.
Cool the medium to 45–50°C and pour aseptically into sterile Petri dishes.
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23. Selenite F broth
It is used as an enrichment medium for the isolation of Salmonella species from fecal specimens and Salmonella typhi from urine. This medium helps in their selective isolation over the commensals.
• Formula and preparation
I. Sodium hydrogen selenite (anhydrous) : 4.0 g
II. Peptone : 5.0 g
III. Lactose : 4.0 g
IV. Disodium hydrogen phosphate : 10.0 g
V. Distilled water : 1000 ml
Dissolve the ingredients in distilled water by gentle heating.
pH of the medium should be adjusted to 7.0.
Dispense in 5 ml amounts in screw-cap bottles or culture tubes.
Sterilize by steaming (with caps loosened) for 20 minutes. Tighten the bottle caps after sterilization. Formation of small amounts of red precipitate will not interfere with the performance of the medium.
24. Simmons citrate agar
Some pathogenic bacteria such as Enterobacter, Serratia and Klebsiella are capable of utilizing citrate and monoammonium phosphate as sole sources of carbon and nitrogen respectively. This is indicated by change in the Simmons citrate agar color which contains bromothymol blue as indicator. The original green color of the medium turns blue.
• Formula and preparation
-
Sodium chloride : 5.0 g
-
Magnesium sulfate : 0.2 g
-
Sodium citrate : 2.0 g
-
Dipotassium phosphate : 1.0 g
-
Ammonium dihydrogen phosphate : 1.0 g
-
Agar : 20.0 g
-
0.4% (w/v) Bromothymol blue : 4.0 ml
-
Distilled water : 1000 ml
-
Dissolve the ingredients in distilled water by gentle heat.
-
pH of the medium should be adjusted to 6.9.
-
Dispense in 5 ml amounts in cotton-plugged tubes.
-
Sterilize by autoclaving at 121°C for 15 minutes.
-
Allow to solidify in slanting position.
25. Tellurite blood agar
It is used as a selective medium for the isolation of Corynebacterium diphtheriae.
• Formula and preparation
-
Blood agar : 100 ml
-
3.5% (w/v) Potassium tellurite 1.0 ml
-
Prepare blood agar.
-
After adding blood add aseptically potassium tellurite solution. Mix well.
-
pH of the medium should be adjusted to 7.4–7.8.
-
Dispense aseptically in about 16 ml amounts in sterile Petri dishes.
26. Thiosulfate citrate bile salt sucrose (TCBS) agar
It is used as a selective medium for the isolation of Vibrio cholerae and other Vibrio species from fecal specimens.
• Formula and preparation
I. Yeast extract powder : 5.0 g
II. Bacteriological peptone : 10.0 g
III. Sodium thiosulfate : 10.0 g
IV. Sodium citrate : 10.0 g
V. Ox bile : 8.0 g .
Sucrose : 20.0g
Sodium cloride : 10.0g
ferric citrate : 1.0g
Bromothymol blue : 0.04g
Thymol blue : 0.04g
Agar : 14.0g
Distilled water : 1000ml
27. Triple sugar iron (TSI) slants
These are used in the first step of identification of Enterobacteriaceae.
• Formula and preparation
-
Peptone : 20.0 g
-
Lactose : 10.0 g
-
Sucrose : 10.0 g
-
Glucose : 1.0 g
-
Sodium chloride : 5.0 g
-
Ferrous ammonium sulfate : 0.2 g
-
Sodium thiosulfate : 0.3 g
-
Phenol red : 0.025 g
-
Distilled water : 1000 ml
-
Dissolve the ingredients in distilled water by heating.
-
pH should be adjusted to 7.3.
-
Dispense in culture tubes in 7 ml amounts and plug with cotton plugs.
-
Sterilize at 121°C for 15 minutes and cool the tubes in a slanted position (so as to get deep butts).
28. Tryptone soya diphasic medium
It is suitable for the growth of a wide range of pathogens. It is specifically used to isolate pathogens from blood.
• Formula and preparation
Tryptone soya agar
-
Tryptone : 15.0 g
-
Soya peptone : 5.0 g
-
Sodium chloride : 5.0 g
-
Agar : 15.0 g
-
Distilled water : 1000 ml
-
Dissolve the ingredients in distilled water by gentle heating.
-
Dispense in 20–25 ml amounts in flat type screw-cap bottles.
-
Sterilize by autoclaving at 121°C for 15 minutes.
-
Allow the medium to cool to obtain an agar slope.
Tryptone soya broth
-
Pancreatic digest of casein : 17.0 g
-
Papaic digest of soya bean meal : 3.0 g
-
Dibasic potassium phosphate : 2.5 g
-
Sodium chloride : 5.0 g
-
Glucose : 2.5 g
-
Distilled water : 1000 ml
-
Dissolve the ingredients in distilled water and add 0.05 g of liquid (sodium polyanethol sulfonate) as blood-culture anticoagulant and 0.05 g of p-aminobenzoic acid to neutralize sulfones.
-
Sterilize the broth by autoclaving at 121°C for 15 minutes, cool to 45–50°C, and dispense aseptically 20 ml on the agar slope in the bottle.