Gayam, Glydenne Glaire P. Bacte MWF 5:30-8:30BS Medical Technology 3A August 19, 2015

Culture Medium Use Preparation Components

1. Nutrient Basal Medium Dissolve 100g agar in 1L DH20. Heat, dispense, autoclave.

ProteinSugarIndicators

2. Acetate Agar Differential Medium Add components, except sodium acetate buffer, to distilled/deionized water and bring volume to 900.0mL. Mix. Heat and bring to boiling. Autoclave and cool. Aseptically add 100.0mL of sterile sodium acetate buffer. Mix.

Peptone SugarMeat extractYeast extractSodium acetate buffer

3. Alkaline Peptone Water Enrichment Medium Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 9.0. Autoclave.

Peptone NaCl

4. Bismuth Sulfite Agar Selective Medium Add components to distilled/deionized water and bring volume to 1.0L. Mix and heat until boiling. Boil for 1 min. Do not autoclave. Cool.

AgarBismuth sulphiteCaseinAnimal tissueGlucoseNa2HPO4

FeSO4·7H2O5. Blood Agar, Sheep Differential Medium Add components, except sheep blood,

to distilled/deionized water and bring volume to 950.0mL. Mix. Heat and bring to boiling. Autoclave and cool Aseptically add 50.0mL of sterile sheep

Trypticase soy agarBrucella agar, or beef heart infusion with 5% sheep blood

blood. Mix. Pour into sterile Petri dishes.

6. Phenylethyl Alcohol Agar Selective Medium Add components to distilled/deionized water and bring volume to 1.0L. Mix, gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave.Pour into sterile Petri dishes or leave in tubes.

AgarTryptoseNaClBeef ExtractPhenylethyl alcohol

7. Buffered Charcoal-Yeast Extract Agar

Enrichment Medium Add components, except L-cysteine solution, to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boil for 1 min. Autoclave and cool. Add 4.0mL of L-cysteine solution. Mix and pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.

Yeast extractAgarCharcoalsalts

8. Chocolate Agar Enrichment medium Add components, except supplement Bsolution and sheep blood, to distilled/deionized water and bring volumeto 890.0mL. Mix and heat until boiling. Autoclave and cool. Asepticallyadd 100.0mL of sterile, defibrinated sheep blood. Heat while stirring and bring to 85°C for 5–10 min. Cool to 50°C. Aseptically add 10.0mL of sterile supplement B. Mix and pour into sterile Petridishes or distribute into sterile tubes.

Peptone base, enriched with solution of 2% hemoglobin or isovitalex (BBL)NaClCornstarchSheep blood

9. Eoisin-Methylene Blue Agar

Selective and Differential medium Add components to distilled/deionized water and bring volume to 1.0L. Mix,

Peptone base (lactose)Eosin Y

heat and bring to boiling. Distribute into tubes or flasks. Autoclave and pour into sterile Petri dishes.

Methylene Blue indicatorSugar

10. Hydrogen Sulfide, Lead Acetate

Differential culture Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Distribute into tubes or flasks. Autoclave and pour into sterile Petri dishes or leave in tubes. Allow tubes to cool in a slanted position.

Lead acetateAgarPeptoneGlucose

11. Kligler’s Iron Agar Differential medium Identify if a gram-negative rod is a glucose or lactose fermenter or both

Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Distribute into tubes. Autoclave. Pour into sterile Petri dishes or leave in tubes.

SugarsIndicatorPeptoneSulfur source

12. Lim Broth Enrichment brothIsolate S. agalactiae

Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave.

Peptones, yeast extract, dextrose

13. Lysine-Iron Sugar Differential mediumIdentify species of Enterobacteriaceae

Add components to distilled/deionized water and bring volume to 1.0L. Mix then heat while stirring and bring to boiling. Distribute into tubes in 10.0mL volumes. Autoclave. Allow tubes to cool in a slanted position.

Amino acidCarbohydrateIndicatorsSulfur

14. MacConkey Agar Selective and Differential medium Add components to distilled/deionized water and bring volume to 1.0L. Mix then heat while stirring until boiling. Autoclave. Pour into sterile Petri dishes or distribute into sterile tubes.

Peptone baseIndicatorNaClBile Salts

15. Mannitol Salt Agar Selective and Differential medium Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat while stirring and bring to boiling.

Peptone baseMannitolIndicator

Distribute into tubes or flasks. Autoclave. Pour into sterile Petri dishes or leave in tubes.

Beef extractNaClCaseinAnimal Tissue

16. Mueller-Hinton Agar Transparent medium Add components to distilled/deionized water and bring to 1.0L. Mix, heat and bring to boiling. Distribute into tubes or flasks. Autoclave. Pour into sterile Petri dishes or leave in tubes.

Animal infusionCasein extractStarchAgar

17. New York City Medium Selective mediumIsolate N. gonorrhoea and N. meningitidis from specimens containing mixed normal flora

Have all solutions prepared and at 45°– 50°C. Aseptically combine components. Mix. Pour into sterile Petri dishes.

Peptone agar base with cornstarchHorse blood cellsHorse plasma, citratedYeast dialysate

18. Selenite Broth Enrichment Broth Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Do not autoclave. Distribute into sterile tubes in 10.0mL volumes.

Peptone base brothSodium biselenite

19. Triple-Sugar Iron Agar Differential mediumIdentification of Glucose-fermenters from Non-glucose-fermenters gram negative rods

Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Distribute into tubes or flasks. Autoclave and cool in a slanted position to form a1.0-inch butt.

SugarsIndicatorPeptoneSulfur source

20. Trypticase Soy Agar All-purpose/ Basal medium Add components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Distribute into tubes or flasks. Autoclave. Do not overheat. Pour into sterile Petri dishes

PeptoneCaseinSodium chloride

or leave in tubes.21. Trypticase Soy Broth All-purpose medium/ Enrichment

mediumAdd components to distilled/deionized water and bring volume to 1.0L. Mix, heat and bring to boiling. Distribute into tubes or flasks. Autoclave then mix thoroughly.

SoybeanCaseinNaClGlucoseDipotassium phosphate

Mahon, C.R. et. al (2015). Textbook of Diagnostic Microbiology (4th edition). Saunders Elsevier.

Tille, P.M. (2014). Bailey & Scott’s Diagnostic Microbiology (13th edition). Elsevier Mosby.

Atlas, R.M. (2010). Handbook of Microbiological Media (4th edition). CRC Press.