Media Preparation

• Culture Media


  • Anything that possess the nutritional needs of microorganisms for growth
  • Those materials that contains nutrients in proper concentrations for growth of bacteria
  • Any material that allow growth of any microorganism
• Inoculum


  Microbes introduced into a culture medium to initiate growth
• Inoculation


  To introduce bacterial suspension in a culture
• Culture


  Microbes that grow and multiply in or on a culture medium
• Fastidious Bacteria


  Organisms that require extensive/complex nutritional requirement
• Non-fastidious bacteria


  Organisms with simple nutritional requirement
• Classification of Culture Media


  • According to consistency
  • According to composition
  • According to function or use of application
  • According to form or desired volume
• According to Consistency

  • Solid Form - in agar/albumin/gelatin
  • Liquid Form - no agar/solidifying agent
  • Semi-Solid - with low concentration of agar

  
• According to Composition

  • Chemically defined
  • Chemically undefined
• According to Function

  • General Isolation Media aka SUPPORTIVE MEDIA
  • Non-Selective Isolation Media aka ENRICHED MEDIA
  • Differential Media
  • Selective Media
  • Special Media
• General Isolation Media aka SUPPORTIVE MEDIA


  Supports growth of most non-fastidious bacteria

  NA, NB, Trypticase Soy agar (TSA)
• Non-Selective Isolation Media aka ENRICHED MEDIA


  Contains nutrient supplements

  Sheep Blood agar (SBA), Chocolate agar (CA)
• Differential Media


  Provide distinct colonial appearances of microorganisms to aid in their identification

  MacConkey agar (MAC), Eosin-Methylene Blue agar (EMB)
• Selective Media


  Allow one to select for pathogens through the inhibition of normal flora

  Hektoen-Enteric agar (HEA), Salmonella-Shigella agar (SSA), Xylosine-lysine-deoxycholate agar (XLD)
• Special Media


  • used to determine the characteristics of microorganisms
  • Transport media – temporary storage
  • Enrichment broth - encourage growth of a particular organism that would likely overgrown by competitors in a sample
  • Cell line and Cell culture – made up of layers of living cells
  • Biochemical Characteristic Media – used for detection of an organism biochemical reaction that aid in its ID
• According to Form/Desired Volume

  • Tubed Media - butt/deep, slant, butt slant, liquid/broth
  • Plated - SBA, PEA, MAC, MSA, MHA
• Plating Media for Routine Bacteriology

  • Bile esculin agar (BEA)
  • Bile esculin azide agar with vancomycin
  • Blood agar
  • Bordet-Gengou agar
  • Brain heart infusion agar or broth
  • Buffered charcoal-yeast extract agar (BCYE)
  • Buffered charcoal-yeast extract (BCYE) agar with antibiotics
  • Burkholderia cepacia selective agar
  • Campy-blood agar
  • Campylobacter thioglycollate broth
  • CDC anaerobe 5% sheep blood agar
  • Cefoperazone, vancomycin, amphotericin (CVA) medium
  • Cefsulodin-irgasan- novobiocin (CIN) agar
  • Chocolate agar
  • Chromogenic media
  • Columbia colistin-nalidixic acid (CNA) agar
  • Cystine-tellurite blood agar
  • Eosin methylene blue (EMB) agar (Levine)
  • Gram-negative broth (GN)
  • Hektoen enteric (HE) agar
  • Loeffler's medium
  • MacConkey agar
  • MacConkey sorbitol agar
  • Mannitol salt agar
  • New York City (NYC) agar
  • Phenylethyl alcohol (PEA) agar
  • Regan Lowe
• Preparation of Mueller-Hinton Agar

  1. Plated media:

  2. Weigh - Weigh 9.5 g MHA using a tared Erlenmeyer flask.

  3. Dissolve - Add 250 mL distilled water aided with constant stirring and heat until cleared.

  4. Plug - Cover flask with cotton plug or aluminum foil. Place autoclave tape.

  5. Sterilize - 121 deg C at 15 psi for 15-20 minutes

  6. Dispense - Dispense 20 mL per plate (ideally it should have a height = 4mm) with constant stirring after pouring.

  7. Form - Allow to cool and solidify. Once solidified, invert plates.
• Agar


  solidifying agent

  complex polysaccharide derived for seaweeds (red algae/Rhodophyte)

  transparent and molten at 100 deg C and solidifies at 24 deg C or RT
• Bile esculin agar (BEA)


  Components: Nutrient agar base with ferric citrate. Hydrolysis of esculin by group D streptococci imparts a brown color to medium; sodium desoxycholate inhibits many bacteria

  Primary Purpose: Differential isolation and presumptive identification of group D streptococci and enterococci
• Bile esculin azide agar with vancomycin

  Components: Contains azide to inhibit gram negative bacteria, vancomycin to select for resistant gram positive bacteria, and bile esculin to differentiate enterococci from other vancomycin resistant bacteria that may grow

  Primary Purpose: Selective and differential for cultivation of vancomycin-resistant enterococci from clinical and surveillance specimens
• Blood agar


  Components: Trypticase soy agar, Brucella agar, or beef heart infusion with 5% sheep blood

  Primary Purpose: Cultivation of non-fastidious microorganisms, determination of hemolytic reactions
• Bordet-Gengou agar


  Components: Potato-glycerol based medium enriched with 15%-20% defibrinated blood; contaminants inhibited by methicillin (final concentration of 2.5 µm/mL)

  Primary Purpose: Isolation of Bordetella pertussis and Bordetella parapertussis
• Brain heart infusion agar or broth


  Components: Dextrose, pork brain and heart dehydrated infusions

  Primary Purpose: Cultivation of fastidious organisms.
• Buffered charcoal-yeast extract agar (BCYE)


  Components: Yeast extract, agar, charcoal, and salts supplemented with L-cysteine HCl, ferric pyrophosphate, ACES buffer, and a-ketoglutarate

  Primary Purpose: Enrichment for Legionella spp. Supports the growth of Francisella and Nocardia spp.
• Buffered charcoal-yeast extract (BCYE) agar with antibiotics


  Components: BCYE supplemented with polymyxin B, vancomycin, and ansamycin, to inhibit gram-negative bacteria, gram-positive bacteria, and yeast, respectively

  Primary purpose: Enrichment and selection for Legionella spp.
• Burkholderia cepacia selective agar


  Components: Bile salts, gentamycin, ticarcillin, polymixin B, Peptone, yeast extract

  Primary purpose: For recovery of B. Cepacia from cystic fibrosis patients
• Campy-blood agar


  Components: Contains vancomycin (10 mg/L), trimethoprim (5 mg/L), polymyxin B (2500 U/L), amphotericin B (2 mg/L), and cephalothin (15 mg/L) in a Brucella agar base with sheep blood

  Primary purpose: Selective for Campylobacter spp.
• Campylobacter Thioglycollate broth


  Components: Thioglycollate broth supplemented with increased agar concentration and antibiotics

  Primary purpose: Selective holding medium for recovery of Campylobacter spp. Incubated at 4° C for cold-enrichment.
• CDC anaerobe 5% sheep blood agar


  Components: Tryptic soy broth, 5% sheep blood and added nutrients

  Primary purpose: Improved growth of obligate, slow growing anaerobes
• Cefoperazone, vancomycin, amphotericin (CVA) medium


  Components: Blood supplemented enrichment medium containing cefoperazone, vancomycin, and amphotericin to inhibit growth of most gram-negative bacteria, gram-positive bacteria, and yeast, respectively

  Primary purpose: Selective medium for isolation of Campylobacter spp.
• Cefsulodin-irgasan-novobiocin (CIN) agar


  Components: Peptone base with yeast extract, mannitol, and bile salts; supplemented with cefsulodin, irgasan, and novobiocin; neutral red and crystal violet

  Primary purpose: Selective for Yersinia spp.; may be useful for isolation of Aeromonas spp.
• Chocolate agar


  Components: Peptone base, enriched with solution of 2% hemoglobin or IsoVitaleX (BBL)

  Primary purpose: Cultivation of fastidious microorganisms such as Haemophilus spp., Brucella spp. and pathogenic Neisseria spp.
• Chromogenic media


  Components: Organism - specific nutrient base, selective supplements and chromogenic substrate

  Primary Purpose: Chromogenic media are designed to optimize growth and differentiate a specific type of organism. Chromagars are routinely used in the identification of yeasts, methicillin - resistant Staphylococcus aureus (MRSA), and a variety of other organisms.
• Columbia colistin - nalidixic acid (CNA) agar


  Components: Columbia agar base with 10 mg colistin per liter, 15 mg nalidixic acid per liter, and 5% sheep blood

  Primary Purpose: Selective isolation of gram - positive cocci
• Cystine - tellurite blood agar


  Components: Infusion agar base with 5% sheep blood; reduction of potassium tellurite by Corynebacterium diphtheriae produces black colonies

  Primary Purpose: Isolation of C. diphtheriae
• Eosin-methylene blue (EMB) agar (Levine)


  Components: Peptone base containing lactose; eosin Y and methylene blue as indicators

  Primary Purpose: Isolation and differentiation of lactose -fermenting and non -lactose - ferment
• Gram - negative broth (GN)


  Components: Peptone base broth with glucose and mannitol; sodium citrate and sodium desoxycholate act as inhibitory agents

  Primary Purpose: Selective (enrichment) liquid medium for enteric pathogens
• Hektoen enteric (HE) agar


  Components: Peptone base agar with bile salts, lactose, sucrose, salicin, and ferric ammonium citrate; indicators include bromthymol blue and acid fuchsin

  Primary Purpose: Differential, selective medium for the isolation and differentiation of Salmonella and Shigella spp. from other gram -negative enteric bacilli
• Loeffler's medium


  Components: Animal tissue (heart muscle), dextrose, eggs and beef serum, and sodium chloride

  Primary Purpose: Isolation and growth of Corynebacterium