Culture Media

ACETATE AGAR

• Purpose 
  
  • differentiate E. coli from Shigella spp.
• Components
  
  • Acetate – carbon source 
  • Bromthymol blue – pH indicator
• RESULT
  • Green – negative (didn’t utilized acetate) 
  • Blue – positive (utilized acetate)

ALKALINE PEPTONE WATER

• Purpose 
  
  • for recovery of Vibro spp. And Aeromonas spp.
• Components
  
  • 0.5 – 1.0% NaCl – to recover Vibrio spp.

BACTEROIDES BILE ESCULIN AGAR

• Purpose 
  
  • for isolation of Bacteroides fragilis group
• Components
  
  • Oxgall – separates bile-resistant & bile-sensitive species. 
  • 1% esculin & ferric ammonium citrate 
    
    • to visualize esculin hydrolysis 
    • (+) reaction – dark brown or black

BILE ESCULIN AGAR

• Purpose – used to isolate & identify group D streptococci and enterococci
• Components
  
  • Oxgall – inhibits most gram (+) organisms 
  • Esculin – differential component
• Esculin —–bacteria—— > Esculetin
• Esculetin + Ferric citrate
• insoluble iron salts (black)
• NOTES
  
  • Addition of vancomycin – used to detect vancomycin-resistant streptococci & enterococci 
  • Addition of azide – inhibits gram-negative organism

BISMUTH SULFITE AGAR

• Purpose – isolation of Salmonella spp.
• Components
  
  • Selective ingredients – inhibits gram (+) bacteria 
    
    • Bismuth sulfite 
    • Brilliant green 
  • Ferrous sulfate – reacts to H2S to produce black ppt. 
• COLONIES
  
  • Salmonella serotype Typhi 
    
    • black surrounded with metallic sheen 
  • Serotype Gallinarum, Cholerasuis, Paratyphi
    
    • light green

KANAMYCIN AND VANCOMYCIN BLOOD AGAR

• Purpose – for isolation of obligate gram (-) anaerobes particularly Bacteroides spp.
• Components
  
  • Antimicrobials 
    
    • Kanamycin 
    • Vancomycin

LAKED BLOOD AGAR W/ KANAMYCIN & VANCOMYCIN & VITAMIN K

• Purpose – for isolation of Bacteroides and Prevotella spp.
• Components
  
  • Antimicrobials 
    
    • Kanamycin 
    • Vancomycin 
  • Laked erythrocytes (lysed by freezing) 
  • Vitamin K
• NOTE
  
  •  helpful in isolation of Prevotella melaninogenica

RABBIT BLOOD AGAR

• Purpose – for recovery and demonstration of beta- hemolysis of Haemophilus
  spp. & Gardnerella vaginalis

BORDET – GENGOU BLOOD AGAR

• Purpose – for isolation of Bordetella pertussis & B. parapertussis
• Components
  
  • Selective agents
    
    • Penicillin 
    • Methicillin 
    • cephalexin 
  • Peptone 
  • Glycerol 
  • Potato infusion 
  • Defibrinated sheep blood (sterile) 
  • 15 – 30% Blood enrichment (3-6 ml/20-ml tube)
• NOTE: plate must be held for 5 days, but not more than 7 days, before regarded as negative

BRAIN-HEART INFUSION BROTH

• Purpose – recommended for cultivation of pneumococci for bile solubility test
• Components
  
  • Brain & Beef heart – provide nutrients 
  • Peptone 
  • Glucose 
  • NaCl 
  • Buffers
• NOTE: 6.5% NaCl can be added – to differentiate salt- tolerant enterococci from streptococci.

BUFFERED CHARCOAL YEAST EXTRACT (BCYE) AGAR

• Purpose – for isolation of Legionella spp.
• Components
  
  • Ferric pyrophosphate – provides Iron 
  • Enhances growth of Legionella 
    
    • Yeast extract 
    • Alpha-ketoglutarate 
    • L-cysteine 
  • Activated charcoal – absorb toxic compounds from organism’s metabolism
• NOTE
  
  • can be used to isolate Francisella & Nocardia spp. 
  • Legionella spp. Not be visible til 3-5 days after inoculation

WADOWSKY – MODIFICATION

• Components
  
  • Inhibitors of Gram (-) organism 
    
    • Glycine o
    • Polymyxin B 
  • Vancomycin – inhibits gram (+) cocci 
  • Anisomycin – inhibits fungi 
  • Differential components
    
    • Bromcresol purple 
    • Bromthymol blue
• RESULT: L. pneumophila colonies – light blue w/ pale green tin

BURKHOLDERIA CEPACIA AGAR

• Purpose – isolate B. cepacian from respiratory spx. of patients with cystic fibrosis
• Components
  
  • Inhibitors – inhibits gram (+) & gram (-)
    
    • Crystal violet 
    • Bile salts 
    • Polymyxin B 
    • Ticarcillin 
  • Inorganic salts 
  • Peptones 
  • Pyruvate 
  • Phenol red – pH indicator

CAMPYLOBACTER BLOOD AGAR

• Purpose – for isolation of Campylobacter spp.
• Components: 
  
  • Brucella agar – base medium 
  • Sodium bisulfite – lowers redox potential, enhancing recovery of microaerophilic organism. 
  • 10% sheep blood 
  • Inhibitors 
    
    • Vancmycin 
    • Trimethoprim – prevents Proteus 
    • Polymyxin B
    • Amphotericin B – prevents fungi 
    • Cefoperazone – antipseudomonal
• Colony characteristic 
  
  • Campylobacter spp. 
    
    • Flat, gray, nonhemolytic, raised or mucoid 
    • Some may be tan or slightly pink 
    • May appear swarming / spreading across surface of plate

CETRIMIDE AGAR (pseudosel agar / Psedomonas- selective agar)

• Purpose – for Pseudomonas spp. (except for P. fluorescens)
• Components: 
  
  • Inhibitor 
    
    • Cetrimide (cetyl trimethyl ammonium bromide / hexamethyltrimethyl ammonium bromide 
    • Pyocyanin production Stimulator – Magnesium chloride 
    • Potassium sulfate 
  • Low Iron content – stimulates pyoverdin production

COOKED MEAT (CHOPPED MEAT GLUCOSE) MEDIUM

• Purpose – useful in cultivation of anaerobes esp. Clostridium spp.
• Components: 
  • Solid Meat Particles – initiates growth from very small inoculum 
  • Peptone 
  • Beef heart 
  • Dextrose

CYCLOSERINE CEFOXITIN FRUCTOSE AGAR (CCFA)

• Purpose – for isolation and identification of Clostridium difficile
• Component: 
  
  • Inhibitors – inhibits intestinal normal flora 
    
    • Cycloserine 
    • Cefoxitin 
  • Fructose 
  • Neutral red – pH indicator
• OTHER VARIATIONS 
  
  • 1st variation 
    
    • Mannitol (instead of fructose) 
    • Bromthymol blue – pH indicator 
  • 2nd variation 
    
    • Addition of egg yolk suspension – Detection of lipase and lecithinase activity 
    • COLONY CHARACTERISTIC = C. difficile – yellow colony and In UV light – gold- yellow

0.4 % DILUTE GELATIN MEDIUM

• Purpose – useful in differentiation of: 
  
  • Nocardia spp. from one another 
  • Streptomyces spp.
• COLONY CHARACTERISTICS 
  
  • N. asteroids – doesn’t grow / grows poor 
  • N. brasiliensis – compact, rounded colonies 
  • Streptomyces spp. 
    
    • Poor to good growth o With stringy or flaky morphology

EGG YOLK AGAR (McClung Toabe Agar)

• Purpose – for detection of lecithinase, lipase, protease activity
• Component – Egg emulsion 
  
  • Provides lecithin, lipids, and proteins
• RESULTS
  
  • Lecithinase activity – zone of opacity 
  • Lipase activity – iridescent sheen around surface of colonies Protease activity – clearing of medium

HAEMOPHILUS TEST MEDIUM

• Purpose 
  
  • for susceptibility testing of Haemophilus 
  • also for broth minimal inhibitory concentration (MIC)
• Components:
  
  • Beef 
  • Yeast 
  • Casein extract 
  • Hematin 
  • NAD

FLETCHER SEMISOLID MEDIUM

• Purpose – for Leptospira spp.
  
• Component: Rabbit serum w/ hemoglobin – enrichment Growth: turbidity (examined in dark-field microscope)

HEKTOEN ENTERIC AGAR

• Purpose 
  
  • for direct isolation of enteric pathogens 
  • for indirect isolation from selective enrichment broth
• Components: 
  
  • bile salt – selective component 
  • lactose 
  • salicin 
  • sucrose 
  • bromthymol blue – pH indicator 
  • for detection of H2S gas 
    
    • sodium thiosulfate 
    • ferric ammonium citrate
• NOTE
  
  • SHOULD NOT BE AUTOCLAVED 
  • AVOID OVERHEATING
• COLONY CHARACTERISTICS 
  
  • Most nonpathogen 
    
    • bright orange to salmon-pink 
  • Salmonella & Shigella spp. 
    
    • Green to blue-green colonies

LIM BROTH (Modified Todd-Hewitt Broth)

• Purpose – for isolation of Streptococcus agalactiae
• Component
  
  • Peptone 
  • Yeast extract 
  • Dextrose 
  • Inhibitors for gram (-) 
    
    • Colistin 
    • Nalidixic acid

LOEFFLER COAGULATED SERUM SLANT

• Purpose – for primary recovery of C. diphtheriae
• Component: 
  
  • Serum (high content) 
  • Animal heart muscle 
  • Dextrose 
  • Egg 
  • NaCl

MacCONKEY AGAR

• Purpose – selects for Enterobacteriaceae & other gram (-) rods
• Components
  
  • Inhibitors 
    
    • Bile salto
    • Crystal violet 
  • Lactose – sole carbohydrate source 
  • Neutral red – pH indicators
• NOTE
  
  • Enterococcus spp. may produce tiny colonies 
  • MAC w/out crystal violet – used to help identify mycobacteria

MacCONKEY SORBITOL AGAR

• Purpose – used to isolate E. coli O157:H7 D-sorbitol is substituted for lactose

MALONATE BROTH

• Purpose – identification of Salmonella spp.
• Component: 
  
  • Sodium malonate – carbon source 
  • Glucose 
  • Yeast extract 
  • Bromthymol blue – pH indicator
• REACTION 
  
  • Prussian blue color – utilized malonate 
  • green (no change of color) – no growth

MANNITOL SALT AGAR

• Purpose – for recovery & identification of staphylococci
• Components: 
  
  • 7.5% NaCl – inhibits gram (-) & (+) except staphylococci 
  • Mannitol – carbohydrate source 
  • Phenol red – pH indicator
• COLONY CHARACTERISTICS 
  
  • S. aureus – yellow zone in colonies
• NOTE
  
  • Enterococcus spp.– may able to grow & weak mannitol fermenter

MOTILITY TEST MEDIUM

• Purpose – to determine if organism is motile or nonmotile
  
• NOTE: add 1% triphenyltetrazolium chloride – to enhance detection of
  motility

MODIFIED THAYER-MARTIN AGAR

• Purpose – for recovery of N. gonorrhoeae & N. meningitidis
• COMPONENTS 
  
  • Hemoglobin 
  • Vitamins 
  • Diphosphopyridine nucleotide 
  • L-cysteine 
  • NAD 
  • Glutamine 
  • Cornstarch – absorb inhibitory substances 
  • Inhibitors 
    
    • Vancomycin 
    • Colistin 
    • Nystatin – prevents fungal growth 
    • Trimethoprim – prevents Proteus swarm

Martin-Lewis Agar

• Components 
  
  • Anisomycin (20ug / mL) – substitute for nystatin 
  • Vancomycin (4ug/mL) – higher conc. Than MTM

MUELLER-HINTON AGAR

• Purpose – for susceptibility testing of organisms in antimicrobial agents.
• COMPONENTS
  
  • Animal infusion
  • Casein extract 
  • Starch
• NOTE
  
  • Add 5% sheep blood – to perform susceptibility testing on streptococci 
  • Add heated / chocolatized SRBC – for fastidious organism (Haemophilus & Neisseria) 
  • Ca2+ & Mg2+ concentration – critical in testing of Pseudomonas isolates w/ aminoglycoside antibiotics

MUELLER-HINTON AGAR W/ 2% NaCl

• Purpose – for detection of MRSA
• Cefoxitin & oxacillin – used for detection of MRSA (in Kirby-Bauer or Etest)

MHA w/ 4% NaCl & 6ug OXACILLIN

• Purpose – to screen S. aureus isolates selectively for resistance to oxacillin or nafcillin

NEW YORK CITY MEDIUM

• Purpose – used also for N. gonorrhoeae & N. meningitidis
• COMPONENTS: 
  
  • Hemoglobin- from lysed horse RBC 
  • Yeast dialysate 
  • Horse plasma 
  • Inhibitors 
    
    • Vancomycin 
    • Colistin 
    • Amphotericin B 
    • Trimethoprim
• NOTE
  
  • also supports growth for Mycoplasma as well as Ureaplasma urealyticum

O-F POLYMYXIN B-BACITRACIN-LACTOSE AGAR

• Purpose – for isolation of Burkholderia cepacia
• INHIBITORS
  
  • Polymyxin B 
  • Bacitracin

PHENYLETHYL ALCOHOL AGAR

• Purpose – for isolation of gram (+) cocci & rods
• COMPONENT: 
  
  • Phenylethyl alcohol – inhibits facultative gram neg. rods
• NOTE
  
  • Bacillus anthracis will not grow in this medium

POTASSIUM TELLURITE BLOOD AGAR

• Purpose – for isolation of C. diphtheriae
• COMPONENTS 
  
  • Cystine 
  • Potassium tellurite – inhibits gram (-) organisms, staph, strep, while allowing growth of C. diphtheriae
• NOTE 
  
  • Some Staphyloccocus, gram (-) bacilli, yeast will overcome inhibition
• COLONY CHARACTERISTICS 
  
  • C. diphtheriae – dull, gray black (reduction of tellurite) 
  • Diphtheroids – light gray-green 
  • Staphylococcus – large, glistening, jet black 
  • Gram (-) bacilli & yeast – dull, gray-black (larger)

PPLO (Pleuropneumonia-like organism) Agar

• Purpose – used to isolate Mycoplasma spp.
• Component: 
  
  • NaCl 
  • Agar 
  • Antimicrobials

REGAN-LOWE MEDIUM

• Purpose – for isolation of B. pertussis & B. parapertussis
• Components: 
  
  • Beef extract 
  • Horse blood 
  • Niacin 
  • Pancreatic digest 
  • Neutralizers 
    
    • Charcoal 
    • Starch 
  • Cephalexin – selective agent
• COLONY CHARACTERISTICS 
  
  • B. pertussis – domed, shiny, transparent, and tiny; mercury droplet appearance

SALMONELLA-SHIGELLA AGAR

• Purpose – selection of Salmonella & some strains of Shigella spp. from stool
• Components: 
  
  • Inhibitors: 
    
    • Bile salts 
    • NaCl 
    • Brilliant green 
  • Lactose – carbohydrate source 
  • Neutral red – pH indicator 
  • For detection of H2S gas 
    
    • Sodium thiosulfate 
    • Ferric ammonium citrate
• NOTE
  
  • HEAVY INOCULUM OF STOOL should be plated on SS agar – because medium is very inhibitory

SELENITE BROTH

• Purpose – recovery of low numbers of Salmonella and some strains of Shigella spp. from stool
• Component: 
  
  • Sodium selenite – inhibitor; effective at neutral pH 
  • Maintains neutral pH 
    
    • Lactose 
    • Phosphate buffers
• NOTE: 1-2g of stool should be inoculated

SP-4 BROTH & AGAR

• Purpose – primary isolation media for Mycoplasma spp.
• Components: 
  
  • Yeast 
  • Preformed nucleic acid 
  • Fetal bovine serum – supplies cholesterol 
  • Inhibitors 
    
    • Penicillin 
    • Amphotericin B 
    • Polymyxin B

STREPTOCOCCUS-SELECTIVE AGAR

• Purpose – for isolation primarily for beta-hemolytic streptococci
• Component: 
  
  • Columbia agar – base 
  • Maltose – enhances prod. Of streptolysin 
  • Inhibitors 
    
    • Polymyxin B 
    • Neomycin
  • In order formulation, with: 
    • Oxolinic acid 
    • Colistin

TETRATHIONATE BROTH

• Purpose – recovery of Salmonella except serotypes 
  
  • Typhi 
  • Arizonae
• Components:
• Inhibitors 
  
  • Iodine-potassium iodide solution(added) 
  • Bile salt in conjunction with thiosulfate 
  • Brilliant green 
  • Crystal violet

THIOSULFATE CITRATE BILE SALT SUCROSE AGAR

• Purpose – isolation of Vibrio spp.
• Components
  • Inhibitors 
    
    • Sodium citrate 
    • Sodium thiosulfate 
    • Oxgall 
  • Bromthymol or thymol blue – pH indicator 
  • For H2S detection 
    
    • Sodium thiosulfate 
    • Ferric citrate 
  • Sucrose – carbohydrate source
• COLONY CHARACTERISTICS 
  
  • Sucrose fermenters – yellow 
    
    • Vibrio cholerae 
    • Vibrio alginolyticus 
  • Non-sucrose fermenters – blue-green colonies 
    
    • Vibrio parahaemolyticus 
    • Vibrio vulnificus 
  • Other organism – blue colonies 
    
    • Pseudomonas 
    • Plesiomonas 
    • Aeromonas
• NOTE
  
  • Heavy inoculum should be applied

TINSDALE AGAR

• Purpose – used for isolation of C. diphtheriae
• COMPONENTS: 
  
  • Inhibitors 
    
    • Potassium tellurite (high concentration) 
  • Cystine 
  • Thiosulfate
• COLONY CHARACTERISTICS 
  
  • Corynebacterium spp. – gray to black colonies 
  • C. diphtheriae – with brown halo 
  • C. ulcerans & C. pseudodiphthericum 
    
    • Dark halo 
  • Proteus – mucoid 
  • Staphylococci & Streptococci (rare) 
    
    • Dark colonies

TODD-HEWITT BROTH W/ GENTAMICIN & NALIDIXIC ACID

• Purpose – used to grow streptococci from vaginal & rectal swab for serotyping
• COMPONENT
  
  • Peptone 
  • Beef heart infusion 
  • Glucose 
  • Inhibitors 
    
    • Gentamicin 
    • Nalidixic acid

VAGINALIS AGAR

• Purpose – isolation of Gardnerella vaginalis
• Components: 
  
  • Columbia agar – base 
  • Inhibitors 
    
    • Colistin 
    • Nalidixic acid 
    • Nystatin

XYLOSE-LYSINE-DESOXYCHOLATE AGAR

• Purpose – used to isolate Salmonella and Shigella
• Components: 
  
  • Sodium desoxycholate – inhibitor 
  • Sucrose 
  • Lactose 
  • Xylose – high concentration 
  • Phenol red – pH indicator
  • For H2S detection 
    
    • Sodium thiosulfate – sulfur source 
    • Ferric ammonium citrate
• COLONY CHARACTERISTICS 
  
  • Yellow colonies – ferments excess carbohydrates 
    
    • E. coli 
  • Yellow colonies w/ black centers – ferments excess carbohydrates; H2S producers 
    
    • Citrobacter spp. 
    • Proteus spp. 
  • Colorless or red colonies 
    
    • Shigella spp. 
  • Red colonies 
    
    • Salmonella spp. 
    • Edwardsiella spp

MEDIA w/ BROMTHYMOL BLUE 

• Acetate agar 
• BCYE 
• CCFA 
• Hektoen Enteric Agar 
• Malonate Broth

MEDIA w/ BROMCRESOL PURPLE 

• Wadowsky-Yee BCYE 
• CCFA 
• Moeller
• LIA 

MEDIA w/ NEUTRAL RED 

• CIN 
• MacConkey 
• Sorbitol McConkey 

MEDIA w/ PHENOL RED 

• Burkholderia cepacia Agar 
• TSI 
• Kligler 
• MSA