Uploaded byVaisHali822687
PPTX, PDF886 views

Culture media part-2

1. The document discusses various culture media used for cultivating microorganisms from clinical specimens. It describes the composition and purpose of different types of media including basal media, enriched media, selective media, and transport media. 2. Specific media are described for cultivating gram positive cocci like Staphylococci and Streptococci. Mannitol salt agar, tellurite glycine agar, and DNase test agar are discussed for isolating and identifying Staphylococcus. Todd Hewitt broth and crystal violet blood agar are mentioned for Streptococci. 3. Modified Thayer Martin medium and modified New York City medium are highlighted as selective media used for isolating Neisseria

Embed presentation

Download to read offline
CULTURE MEDIA – PART 2 MODERATOR : Dr. S. K. SINGH PRESENTED BY: Dr. SAVITA CHOUDHARY DR. SUMEDHA THANVI
INTRODUCTION DEFINITION: • CULTURE MEDIA: A medium used for cultivation of microorganisms. • CULTIVATION: Cultivation is a process of growing microorganisms in culture by taking bacteria from infection site (i.e. the in vivo environment ) by some means of specimen collection and growing them in artificial environment of the lab (i.e. in vitro environment ).
TYPES OF CULTURE MEDIA • BASED ON FUNCTION: 1.Basal Media : Basic media with minimum basic ingredients that supports growth of non-fastidious bacteria. e.g: Peptone water, Nutrient broth, Nutrient agar. 2. Enriched media : Media prepared to meet the nutritional requirements of more exacting bacteria by addition of substances such as blood,serum or egg to a basal medium. e.g: Blood agar, Chocolate agar, Loeffler’s serum slope ,etc. 3. Enrichment Media: Liquid media added with some inhibitory agents which selectively allow certain organisms to grow and inhibit others. e.g: Tetrathionate broth ,Selenite F broth etc.
TYPES OF CULTURE MEDIA (…contd) • 4. Selective Media: This media contains one or more agents that are inhibitory to all organisms except those “ selected” by the specific growth condition or chemical. E.g: Lowenstein- Jensen medium,Thiosulfate Citrate Bile Salt (TCBS) agar, etc. • 5. Differential Media: These media differentiate between two groups of bacteria by using an indicator. E.g: Mac conkey agar, CLED agar, etc • 6. Transport Media : Media which are devised to maintain the viability of a pathogen and to avoid overgrowth of other contaminants during transit from the patient to laboratory. e.g: Stuart’s medium, Venkatraman- Ramakrishnan medium, Cary Blair medium, etc.
Gram positive cocci • Mannitol Salt Agar • Tellurite Glycine Agar • Salt cooked meat broth • DNase Test Agar • Milk Salt Agar Staphylococci • Todd Hewitt broth • Crystal violet blood agar • PNF medium • Nichols & Freeman Medium Streptococci
Mannitol Salt Agar • Mannitol Salt Agar is used as a selective media for the isolation of pathogenic Staphylococci aureus from clinical and non clinical (soil, faeces etc.) specimens. PRINCIPLE: i. Beef extract and proteose peptone supply essential growth factors and trace nutrients to the growing bacteria. ii. Sodium chloride serve as an inhibitory agent against bacteria other than Staphylococci. iii. Mannitol is the fermentable carbohydrate ,fermentation of which leads to acid production which is detected by phenol red indicator. Ingredients Quantity (gm/l) Proteose peptone 10 Beef extract 1 Sodium chloride 75 D-Mannitol 10 Phenol Red 0.025 Agar 15 Final pH 7.4± 0.2
Mannitol Salt Agar (…contd) INTERPRETATION: • S.aureus ferments mannitol and produce yellow coloured colonies surrounded by yellow zones. • Coagulase negative strains of Staphylococci are usually mannitol non fermenters and therefore produce pink to red colonies surrounded by red purple zone. • MSA selective for MRSA: - To MSA add oxacillin 6 mg/litre or methicillin 10 mg/litre. - To MSA add oxacillin 6 mg/litre and polymixin B 10 mg/litre. Organism Growth Colour of colonies S. aureus ATCC 6538 luxuriant Yellow /white colonies surrounded by yellow zone E. coli ATCC 25923 Inhibited -
Tellurite Glycine Agar Used for quantitative detection of Coagulase positive Staphylococci. • This medium supports better growth of coagulase positive cocci even if present in small numbers. PRINCIPLE  Casein enzymic hydrolysate and yeast extract provide nitrogenous compounds and vitamin B complex.  Lithium chloride and potassium tellurite inhibits coagulase negative Staphylococci.  Mannitol is a source of fermentable carbohydrate INTERPRETATION  Occasional Coagulase-negative strain that may produce small grey colonies.  Coagulase-positive Staphylococci reduce tellurite and form black colonies Ingredients Quantity (gm/l) Casein enzymic hydrolysate 10 Yeast extract 5 Mannitol 5 Dipotassium phosphate 5 Lithium chloride 5 Glycine 10 Agar 16 Final pH 7.2±0.2 S.aureus ATCC 25923 Good luxuriant Black S.epidermidis ATCC 12228 Poor- Fair Grey E.coli ATCC 25922 Inhibited
DNase Test Agar • This agar is recommended for the detection of deoxyribonuclease activity of bacteria and fungi, specially for the identification of pathogenic Staphylococci. PRINCIPLE AND INTERPRETATION: • Detection of deoxyribonuclease activity of bacteria and fungi. • With added toluidine blue,it is used in the identification and differentiation of non pigmented Serratia spp. from Enterobacter and Klebsiella. • Positive DNase activity is visualised as clear zones around colonies when plate is flooded with 1 N hydrochloric acid Organism Growth DNase activity S. aureus ATCC 25923 Luxuriant Positive Serratia marcescens ATCC 81005 Luxuriant Positive S.epidermidis ATCC 12228 Luxuriant Negative Ingredients Quantity (gm/l) Tryptone 15 Soya Peptone 5 Deoxyribonucleic acid (DNA) 2 Sodium Chloride 5 Agar 15 Final pH 7.3±0.2 Water 1 L
Salt Cooked Meat Broth Used as an enrichment medium for isolation of Staphylococcus from grossly contaminated specimens. PRINCIPLE AND INTERPRETATION: • Selective for Staphylococci due to presence of sodium chloride in high concentration. • Peptic digest of animal tissue and beef extract provide essential nutrients. • Sodium chloride maintains osmotic equilibrium. Organism Growth S.Aureus ATCC 25923 Good luxuriant E.coli ATCC 25922 Inhibited Ingredients Quantity(gm/l) Beef Extract 10 Peptic digest of animal tissue 10 Neutral ox- heart tissue 30 Sodium chloride 100 Final pH 7.6±0.2 P.vulgaris ATCC 13315 Inhibited
Milk Salt Agar • Used for selective isolation and cultivation of Staphylococcus. PRINCIPLE AND INTERPRETATION:  Beef extract, peptic digest of animal tissue and skim milk supply essential nutrients  Highly selective as majority of the contaminating organisms are inhibited by the high salt concentration (6.5%) Organism Growth Recovery S.aureus ATCC 25923 Good Luxuriant >=50% E.coli ATCC 25922 Inhibited 0% Ingredients Gms/L Peptic digest of animal tissue 5 Beef extract 3 Sodium chloride 65 Skim Milk 10% Agar 15
Todd Hewitt broth •This enriched broth is used for luxuriant growth of organisms such as group A haemolytic Streptococci PRINCIPLE & INTERPRETATION: •Todd Hewitt Broth medium is very nutritious due to the presence of peptic digest of animal tissue and beef heart infusion. •Dextrose stimulates haemolysin production •The medium is well buffered by sodium phosphate and sodium carbonate to neutralize the acid produced during dextrose fermentation. This restricts destruction of antigenic streptococcal haemolysin. Selective medium for group B Streptococci : Prepare a solution of 15µg/l nalidixic acid and 8µg/l gentamicin and add .5ml of it and .25ml sterile defibrinated sheep blood to 4.75 ml of todd Hewwit broth. Ingredients Quantity (gm/l) Beef heart, infusion from 500 Peptic digest of animal tissue 20 Dextrose 2 Sodium chloride 2 Disodium phosphate .4 Sodium carbonate 2.5 Final pH 7.8±2
Crystal violet blood agar • Selective for Streptococus pyogenes and other Streptococci •At a concentration of 1 in 500000, crystal violet inhibits the growth of Staphylococcus aureus and some other bacteria found in clinical specimen PNF Medium Selective for Beta hemolytic Streptococci  Inhibit the growth of Staphylococci and Coliforms  Melt the Nutrient Agar, cool to 50ºC, add the ingredients and pour plates Ingredients Per Litre Sterile Nutrient Agar 90 ml Sterile Blood 10 ml Crystal Violet 0.1 % 0.2 ml Ingredients Per Litre Sterile Nutrient Agar Sterile Blood 50 ml Polymyxin B Sulphate 17000 units
Agar of Nicholas and Freeman (Crystal Violet- Nalidixic Acid- Gentamycin Agar (CVNG) • This is a new selective medium for the detection of Streptococcus pneumonia • Highly selective for Strept. pneumoniae in sputum samples. • This medium is inhibitory to many respiratory bacteria other then the Pneumococcus,Viridans Streptococci and Enterococci. Ingredients Quantity Columbia agar Defibrinated horse blood 10% Crystal violet 2 mg/l Nalidixic acid 50mg/l Gentamycin 2mg/l
Gram Negative Cocci Neisseria: Neisseria • Modified Thayer Martin Medium • Modified New York City Medium
Modified Thayer Martin Medium  Selective isolation & enumeration of Neisseria gonorrhoeae PRINCIPLE & INTERPRETATION:  Vancomycin, Colistin, Nystatin and Trimethoprim make medium selective  Peptic digest of animal tissue provides nutrients while starch neutralizes the toxic fatty acid.  Vancomycin inhibits gram-positive, Colistin inhibits gram-negative bacteria and Nystatin inhibits fungi.  If chocolate agar and antibiotics added-suppress the growth of contaminants , saprophytic Neisseria species ; enhace the growth of pathogenic Neisseria.  Small grey convex colonies with irregular outlines seen. Growth is slower as compared to other selective media. Ingredients Gms/L Peptone 23 Starch 1 Sodium Chloride 5 Dextrose 2.5 Agar 20 Organism Growth Colour of colony N.gonorrhoeae ATCC 19424 Good-luxuriant Small, greyishwhite to colourless, mucoid E.coli ATCC 25922 Inhibited
Modified New York City Medium  Enriched selective medium for Neisseria sp.  Preferred medium as it gives better growth ; use of lincomycin as selective agent avoid problem of vancomycin sensitivity. PRINCIPLE & INTERPRETATION:  Lysed horse blood, dextrose and yeast dialysate are the enrichments; proteose peptone,horse plasma, hemoglobin provide nutrients. • Colistin inhibits gram-negative bacilli, amphotericin B suppress yeast growth and trimethoprim prevents Proteus sp. swarming • After incubation for 24 hours in moist aerobic environment,5-10% CO2- small( 1mm) ,grey,convex colonies; after 48 hours, larger colonies with crenated margins & opaque raised centre. GC Medium Base 36 g Yeast dialysate 25 ml Blood (100ml) lysed with 5 ml of 10% saponin 105 ml Glucose 10% sterlized at 115ºC for 10 min. 10 ml Colistin (6mg/L) 1ml Lincomycin (1mg/L) 1ml Trimethoprim (5mg/L) 1ml Amphoterecin B (1mg/L) 1ml Organisms Growth Recovery Neisseria gonorrheae ATCC 19424 Good-luxuriant >=50% Proteus mirabilis ATCC 13883 None- poor <=10%
Gram Positive Bacilli • Gram positive bacilli: Corynebacterium Bacillus Listeria • Loeffler’s Serum Slope • Hoyle’s Tellurite Lysed Blood Agar • Tinsdale Medium • Hiss’s Serum Sugar Media • Knisely’s PLET Medium • Phenol red-Egg yolk- Polymyxin agar • PEMBA Medium • PALCAM Agar
Hoyle’s Tellurite Lysed Blood Agar  Selective medium for Corynebacterium species PRINCIPLE & INTERPRETATION:  Biopeptone provides nitrogenous compounds  Haemoglobin stimulates good growth of Corynebacterium  Potassium tellurite acts as a selective inhibitor against most gram-positive and gram-negative bacteria except Corynebacterium species  C.diphtheriae reduces potassium tellurite to tellurium and thereby produce grey-black coloured colonies Agar Base Meat Extract 10 g Proteose Peptone 10 g Sodium Chloride 5 g Agar Powder 15 g Water 1 L Tellurite Solution Potassium Tellurite 0.7 g Water 20 ml Complete Medium Agar Base 200 ml Lysed Blood 10 ml Tellurite Solution 2 ml Organism Growth Colour of colony C.diphtheriae ATCC 11913 Good-luxuriant Grey-black E.coli ATCC 25922 Inhibited -
Tinsdale Medium  For selective isolation and differentiation of Corynebacterium diphtheria. PRINCIPLE & INTERPRETATION:  Peptic digest provides nitrogenous compounds. L-cystine and sodium thiosulphate form the H₂S indicator system.  Potassium tellurite inhibits all gram-negative bacteria and most of the upper respiratory tract normal flora.  C.diphtheriae forms greyish black colonies surrounded by a dark brown halo. Halo is formed due to H₂S production from cystine combining with the tellurite salt. Agar Base Proteose Peptone 20 g Sodium Chloride 5 g Agar Powder 19 g Water 1 L Complete Medium Agar Base 200 ml Ox Serum 20 ml Sodium Hydroxide 0.1 mol/L 12 ml L-Cystine, 0.4% in Hcl, 0.1 mol/L 12 ml Potassium Tellurite 1% in Sterile Water 6 ml Sodium Thiosulphate 2.5% solution 3.4 ml Organism Growth Colony characteristics C.diphtheriae gravis Good-luxuriant Brown-black with halo K.pneumoniae ATCC 13883 Inhibited
Knisely’s PLET Medium  For selective culture of small numbers of Bacillus anthracis from soil and other materials containing spore formers of other species.  On incubation at 37ºC for 24 hours, colonies develop from 30-100% of the B. anthracis spores that would grow on unselective heart infusion agar. PRINCIPLE & INTERPRETATION:  PLET medium inhibits the growth of most strains of B.cereus, B.subtilis, Enterobacteria and Pseudomonas  Some strains of B.cereus from soil form colonies, smaller than B.anthracis, minute after 24 h and moderately sized only after 48 h, roughly circular creamy- white colonies with a ground glass texture. Heart Infusion Agar 40 g Thallous acetate 0.04 g EDTA 0.3 g Polymyxin 30,000 units Lysozyme 0.04 g Deionized Water 1 L Organism Growth B.anthracis ATCC 14578 Luxuriant B.cereus ATCC 10876 Inhibited
Phenol Red Egg Yolk Polymyxin Agar • Relatively selective for B.cereus ; used to isolate it from food,faeces and vomitus. PRINCIPLE & INTERPRETATION:  Colonies recognized by their opacity in the egg-yolk, pink color with phenol red and failure to ferment mannitol.  Egg yolk emulsion helps in differentiation of lecithinase producing colonies which are surrounded by a zone of white precipitate  Polymyxin B Sulphate restricts growth of gram-negative bacteria such as E.coli and Pseudomonas aeruginosa  Differentiation of Bacillus cereus from other Bacillus species by its inability to ferment mannitol and poor sporulation Peptone 10 g Meat Extract 1 g Mannitol 10 g Sodium Chloride 10 g Phenol red 0.025 g Agar 15 g Egg-yolk emulsion 100 ml Polymyxin B sulphate 0.01 g Deionized water 1 L Organism Growth Recovery Colour Lecithinase activity B.cereus ATCC 10876 Luxuriant >=50% Red Positive,opaque zone around the colony E.coli ATCC 25922 None-poor <=10%
PEMBA Medium (Polymyxin Pyruvate Egg Yolk Mannitol Bromothymol Blue Agar) • Recommended for enumeration of B.cereus in foods. • Supports growth of even a small number of B.cereus. PRINCIPLE & INTERPRETATION:  Low peptone content promotes sporulation,sodium pyruvate reduces the colony size of the organisms.  Egg yolk emulsion demonstrates the strong lecithinase opacity reaction  Bromothymol blue acts as pH indicator to detect mannitol fermentation.  B.cereus forms crenated blue colonies surrounded by a zone of opacity in the medium Peptone 1 g D- Mannitol 10 g Magnesium sulphate.heptahydrate 0.1 g Disodium phosphate 2.5 g Potassium dihydrogen phosphate 0.25 g Sodium chloride 2 g Sodium pyruvate 10 g Polymyxin B sulphate 1 lac U Bromothymol blue 0.1 g Egg-yolk emulsion 50 ml Agar 18 g Deionized water 1 L Organism Growth Colour of colony Egg Yolk Reaction B.cereus ATCC 10876 Good-luxuriant Blue Positive, precipitation E.coli ATCC 25922 Inhibited
PALCAM Agar (Polymyxin-Acriflavin-Lithium chloride-Ceftazidime-Aesculin-Mannitol) • For selective isolation and identification of Listeria species. PRINCIPLE & INTERPRETATION:  Highly selective due to the presence of lithium chloride, ceftazidime, polymyxin B and acriflavin.  Differential diagnostic medium utilizing two indicator systems, esculin-ferric citrate and mannitol-phenol red.  L. monocytogenes hydrolyzes esculin forms esculetin and dextrose. Esculetin reacts with ammonium ferric citrate and forms a brown-black complex seen as a black halo around colonies.  On PALCAM Agar,after inoculation and incubation at 35 degrees for 24-48 hours in aerobic or microaerophillic condition, Listeria colonies appear grey-green with a black precipitate. Ingredients g/L Peptone 23 Starch 1 Sodium Chloride 5 Mannitol 10 Ammonium ferric citrate 0.5 Esculin 0.8 Dextrose 0.5 Lithium Chloride 15 Phenol red 0.08 Agar 13
Gram Negative Bacilli • . Coliforms Salmonella & Shigella Yersinia EMB Agar • Bismuth Sulphite Agar • DCA • XLD • Hektoen Agar • SS Agar Schiemann CIN Agar Vibrio • TCBS Agar • Bile Salt Agar • Monsur’s Tellurite Taurocholate Gelatin Agar
EMB Agar  Eosin Methylene Blue Agar is a selective and differential medium for Coliforms. PRINCIPLE & INTERPRETATION:  Eosin-Y and Methylene blue (ratio 6:1) inhibit gram-positive bacteria, serve as differential indicators in response to the fermentation of carbohydrates  Coliforms produce purplish black colonies due to taking up of methylene blue-eosin dye complex, when the pH drops.  Nonfermenters raise the pH by oxidative deamination of protein, which solubilizes the methylene blue-eosin complex resulting in colourless colonies. Organism Growth Color of Colony E.coli ATCC 25922 Luxuriant Purple with black centre and green metallic sheen P.mirabilis ATCC 25933 Luxuriant Colourless S.aureus ATCC 25923 Inhibited Peptic digest 10 g Dipotassium phosphate 2 g Lactose 5 g Sucrose 5 g Eosin - Y 0.4 g Methylene blue 0.065 g Agar 13.5 g Distilled water 1 L
Wilson & Blair’s Brilliant green Bismuth Sulphite Agar • Particularly used for the isolation of Salmonella typhi. • Highly selective for Salmonella; inhibitory to Coliforms, Proteus & Shigella. PRINCIPLE & INTERPRETATION: • Sulphur compounds provide a substrate for hydrogen sulphide production, while the metallic salts stain the colony and surrounding medium black or brown in the presence of hydrogen sulphide. • Cultures examined after 24 hours, then again after 48 hours. Crowded, green or pale brown colonies, 1 mm in diameter seen. Larger ,discrete colonies have a black centre and clear edge. Bismuth sulphite glucose phosphate mixture Bismuth ammonio citrate scales 30 g Sodium sulphite 100 g Disodium hydrogen phosphate 100 g Glucose 50 g Sterile water 1 L Iron citrate brilliant green mixture Ferric citrate, brown scales 2 g Brilliant green 0.25 g Sterile water 225 ml Complete medium Sterile nutrient agar 100 ml Bismuth sulphite glucose phosphate mixture 20 ml Iron citrate brilliant green mixture 4.5 ml Organism Growth Color of colony S.typhi ATCC 6539 Good-luxuriant Black with metallic sheen E.faecalis ATCC 29212 Inhibited
Leifson’s Deoxycholate Citrate Agar (DCA) • Most widely used plating medium for isolation of intestinal pathogens from faeces; pathogens that cause bacilliary dysentery, Salmonella strains that cause food poisoning and Salmonella paratyphi. PRINCIPLE & INTERPRETATION:  Coliforms and gram-positive bacteria are inhibited due to sodium deoxycholate, sodium citrate and ferric ammonium citrate  Lactose helps in differentiating enteric bacilli, as lactose fermenters produce red colonies while lactose non-fermenters produce colorless colonies  Coliforms, if present form pink colonies  Salmonella and Shigella species do not ferment lactose but Salmonella may produce H2S, forming colourless colonies with or without black centers. Neutral Red Lactose Agar Meat Extract 20 g Peptone 20 g Agar 90 g Neutral Red 2% in 50% ethanol 5 ml Lactose 40 g Water 4 L Solution A Sodium citrate 17 g Sodium thiosulphate 17 g Ferric ammonium citrate 4 g Sterile water 100 ml Solution B Sodium deoxycholate 10 g Sterile water 100 ml Complete medium Neutral red lactose agar 100 ml Solution A 5 ml Solution B 5 ml
Taylor’s Xylose Lysine Deoxycholate (XLD)  Selective for Salmonella and Shigella PRINCIPLE & INTERPRETATION:  Sodium deoxycholate acts as a selective agent; restrain the growth of E.coli  Colony turns red due to fermentation of Xylose, detected by indicator Phenol red  Lactose and Sucrose are fermentable sugars  L-lysine, essential amino acid source, differentiates Salmonella Sp.  Sodium thiosulphate and Ferric ammonium citrate helps visualizing the black centered colonies on production of H2S.  Salmonella produce red colonies with black centres whereas S.paratyphi,Shigella & Providenciae form red colonies without black centres. Yeast Extract 3 g Xylose 3.75 g Lactose 7.5 g Sucrose 7.5 g L- Lysine HCl 5 g Sodium chloride 5 g Sodium deoxycholate 2.5 g Sodium thiosulphate 6.8 g Ferric ammonium citrate 0.8 g Phenol red 0.08 g Agar 15 g Water 1 L
Hektoen Enteric Agar Medium  For differential and selective isolation of Salmonella and Shigella PRINCIPLE & INTERPRETATION:  Bile salts, bromothymol blue and acid fuchsin inhibit the growth of most gram positive organisms  Ferric ammonium citrate as iron source, cause production of hydrogen sulfide from sodium thiosulphate and by reacting with hydrogen sulfide, forms a black precipitate.  Enterobacters other than Salmonella and Shigella are capable of fermenting one or more of the carbohydrates produce yellow or salmon-orange coloured colonies.  Non-fermenters will produce blue-green colonies  Salmonella reduce sulfur to hydrogen sulfide, producing a black precipitate Peptone 12 g Yeast extract 3 g Lactose 12 g Sucrose 2 g Salicin 9 g Bile Salts mixture 9 g Sodium chloride 5 g Sodium thiosulfate 5 g Ferric ammonium citrate 1.5 Acid fuchsin 0.1 g Bromothymol blue 0.065g Agar 14 g Water 1 L Organism Colour of colony S. typhimurium ATCC 14028 Blue-green with or without black centres Shigella flexneri ATCC 12022 Greenish blue E. faecalis ATCC 29212 -
SS Agar  Moderately selective and differential for Salmonella spp. and some Shigella spp.  Modification of the Deoxycholate Citrate Agar PRINCIPLE & INTERPRETATION:  Bile Salts, Sodium Citrate and Brilliant Green inhibit gram- positive, Coliforms and inhibit swarming Proteus spp.  Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide by the production of colonies with black centers  Salmonella will not ferment lactose, but produce hydrogen sulfide gas; colonies appear colorless with black centres.  Shigella do not ferment lactose or produce hydrogen sulfide gas, hence colonies appear colourless. Beef Extract 5 g Enzymatic Digest of Casein 2.5 g Enzymatic Digest of Animal Tissue 2.5 g Lactose 10 g Bile Salts 8.5 g Sodium Citrate 8.5 g Sodium Thiosulfate 8.5 g Ferric Citrate 1.0 g Brilliant Green 0.00033 g Neutral Red 0.025 g Agar 13.5 Distilled Water 1 L S. typhi ATCC 6539 Colorless colonies with black center S. flexneri ATCC 12022 Colorless colonies E. faecalis ATCC 19433 Inhibited
Special Medias for Salmonella & Shigella • . BISMUTH SULPHITE AGAR HEKTOEN ENTERIC AGAR XLD DCA SS AGAR
Schiemann CIN Agar Cefsulodin Irgasan Novobiocin  Selective isolation of Yersinia enterocolitica  Differentiates between mannitol fermenting and non-fermenting bacteria PRINCIPLE & INTERPRETATION:  Mannitol fermenting colonies take red colour, while non-fermenting form colourless and translucent colonies  Sodium deoxycholate and crystal violet, selectively inhibit gram- positive and gram-negative bacteria  Antibiotic supplement makes it highly selective for Yersinia  Dark red colonies resembling bulls eye, surrounded by a transparent border  Serotyping is based on colony size, smoothness and ratio of the border to centre diameter Yersinia Selective Agar Base Peptone 20 g/L Yeast Extract 2.0 g/L Mannitol 20 g/L Sodium pyruvate 2.0 g/L Sodium chloride 1.0 g/L Magnesium sulphate 0.01 g/L Sodium deoxycholate 0.5 g/L Neutral red 0.03 g/L Crystal violet 0.001 g/L Agar 12.5 g/L Complete medium Agar Base 500ml Cefsulodin 7.5 mg Irgasan 2.0 mg Novobiocin 1.25 mg Organism Growth Colour of colony Y.enterocolitica ATCC 27729 Good- luxuriant Transluscent with dark pink/red centre & bile precipitate E.faecalis ATCC 29212 Inhibited
TCBS Agar (Thiosulphate Citrate Bile Sucrose)  Highly selective and differential for Vibrio species PRINCIPLE & INTERPRETATION:  Sucrose fermentation produces acid, which converts the colour of bromothymol blue or thymol blue  High concentrations of sodium thiosulfate and sodium citrate inhibit the growth of Enterobacteriaceae  Inhibition of Gram-positive bacteria is achieved by the incorporation of ox bile  Sodium thiosulfate as a sulfur source, with ferric citrate, allows easy detection of hydrogen sulfide production  Alkaline pH of the medium enhances the recovery of V.cholerae and inhibits the growth of others Yeast extract 5 g Peptone 10 g Sodium thiosulfate 10 g Sodium citrate 10 g Ox bile 8 g Sucrose 20 g Sodium chloride 10 g Ferric citrate 1 g Bromothymol blue 0.04 g Thymol blue 0.04 g Agar 15 g Water 1 L Organism Growth Colour of colony V.cholerae ATCC 15748 Good-luxuriant Yellow E.faecalis ATCC 29212 Inhibited
Bile Salt Agar (BSA)  For isolation and identification of bile tolerant bacteria responsible for food poisoning. PRINCIPLE & INTERPRETATION:  Vibrio species, like many other gram-negative bacteria, grow in the presence of relatively high levels of bile salts  Sodium chloride buffers the medium well. Sodium taurocholate inhibits most of the gram-negative organisms  Colonies of Vibrios have a distinctive appearance which may be seen by comparing a known strain of Vibrio cholerae with Escherichia coli. Peptone 10 g Meat Extract 5 g Sodium Chloride 5 g Sodium taurocholate 5 g Agar 15 g Water 1 L Organism Growth Vibrio cholerae ATCC15748 Luxuriant Staphylococcus aureus ATCC 25923 Inhibited
Monsur's tellurite taurocholate gelatin agar  Selective isolation and differentiation of Vibrio cholerae and other species of Vibrios. PRINCIPLE & INTERPRETATION:  Sodium chloride maintains the osmotic equilibrium ; sodium carbonate maintains the elevated pH  Gelatin acts as an additional carbon and energy source  The high pH and potassium tellurite are inhibitory to most Enterobacteriaceae and gram-positive bacteria, though Proteus may form grey centered colonies without a halo  Colonies are often surrounded by a gelatin liquefaction halo. Organism Growth Color Vibrio cholerae ATCC 15748 Good-luxuriant Grey Proteus mirabilis ATCC 25933 None-poor Black Taurocholate Gelatin Agar Peptone 10 g Sodium chloride 10 g Sodium taurocholate 5 g Sodium carbonate 1 g Gelatin 30 g Agar 15 g Distilled water 1 L Complete Medium Taurocholate Gelatin Agar 100 ml Diluted Potassium tellurite (0.05%) 1 ml
Pseudomonas and Burkholderia P. aeruginosa B. pseudomallei B.cepacia • Cetrimide Agar • Pseudomonas Isolation Agar • Pseudomonas Enrichment Broth • Ashdown’s Medium • Burkholderia pseudomallei Selective Agar (BPSA) • Burkholderia cepacia Selective Agar (BCSA) • Pseudomonas cepacia (PC) agar • Oxidative–fermentative base–polymyxin B– bacitracin–lactose (OFPBL) agar
Cetrimide Agar • For isolation of Pseudomonas aeruginosa. • P. aeruginosa produces water soluble pigments, including yellow-green fluorescent pigment pyoverdin and blue pigment pyocyanin, together creating bright green colour. PRINCIPLE & INTERPRETATION: • Cetyltrimethylammonium bromide (Cetrimide) acts as cationic detergent and causes denaturation of bacterial membrane proteins . • Gelatin peptone provides nutrition. • Sodium chloride maintains osmotic equilibrium. • Magnesium chloride and potassium sulphate act as pigment-enhancers. • Colonies exhibit a yellow-green to blue colour and show fluorescence under short wavelength (254 nm) ultraviolet light, with a characteristic grape like smell of aminoacetophenone. Pseudomonas aeruginosa ATCC 9027 luxuriant Escherichia coli ATCC 8739 inhibited Ingredients Gelatin peptone Magnesium chloride Potassium sulphate Cetrimide (0.03%) Agar (1.5%)
Ashdown's medium • For isolation of Burkholderia pseudomallei. PRINCIPLE & INTERPRETATION: • Crystal violet and Gentamicin act as selective agents. • Neutral red – indicator. • Glycerol provides nutrition. • Colonies appear flat, wrinkled, slightly dry, having pinkish purple colour with a metallic sheen. B.pseudomallei ATCC 23343 Growth P. aeruginosa ATCC 27853 No growth INGREDIENTS- Tryptone soya broth Gentamicin Crystal violet (0.1%) Neutral Red (1%) Glycerol Agar (1.5%)
Burkholderia cepacia Selective Agar (BCSA) • For isolation of Burkholderia cepacia. • Better selectivity ,quicker growth and lower false positivity rates than PC Agar and OFPBL Agar. PRINCIPLE & INTERPRETATION: • Pancreatic digest of casein ,yeast extract, lactose and sucrose provide nutrition. • Phenol Red -pH indicator, changes colour of medium from orange to yellow due to acid production by fermentation of lactose and sucrose. • Crystal violet, vancomycin, polymyxin and gentamicin- Selective Agents. • Colonies appear translucent and rough with yellow halos (d/t carbohydrate fermentation) • Burkholderia cepacia ATCC 25416 • Growth; color change in media from red-orange to yellow • Pseudomonas aeruginosa ATCC 27853 • Complete inhibition Pancreatic Digest of Casein Lactose Sucrose Sodium Chloride Yeast Extract Phenol Red Gentamicin Vancomycin Crystal Violet Polymyxin B Agar Ingredients:
Haemophilus species • Fildes Blood-Digest Agar • Levinthal’s Medium • Haemophilus Isolation Agar • Haemophilus Differential Selective medium H.influanzae, H.parainfluanzae,H. parahaemolyticus • Mueller-Hinton–based chocolate agar (supplemented with 1% IsoVitaleX and 3 mg/mL vancomycin) • Heart infusion–based agar (supplemented with 10% fetal bovine serum and 3 mg/mL vancomycin) H.ducreyi
Fildes Blood-Digest Agar Used for isolation of Haemophilus species. PRINCIPLE & INTERPRETATION: •Pepsin causes sterile digestion of Defibrinated Sheep Blood which causes release of factor x (haemin) and factor V(NAD) •High concentration of haemin is inhibitory to many throat commensals •Colonies of Non capsulated strains 0.5-1 mm circular, low convex, smooth, pale grey and transparent with slightly blue iridescence and a fishy, seminal odour. •Capsulated strains = Larger (1-3mm), high convex, mucoid with strong irridescence of red, orange, green and blue shades which alter with angle of observation. INGREDIENTS Qnt. NaCl 150ml HCl 6ml Defibrinated Sheep Blood 50ml Pepsin 1g NaOH 12ml Chloroform .5ml Agar
Levinthal’s Medium For cultivation of Haemophilus species PRINCIPLE & INTERPRETATION: •Blood provides factor-X (Heat stable) and factor-V (Heat labile). •Peptic digest of animal tissue and beef extract act as source of nitrogen. •Sodium chloride maintains osmotic balance. Capsulated strains show translucent colonies with distinctive iridescence while non-capsulated strains are transparent and bluish . Composition Quantity Peptic digest of animal tissue 10 Beef extract 10 sterile rabbit or human blood 5 sterile rabbit or human blood Agar 20 H.influenzae ATCC 35056 Luxuriant H. influenzae ATCC 35056 Luxuriant H. influenzae ATCC 35056 Luxuriant
Haemophilus Isolation Agar For isolation of Haemophilus species. PRINCIPLE & INTERPRETATION: • Horse blood provides factor-X, factor-V and other Growth Factors. •Intact erythrocytes enable differentiation of hemolytic and non- hemolytic Haemophilus spp. •Beef Heart Infusion and Casein Peptone provide nutrition. •Bacitracin (300 μg/mL) inhibits the normal respiratory tract microbes (i.e., Staphylococci, Micrococci, Neisseriae , Streptococci). Haemophilus influenzae ATCC 10211 Growth Haemophilus parahaemolyticus ATCC 10014 Growth, Beta hemolysis Staphylococcus aureus ATCC 25923 Inhibition (partial to complete) Composition- Beef Heart Infusion Yeast extract Casein Peptone Sodium Chloride NAD Bacitracin Defibrinated Horse Blood (5%) Agar
Haemophilus Differential Selective medium •This medium contains bacitracin as a selective agent •Sucrose and phenol red are used to distinguish H.parainfluenzae from H.influenzae •H.parainfuenzae ferments sucrose and forms yellow colonies whereas H.influenzae forms smaller, colourless non-fermenting colonies. Levinthal’s medium H. Influenzae colonies on Haemohillus isolation agar
Bordetella • Bordet Gengou Medium • Regan-Lowe Agar (Charcoal Blood Agar) • Stainer Scholte synthetic medium • Jones-Kendrick Medium(Blood-free solid medium for transport and cultivation) Bordetella
Bordet Gengou Medium Recommended for the detection and isolation of Bordetella pertussis and Bordetella parapertussis . PRINCIPLE & INTERPRETATION: •Potato infusion and peptic digest of animal tissue serve as carbon and nitrogen source while glycerol and blood enrichment provides additional nutrients. •Sodium chloride maintains osmotic equilibrium • Colonies appear small, smooth, dome shaped, greyish-white with a shiny surface resembling ‘mercury drops’ or ‘bisected pearls’ surrounded by a hazy zone of haemolysis. Bordetella pertussis ATCC 8467 Luxuriant growth with beta-hemolysis Bordetella parapertussis ATCC 15311 Luxuriant growth with gamma-hemolysis Staphylococcus aureus ATCC 25923 Inhibited Ingredients Qnt. (gm/l) Potatoes, infusion from 125 Peptic digest of animal tissue 10 Sodium chloride 5 Agar 20 Final pH 6.7±0.2
Regan-Lowe Agar(Charcoal Blood Agar) This media has largely replaced traditional Bordet Gongou media PRINCIPLE & INTERPRETATION: • Peptic digest of animal tissue, beef extract and yeast extract provide essential nutrients to the organisms •Sodium chloride maintains osmotic balance • Starch soluble and charcoal neutralizes substances toxic to Bordetella species such as fatty acid Bordetella pertussis ATCC 8467 Growth Staphylococcus aureus ATCC 25923 Inhibition Ingredients Qnt. (gm/l) Peptic digest of animal tissue 10 Beef extract 10 Starch soluble 10 Sodium chloride 5 charcol 4 Yeast extract 3.5 Agar 12 Final pH 7.5±0.2 Both media contain cephalaxin as an additive for suppression of contaminating microorganism
Regan Lowe agar and typical “mercury drop colonies” of Bordetella pertussis Bordet Gengou agar and colonies of Bordetella pertussis surrounded by beta hemolysis
Campylobacter and Helicobacter • Skirrow Campylobcter medium • Preston Campylobacter selective medium • Charcoal-based blood-free selective medium • Blaser’s medium(Campy-BAP) Campylobacter • Modified Thayer Martin agar • Skirrow medium • BHI Agar with Vancomycin, Nalidixic acid and AmphotericinB Helicobacter
Skirrow Campylobacter selective agar •For isolation of campylobacter species PRINCIPLE & INTERPRETATION: •Trimethoprim , Vancomycin and Polymyxin - Selective agents. •Blood serves as an additional source of nutrients including X-factor •Colonies are effuse, droplet like with a tendency to form a spreading film. Also suitable for isolation of H.pylori colonies of which are small(<2mm), grey and translucent. Campylobacter jejuni ATCC 33291 Growth E.Coli ATCC 25922 Inhibition Ingredients Proteose peptone Liver digest Yeast extract Sodium chloride Agar Lysed horse blood Vancomycin Polymyxin-B Trimethoprim Agar
Charcoal-based blood-free selective medium More selective than Skirrow’s medium and has a higher isolation rate of C.jejuni from mixed cultures. PRINCIPLE & INTERPRETATION: •Charcoal, hematin, ferrous sulfate and sodium pyruvate serve as substitutes for blood. They enhance the growth and aerotolerance of campylobacters by quenching photochemically generated toxic oxygen derivatives. •Addition of cefoperazone increases the selectivity of the medium. C.jejuni produces grey, moist, flat-spreading colonies. Some strains may have green hue or a dry appearance with or without metallic sheen. C. jejuni ATCC 33291 Growth E. coli ATCC 25922 Partial to complete inhibition Beef extract 10 Peptone 10 Casein enzyme hydrolysate 3 Sodium chloride 5 Sodium deoxycholate 1 Ferrous sulphate .25 Sodium pyruvate .25 charcol 4 Agar 12
Translucent droplet colonies of Campylobacter on Skirrow medium Colonies of H.pylori on Skirrow medium C.jejuni on Charcoal based selective medium
Legionella and Francisella • Buffered Charcoal Yeast Extract Agar (BCYE) • Selective BCYE Agar • BMPA agar • Modified Wadowsky–Yee Medium (MWY medium) (Contains glycine, vancomycin, polymyxin B and anisomycin.) Legionella • Glucose cystine agar (BCG) • Cystine-heart agar (CHAB) • BCYE agar • Modified Thayer-Martin agar Francisella
Buffered Charcoal Yeast Extract Agar (BCYE) Selective isolation and cultivation of Legionella species PRINCIPLE & INTERPRETATION: •Charcoal acts as a detoxicant. •Yeast extract acts as a rich source of vitamins, nitrogen as well as carbon. •L-cystine hydrochloride; ferric pyrophosphate and a-ketoglutarate stimulate growth of Legionella species •ACES buffer maintains optimal pH. Legionella colonies are gray-white to blue-green, glistening, convex, and circular and may exhibit a cut-glass type of internal granular speckling. Legionella pneumophila ATCC 33153 luxuriant Enterococcus faecalis ATCC 29212 inhibited Ingredients Qty. (gm/l) Yeast extract 10 Activated charcol 2 Alpha ketoglutarate, mono potassium salt 1 ACES buffer 10 Agar 12 Final pH 6.8±0.2
Selective BCYE Agar- BCYE+ Glycine+ Polymyxin-B (79,200 IU/L)+ Cycloheximide(80mg/L)+ Vancomycin(5mg/L). BMPA Medium- BCYE base supplemented with cefamandole, polymyxin-B and anisomycin BYCE Agar also supports growth of Francisella tularensis and Bordetella pertussis. Colonies of Legionella on BCYE Agar
Media for Isolation of Francisella •The organism is strictly aerobic and is enhanced by enriched medium containing sulfhydryl compound (cystine, cystein, thiosulphate, IsoVitalex) •Two commercial media available: Glucose cystine agar (BCG) and Cystine-heart agar (CHAB), both require the addition of 5% sheep or rabbit blood. •Colonies are minute droplet like (2-4mm), butyrous and smooth with an opalescent sheen. glucose 2.5% Cystine HCL 0.1% Blood(human /Sheep/rabbit) 5% Nutrient agar Francisella tularensis ATCC 29684 luxuriant
Spirochaetes • Modified Korthof Medium • Fletcher’s Medium • Ellinghausen-McCullough- Johnson-Harris (EMJH) Medium Leptospira • Barbour-Stoenner-Kelly Medium (BSK II) Borrelia
Modified Korthof Medium Liquid media used for isolation, cultivation and maintenance of Leptospira species. PRINCIPLE & INTERPRETATION: •Peptic digest of animal tissue provide amino acids and other nitrogenous substances •The salts supply essential nutrients •Phosphates form buffering system while sodium chloride maintains osmotic equilibrium and provides essential ions. Growth occurs as a ringed area (disc) 1-3 cm below the surface of the medium. Growth is examined using a direct wet preparation under dark field illumination. Peptic digest of animal tissue 0.8 Sodium chloride 1.4 Sodium bicarbonate 0.02 Pottassium chloride 0.04 Calcium chloride 0.04 Monopotassium hydrogen phosphate 0.240 Disodium hydrogen phosphate 0.880 Final pH 7.2±.2
Fletcher’s Medium Semi-solid Medium for isolation of Leptospira species. Advantages over Liquid medium- 1. Evaporates less rapidly 2. Helps to maintain the virulence of freshly isolated strains 3. Used for preparing cultures for iv inoculation into rabbits for production of hyperimmune serum. • Low concentration of agar helps in detecting motility • Leptospira multiply within the upper part of the tube forming zones of turbidity at varying depths, known as Dinger’s Rings. ingredients Qty. (gm/l) Peptic digest of animal tissue 0.3 Beef extract 0.2 Sodium chloride 0.5 Agar 1.5 Final pH 7.9±.2 DINGER’S RING
EMJH Medium (Ellinghausen-McCullough-Johnson-Harris) Semi-solid medium where rabbit whole serum is replaced by bovine serum albumin (fraction V) and polysorbate (Tween 80). Thiamine, Albumin and polysorbate 80 are growth factors Growth occurs as a band 0.5 to 1.0 cm below the surface of semisolid media, known as Dinger’s ring.
Barbour-Stoenner-Kelly Medium (BSK II) •This medium is complex mixture of different amino acids, vitamins and growth factors which are required for the growth of Borrelia and Spirochaete PRINCIPLE & INTERPRETATION: • Peptone, special serves as nitrogen source while glucose as energy source. •Cholesterol acts as source of lipids. • HEPES provides buffering capacity . •Salts of Magnesium, sodium, calcium and potassium maintain the ionic balance. •Cultures are examined by dark-field microscopy or by fluorescence microscopy . B. burgdorferi ATCC 35210 Fair to good S. aureus ATCC 25923 Inhibited Ingredients- CMRL 1066 medium Neopeptone Bovine serum albumin, fraction V yeastolate HEPES glucose Sodium pyruvate Sodium Citrate N-acetylglucosamine Sodium bicarbonate 7% gelatin Unheated rabbit serum
Mycoplasmas and Ureaplasmas • Methylene Blue-Glucose Biphasic Medium • Mycoplasma Glucose Agar Medium • SP-4 Medium • Hayflick’s Medium Mycoplasma pneumoniae • A-8 Agar • 10B Broth • SP-4 Broth or Agar • Bromothymol Blue Broth • Shepard’s 10B broth Mycoplasma hominis • SP-4 Broth or Agar • A-8 Agar • 10B Broth • Bromthymol Blue Broth • Shepard’s 10B broth Ureaplasma
Methylene Blue-Glucose Biphasic Medium •Biphasic medium for isolation of M.pneumoniae •Solid phase is dispensed in sterile bottles ,allowed to set and then overlayed with liquid phase. PRINCIPLE & INTERPRETATION: •Methylene blue acts as selective agent •During growth , the medium becomes acidic and phenol red turns colour from salmon to yellow. •Reduction of methylene blue turns medium from blue to colourless. •Hence, colour of broth phase changes from purple to green, while the agar phase turns from purple to yellow. Mycoplasma pneumoniae ATCC 15531 Growth; colour change Escherichia coli ATCC 25922 Inhibited Ingredients PPLO (agar base/broth) w/o crystal violet Yeast extract Sodiumdeoxyribonucleate (calf thymus) solution (0.2%) Thallous acetate solution Penicillin solution Horse serum (unheated) Glucose solution (10%) Phenol Red solution (0.2%) Methylene blue solution(0.1%)
SP 4 Medium •For isolation of M. pneumoniae, M. hominis, and Ureaplasma spp. •Most commonly used media for isolation of genital mycoplasmas PRINCIPLE & INTERPRETATION: •May be used as liquid/solid form or as Biphasic medium. •CMRL 1066 (a cell culture medium) containing 21 amino acids, 13 vitamins, nucleic acid precursors, enzyme co-factors and cholesterol is added. •Arginine, urea, and antibiotics are selective agents. • Mycoplasma appear as tiny "fried-egg" colonies. Colonies range from 20-300 µm in diameter. Glucose fermenting mycoplasma SP4 with Glucose Red color to yellow Ureaplasma SP4 with Urea Yellow-orange to red Arginine using mycoplasma SP4 with Arginine Orange to red Ingredients- Mycoplasma broth base Tryptone Peptone Glucose Fetal Bovine Serum CMRL 1066 medium 10x concentrate with glutamine Fresh yeast extract (25%) Yeastolate (2%) Penicillin (100,000 units/ml) Phenol Red solution (0.1%)
Growth of M.pneumoniae on Methylene Blue-Glucose Biphasic Medium Fried egg colonies of Mycoplasma SP-4 Medium
REFERENCES • Mackie & McCartney Practical Medical Microbiology (14th edition) • Bailey and Scott’s Diagnostic Microbiology (15th edition) • HIMEDIA • Nichols T, Freeman R. A new selective medium Streptococcus pneumoniae. J Clin Pathol. 1980 Aug;33(8):770-3. doi: 10.1136/jcp.33.8.770. PMID: 7430390; PMCID: PMC1146214.
THANK YOU!! .

More Related Content

PPTX
Special stains useful in Microbiology laboratory
by9925752690
 
PPTX
Camp test
byDr Abhijith S.P.
 
PPT
salmonella spp.ppt
byobedcudjoe1
 
PPT
Trypanosoma
byAppy Akshay Agarwal
 
PDF
BLOOD AND TISSUES FLAGELLATES/ HAEMOFLAGELLATES OF HUMAN BEINGS
bymed zar
 
PPTX
stains
byMLT LECTURES BY TANVEER TARA
 
PPTX
Taenia solium and taenia saginata
bySaad Ijaz
 
PPTX
Culture of fungus
byviveksanjumt
 
Special stains useful in Microbiology laboratory
by9925752690
 
Camp test
byDr Abhijith S.P.
 
salmonella spp.ppt
byobedcudjoe1
 
Trypanosoma
byAppy Akshay Agarwal
 
BLOOD AND TISSUES FLAGELLATES/ HAEMOFLAGELLATES OF HUMAN BEINGS
bymed zar
 
stains
byMLT LECTURES BY TANVEER TARA
 
Taenia solium and taenia saginata
bySaad Ijaz
 
Culture of fungus
byviveksanjumt
 

What's hot

PDF
PROTEUS
byManeesha M Joseph
 
PDF
L13 medical microbiology laboratory (staphylococcus spp.)
byHussein Al-tameemi
 
PPTX
Brucella
bydrakmane
 
PPT
Genus staphylococcus
byRavi Kant Agrawal
 
PPT
Bacillus anthracis
byKamran Afzal, PhD.
 
PDF
Fastidious Bacteria.pdf
bynedalalazzwy
 
PPTX
Catalase test
byRebika Rajbanshi
 
PPTX
Staphylococcus aureus
byDr. Samira Fattah
 
PPTX
Z n staining
bymicroarunkumar
 
PPT
Spirochaetes
byAlia Najiha
 
PPTX
Culture media
byFasahat Butt
 
PPT
Genus Streptococcus
byRavi Kant Agrawal
 
PPTX
E coli lab types and laboratory diagnosis
byDevlop Shrestha
 
PDF
Haemagglutination test poultry
byanw001
 
PPTX
Leishmania donovani
byBishal Chauhan
 
PPTX
Classification of medical parasites
byshahiraahmed
 
PPTX
Dermatophytes
byrubaiya kabir
 
PPTX
Blood smear staining
byankur16491
 
PPTX
Staphylococcus
bySHALINI BISHT
 
PPTX
Tsi test
byMehraj Alam
 
PROTEUS
byManeesha M Joseph
 
L13 medical microbiology laboratory (staphylococcus spp.)
byHussein Al-tameemi
 
Brucella
bydrakmane
 
Genus staphylococcus
byRavi Kant Agrawal
 
Bacillus anthracis
byKamran Afzal, PhD.
 
Fastidious Bacteria.pdf
bynedalalazzwy
 
Catalase test
byRebika Rajbanshi
 
Staphylococcus aureus
byDr. Samira Fattah
 
Z n staining
bymicroarunkumar
 
Spirochaetes
byAlia Najiha
 
Culture media
byFasahat Butt
 
Genus Streptococcus
byRavi Kant Agrawal
 
E coli lab types and laboratory diagnosis
byDevlop Shrestha
 
Haemagglutination test poultry
byanw001
 
Leishmania donovani
byBishal Chauhan
 
Classification of medical parasites
byshahiraahmed
 
Dermatophytes
byrubaiya kabir
 
Blood smear staining
byankur16491
 
Staphylococcus
bySHALINI BISHT
 
Tsi test
byMehraj Alam
 

Similar to Culture media part-2

PDF
Microbiology - Types of Culture Media.pdf
bygirlwithbrowneyes
 
PPTX
culture media detailed information based on consistency and Growth requiremen...
bySachin Kumar Sharma
 
PPT
1.culture media and culture methods
byssuser25c093
 
PPTX
Rangkuman Beberapa Media Kultur untuk Bakteri.pptx
byAILEENALESS
 
PDF
Culture media
bypiyushparashar13
 
PPTX
Final presentation CULTURE MEDIA.pptx
byVaisHali822687
 
PPTX
Culture media
byAjay Kumar Chaurasiya
 
PPTX
CULTURE MEDIA.pptx
byADILRAZA15
 
PPTX
CULTURE MEDIA-1.pptx media ingredients and preparation
byashishpatelod98
 
PDF
Microbiology lab session 3 Types of Media Used in Microbiology Lab.pdf
byMohammadBakari
 
PDF
Bacterial culture media بكتريا عملي
byفي رحاب الله
 
PPT
7_CULTURE_MEDIA.pptcvvc f
byGeoffreyOkelo1
 
PPTX
culture media
byromagoyal37
 
PPTX
BACTERIAL CULTURE MEDIA CLASS based on various factors
byMeeraKrishnan32
 
PPTX
Types of culture media
byabdulmonem Elmakki
 
PPT
Culture media
byDana Sinziana Brehar-Cioflec
 
PDF
Invaders 09 10-2012
byMohamed Taha
 
PPTX
Culture Media.pptx culture media use in
bySantoshBhandari52
 
PDF
culturemedia-170404072433.pdf
bydawitg2
 
PPT
Micro Practical Aid that guide eveberyr.ppt
byEgbinadeVictor
 
Microbiology - Types of Culture Media.pdf
bygirlwithbrowneyes
 
culture media detailed information based on consistency and Growth requiremen...
bySachin Kumar Sharma
 
1.culture media and culture methods
byssuser25c093
 
Rangkuman Beberapa Media Kultur untuk Bakteri.pptx
byAILEENALESS
 
Culture media
bypiyushparashar13
 
Final presentation CULTURE MEDIA.pptx
byVaisHali822687
 
Culture media
byAjay Kumar Chaurasiya
 
CULTURE MEDIA.pptx
byADILRAZA15
 
CULTURE MEDIA-1.pptx media ingredients and preparation
byashishpatelod98
 
Microbiology lab session 3 Types of Media Used in Microbiology Lab.pdf
byMohammadBakari
 
Bacterial culture media بكتريا عملي
byفي رحاب الله
 
7_CULTURE_MEDIA.pptcvvc f
byGeoffreyOkelo1
 
culture media
byromagoyal37
 
BACTERIAL CULTURE MEDIA CLASS based on various factors
byMeeraKrishnan32
 
Types of culture media
byabdulmonem Elmakki
 
Culture media
byDana Sinziana Brehar-Cioflec
 
Invaders 09 10-2012
byMohamed Taha
 
Culture Media.pptx culture media use in
bySantoshBhandari52
 
culturemedia-170404072433.pdf
bydawitg2
 
Micro Practical Aid that guide eveberyr.ppt
byEgbinadeVictor
 

More from VaisHali822687

PPTX
entamoeba, giardia. pptx
byVaisHali822687
 
PPTX
ascaris,hookworm .pptx
byVaisHali822687
 
PPTX
Overview of Fungal Infections[1].ppt pptx
byVaisHali822687
 
PPTX
ACTINOMYCETES DMLT powerpoint presentation
byVaisHali822687
 
PPTX
Hypersensitivity.pptxHypersensitivity.pptx
byVaisHali822687
 
PPTX
gastrointestinal infective syndromes.pptx
byVaisHali822687
 
PPTX
cryptococcus.pptxcryptococcus.pptxcryptococcus.pptx
byVaisHali822687
 
PPTX
Gas gangrene [Autosaved].pptxGas gangrene [Autosaved].pptx
byVaisHali822687
 
PPTX
MONKEYPOX PRESENTATION power point presentation
byVaisHali822687
 
PPTX
Sexually transmitted infection power point
byVaisHali822687
 
PPT
biomedical waste management power point ppt
byVaisHali822687
 
PPTX
mycotoxins, mycology, mycelium, aflatoxin
byVaisHali822687
 
PPTX
Operation theatre sterilization powerpoint
byVaisHali822687
 
PPTX
Parasitic Infections of CNS. Free living amoeba and neurocysticercosispptx
byVaisHali822687
 
PPTX
Sample collection and transport ppt 1234
byVaisHali822687
 
PPTX
Capanna technique for chronic osteomyelitis
byVaisHali822687
 
PPTX
chronicosteomyelitis Powerpt power point
byVaisHali822687
 
PPTX
Hemiarthoplasty debate. hemiarthroplasty
byVaisHali822687
 
PPTX
viral hemorrhagic fever -dengue, chikungunya.
byVaisHali822687
 
PPTX
Complement system and its synthesis + activation
byVaisHali822687
 
entamoeba, giardia. pptx
byVaisHali822687
 
ascaris,hookworm .pptx
byVaisHali822687
 
Overview of Fungal Infections[1].ppt pptx
byVaisHali822687
 
ACTINOMYCETES DMLT powerpoint presentation
byVaisHali822687
 
Hypersensitivity.pptxHypersensitivity.pptx
byVaisHali822687
 
gastrointestinal infective syndromes.pptx
byVaisHali822687
 
cryptococcus.pptxcryptococcus.pptxcryptococcus.pptx
byVaisHali822687
 
Gas gangrene [Autosaved].pptxGas gangrene [Autosaved].pptx
byVaisHali822687
 
MONKEYPOX PRESENTATION power point presentation
byVaisHali822687
 
Sexually transmitted infection power point
byVaisHali822687
 
biomedical waste management power point ppt
byVaisHali822687
 
mycotoxins, mycology, mycelium, aflatoxin
byVaisHali822687
 
Operation theatre sterilization powerpoint
byVaisHali822687
 
Parasitic Infections of CNS. Free living amoeba and neurocysticercosispptx
byVaisHali822687
 
Sample collection and transport ppt 1234
byVaisHali822687
 
Capanna technique for chronic osteomyelitis
byVaisHali822687
 
chronicosteomyelitis Powerpt power point
byVaisHali822687
 
Hemiarthoplasty debate. hemiarthroplasty
byVaisHali822687
 
viral hemorrhagic fever -dengue, chikungunya.
byVaisHali822687
 
Complement system and its synthesis + activation
byVaisHali822687
 

Recently uploaded

PPTX
Benign and Artifacts in EEG- wubsew.pptx
byssuser158ee91
 
PPTX
Full Endoscopic Interlaminar Lumbar Discectomy
byNtambi Rogers
 
PPTX
Phytochemical Analysis And Antimicrobial Screening Studies Of Calotropis Giga...
bySoukat Garain
 
PDF
MIndsets in Career of Emergency Medicine
byChing-Hsing Lee
 
PDF
Lenvatinib Tablets: Uses, Benefits, Diseases Treated & Availability in the Ph...
byLetsMeds
 
PDF
MAMMARY GLAND -Prof.Dr.N.Mugunthan.Professor of Anatomy, KMMC Medical College...
byKanyakumari Medical Mission Research Center, Muttom
 
PPTX
Central Venous Line: Insertion Technique & Complications
byDr Jebish Pradhan
 
PPTX
Complications of Coronary angiography and PCI.pptx
byVishal Gaurab
 
PPTX
Implant roadmap by Dr mohamed sidky.pptx
byssuseraa4a1e
 
PPTX
Human Digestive System (AOTT): Structure, Functions & Digestion.pptx
byBALAJI SOMA
 
PPTX
2025 APVRS-PAO Diplopia After Cataract & Retina Surgery.pptx
byAlvina Pauline Santiago, MD
 
PDF
Prosthetic Solutions Cranio-Maxillo-Facial
byDavide Decesari
 
PPTX
FACIAL NERVE AND ITS APPLIED ANATOMY BY Dr. AASTHA DUBEY
byAASTHA DUBEY
 
PDF
COMPREHENSIVE SURVEY OF UPPER LIMB NERVES-Prof.Dr.N.Mugunthan.pdf
byKanyakumari Medical Mission Research Center, Muttom
 
PDF
Partograph: It is a composite graphical recording of cervical dilatation and ...
byMayuriGamit2
 
PDF
classification & denaturation and structural -functional relationship of Prot...
byDr. Santhosh Kumar. N
 
PDF
UNIT-II Acid Base Titration neutralization curves
bysanjayudps2016
 
PPTX
blood banking and blood component. New onepptx
byRayAdhikariRatnesh
 
PPTX
SCOPE AND LIMITATIONS OF HOMOEOPATHY IN SURGICAL CONDITIONS
byDR.P.S SUDHAKAR
 
PDF
Technodelics.tech l Consciousness Hacking Through Light, Sound & Vibration l ...
byVladislav Solodkiy
 
Benign and Artifacts in EEG- wubsew.pptx
byssuser158ee91
 
Full Endoscopic Interlaminar Lumbar Discectomy
byNtambi Rogers
 
Phytochemical Analysis And Antimicrobial Screening Studies Of Calotropis Giga...
bySoukat Garain
 
MIndsets in Career of Emergency Medicine
byChing-Hsing Lee
 
Lenvatinib Tablets: Uses, Benefits, Diseases Treated & Availability in the Ph...
byLetsMeds
 
MAMMARY GLAND -Prof.Dr.N.Mugunthan.Professor of Anatomy, KMMC Medical College...
byKanyakumari Medical Mission Research Center, Muttom
 
Central Venous Line: Insertion Technique & Complications
byDr Jebish Pradhan
 
Complications of Coronary angiography and PCI.pptx
byVishal Gaurab
 
Implant roadmap by Dr mohamed sidky.pptx
byssuseraa4a1e
 
Human Digestive System (AOTT): Structure, Functions & Digestion.pptx
byBALAJI SOMA
 
2025 APVRS-PAO Diplopia After Cataract & Retina Surgery.pptx
byAlvina Pauline Santiago, MD
 
Prosthetic Solutions Cranio-Maxillo-Facial
byDavide Decesari
 
FACIAL NERVE AND ITS APPLIED ANATOMY BY Dr. AASTHA DUBEY
byAASTHA DUBEY
 
COMPREHENSIVE SURVEY OF UPPER LIMB NERVES-Prof.Dr.N.Mugunthan.pdf
byKanyakumari Medical Mission Research Center, Muttom
 
Partograph: It is a composite graphical recording of cervical dilatation and ...
byMayuriGamit2
 
classification & denaturation and structural -functional relationship of Prot...
byDr. Santhosh Kumar. N
 
UNIT-II Acid Base Titration neutralization curves
bysanjayudps2016
 
blood banking and blood component. New onepptx
byRayAdhikariRatnesh
 
SCOPE AND LIMITATIONS OF HOMOEOPATHY IN SURGICAL CONDITIONS
byDR.P.S SUDHAKAR
 
Technodelics.tech l Consciousness Hacking Through Light, Sound & Vibration l ...
byVladislav Solodkiy
 

Culture media part-2

  • 1.
    CULTURE MEDIA –PART 2 MODERATOR : Dr. S. K. SINGH PRESENTED BY: Dr. SAVITA CHOUDHARY DR. SUMEDHA THANVI
  • 2.
    INTRODUCTION DEFINITION: • CULTURE MEDIA:A medium used for cultivation of microorganisms. • CULTIVATION: Cultivation is a process of growing microorganisms in culture by taking bacteria from infection site (i.e. the in vivo environment ) by some means of specimen collection and growing them in artificial environment of the lab (i.e. in vitro environment ).
  • 3.
    TYPES OF CULTUREMEDIA • BASED ON FUNCTION: 1.Basal Media : Basic media with minimum basic ingredients that supports growth of non-fastidious bacteria. e.g: Peptone water, Nutrient broth, Nutrient agar. 2. Enriched media : Media prepared to meet the nutritional requirements of more exacting bacteria by addition of substances such as blood,serum or egg to a basal medium. e.g: Blood agar, Chocolate agar, Loeffler’s serum slope ,etc. 3. Enrichment Media: Liquid media added with some inhibitory agents which selectively allow certain organisms to grow and inhibit others. e.g: Tetrathionate broth ,Selenite F broth etc.
  • 4.
    TYPES OF CULTUREMEDIA (…contd) • 4. Selective Media: This media contains one or more agents that are inhibitory to all organisms except those “ selected” by the specific growth condition or chemical. E.g: Lowenstein- Jensen medium,Thiosulfate Citrate Bile Salt (TCBS) agar, etc. • 5. Differential Media: These media differentiate between two groups of bacteria by using an indicator. E.g: Mac conkey agar, CLED agar, etc • 6. Transport Media : Media which are devised to maintain the viability of a pathogen and to avoid overgrowth of other contaminants during transit from the patient to laboratory. e.g: Stuart’s medium, Venkatraman- Ramakrishnan medium, Cary Blair medium, etc.
  • 5.
    Gram positive cocci •Mannitol Salt Agar • Tellurite Glycine Agar • Salt cooked meat broth • DNase Test Agar • Milk Salt Agar Staphylococci • Todd Hewitt broth • Crystal violet blood agar • PNF medium • Nichols & Freeman Medium Streptococci
  • 6.
    Mannitol Salt Agar •Mannitol Salt Agar is used as a selective media for the isolation of pathogenic Staphylococci aureus from clinical and non clinical (soil, faeces etc.) specimens. PRINCIPLE: i. Beef extract and proteose peptone supply essential growth factors and trace nutrients to the growing bacteria. ii. Sodium chloride serve as an inhibitory agent against bacteria other than Staphylococci. iii. Mannitol is the fermentable carbohydrate ,fermentation of which leads to acid production which is detected by phenol red indicator. Ingredients Quantity (gm/l) Proteose peptone 10 Beef extract 1 Sodium chloride 75 D-Mannitol 10 Phenol Red 0.025 Agar 15 Final pH 7.4± 0.2
  • 7.
    Mannitol Salt Agar(…contd) INTERPRETATION: • S.aureus ferments mannitol and produce yellow coloured colonies surrounded by yellow zones. • Coagulase negative strains of Staphylococci are usually mannitol non fermenters and therefore produce pink to red colonies surrounded by red purple zone. • MSA selective for MRSA: - To MSA add oxacillin 6 mg/litre or methicillin 10 mg/litre. - To MSA add oxacillin 6 mg/litre and polymixin B 10 mg/litre. Organism Growth Colour of colonies S. aureus ATCC 6538 luxuriant Yellow /white colonies surrounded by yellow zone E. coli ATCC 25923 Inhibited -
  • 8.
    Tellurite Glycine Agar Usedfor quantitative detection of Coagulase positive Staphylococci. • This medium supports better growth of coagulase positive cocci even if present in small numbers. PRINCIPLE  Casein enzymic hydrolysate and yeast extract provide nitrogenous compounds and vitamin B complex.  Lithium chloride and potassium tellurite inhibits coagulase negative Staphylococci.  Mannitol is a source of fermentable carbohydrate INTERPRETATION  Occasional Coagulase-negative strain that may produce small grey colonies.  Coagulase-positive Staphylococci reduce tellurite and form black colonies Ingredients Quantity (gm/l) Casein enzymic hydrolysate 10 Yeast extract 5 Mannitol 5 Dipotassium phosphate 5 Lithium chloride 5 Glycine 10 Agar 16 Final pH 7.2±0.2 S.aureus ATCC 25923 Good luxuriant Black S.epidermidis ATCC 12228 Poor- Fair Grey E.coli ATCC 25922 Inhibited
  • 9.
    DNase Test Agar •This agar is recommended for the detection of deoxyribonuclease activity of bacteria and fungi, specially for the identification of pathogenic Staphylococci. PRINCIPLE AND INTERPRETATION: • Detection of deoxyribonuclease activity of bacteria and fungi. • With added toluidine blue,it is used in the identification and differentiation of non pigmented Serratia spp. from Enterobacter and Klebsiella. • Positive DNase activity is visualised as clear zones around colonies when plate is flooded with 1 N hydrochloric acid Organism Growth DNase activity S. aureus ATCC 25923 Luxuriant Positive Serratia marcescens ATCC 81005 Luxuriant Positive S.epidermidis ATCC 12228 Luxuriant Negative Ingredients Quantity (gm/l) Tryptone 15 Soya Peptone 5 Deoxyribonucleic acid (DNA) 2 Sodium Chloride 5 Agar 15 Final pH 7.3±0.2 Water 1 L
  • 10.
    Salt Cooked MeatBroth Used as an enrichment medium for isolation of Staphylococcus from grossly contaminated specimens. PRINCIPLE AND INTERPRETATION: • Selective for Staphylococci due to presence of sodium chloride in high concentration. • Peptic digest of animal tissue and beef extract provide essential nutrients. • Sodium chloride maintains osmotic equilibrium. Organism Growth S.Aureus ATCC 25923 Good luxuriant E.coli ATCC 25922 Inhibited Ingredients Quantity(gm/l) Beef Extract 10 Peptic digest of animal tissue 10 Neutral ox- heart tissue 30 Sodium chloride 100 Final pH 7.6±0.2 P.vulgaris ATCC 13315 Inhibited
  • 11.
    Milk Salt Agar •Used for selective isolation and cultivation of Staphylococcus. PRINCIPLE AND INTERPRETATION:  Beef extract, peptic digest of animal tissue and skim milk supply essential nutrients  Highly selective as majority of the contaminating organisms are inhibited by the high salt concentration (6.5%) Organism Growth Recovery S.aureus ATCC 25923 Good Luxuriant >=50% E.coli ATCC 25922 Inhibited 0% Ingredients Gms/L Peptic digest of animal tissue 5 Beef extract 3 Sodium chloride 65 Skim Milk 10% Agar 15
  • 12.
    Todd Hewitt broth •Thisenriched broth is used for luxuriant growth of organisms such as group A haemolytic Streptococci PRINCIPLE & INTERPRETATION: •Todd Hewitt Broth medium is very nutritious due to the presence of peptic digest of animal tissue and beef heart infusion. •Dextrose stimulates haemolysin production •The medium is well buffered by sodium phosphate and sodium carbonate to neutralize the acid produced during dextrose fermentation. This restricts destruction of antigenic streptococcal haemolysin. Selective medium for group B Streptococci : Prepare a solution of 15µg/l nalidixic acid and 8µg/l gentamicin and add .5ml of it and .25ml sterile defibrinated sheep blood to 4.75 ml of todd Hewwit broth. Ingredients Quantity (gm/l) Beef heart, infusion from 500 Peptic digest of animal tissue 20 Dextrose 2 Sodium chloride 2 Disodium phosphate .4 Sodium carbonate 2.5 Final pH 7.8±2
  • 13.
    Crystal violet bloodagar • Selective for Streptococus pyogenes and other Streptococci •At a concentration of 1 in 500000, crystal violet inhibits the growth of Staphylococcus aureus and some other bacteria found in clinical specimen PNF Medium Selective for Beta hemolytic Streptococci  Inhibit the growth of Staphylococci and Coliforms  Melt the Nutrient Agar, cool to 50ºC, add the ingredients and pour plates Ingredients Per Litre Sterile Nutrient Agar 90 ml Sterile Blood 10 ml Crystal Violet 0.1 % 0.2 ml Ingredients Per Litre Sterile Nutrient Agar Sterile Blood 50 ml Polymyxin B Sulphate 17000 units
  • 14.
    Agar of Nicholasand Freeman (Crystal Violet- Nalidixic Acid- Gentamycin Agar (CVNG) • This is a new selective medium for the detection of Streptococcus pneumonia • Highly selective for Strept. pneumoniae in sputum samples. • This medium is inhibitory to many respiratory bacteria other then the Pneumococcus,Viridans Streptococci and Enterococci. Ingredients Quantity Columbia agar Defibrinated horse blood 10% Crystal violet 2 mg/l Nalidixic acid 50mg/l Gentamycin 2mg/l
  • 15.
    Gram Negative Cocci Neisseria: Neisseria •Modified Thayer Martin Medium • Modified New York City Medium
  • 16.
    Modified Thayer MartinMedium  Selective isolation & enumeration of Neisseria gonorrhoeae PRINCIPLE & INTERPRETATION:  Vancomycin, Colistin, Nystatin and Trimethoprim make medium selective  Peptic digest of animal tissue provides nutrients while starch neutralizes the toxic fatty acid.  Vancomycin inhibits gram-positive, Colistin inhibits gram-negative bacteria and Nystatin inhibits fungi.  If chocolate agar and antibiotics added-suppress the growth of contaminants , saprophytic Neisseria species ; enhace the growth of pathogenic Neisseria.  Small grey convex colonies with irregular outlines seen. Growth is slower as compared to other selective media. Ingredients Gms/L Peptone 23 Starch 1 Sodium Chloride 5 Dextrose 2.5 Agar 20 Organism Growth Colour of colony N.gonorrhoeae ATCC 19424 Good-luxuriant Small, greyishwhite to colourless, mucoid E.coli ATCC 25922 Inhibited
  • 17.
    Modified New YorkCity Medium  Enriched selective medium for Neisseria sp.  Preferred medium as it gives better growth ; use of lincomycin as selective agent avoid problem of vancomycin sensitivity. PRINCIPLE & INTERPRETATION:  Lysed horse blood, dextrose and yeast dialysate are the enrichments; proteose peptone,horse plasma, hemoglobin provide nutrients. • Colistin inhibits gram-negative bacilli, amphotericin B suppress yeast growth and trimethoprim prevents Proteus sp. swarming • After incubation for 24 hours in moist aerobic environment,5-10% CO2- small( 1mm) ,grey,convex colonies; after 48 hours, larger colonies with crenated margins & opaque raised centre. GC Medium Base 36 g Yeast dialysate 25 ml Blood (100ml) lysed with 5 ml of 10% saponin 105 ml Glucose 10% sterlized at 115ºC for 10 min. 10 ml Colistin (6mg/L) 1ml Lincomycin (1mg/L) 1ml Trimethoprim (5mg/L) 1ml Amphoterecin B (1mg/L) 1ml Organisms Growth Recovery Neisseria gonorrheae ATCC 19424 Good-luxuriant >=50% Proteus mirabilis ATCC 13883 None- poor <=10%
  • 18.
    Gram Positive Bacilli •Gram positive bacilli: Corynebacterium Bacillus Listeria • Loeffler’s Serum Slope • Hoyle’s Tellurite Lysed Blood Agar • Tinsdale Medium • Hiss’s Serum Sugar Media • Knisely’s PLET Medium • Phenol red-Egg yolk- Polymyxin agar • PEMBA Medium • PALCAM Agar
  • 19.
    Hoyle’s Tellurite LysedBlood Agar  Selective medium for Corynebacterium species PRINCIPLE & INTERPRETATION:  Biopeptone provides nitrogenous compounds  Haemoglobin stimulates good growth of Corynebacterium  Potassium tellurite acts as a selective inhibitor against most gram-positive and gram-negative bacteria except Corynebacterium species  C.diphtheriae reduces potassium tellurite to tellurium and thereby produce grey-black coloured colonies Agar Base Meat Extract 10 g Proteose Peptone 10 g Sodium Chloride 5 g Agar Powder 15 g Water 1 L Tellurite Solution Potassium Tellurite 0.7 g Water 20 ml Complete Medium Agar Base 200 ml Lysed Blood 10 ml Tellurite Solution 2 ml Organism Growth Colour of colony C.diphtheriae ATCC 11913 Good-luxuriant Grey-black E.coli ATCC 25922 Inhibited -
  • 20.
    Tinsdale Medium  Forselective isolation and differentiation of Corynebacterium diphtheria. PRINCIPLE & INTERPRETATION:  Peptic digest provides nitrogenous compounds. L-cystine and sodium thiosulphate form the H₂S indicator system.  Potassium tellurite inhibits all gram-negative bacteria and most of the upper respiratory tract normal flora.  C.diphtheriae forms greyish black colonies surrounded by a dark brown halo. Halo is formed due to H₂S production from cystine combining with the tellurite salt. Agar Base Proteose Peptone 20 g Sodium Chloride 5 g Agar Powder 19 g Water 1 L Complete Medium Agar Base 200 ml Ox Serum 20 ml Sodium Hydroxide 0.1 mol/L 12 ml L-Cystine, 0.4% in Hcl, 0.1 mol/L 12 ml Potassium Tellurite 1% in Sterile Water 6 ml Sodium Thiosulphate 2.5% solution 3.4 ml Organism Growth Colony characteristics C.diphtheriae gravis Good-luxuriant Brown-black with halo K.pneumoniae ATCC 13883 Inhibited
  • 21.
    Knisely’s PLET Medium For selective culture of small numbers of Bacillus anthracis from soil and other materials containing spore formers of other species.  On incubation at 37ºC for 24 hours, colonies develop from 30-100% of the B. anthracis spores that would grow on unselective heart infusion agar. PRINCIPLE & INTERPRETATION:  PLET medium inhibits the growth of most strains of B.cereus, B.subtilis, Enterobacteria and Pseudomonas  Some strains of B.cereus from soil form colonies, smaller than B.anthracis, minute after 24 h and moderately sized only after 48 h, roughly circular creamy- white colonies with a ground glass texture. Heart Infusion Agar 40 g Thallous acetate 0.04 g EDTA 0.3 g Polymyxin 30,000 units Lysozyme 0.04 g Deionized Water 1 L Organism Growth B.anthracis ATCC 14578 Luxuriant B.cereus ATCC 10876 Inhibited
  • 22.
    Phenol Red EggYolk Polymyxin Agar • Relatively selective for B.cereus ; used to isolate it from food,faeces and vomitus. PRINCIPLE & INTERPRETATION:  Colonies recognized by their opacity in the egg-yolk, pink color with phenol red and failure to ferment mannitol.  Egg yolk emulsion helps in differentiation of lecithinase producing colonies which are surrounded by a zone of white precipitate  Polymyxin B Sulphate restricts growth of gram-negative bacteria such as E.coli and Pseudomonas aeruginosa  Differentiation of Bacillus cereus from other Bacillus species by its inability to ferment mannitol and poor sporulation Peptone 10 g Meat Extract 1 g Mannitol 10 g Sodium Chloride 10 g Phenol red 0.025 g Agar 15 g Egg-yolk emulsion 100 ml Polymyxin B sulphate 0.01 g Deionized water 1 L Organism Growth Recovery Colour Lecithinase activity B.cereus ATCC 10876 Luxuriant >=50% Red Positive,opaque zone around the colony E.coli ATCC 25922 None-poor <=10%
  • 23.
    PEMBA Medium (Polymyxin PyruvateEgg Yolk Mannitol Bromothymol Blue Agar) • Recommended for enumeration of B.cereus in foods. • Supports growth of even a small number of B.cereus. PRINCIPLE & INTERPRETATION:  Low peptone content promotes sporulation,sodium pyruvate reduces the colony size of the organisms.  Egg yolk emulsion demonstrates the strong lecithinase opacity reaction  Bromothymol blue acts as pH indicator to detect mannitol fermentation.  B.cereus forms crenated blue colonies surrounded by a zone of opacity in the medium Peptone 1 g D- Mannitol 10 g Magnesium sulphate.heptahydrate 0.1 g Disodium phosphate 2.5 g Potassium dihydrogen phosphate 0.25 g Sodium chloride 2 g Sodium pyruvate 10 g Polymyxin B sulphate 1 lac U Bromothymol blue 0.1 g Egg-yolk emulsion 50 ml Agar 18 g Deionized water 1 L Organism Growth Colour of colony Egg Yolk Reaction B.cereus ATCC 10876 Good-luxuriant Blue Positive, precipitation E.coli ATCC 25922 Inhibited
  • 24.
    PALCAM Agar (Polymyxin-Acriflavin-Lithium chloride-Ceftazidime-Aesculin-Mannitol) •For selective isolation and identification of Listeria species. PRINCIPLE & INTERPRETATION:  Highly selective due to the presence of lithium chloride, ceftazidime, polymyxin B and acriflavin.  Differential diagnostic medium utilizing two indicator systems, esculin-ferric citrate and mannitol-phenol red.  L. monocytogenes hydrolyzes esculin forms esculetin and dextrose. Esculetin reacts with ammonium ferric citrate and forms a brown-black complex seen as a black halo around colonies.  On PALCAM Agar,after inoculation and incubation at 35 degrees for 24-48 hours in aerobic or microaerophillic condition, Listeria colonies appear grey-green with a black precipitate. Ingredients g/L Peptone 23 Starch 1 Sodium Chloride 5 Mannitol 10 Ammonium ferric citrate 0.5 Esculin 0.8 Dextrose 0.5 Lithium Chloride 15 Phenol red 0.08 Agar 13
  • 25.
    Gram Negative Bacilli •. Coliforms Salmonella & Shigella Yersinia EMB Agar • Bismuth Sulphite Agar • DCA • XLD • Hektoen Agar • SS Agar Schiemann CIN Agar Vibrio • TCBS Agar • Bile Salt Agar • Monsur’s Tellurite Taurocholate Gelatin Agar
  • 26.
    EMB Agar  EosinMethylene Blue Agar is a selective and differential medium for Coliforms. PRINCIPLE & INTERPRETATION:  Eosin-Y and Methylene blue (ratio 6:1) inhibit gram-positive bacteria, serve as differential indicators in response to the fermentation of carbohydrates  Coliforms produce purplish black colonies due to taking up of methylene blue-eosin dye complex, when the pH drops.  Nonfermenters raise the pH by oxidative deamination of protein, which solubilizes the methylene blue-eosin complex resulting in colourless colonies. Organism Growth Color of Colony E.coli ATCC 25922 Luxuriant Purple with black centre and green metallic sheen P.mirabilis ATCC 25933 Luxuriant Colourless S.aureus ATCC 25923 Inhibited Peptic digest 10 g Dipotassium phosphate 2 g Lactose 5 g Sucrose 5 g Eosin - Y 0.4 g Methylene blue 0.065 g Agar 13.5 g Distilled water 1 L
  • 27.
    Wilson & Blair’sBrilliant green Bismuth Sulphite Agar • Particularly used for the isolation of Salmonella typhi. • Highly selective for Salmonella; inhibitory to Coliforms, Proteus & Shigella. PRINCIPLE & INTERPRETATION: • Sulphur compounds provide a substrate for hydrogen sulphide production, while the metallic salts stain the colony and surrounding medium black or brown in the presence of hydrogen sulphide. • Cultures examined after 24 hours, then again after 48 hours. Crowded, green or pale brown colonies, 1 mm in diameter seen. Larger ,discrete colonies have a black centre and clear edge. Bismuth sulphite glucose phosphate mixture Bismuth ammonio citrate scales 30 g Sodium sulphite 100 g Disodium hydrogen phosphate 100 g Glucose 50 g Sterile water 1 L Iron citrate brilliant green mixture Ferric citrate, brown scales 2 g Brilliant green 0.25 g Sterile water 225 ml Complete medium Sterile nutrient agar 100 ml Bismuth sulphite glucose phosphate mixture 20 ml Iron citrate brilliant green mixture 4.5 ml Organism Growth Color of colony S.typhi ATCC 6539 Good-luxuriant Black with metallic sheen E.faecalis ATCC 29212 Inhibited
  • 28.
    Leifson’s Deoxycholate CitrateAgar (DCA) • Most widely used plating medium for isolation of intestinal pathogens from faeces; pathogens that cause bacilliary dysentery, Salmonella strains that cause food poisoning and Salmonella paratyphi. PRINCIPLE & INTERPRETATION:  Coliforms and gram-positive bacteria are inhibited due to sodium deoxycholate, sodium citrate and ferric ammonium citrate  Lactose helps in differentiating enteric bacilli, as lactose fermenters produce red colonies while lactose non-fermenters produce colorless colonies  Coliforms, if present form pink colonies  Salmonella and Shigella species do not ferment lactose but Salmonella may produce H2S, forming colourless colonies with or without black centers. Neutral Red Lactose Agar Meat Extract 20 g Peptone 20 g Agar 90 g Neutral Red 2% in 50% ethanol 5 ml Lactose 40 g Water 4 L Solution A Sodium citrate 17 g Sodium thiosulphate 17 g Ferric ammonium citrate 4 g Sterile water 100 ml Solution B Sodium deoxycholate 10 g Sterile water 100 ml Complete medium Neutral red lactose agar 100 ml Solution A 5 ml Solution B 5 ml
  • 29.
    Taylor’s Xylose LysineDeoxycholate (XLD)  Selective for Salmonella and Shigella PRINCIPLE & INTERPRETATION:  Sodium deoxycholate acts as a selective agent; restrain the growth of E.coli  Colony turns red due to fermentation of Xylose, detected by indicator Phenol red  Lactose and Sucrose are fermentable sugars  L-lysine, essential amino acid source, differentiates Salmonella Sp.  Sodium thiosulphate and Ferric ammonium citrate helps visualizing the black centered colonies on production of H2S.  Salmonella produce red colonies with black centres whereas S.paratyphi,Shigella & Providenciae form red colonies without black centres. Yeast Extract 3 g Xylose 3.75 g Lactose 7.5 g Sucrose 7.5 g L- Lysine HCl 5 g Sodium chloride 5 g Sodium deoxycholate 2.5 g Sodium thiosulphate 6.8 g Ferric ammonium citrate 0.8 g Phenol red 0.08 g Agar 15 g Water 1 L
  • 30.
    Hektoen Enteric AgarMedium  For differential and selective isolation of Salmonella and Shigella PRINCIPLE & INTERPRETATION:  Bile salts, bromothymol blue and acid fuchsin inhibit the growth of most gram positive organisms  Ferric ammonium citrate as iron source, cause production of hydrogen sulfide from sodium thiosulphate and by reacting with hydrogen sulfide, forms a black precipitate.  Enterobacters other than Salmonella and Shigella are capable of fermenting one or more of the carbohydrates produce yellow or salmon-orange coloured colonies.  Non-fermenters will produce blue-green colonies  Salmonella reduce sulfur to hydrogen sulfide, producing a black precipitate Peptone 12 g Yeast extract 3 g Lactose 12 g Sucrose 2 g Salicin 9 g Bile Salts mixture 9 g Sodium chloride 5 g Sodium thiosulfate 5 g Ferric ammonium citrate 1.5 Acid fuchsin 0.1 g Bromothymol blue 0.065g Agar 14 g Water 1 L Organism Colour of colony S. typhimurium ATCC 14028 Blue-green with or without black centres Shigella flexneri ATCC 12022 Greenish blue E. faecalis ATCC 29212 -
  • 31.
    SS Agar  Moderatelyselective and differential for Salmonella spp. and some Shigella spp.  Modification of the Deoxycholate Citrate Agar PRINCIPLE & INTERPRETATION:  Bile Salts, Sodium Citrate and Brilliant Green inhibit gram- positive, Coliforms and inhibit swarming Proteus spp.  Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide by the production of colonies with black centers  Salmonella will not ferment lactose, but produce hydrogen sulfide gas; colonies appear colorless with black centres.  Shigella do not ferment lactose or produce hydrogen sulfide gas, hence colonies appear colourless. Beef Extract 5 g Enzymatic Digest of Casein 2.5 g Enzymatic Digest of Animal Tissue 2.5 g Lactose 10 g Bile Salts 8.5 g Sodium Citrate 8.5 g Sodium Thiosulfate 8.5 g Ferric Citrate 1.0 g Brilliant Green 0.00033 g Neutral Red 0.025 g Agar 13.5 Distilled Water 1 L S. typhi ATCC 6539 Colorless colonies with black center S. flexneri ATCC 12022 Colorless colonies E. faecalis ATCC 19433 Inhibited
  • 32.
    Special Medias forSalmonella & Shigella • . BISMUTH SULPHITE AGAR HEKTOEN ENTERIC AGAR XLD DCA SS AGAR
  • 33.
    Schiemann CIN Agar CefsulodinIrgasan Novobiocin  Selective isolation of Yersinia enterocolitica  Differentiates between mannitol fermenting and non-fermenting bacteria PRINCIPLE & INTERPRETATION:  Mannitol fermenting colonies take red colour, while non-fermenting form colourless and translucent colonies  Sodium deoxycholate and crystal violet, selectively inhibit gram- positive and gram-negative bacteria  Antibiotic supplement makes it highly selective for Yersinia  Dark red colonies resembling bulls eye, surrounded by a transparent border  Serotyping is based on colony size, smoothness and ratio of the border to centre diameter Yersinia Selective Agar Base Peptone 20 g/L Yeast Extract 2.0 g/L Mannitol 20 g/L Sodium pyruvate 2.0 g/L Sodium chloride 1.0 g/L Magnesium sulphate 0.01 g/L Sodium deoxycholate 0.5 g/L Neutral red 0.03 g/L Crystal violet 0.001 g/L Agar 12.5 g/L Complete medium Agar Base 500ml Cefsulodin 7.5 mg Irgasan 2.0 mg Novobiocin 1.25 mg Organism Growth Colour of colony Y.enterocolitica ATCC 27729 Good- luxuriant Transluscent with dark pink/red centre & bile precipitate E.faecalis ATCC 29212 Inhibited
  • 34.
    TCBS Agar (Thiosulphate CitrateBile Sucrose)  Highly selective and differential for Vibrio species PRINCIPLE & INTERPRETATION:  Sucrose fermentation produces acid, which converts the colour of bromothymol blue or thymol blue  High concentrations of sodium thiosulfate and sodium citrate inhibit the growth of Enterobacteriaceae  Inhibition of Gram-positive bacteria is achieved by the incorporation of ox bile  Sodium thiosulfate as a sulfur source, with ferric citrate, allows easy detection of hydrogen sulfide production  Alkaline pH of the medium enhances the recovery of V.cholerae and inhibits the growth of others Yeast extract 5 g Peptone 10 g Sodium thiosulfate 10 g Sodium citrate 10 g Ox bile 8 g Sucrose 20 g Sodium chloride 10 g Ferric citrate 1 g Bromothymol blue 0.04 g Thymol blue 0.04 g Agar 15 g Water 1 L Organism Growth Colour of colony V.cholerae ATCC 15748 Good-luxuriant Yellow E.faecalis ATCC 29212 Inhibited
  • 35.
    Bile Salt Agar(BSA)  For isolation and identification of bile tolerant bacteria responsible for food poisoning. PRINCIPLE & INTERPRETATION:  Vibrio species, like many other gram-negative bacteria, grow in the presence of relatively high levels of bile salts  Sodium chloride buffers the medium well. Sodium taurocholate inhibits most of the gram-negative organisms  Colonies of Vibrios have a distinctive appearance which may be seen by comparing a known strain of Vibrio cholerae with Escherichia coli. Peptone 10 g Meat Extract 5 g Sodium Chloride 5 g Sodium taurocholate 5 g Agar 15 g Water 1 L Organism Growth Vibrio cholerae ATCC15748 Luxuriant Staphylococcus aureus ATCC 25923 Inhibited
  • 36.
    Monsur's tellurite taurocholategelatin agar  Selective isolation and differentiation of Vibrio cholerae and other species of Vibrios. PRINCIPLE & INTERPRETATION:  Sodium chloride maintains the osmotic equilibrium ; sodium carbonate maintains the elevated pH  Gelatin acts as an additional carbon and energy source  The high pH and potassium tellurite are inhibitory to most Enterobacteriaceae and gram-positive bacteria, though Proteus may form grey centered colonies without a halo  Colonies are often surrounded by a gelatin liquefaction halo. Organism Growth Color Vibrio cholerae ATCC 15748 Good-luxuriant Grey Proteus mirabilis ATCC 25933 None-poor Black Taurocholate Gelatin Agar Peptone 10 g Sodium chloride 10 g Sodium taurocholate 5 g Sodium carbonate 1 g Gelatin 30 g Agar 15 g Distilled water 1 L Complete Medium Taurocholate Gelatin Agar 100 ml Diluted Potassium tellurite (0.05%) 1 ml
  • 37.
    Pseudomonas and Burkholderia P.aeruginosa B. pseudomallei B.cepacia • Cetrimide Agar • Pseudomonas Isolation Agar • Pseudomonas Enrichment Broth • Ashdown’s Medium • Burkholderia pseudomallei Selective Agar (BPSA) • Burkholderia cepacia Selective Agar (BCSA) • Pseudomonas cepacia (PC) agar • Oxidative–fermentative base–polymyxin B– bacitracin–lactose (OFPBL) agar
  • 38.
    Cetrimide Agar • Forisolation of Pseudomonas aeruginosa. • P. aeruginosa produces water soluble pigments, including yellow-green fluorescent pigment pyoverdin and blue pigment pyocyanin, together creating bright green colour. PRINCIPLE & INTERPRETATION: • Cetyltrimethylammonium bromide (Cetrimide) acts as cationic detergent and causes denaturation of bacterial membrane proteins . • Gelatin peptone provides nutrition. • Sodium chloride maintains osmotic equilibrium. • Magnesium chloride and potassium sulphate act as pigment-enhancers. • Colonies exhibit a yellow-green to blue colour and show fluorescence under short wavelength (254 nm) ultraviolet light, with a characteristic grape like smell of aminoacetophenone. Pseudomonas aeruginosa ATCC 9027 luxuriant Escherichia coli ATCC 8739 inhibited Ingredients Gelatin peptone Magnesium chloride Potassium sulphate Cetrimide (0.03%) Agar (1.5%)
  • 39.
    Ashdown's medium • Forisolation of Burkholderia pseudomallei. PRINCIPLE & INTERPRETATION: • Crystal violet and Gentamicin act as selective agents. • Neutral red – indicator. • Glycerol provides nutrition. • Colonies appear flat, wrinkled, slightly dry, having pinkish purple colour with a metallic sheen. B.pseudomallei ATCC 23343 Growth P. aeruginosa ATCC 27853 No growth INGREDIENTS- Tryptone soya broth Gentamicin Crystal violet (0.1%) Neutral Red (1%) Glycerol Agar (1.5%)
  • 40.
    Burkholderia cepacia SelectiveAgar (BCSA) • For isolation of Burkholderia cepacia. • Better selectivity ,quicker growth and lower false positivity rates than PC Agar and OFPBL Agar. PRINCIPLE & INTERPRETATION: • Pancreatic digest of casein ,yeast extract, lactose and sucrose provide nutrition. • Phenol Red -pH indicator, changes colour of medium from orange to yellow due to acid production by fermentation of lactose and sucrose. • Crystal violet, vancomycin, polymyxin and gentamicin- Selective Agents. • Colonies appear translucent and rough with yellow halos (d/t carbohydrate fermentation) • Burkholderia cepacia ATCC 25416 • Growth; color change in media from red-orange to yellow • Pseudomonas aeruginosa ATCC 27853 • Complete inhibition Pancreatic Digest of Casein Lactose Sucrose Sodium Chloride Yeast Extract Phenol Red Gentamicin Vancomycin Crystal Violet Polymyxin B Agar Ingredients:
  • 41.
    Haemophilus species • FildesBlood-Digest Agar • Levinthal’s Medium • Haemophilus Isolation Agar • Haemophilus Differential Selective medium H.influanzae, H.parainfluanzae,H. parahaemolyticus • Mueller-Hinton–based chocolate agar (supplemented with 1% IsoVitaleX and 3 mg/mL vancomycin) • Heart infusion–based agar (supplemented with 10% fetal bovine serum and 3 mg/mL vancomycin) H.ducreyi
  • 42.
    Fildes Blood-Digest Agar Usedfor isolation of Haemophilus species. PRINCIPLE & INTERPRETATION: •Pepsin causes sterile digestion of Defibrinated Sheep Blood which causes release of factor x (haemin) and factor V(NAD) •High concentration of haemin is inhibitory to many throat commensals •Colonies of Non capsulated strains 0.5-1 mm circular, low convex, smooth, pale grey and transparent with slightly blue iridescence and a fishy, seminal odour. •Capsulated strains = Larger (1-3mm), high convex, mucoid with strong irridescence of red, orange, green and blue shades which alter with angle of observation. INGREDIENTS Qnt. NaCl 150ml HCl 6ml Defibrinated Sheep Blood 50ml Pepsin 1g NaOH 12ml Chloroform .5ml Agar
  • 43.
    Levinthal’s Medium For cultivationof Haemophilus species PRINCIPLE & INTERPRETATION: •Blood provides factor-X (Heat stable) and factor-V (Heat labile). •Peptic digest of animal tissue and beef extract act as source of nitrogen. •Sodium chloride maintains osmotic balance. Capsulated strains show translucent colonies with distinctive iridescence while non-capsulated strains are transparent and bluish . Composition Quantity Peptic digest of animal tissue 10 Beef extract 10 sterile rabbit or human blood 5 sterile rabbit or human blood Agar 20 H.influenzae ATCC 35056 Luxuriant H. influenzae ATCC 35056 Luxuriant H. influenzae ATCC 35056 Luxuriant
  • 44.
    Haemophilus Isolation Agar Forisolation of Haemophilus species. PRINCIPLE & INTERPRETATION: • Horse blood provides factor-X, factor-V and other Growth Factors. •Intact erythrocytes enable differentiation of hemolytic and non- hemolytic Haemophilus spp. •Beef Heart Infusion and Casein Peptone provide nutrition. •Bacitracin (300 μg/mL) inhibits the normal respiratory tract microbes (i.e., Staphylococci, Micrococci, Neisseriae , Streptococci). Haemophilus influenzae ATCC 10211 Growth Haemophilus parahaemolyticus ATCC 10014 Growth, Beta hemolysis Staphylococcus aureus ATCC 25923 Inhibition (partial to complete) Composition- Beef Heart Infusion Yeast extract Casein Peptone Sodium Chloride NAD Bacitracin Defibrinated Horse Blood (5%) Agar
  • 45.
    Haemophilus Differential Selectivemedium •This medium contains bacitracin as a selective agent •Sucrose and phenol red are used to distinguish H.parainfluenzae from H.influenzae •H.parainfuenzae ferments sucrose and forms yellow colonies whereas H.influenzae forms smaller, colourless non-fermenting colonies. Levinthal’s medium H. Influenzae colonies on Haemohillus isolation agar
  • 46.
    Bordetella • Bordet GengouMedium • Regan-Lowe Agar (Charcoal Blood Agar) • Stainer Scholte synthetic medium • Jones-Kendrick Medium(Blood-free solid medium for transport and cultivation) Bordetella
  • 47.
    Bordet Gengou Medium Recommendedfor the detection and isolation of Bordetella pertussis and Bordetella parapertussis . PRINCIPLE & INTERPRETATION: •Potato infusion and peptic digest of animal tissue serve as carbon and nitrogen source while glycerol and blood enrichment provides additional nutrients. •Sodium chloride maintains osmotic equilibrium • Colonies appear small, smooth, dome shaped, greyish-white with a shiny surface resembling ‘mercury drops’ or ‘bisected pearls’ surrounded by a hazy zone of haemolysis. Bordetella pertussis ATCC 8467 Luxuriant growth with beta-hemolysis Bordetella parapertussis ATCC 15311 Luxuriant growth with gamma-hemolysis Staphylococcus aureus ATCC 25923 Inhibited Ingredients Qnt. (gm/l) Potatoes, infusion from 125 Peptic digest of animal tissue 10 Sodium chloride 5 Agar 20 Final pH 6.7±0.2
  • 48.
    Regan-Lowe Agar(Charcoal BloodAgar) This media has largely replaced traditional Bordet Gongou media PRINCIPLE & INTERPRETATION: • Peptic digest of animal tissue, beef extract and yeast extract provide essential nutrients to the organisms •Sodium chloride maintains osmotic balance • Starch soluble and charcoal neutralizes substances toxic to Bordetella species such as fatty acid Bordetella pertussis ATCC 8467 Growth Staphylococcus aureus ATCC 25923 Inhibition Ingredients Qnt. (gm/l) Peptic digest of animal tissue 10 Beef extract 10 Starch soluble 10 Sodium chloride 5 charcol 4 Yeast extract 3.5 Agar 12 Final pH 7.5±0.2 Both media contain cephalaxin as an additive for suppression of contaminating microorganism
  • 49.
    Regan Lowe agarand typical “mercury drop colonies” of Bordetella pertussis Bordet Gengou agar and colonies of Bordetella pertussis surrounded by beta hemolysis
  • 50.
    Campylobacter and Helicobacter •Skirrow Campylobcter medium • Preston Campylobacter selective medium • Charcoal-based blood-free selective medium • Blaser’s medium(Campy-BAP) Campylobacter • Modified Thayer Martin agar • Skirrow medium • BHI Agar with Vancomycin, Nalidixic acid and AmphotericinB Helicobacter
  • 51.
    Skirrow Campylobacter selectiveagar •For isolation of campylobacter species PRINCIPLE & INTERPRETATION: •Trimethoprim , Vancomycin and Polymyxin - Selective agents. •Blood serves as an additional source of nutrients including X-factor •Colonies are effuse, droplet like with a tendency to form a spreading film. Also suitable for isolation of H.pylori colonies of which are small(<2mm), grey and translucent. Campylobacter jejuni ATCC 33291 Growth E.Coli ATCC 25922 Inhibition Ingredients Proteose peptone Liver digest Yeast extract Sodium chloride Agar Lysed horse blood Vancomycin Polymyxin-B Trimethoprim Agar
  • 52.
    Charcoal-based blood-free selectivemedium More selective than Skirrow’s medium and has a higher isolation rate of C.jejuni from mixed cultures. PRINCIPLE & INTERPRETATION: •Charcoal, hematin, ferrous sulfate and sodium pyruvate serve as substitutes for blood. They enhance the growth and aerotolerance of campylobacters by quenching photochemically generated toxic oxygen derivatives. •Addition of cefoperazone increases the selectivity of the medium. C.jejuni produces grey, moist, flat-spreading colonies. Some strains may have green hue or a dry appearance with or without metallic sheen. C. jejuni ATCC 33291 Growth E. coli ATCC 25922 Partial to complete inhibition Beef extract 10 Peptone 10 Casein enzyme hydrolysate 3 Sodium chloride 5 Sodium deoxycholate 1 Ferrous sulphate .25 Sodium pyruvate .25 charcol 4 Agar 12
  • 53.
    Translucent droplet coloniesof Campylobacter on Skirrow medium Colonies of H.pylori on Skirrow medium C.jejuni on Charcoal based selective medium
  • 54.
    Legionella and Francisella •Buffered Charcoal Yeast Extract Agar (BCYE) • Selective BCYE Agar • BMPA agar • Modified Wadowsky–Yee Medium (MWY medium) (Contains glycine, vancomycin, polymyxin B and anisomycin.) Legionella • Glucose cystine agar (BCG) • Cystine-heart agar (CHAB) • BCYE agar • Modified Thayer-Martin agar Francisella
  • 55.
    Buffered Charcoal YeastExtract Agar (BCYE) Selective isolation and cultivation of Legionella species PRINCIPLE & INTERPRETATION: •Charcoal acts as a detoxicant. •Yeast extract acts as a rich source of vitamins, nitrogen as well as carbon. •L-cystine hydrochloride; ferric pyrophosphate and a-ketoglutarate stimulate growth of Legionella species •ACES buffer maintains optimal pH. Legionella colonies are gray-white to blue-green, glistening, convex, and circular and may exhibit a cut-glass type of internal granular speckling. Legionella pneumophila ATCC 33153 luxuriant Enterococcus faecalis ATCC 29212 inhibited Ingredients Qty. (gm/l) Yeast extract 10 Activated charcol 2 Alpha ketoglutarate, mono potassium salt 1 ACES buffer 10 Agar 12 Final pH 6.8±0.2
  • 56.
    Selective BCYE Agar-BCYE+ Glycine+ Polymyxin-B (79,200 IU/L)+ Cycloheximide(80mg/L)+ Vancomycin(5mg/L). BMPA Medium- BCYE base supplemented with cefamandole, polymyxin-B and anisomycin BYCE Agar also supports growth of Francisella tularensis and Bordetella pertussis. Colonies of Legionella on BCYE Agar
  • 57.
    Media for Isolationof Francisella •The organism is strictly aerobic and is enhanced by enriched medium containing sulfhydryl compound (cystine, cystein, thiosulphate, IsoVitalex) •Two commercial media available: Glucose cystine agar (BCG) and Cystine-heart agar (CHAB), both require the addition of 5% sheep or rabbit blood. •Colonies are minute droplet like (2-4mm), butyrous and smooth with an opalescent sheen. glucose 2.5% Cystine HCL 0.1% Blood(human /Sheep/rabbit) 5% Nutrient agar Francisella tularensis ATCC 29684 luxuriant
  • 58.
    Spirochaetes • Modified KorthofMedium • Fletcher’s Medium • Ellinghausen-McCullough- Johnson-Harris (EMJH) Medium Leptospira • Barbour-Stoenner-Kelly Medium (BSK II) Borrelia
  • 59.
    Modified Korthof Medium Liquidmedia used for isolation, cultivation and maintenance of Leptospira species. PRINCIPLE & INTERPRETATION: •Peptic digest of animal tissue provide amino acids and other nitrogenous substances •The salts supply essential nutrients •Phosphates form buffering system while sodium chloride maintains osmotic equilibrium and provides essential ions. Growth occurs as a ringed area (disc) 1-3 cm below the surface of the medium. Growth is examined using a direct wet preparation under dark field illumination. Peptic digest of animal tissue 0.8 Sodium chloride 1.4 Sodium bicarbonate 0.02 Pottassium chloride 0.04 Calcium chloride 0.04 Monopotassium hydrogen phosphate 0.240 Disodium hydrogen phosphate 0.880 Final pH 7.2±.2
  • 60.
    Fletcher’s Medium Semi-solid Mediumfor isolation of Leptospira species. Advantages over Liquid medium- 1. Evaporates less rapidly 2. Helps to maintain the virulence of freshly isolated strains 3. Used for preparing cultures for iv inoculation into rabbits for production of hyperimmune serum. • Low concentration of agar helps in detecting motility • Leptospira multiply within the upper part of the tube forming zones of turbidity at varying depths, known as Dinger’s Rings. ingredients Qty. (gm/l) Peptic digest of animal tissue 0.3 Beef extract 0.2 Sodium chloride 0.5 Agar 1.5 Final pH 7.9±.2 DINGER’S RING
  • 61.
    EMJH Medium (Ellinghausen-McCullough-Johnson-Harris) Semi-solid mediumwhere rabbit whole serum is replaced by bovine serum albumin (fraction V) and polysorbate (Tween 80). Thiamine, Albumin and polysorbate 80 are growth factors Growth occurs as a band 0.5 to 1.0 cm below the surface of semisolid media, known as Dinger’s ring.
  • 62.
    Barbour-Stoenner-Kelly Medium (BSKII) •This medium is complex mixture of different amino acids, vitamins and growth factors which are required for the growth of Borrelia and Spirochaete PRINCIPLE & INTERPRETATION: • Peptone, special serves as nitrogen source while glucose as energy source. •Cholesterol acts as source of lipids. • HEPES provides buffering capacity . •Salts of Magnesium, sodium, calcium and potassium maintain the ionic balance. •Cultures are examined by dark-field microscopy or by fluorescence microscopy . B. burgdorferi ATCC 35210 Fair to good S. aureus ATCC 25923 Inhibited Ingredients- CMRL 1066 medium Neopeptone Bovine serum albumin, fraction V yeastolate HEPES glucose Sodium pyruvate Sodium Citrate N-acetylglucosamine Sodium bicarbonate 7% gelatin Unheated rabbit serum
  • 63.
    Mycoplasmas and Ureaplasmas •Methylene Blue-Glucose Biphasic Medium • Mycoplasma Glucose Agar Medium • SP-4 Medium • Hayflick’s Medium Mycoplasma pneumoniae • A-8 Agar • 10B Broth • SP-4 Broth or Agar • Bromothymol Blue Broth • Shepard’s 10B broth Mycoplasma hominis • SP-4 Broth or Agar • A-8 Agar • 10B Broth • Bromthymol Blue Broth • Shepard’s 10B broth Ureaplasma
  • 64.
    Methylene Blue-Glucose BiphasicMedium •Biphasic medium for isolation of M.pneumoniae •Solid phase is dispensed in sterile bottles ,allowed to set and then overlayed with liquid phase. PRINCIPLE & INTERPRETATION: •Methylene blue acts as selective agent •During growth , the medium becomes acidic and phenol red turns colour from salmon to yellow. •Reduction of methylene blue turns medium from blue to colourless. •Hence, colour of broth phase changes from purple to green, while the agar phase turns from purple to yellow. Mycoplasma pneumoniae ATCC 15531 Growth; colour change Escherichia coli ATCC 25922 Inhibited Ingredients PPLO (agar base/broth) w/o crystal violet Yeast extract Sodiumdeoxyribonucleate (calf thymus) solution (0.2%) Thallous acetate solution Penicillin solution Horse serum (unheated) Glucose solution (10%) Phenol Red solution (0.2%) Methylene blue solution(0.1%)
  • 65.
    SP 4 Medium •Forisolation of M. pneumoniae, M. hominis, and Ureaplasma spp. •Most commonly used media for isolation of genital mycoplasmas PRINCIPLE & INTERPRETATION: •May be used as liquid/solid form or as Biphasic medium. •CMRL 1066 (a cell culture medium) containing 21 amino acids, 13 vitamins, nucleic acid precursors, enzyme co-factors and cholesterol is added. •Arginine, urea, and antibiotics are selective agents. • Mycoplasma appear as tiny "fried-egg" colonies. Colonies range from 20-300 µm in diameter. Glucose fermenting mycoplasma SP4 with Glucose Red color to yellow Ureaplasma SP4 with Urea Yellow-orange to red Arginine using mycoplasma SP4 with Arginine Orange to red Ingredients- Mycoplasma broth base Tryptone Peptone Glucose Fetal Bovine Serum CMRL 1066 medium 10x concentrate with glutamine Fresh yeast extract (25%) Yeastolate (2%) Penicillin (100,000 units/ml) Phenol Red solution (0.1%)
  • 66.
    Growth of M.pneumoniae on MethyleneBlue-Glucose Biphasic Medium Fried egg colonies of Mycoplasma SP-4 Medium
  • 67.
    REFERENCES • Mackie &McCartney Practical Medical Microbiology (14th edition) • Bailey and Scott’s Diagnostic Microbiology (15th edition) • HIMEDIA • Nichols T, Freeman R. A new selective medium Streptococcus pneumoniae. J Clin Pathol. 1980 Aug;33(8):770-3. doi: 10.1136/jcp.33.8.770. PMID: 7430390; PMCID: PMC1146214.
  • 68.