Final presentation CULTURE MEDIA.pptx

Culture media

1. CULTURE MEDIA By: Dr Mansi, Dr Preeti, Dr Savita Guide: Dr Vaishali Wabale

2. Index Introduction Definition History Classification Preparation, sterilization and testing of media Simple media, Enriched media and Enrichment media Selective and Differential media Transport media Biochemical media Fungal, Parasitic and Viral media Anaerobic media Disposal of media Non culturable microorganisms

3. Introduction Microorganisms have to be grown (cultured)for them to be identified. Cultivation is growing microorganisms in culture by taking bacteria from infection site by specimen collection and growing them in artificial environment. Culture is the most common diagnostic method used for detection of bacterial infections.

4. History Robert Koch Louis Pasteur

5. Culture M edia : Classification Based on consistency Based on method of growth detection

6. A. Based on consistency Liquid media or broth :no agar Semi solid media : 0.5%agar Solid media : 2%agar

7. B. Based on the method of growth detection Conventional culture media Simple or ba s al media Enriched media Enrichment broth Selective media Differential media Transport media Anaerobic media Automated culture media

8. Preparation of culture media Weigh the dehydrated media in required quantity. Dissolve in purified/distilled water as per the manufacturer instruction Heat to boiling to dissolve the medium completely Check the pH of the media by using narrow range pH paper Compare the colour of the pH against the chart provided.

9. Sterilization of culture media The following methods are used to sterilize culture media: >Autoclaving >Steaming at 100⁰C >Inspissation >Filtration

10. Dispensing sterile media into petri dishes Pour plates under sterile conditions within the hood Lay out the sterile petri dishes on clean surfaces Name of the medium and date of preparation should be mentioned on the plate. Media should not be warmer than 60⁰C when poured Mix the medium gently by rotating the flask,avoid air bubbles Hold the plug of flask in such a position that it does not get contaminated Flame sterilize the neck of the flask Pour 15-20ml of medium into each disc(90-100mm diameter)

11. Storage of culture media >Stored at dark place >At 2-8⁰C Testing of culture media >Sterility testing- Done for routine media to which sheep blood or other substances are added after autoclaving. 5% batch incubated at 35-37⁰C overnight. All media to be checked visually before using.

12. Name of the medium Controls Sheep blood agar Alpha hemolysis -Streptococcus viridans Beta hemolysis - Streptococcus pyogens Gamma hemolysis - Enterococcus spp Chocolate agar Alpha hemolysis - Streptococcus pneumoniae Nutrient agar Pigmented strain of Pseudomonas MacConkey agar LF –Escherichia coli NLF -Pseudomonas TCBS(Thiosulfate-citrate-bile salts-sucrose agar) Sucrose fermenting- Vibrio cholerae Wilson Blair H2S producing- Salmonella typhi H2S non producing- Salmonella para typhi A Alkaline peptone water Positive control- Vibrio cholerae Negative control- Escherichia coli >Performance testing

13. Name of the medium Controls Selenite F broth Positive control- Salmonella Negative control- Escherichia coli Potassium tellurite agar Black coloured colonies -Corynebacterium diphtheriae CLED(Cystine-Lactose-Electrolyte-Deficient agar) Staphylococcus aureus -yellow colonies Escherichia coli- yellow colonies Pseudomonas -green colonies Triple sugar iron agar Acid/acid , with/without gas -Escherichia coli Acid/acid,H2S- Proteus Alkaline/no change- Pseudomonas Simmon’s citrate medium Positive control- Klebsiella Pneumoniae Negative control- Escherichia coli Christensen’s urea medium Positive control- Proteus spp Negative control- Escherichia coli Bile esculin agar Positive control- Enterococcus species Negative control- Streptococcus viridans Cetrimide agar Positive control- Pseudomonas aeruginosa Negative control- Escherichia coli

14. Simple /basal media : They contain minimum ingredients that support the growth of non fastidious bacteria. Name of media Components Primary purpose Peptone water Peptone(1%)NACI,(0.5%)water >Used as growth medium >Base for carbohydrate media >Used for indole test Nutrient broth Peptone water , meat extract(1%) Isolation of non-fastidious organisms Nutrient agar Nutrient broth ,2%agar Isolation of non-fastidious organisms Semi-solid agar Nutrient broth,0 .2 to 0.5%agar Determination of bacterial motility

15. Nutrient broth Nutrient agar Semisolid medium demonstrating bacterial motility

16. Enriched media: Substances like blood , serum , egg are added to the simple/basal medium to support growth of fastidious bacteria. Enriched media components Primary purpose Blood agar 5-10%sheep blood added to molten NA at 45⁰C- 55⁰C Isolation of Staphylococcus , Streptococcus. Chocolate agar 5-10%sheep blood added to molten NA at 72⁰C Hemoglobin , hemin(X factor) and Co-enzyme nicotinamide adenine dinucleotide(NAD or V factor) Isolation of Streptococcus pneumonae , Neisseria gonorrhoeae , Hemophilus spp.

17. Blood agar Chocolate agar alpha- hemolysis Streptococcus viridans

18. Enriched media Components Primary purpose Brain heart infusion broth Calf brain , Beef heart , proteose peptone , dextrose , sodium chloride , disodium hydrogen phosphate Isolation of fastidious pathogenic microorganisms like streptococcus , meningococci and pneumococci BACTEC automated blood culture bottle (BD BACTEC™ Automated Blood Culture System ) Soybean casein digest broth, yeast , amino acids , sugar , vitamins , sodium polyanetholsulfonate and Non-ionic adsorbing resins and cationic-exchange resins Detection of bacteria , fungus and viruses Castaneda’s biphasic media Trypticase soy agar,Trypticase soy broth Isolation of Brucellae from blood

19. Castaneda’s biphasic media BACTEC automated blood culture bottle Brain heart infusion broth

20. Name of the media Components Primary purpose Serum containing enriched medium Loeffler serum slope Nutrient broth, glucose, ox/sheep/horse serum Isolation of Corynebacterium diphtheriae. Egg containing enriched medium Lowenstein Jensen medium Dorset egg medium >Potato flour, L-Asparagine , Monopotassium phosphate , Magnesium citrate , Malachite green , glycerol , Egg suspension , distilled water >Nutrient broth , whole fresh eggs. >Isolation of Mycobacterium tuberculosis and Atypical mycobacteria . > Isolation of Corynebacterium diphtheriae. Buffered charcoal yeast extract agar Yeast extract, agar, charcoal and salts supplemented with L-cysteine , HCL , ferric pyrophosphate and alpha-ketoglutarate. >Enrichment media for Legionella spp. >Supports growth of Nocardia.

21. Loefflers serum slope Lowenstein-Jensen medium Dorset egg agar Buffered charcoal yeast extract agar

22. Enrichment media: They are liquid media added with some inhibitory agents which selectively allow certain organism to grow and inhibit others.

23. Enrichment media components Primary purpose Selenite F broth Peptone-base broth ; sodium selenite toxic for most Enterobacteriaceae Isolation of Samonella species Tetrathionate broth Peptone-base broth;iodine and potassium iodide,bile salts and sodium thiosulfate inhibit Gram positive organisms and Enterobacteriaceae Isolation of Salmonella and Shigella spp. Alkaline peptone water Peptone ,sodium chloride,distilled water. Isolation of Vibrio cholerae Gram negative broth Peptone-base broth with glucose and mannitol;sodium citrate and sodium deoxycholate act as inhibitory agents Isolation of Enteric pathogens

24. SELECTIVE AND DIFFERENTIAL MEDIA

25. NAME OF THE MEDIUM CONSTITUENTS USE MacConkey Agar Peptone base with lactose Bile salts (Sodium taurocholate) Indicator: Neutral Red SELECTIVE - Gram Negative bacilli DIFFERENTIAL - Lactose fermentation CLED (Cystine lysine electrolyte deficient agar) Peptone base with lactose, tryptone, L-cystine Indicator: Bromothymol blue DIFFERENTIAL - Lactose fermentation (mostly for bacteria from the urinary tract) EMB AGAR (Eosin Methylene Blue) Peptone base with lactose Indicators: Eosin Y and Methylene blue DIFFERENTIAL - Lactose fermentation (for Enteric bacilli)

26. MacConkey Agar CLED AGAR Levine EMB Agar

27. NAME OF THE MEDIUM CONSTITUENTS USE BEA (Bile Esculin Agar) Nutrient agar base with ferric citrate Brown colour of the medium is due to hydrolysis of esculin Indicator: Sodium Desoxycholate (Bile salt) DIFFERENTIAL - Isolation and presumptive identification of group D streptococci and enterococci POTASSIUM TELLURITE AGAR Sheep blood agar base with potassium tellurite SELECTIVE: Corynebacterium diphtheriae (Black coloured colonies) TCBS AGAR (Thiosulfate citrate bile sucrose) Peptone base agar with yeast extract, bile salts, citrate, sucrose, ferric citrate and sodium thiosulfate Indicator: Bromothymol blue SELECTIVE: Vibrio spp (Yellow coloured colonies) DIFFERENTIAL: Sucrose fermentation

28. Bile Esculin Agar Potassium Tellurite Agar TCBS Agar

29. NAME OF THE MEDIUM CONSTITUENTS USE SS AGAR (Salmonella Shigella Agar) Peptone base with lactose, ferric citrate and sodium citrate Inhibitors: Brilliant green and bile salts Indicator: Neutral red SELECTIVE - Salmonella and some Shigella spp WILSON BLAIR AGAR Peptone agar base with beef extract, dextrose, disodium phosphate and Bismuth sulphite Inhibitor: Brilliant green dye Indicators: Ferrous sulphate SELECTIVE – Isolation and preliminary identification of Salmonella spp from clinical specimens DIFFERENTIAL – Dextrose fermentation DCA (Deoxycholate citrate agar) Peptone base agar with lactose, sodium citrate, sodium thiosulfate, ferric citrate Inhibitor: Sodium deoxycholate (Inhibits Gram positive organisms) Indicator: Neutral Red DIFFERENTIAL - Salmonella and Shigella spp from other Gram negative enteric bacilli XLD AGAR (Xylose Lysine Deoxycholate) Yeast extract agar with lysine, xylose, lactose, sucrose and ferric ammonium citrate Inhibitor: Sodium deoxycholate (Inhibits Gram positive organisms) Indicator: Phenol Red DIFFERENTIAL - Salmonella and Shigella spp from other Gram negative enteric bacilli HE AGAR (Hektoen Enteric Agar) Peptone base agar with bile salts, lactose, sucrose, salicin and ferric ammonium citrate Indicators: Bromothymol blue and acid fuschin SELECTIVE - Salmonella and some Shigella spp DIFFERENTIAL - Salmonella and Shigella spp from other Gram negative enteric bacilli

30. Wilson Blair Agar Salmonella colonies on Wilson Blair Agar

31. Salmonella-Shigella Agar Hektoen Enteric Agar

32. DCA Salmonella on DCA XLD AGAR Salmonella on XLD Shigella on XLD

33. NAME OF THE MEDIUM CONSTITUENTS USE BORDET-GENGOU AGAR Potato-glycerol-based medium enriched with 15 to 20% defibrinated blood Inhibitor: Methicillin (2.5 microgram/mL) SELECTIVE – Bordetella pertussis and Bordetella parapertussis REGAN LOWE AGAR Charcoal agar with horse blood, cephalexin and amphotericin B ENRICHMENT and SELECTIVE – Isolation of Bordetella pertussis THAYER MARTIN AGAR Blood agar base enriched with haemoglobin and supplement B Inhibitors: Colistin, Nystatin, Vancomycin, Trimethoprim SELECTIVE – N. gonorrhoeae and N. meningitidis NYC AGAR Peptone agar base with cornstarch supplemented with yeast dialysate, 3% hemoglobin and horse plasma Antibiotic supplements include: Vancomycin (2 microgram/mL) Colistin (5.5 microgram/mL) Amphoterecin B (1.2 microgram/mL) Trimethoprim (3 microgram/mL) SELECTIVE – Neisseria gonorrhoeae Also supports growth of Ureaplasma urealyticum and some Mycoplasma spp.

34. THAYER MARTIN AGAR

35. NAME OF THE MEDIUM CONSTITUENTS USE CAMPY BLOOD AGAR Brucella agar base with sheep blood and the following: Vancomycin(10 mg/L) Trimethoprim(5 mg/L) Polymyxin B(2500 U/L) Amphoterecin B(2 mg/L) Cephalothin(15 mg/L) SELECTIVE – Campylobacter spp. CVA AGAR ( Cefoperazone , Vancomycin, Amphoterecin meduim ) Blood supplemented with the 3 drugs to inhibit growth of most Gram-negative bacteria, Gram-positive bacteria and yeast, respectively. SELECTIVE – C ampylobacter spp SKIRROW AGAR Peptone and soy protein base agar with lysed horse blood Inhibitors: Vancomycin – Gram positive organisms Polymyxin B and Trimethoprim – most Gram negative organisms SELECTIVE – Campylobacter spp

36. Campy Blood agar Skirrow agar

37. NAME OF THE MEDIUM CONSTITUENTS USE BURKHOLDERIA CEPACIA SELECTIVE AGAR Bile salts Gentamicin Ticarcillin Polymyxin B Peptone Yeast extract SELECTIVE - B urkholderia cepacia (Cystic fibrosis patients) CNA AGAR (Columbia Colistin-Nalidixic acid agar) Columbia agar base with 10 mg/L colistin 15 mg/L Nalidixic acid 5% sheep blood SELECTIVE - Isolation of Gram-positive cocci CIN AGAR ( Cefsulodin - Irgasan -Novobiocin agar) Peptone base with yeast extract, mannitol and bile salts Inhibitors: drugs Indicator: Neutral red and Crystal violet SELECTIVE - Isolation of Yersinia spp and Aeromonas spp DNase agar Tryptose, Deoxyribonucleic acid, Sodium Chloride, Agar DIFFERENTIAL – Deoxyribonuclease enzyme producing property Eg. Serratia marcescens

38. BCSA CIN Dnase AGAR

39. NAME OF THE MEDIUM CONSTITUENTS USE MANNITOL SALT AGAR Peptone base, mannitol Inhibitor: 7.5% concentrated salt Indicator: Phenol red SELECTIVE DIFFERENTIATION - Staphylococci PEA (Phenylethyl alcohol agar) Nutrient agar base Inhibitor: Phenylmethanol (inhibits Gram negative organisms) SELECTIVE - Isolation of: Aerobic Gram positive cocci and bacilli Anaerobic Gram positive cocci and negative bacilli SSA (Streptococcal selective agar) 5% sheep blood agar base with colistin and trimethoprim-sulfamethoxazole Indicator: Crystal violet SELECTIVE - Streptococcus pyogenes and Streptococcus agalactiae

40. STREPTOCOCCAL SELECTIVE AGAR

41. MILK AGAR: NON-SELECTIVE SOLID MEDIUM CONSTITUENTS: Peptone agar base with yeast extract and milk solids USE: Identification of organisms that can hydrolyse casein eg. Streptomyces, Pseudomonas and Actinomadura. For differentiation of aerobic actinomycetes based on casein proteolysis. It is used to demonstrate the pigment producing properties of certain organisms.

42. MUELLER HINTON AGAR N on-selective, non-differential medium Constituents : Beef Extract, Acid Hydrolysate of Casein, Starch and Agar. Uses  1. Routine susceptibility testing of non-fastidious microorganisms by the Kirby-Bauer disk diffusion technique. 2. It can be used to cultivate  Neisseria 3. It is specified in FDA Bacteriological Analytical Manual for food testing, and procedures commonly performed on aerobic and facultative anaerobic bacteria .

43. TRANSPORT MEDIA

44. NAME OF THE MEDIUM CONSTITUENTS USE CARY-BLAIR Soft agar semisolid medium with sodium thioglycolate, disodium hydrogen phosphate, NaCl and CaCl2 To transport enteric pathogens Eg. Shigella, Salmonella, Vibrio cholerae and E. coli For detection of Campylobacter spp from faeces (when transport time < 2 hours) STUART’S Soft agar medium with sodium thioglycolate ( mercaptoacetate ), sodium glycerophosphate, CaCl2, Distilled water and methylene blue To maintain viability of gonococci on swabs during their transmission. Also for Shigella and E. coli AMIES Soft agar medium with sodium thioglycolate ( mercaptoacetate ), NaCl, KCl , CaCl2, MgCl2, Na2PO4, Potassium dihydrogen phosphate, CHARCOAL (finely powdered), distilled water To maintain viability of gonococci on swabs during their transmission. Also for Shigella and E. coli

45. STUART’S

46. NAME OF THE MEDIUM CONSTITUENTS USE PIKE’S Blood agar containing 1 in 10 lakh Crystal violet 1 in 16000 Sodium azide Distributed as for stab cultures in tubes or bottles Preservation of Streptococcus pyogenes , pneumococci and Haemophilus influenzae in nose and throat swabs SACH’S BUFFERED GLYCEROL SALINE Distilled water and glycerol with NaCl, Disodium hydrogen phosphate, potassium dihydrogen phosphate and phenol red Preservation and transport of  Salmonella  and  Shigella   species and  Escherichia coli  in facial specimens. It is not suitable for  Vibrio cholera, Campylobacter species or  Yersinia enterocolitica.

47. PIKE’S MEDIUM

48. BIOCHEMICAL MEDIA

49. FERMENTATION TEST MEDIA Constituents: 1. Nutrient medium base: Peptone water, serum peptone water and serum agar 2. Carbohydrate or related compound under test (SUGARS): MONOSACCHARIDES Pentoses (Arabinose/ Xylose/ Rhamnose) Hexoses (Glucose/ Fructose/ Mannose/ Sorbose/ Galactose) DISACCHARIDES Sucrose/ inulin/ Dextrin/ Glycogen POLYHYDRIC ALCOHOLS Glycerol/ Erythritol/ Adonitol/ Mannitol/ Dulcitol/ Sorbitol/ Inositol GLYCOSIDES Salicin / Coniferin/ Aesculin ORGANIC ACIDS Tartrate( Dextro / Laevo / Meso ) / Citrate/ Mucate / Gluconate/ Malonate 3. Suitable Indicator: ANDRADE’S INDICATOR (NaOH with acid fuchsin till colour becomes yellow) BROMOCRESOL PURPLE PHENOL RED BROMOTHYMOL BLUE 4. A small inverted tube (DURHAM TUBE) completely filled with liquid and containing no air bubbles is usually included in each culture tube or bottle to detect gas.

50. TSI (Triple Sugar Iron): DIFFERENTIAL MEDIUM CONSTITUENTS : Agar slant of special medium with Phenol Red (pH sensitive dye), 1% lactose, 1% sucrose, 0.1% glucose, sodium thiosulfate and ferrous sulfate. Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow. USES: D ifferentiate members of the Enterobacteriaceae family from other gram-negative rods Differentiation among Enterobacteriaceae on the basis of their sugar fermentation patterns

51. INTERPRETATION OF TSI: An alkaline/acid (red slant/yellow butt) reaction:  Indicative of dextrose fermentation only An acid/acid (yellow slant/yellow butt) reaction:   F ermentation of dextrose, lactose and/or sucrose An alkaline/alkaline (red slant, red butt) reaction:  Absence of carbohydrate fermentation Blackening of the medium:  production of hydrogen sulphide Gas production : Bubbles or cracks (formation of CO 2 and H 2 )

52. OXIDATIVE FERMENTIVE MEDIA A semi-solid tubed medium containing the carbohydrate along with a pH indicator. Constituents : Peptone base agar with NaCl, Dipotassium hydrogen phosphate, distilled water and bromothymol blue. Fermenting organisms: Enterobacteriaceae, Aeromonas , Vibrio Oxidizing organisms: Pseudomonas Non-fermenting organisms: Alcaligenes faecalis

53. DECAROXYLASE MEDIUM Constituents: Decarboxylase test medium base (Peptone, meat extract, glucose, pyridoxal, bromocresol blue, cresol red and distilled water) The media is divided into four equal parts. One part is tubed without the addition of any amino acid, and the tube is labeled as ‘Control’. The remaining three parts are dispensed onto three tubes, to which L-lysine hydrochloride, L-arginine hydrochloride, and L-ornithine hydrochloride are added separately. Uses: Decarboxylase test is used to differentiate the members of the Enterobacteriaceae family with closely related physiological characteristics on the basis of their ability to produce the enzyme decarboxylase

54. Arginine decarboxylase POSITIVE: Enterococcus faecalis and Enterococcus faecium NEGATIVE: Enterococcus avium Lysine decarboxylase POSITIVE: Salmonella NEGATIVE: Shigella Ornithine decarboxylase POSITIVE: E. coli, Enterobacter cloacae NEGATIVE: Klebsiella pneumoniae, Klebsiella oxytoca

55. NAME OF THE MEDIUM CONSTITUENTS USE MR (METHYL RED) Ingredients per liter of deionized water: buffered peptone= 7.0 gm glucose= 5.0 gm dipotassium phosphate Positive Reaction:  A distinct red color   Examples:   E. coli, Yersinia   sps , etc. Negative Reaction:  A yellow color Examples:   Enterobacter aerogenes, Klebsiella pneumoniae,  etc INDOLE KOVACS MEDIUM P- dimethylaminobenzaldehyde Hydrochloric acid (37%) Amyl alcohol Positive:  Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent. Examples:   Escherichia coli ,  Haemophilus influenzae ,  Klebsiella oxytoca ,  Proteus  sp., Enterococcus faecalis , and  Vibrio  sp. Negative:  No color change Eg most  Haemophilus  sp., most  Klebsiella  sp.,  Neisseria  sp., Proteus mirabilis ,  P. penneri ,  Pseudomonas   sp ., Salmonella  sp.,  Serratia  sp.,  Yersinia   sp

56. NAME OF THE MEDIUM CONSTITUENTS USE VP (VOGES-PROSKAUER) VP REAGENT A: BARRITT’S REAGENT A Alpha naphthol 5% Absolute alcohol VP REAGENT B: BARITT’S REAGENT B Potassium hydroxide Deionized water Positive Reaction:  A pink-red color at the surface Examples:   Viridans  group streptococci, Enterobacter, Klebsiella, Serratia marcescens, Vibrio eltor . Negative Reaction:  A lack of a pink-red color Examples:   Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Salmonella, Vibrio parahaemolyticus 

57. NAME OF THE MEDIUM CONSTITUENTS USE SIMMONS’ CITRATE MEDIUM Koser’s medium with agar and Bromothymol blue as the indicator. Koser’s medium includes: NaCl, MgSO4, Ammonium dihydrogen phosphate, potassium dihydrogen phosphate and sodium citrate Positive Reaction:  Growth with color change from green to intense blue along the slant. Examples:   Salmonella, Edwardsiella , Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia , etc. Negative Reaction:  No growth and No color change; Slant remains green. Examples:   Escherichia, Shigella, Morganella , Yersinia  etc. CHRISTENSEN’S UREASE MEDIUM Peptone agar base with NaCl, dipotassium hydrogen phosphate, Glucose (10%), Urea (20%) Indicator: Phenol red to distinguish urease-positive Enterobacteriaceae .  can also be used to  differentiate between yeasts  : Candida albicans (negative urease) and Cryptococcus neoformans (rapid production of urease). detect the presence of  Helicobacter pylori

58. UREASE UTILIZATION TEST

59. NAME OF THE MEDIUM CONSTITUENTS USE MALONATE AGAR Distilled water with yeast extract, ammonium sulphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, NaCl and sodium malonate Indicator: Bromothyol Blue Differentiate among Enterobacteriaceae: Klebsiella pneumoniae is positive (blue at 24 hours), Escherichia coli is negative(culture remains green). Also used to separate Salmonella  arizonae  (positive) from other Salmonella  spp (negative). Differentiate Enterobacteriaceae in food and dairy products. CETRIMIDE AGAR Distilled water, agar, glycerine, MgCl2, Potassium sulfate, pancreatic digest of gelatin , cetyltrimethylammonium bromide selective medium for the isolation of  Pseudomonas aeruginosa used for determining the ability of an organism to produce fluorescein and pyocyanin ( Antibiotica )

60. NAME OF THE MEDIUM CONSTITUENTS USE HYDROLYSIS OF TRIBUTYRIN Peptone agar base with water, yeast extract and Tributyrin Lipolytic organisms remove the opacity by converting the fat to water soluble butyric acid