aerobic culture media-1 .........................................................

aerobic culture media/gram positive gram negative........

1. AEROBIC CULTURE MEDIA DR . LIKHITA 1 st YEAR PG DEPT.OF MICROBIOLOGY GOVT.MEDICAL COLLEGE ANANTAPURAMU

2. SPECIFIC LEARNING OBJECTIVES DEFINITION HISTORICAL INTRODUCTION COMMON MEDIA INGREDIENTS MEDIA PREPARATION TYPES OF CULTURE MEDIA APPLICATIONS REFERENCES

3. CULTURE AND MEDIUM CULTURE : Process of growing microorganisms by taking organisms from the infectious site (in vivo) and growing on the artificial environment of lab (in vitro) MEDIUM : Combination of ingredients that will support growth and cultivation of organisms by providing all the essential nutrients required for growth

4. MAJOR CONTRIBUTIONS 1860-LOUIS PASTEUR-EARLIEST-LIQUID ARTIFICIAL MEDIA

5. 1881-ROBERT KOCH DEVOLOPED TECHNIQUES TO GROW ORGANISMS ON SOLID MEDIA

6. 1881 FREDERICK LOEFFLER-ASSOCIATE OF KOCH DEVELOPED MEAT EXTRACT PEPTONE MEDIUM KOCH SOLIDIFIED THE MEDIUM WITH GELATIN COULD NOT BE INCUBATED AT 37 °C TEMP AS GELATIN WOULD MELT AND SOME BACTERIA DIGESTED THE GELATIN

7. 1882-MINORA TARAZAEMON- japanese innkeeper –AGAR FANNIE ELSHEMIUS HESSE –suggested use of AGAR in solidifying medium-instant success

8. 1887-JULIUS RICHARD PETRI-replaced glass plate with a circular culture box the PETRI BOX

9. Basic ingredients of culture media Water- water with low mineral content - Distilled water

10. AGAR Agar-agar, the Malay name for red algae ( Gigartina , Eucheuma , Gracilaria ) from which the jelly is produced.  Derived from the cell wall of sea weeds( Rhodophyceae ), available commercially in powder forms Forms base of solid media Bacteriologically inert Does not add nutritive properties Used at conc of 1-2% Melts at 98°Cand solidifies at 42°C Doesn’t alter the PH of the medium

11. AGAR Chief components: Long chain polysaccharides(D- galacto pyranose ) Minerals –mg2+ Ca2+ Inorganic salts Long chain fatty acids Concentration : 1-2% = solid medium 0.5% = semisolid medium 0.7-2% = agarose gel 6% = inhibit proteus swarming

12. PEPTONE Complex mixture of partially digested proteins Consists of water soluble products obtained from lean meat or other protein material – heart muscle, casein, fibrin or soya flour Usually with proteolytic enzymes- pepsin ,trypsin or papain IMPORTANT CONSTITUENTS : Peptones, proteases, amino acids, inorganic salts, accessory growth factors – nicotinic acid, riboflavin

13. PEPTONE Commercially – golden granular powder with < 5% moisture content Special grades of commercially available peptones 1. Neopetone 2. Protease peptone 3. Mycological peptone

14. CASEIN HYDROLYSATE Consists of amino acids obtained by the hydrolysis of the milk protein casein, phosphates and other salts. Tryptophan must be added May be substituted for peptone in broth /media

15. MEAT EXTRACT Contains several water soluble compounds including protein degradation products Gelatin, albumoses, peptones, proteoses and amino acids Nitrogen compounds – creatinine , creatinine carnosine, anserine, purines and glutathione's. Mineral salts – KH2PO4, NaCl Acc.GF – thiamine, nicotinic acid, riboflavin, pyridoxine, pantothenic acid and choline Commercial – lab- lemco , oxoid unipath

16. YEAST EXTRACT Prepared from washed cells of brewers or bakers yeast Wide range of amino acids Growth factors- VIT B GROUP, K+ and phosphate 10% is carbohydrate –glycogen, trehalose, pentose's

17. MALT EXTRACT Prepared by extracting the soluble materials from sprouted barley in water at about 55°C Liquor is strained & concentrated by evaporation at a temp below 55°C to yield brown viscous material Consists of maltose about 50% starch, dextrin's, glucose, and about 5% of proteins mineral salts GF’S – Thiamine, nicotinic acid, riboflavin, biotin, pantothenic acid, pyridoxin,folic acid , inositol

18. BLOOD Collected with aseptic precautions Must be rendered non coagulation by 1. defibrination 2. addition of citrate/oxalate DEFIBRINATION: Bottle containing glass beads is half filled with blood, stoppered at once and shaken continuously for 5 min OXALATED BLOOD: 10ml of 10% neutral potassium oxalate for 1 lit blood CITRATED BLOOD: Thoroughly shaken in a bottle with sodium citrate 60mg per 10ml of blood Source: horse ,rabbits, ox , sheep

19. SERUM Prepared from unsterile defibrinated / oxalated blood Filtered –sterilised-through a membrane filter / seitz filter Stored at 3-5°C

20. READYMADE MEDIA Ready prepared media or ready poured plates or as dehydrated powders More stringent quality control tests are advisable before using examples 1.Nutrient broth 2.Nutrient agar 3.Mac Conkey agar 4.Antibiotic sensitivity test agar

21. MEDIA PREPARATION Media power dissolved with water, usually by boiling. Sterilization by autoclave at 121°C for 15 minutes. Molten agar cooled to 45°C Poured into petri dishes(20 -25mL ) COMPONENTS CANNOT WITHSTAND AUTOCLAVING: Eg . Serum, carbohydrate solutions, etc, Sterilized by membrane filtration. Pore size of 0.2 -0.45micrometer.

22. ENVIRONMENTAL REQUIREMENTS: OXYGEN & CO2 : Aerobic – grow well in room air. e.g. : Pseudomonas, Neisseriae, brucella, etc MICROAEROPHILIC: Low levels of O2 (20%) CAPNOPHILIC: grow at higher co2 conc. (5-10%) TEMPERATURE: 35°TO 37°C – Internal human host tissues. Campylobacter jejuni – 42°C

23. ENVIRONMENTAL REQUIREMENTS: pH: Neutral pH – 6.5-7.5 MOISTURE: Water- major constituent of both agar & broth. Loss of water – deleterious to bacterial growth in 2 ways: 1) less water is available for bacterial metabolic pathways, 2)increased solute conc. of media bacterial cell lysis. Measures: sealing agar plates, humidified incubators.

24. BASED ON PHYSICAL NATURE SOLID MEDIUM - DISTINCT CULTURE MORPHOLOGY. CHARACTERISTICS - EASY TO IDENTIFY. MACROSCOPICALLY VISIBLE COLLECTIONS OF BACTERIA. EARLIEST MEDIUM: COOKED CUT POTATO BY ROBERT KOCH GELATIN – NOT SATISFACTORY LIQUIFY AT 24 C. LIQUID MEDIUM DIFFUSED GROWTH - NO CHARACTERISTIC FOR IDENTIFICATION. DIFFICULT TO ISOLATE. EARLIEST MEDIUM: URINE OR MEAT BROTH USED BY LOUIS PASTEUR.

25. SIMPLE /BASAL MEDIA PEPTONE WATER : Peptone -10 g NaCl -5g Water - 1 litre Dissolve the above ingredients in warm water, adjust the pH to 7.4-7.5 and filter. Distribute as required and autoclave at 121° C for 15 min USES For the growth of gram negative bacilli, to test indole production, used to prepare sugar media Alkaline peptone water (8.6)-enrichment of vibrio cholerae from faeces samples

26. SIMPLE /BASAL MEDIA NUTRIENT BROTH – peptone water + meat extract 1% Types – 1. Meat infusion broth 2. Meat extract broth 3. Hartley's digest broth 4. Horse flesh digest broth( hemolytic streptococci)

27. NUTRIENT BROTH INGREDIENTS GRAM/LITER PEPTONES 10g BEEF EXTRACT 1g YEAST EXTRACT 2g SODIUM CHLORIDE 5g pH final 7.4

28. SIMPLE /BASAL MEDIA NUTRIENT AGAR: Nutrient broth + 2% agar New Zealand agar is superior to Japanese agar Uses: To study colony characters To detect pigment formation To maintain stock cultures , AST Pure growths are for slide agglutination

29. NUTRIENT AGAR INGREDIENTS CONCENTRATION PEPTONE 1% MEAT EXTRACT 1% DISTILLED WATER 100mL SODIUM CHLORIDE 0.5% AGAR 2%

30. SIMPLE /BASAL MEDIA SEMI SOLID AGAR: Prepared by reducing the concentration of agar to 0.2-0.5% Uses – demonstrate the motility maintaining stock culture

31. USES OF BASAL MEDIA Testing the non-fastidiousness of bacteria They serve as the base for the preparation of many other media Nutrient broth is used for studying the bacterial growth curve Nutrient agar is the preferred medium for : Performing bio-chemical tests- oxidase , catalase and slide agglutination test, etc. To study the colony morphology Pigment demonstration

32. COMPLEX MEDIA has added ingredients to bring the characteristics of microorganisms with unique nutrients. e.g. :1) Tryptic soy broth, (soybeans) 2)Blood agar and 3)Nutrient broth. The exact composition of the medium is not known.

33. SYNTHETIC MEDIA Chemically defined media and is produced from pure chemical substances A defined media refers to a medium having a known concentration of ingredients, like sugar (glucose or glycerol) and nitrogen source (such as ammonium salt or nitrate as inorganic nitrogen). It is generally used in scientific research, and an example is Czapek Dox Medium . (medium containing sodium nitrate as the sole source of nitrogen, most useful media for the general cultivation of fungi. highly satisfactory substrate for chlamydospore production by Candida albicans .)

34. ENRICHED MEDIA Basal medium added with additional nutrients such as blood ,serum, egg. Supports the growth of fastidious organisms Examples : Blood agar: prepared by adding 5-10% of sheep blood to the molten nutrient agar at 45°C To test the haemolytic property of the bacteria Either 1. partial / alpha/ green hemolysis 2. complete / beta hemolysis

35. BLOOD AGAR : Protein source – tryptones Soybean protein digest NaCl , agar Defibrinated blood is preferred over citrate blood Haemophilus sps – rabbit or horse blood Wagatsuma agar- special high salt(7%) blood agar -to differentiate pathogenic and non pathogenic strains of vibrio parahemolyticus – kanagawa phenomenon

36. kanagawa

37. LYSED BLOOD AGAR: heating at 55-56°C for app 1 hour NA-100ml, lysed blood – 10-20 ml Melt the NA, cool to 50°C, add the lysed blood Suitable for the culture of gonococci - sulphonamide sensitivity tests

38. CHOCOLATE AGAR/HEATED BLOOD AGAR PREPARATION: 1)Add 5-10% defribinated blood to sterile melted nutrient agar at 50-55°C in a boiling water bath at 75°C 2)Gentle agitation of blood and agar until medium becomes chocolate brown Lysed RBC release intracellular nutrients such as heme , hemin & (NAD) V factor – Favourable for the growth of fastidious organisms eg : neisseria , Haemophilus , Pnuemococci , Meningococci & Other fastidious organisms

39. LOEFFLER’S SERUM SLOPE: -contains serum -isolation of corynebacterium diphtheriae -10% sterile serum is added to sterile nutrient broth and glucose ,sterilized and solidified by inspissation - growth is rapid 6-8 hrs – yellow tint colonies

40. FILDES AGAR AND BROTH: fildes peptic digest to nutrient broth or agar in proportion of 2-5% after heating at 55°C for 30 min stimulates Haemophilus sps DORSETTS EGG MEDIUM: preservation of cultures diptheria and tubercle bacilli MILK AGAR: Staphylococci – pigment production 1% glycerol monoacetate pigment production

41. Bordet – Gengou Glycerine potato BA : Bordotella pertusis Serum dextrose and serum potato infusion agar : Brucella sps Yeast extract serum agar : Mycoplasma Egg yolk agar and Serum agar : Clostridium perfringens – nagler reaction Bacterial lecithinases breakdown the lecithin into insoluble diglycerides , resulting with an opaque and halo around the colony, when it is grown on egg yolk medium. Mannitol - egg yolk- phenolred - polymyxin (MYPA) agar : Bacillus cereus

42. (a),(b) Typical colonies of B. cereus grown on MYP agar plate

43. MEDIA FOR BLOOD CULTURE Monophasic medium- BHI broth nutritionally rich medium for fastidious and non fastidious organisms INGREDIENTS: Several animal tissue sources peptone phosphate buffer dextrose USE – major component of the media developed for culturing a patient blood for bacteria

44. MEDIA FOR BLOOD CULTURE Biphasic medium – BHI broth and BHI agar e.g. : Casteneda medium - solid phase -30ml sterile glucose serum agar - liquid phase – sterile glucose serum broth - Less chances of contamination - Isolation of brucella from blood

45. ENRICHMENT MEDIA Essentially liquid media Enhances the growth of particular bacterial pathogen from a mixture of organisms by providing specific nutrients for growth and inhibits the growth of non pathogens –sputum/faeces

46. ENRICHMENT MEDIA Back up broths/Enrichment broth: used to ensure growth of organism when there is no growth on solid media and prevent growth of contaminant organisms eg : Thioglycollate – anaerobes Selenite F broth- isolation of salmonella - peptone, sodium acid selenite, phosphate buffer with pH7.2 - incubated at 37°C for 6-8 hrs - sodium selenite is toxic to E.coli and Enterobacteria

47. ENRICHMENT MEDIA TETRATHIONATE BROTH : Nutrient broth , calcium carbonate, sodium thiosulphate iodine and phenol red indicator Enrichment media for salmonella and shigella from feaces samples Inhibits the growth of coliform bacilli Permits proteus Procedure : faeces inoculated on tetrathionate broth & incubated for 6-18hours & a subculture is made on MaC conkey agar & DCA medium

48. ENRICHMENT MEDIA GRAM NEGATIVE BROTH : Selective broth for cultivation of Salmonella and Shigella species -sodium citrate and sodium deoxycholate inhibits gram positive organisms and early multiplication of non enteric GN pathogens - mannitol – source of carbon MONSURS TAUROCHOLATE TELLURITE PEPTONE WATER(9.2): - Enrichment of vibrio cholera from faeces BILE BROTH : isolation of salmonella from blood SALT BROTH : 8-10% NaCl – enrichment of Staph aureus

49. SELECTIVE MEDIA Solid media in which chemical substance are incorporated which promotes the growth of required bacterial pathogens selectively and suppresses the growth of unwanted commensal non pathogenic bacteria Wilson and Blair’s bismuth sulphite Agar : For isolation of typhoid and para -typhoid bacilli from faces Addition of Bismuth-sulphite-phosphate-glucose mixture and iron citrate- brilliant green mixture to nutrient Agar Bismuth sulphite and brilliant green are selective agents for salmonella and inhibits the growth of intestinal organisms Salmonella typhi and paratyphi both reduces sulphide to sulphide in the presence of glucose and produce black colonies with a metallic sheen due to formation of H2S Salmonella A and salmonella cholera suis –green colored colonies

50. SELECTIVE MEDIA 2) TCBS AGAR : Thiosulphate Citrate Bile Sucrose Agar Isolation of vibrio Cholera and vibrio para hemolyticus inhibits growth of Enterobacteria and Gram Positive bacteria pH 8.6 , indicator - bromo thymol blue Vibrio Cholera produces large yellow convex colonies due to sucrose fermentation vibrio para hemolyticus does not ferment sucrose and produce green colour colonies

51. SELECTIVE MEDIA 3) Lowenstein Jensen(LJ) medium : Cultivation and differentiation of human and bovine types of tubercle bacilli and atypical mycobacteria Mineral salts, glycerol(carbon) aspergine (N2 ) malachite green-selective agent Medium is sterilized and solidified simultaneously by inspissation and inspissator egg coagulation solidifies instead of agar

52. TCBS Agar L J medium

53. SELECTIVE MEDIA 4)Potassium Tellurite Blood Agar (or) McLeod's medium Cultivation of Corynebacterium diphtheria McLeod- heated rabbit blood and potassium Telliurite called chocolate telliurite Agar Diphtheria bacilli reduce telliurite to metallic tellurium and give black colour colonies Based on potassium Telliurite chocolate Agar medium McLeod classified diphtheria into three types Gravis- Daisy head colonies Intermediate – frog’s egg colonies Mitis - poached egg Colonies

54. Wilson -Blair Pottasium tellurite choclate agar salmonella Corynebacterium

55. SELECTIVE MEDIA Deoxycholate citrate Agar: For isolation of Salmonella and Shigella Sodium citrate, ferric citrate, sodium thiosulphate, lactose, deoxycholate added to nutrient Agar Neutral red- indicator Colony shows Central black dot( H2S and Ferric citrate produce iron sulphide)

56. SELECTIVE MEDIA XLD( Xylose -Lysine- Deoxycholate)AGAR: 1) isolation of Salmonella and Shigella species 2) xylose is not fermented by Shigella but practically by all enterics 3) Lysine to differentiate the Salmonella group from the non-pathogens SS(salmonella – shigella )AGAR: For the isolation of salmonellae and shigellae

57. DIFFERENTIAL MEDIA MAC CONKEY AGAR: Differential & indicator medium used for isolation of enteric GNB. CONTENTS: P eptone L actose A gar N eutral red – indicator T aurocholate

58. DIFFERENTIAL MEDIA Cystine Lactose Electrolyte Deficient (CLED) Agar Culture of urine specimens Absence of electrolytes inhibits swarming of proteus Alternative to Blood agar and MacConkey agar Differentiates lactose and non lactose fermenting bacteria

59. INDICATOR MEDIA Indicate a property of the bacterium Media contains a indicator which changes color when bacteria grows in it

60. TRANSPORT MEDIA When there is a risk of pathogen survival or overgrowth of non-pathogens, during the time of transport of specimen to the laboratory Stuarts transport medium- Gonococci Sachs buffered glycerol saline – Enteric bacilli Cary blair medium – Vibrio cholera V.R. Fluid (pH- 9.2) – Vibrio cholera Alkaline peptone water - Vibrio cholera Pikes medium – streptococcus pyogenes , - pnuemococci - H.influenza Amies transport medium - Neisseria gonorrhoeae

61. Mueller Hinton Agar (MHA) Contains Beef Extract, Acid Hydro lysate of Casein, Starch and Agar. For antimicrobial susceptibility testing. Used to cultivate  Neisseria Addition of 2% glucose and methylene blue for antifungal disc diffusion susceptibility test Mueller Hinton broth – determining MIC

62. APPLICATIONS Demonstrate characteristic properties that have clinical significance and helps in identification Determine antibiotic sensitivity Study their physiological virulence and genetic properties To obtain sufficient growth for preparing antigens and vaccines Research and epidemiological purposes

63. references

64. THANK YOU