Bacterial culture media بكتريا عملي
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This document discusses different types of culture media used to grow microorganisms outside of their natural habitats. It describes various media including basic media, enriched media containing additional nutrients, selective and differential media that inhibit some bacteria and allow easy identification of others based on colony characteristics. Transport media are also discussed which maintain specimens and prevent overgrowth until laboratory analysis. The document provides examples of commonly used media for different applications and microorganisms.
Bacterial culture media بكتريا عملي
- 1. Bacterial Culture Medium By Dr. Nabil El Aila Assistant Professor of Molecular Microbiology Medical Technology Department Al -Aqsa University
- 2. Culture and Medium •Culture is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them. •Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth. - Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats Dr. Nabil El Aila General Microbiology
- 3. Basic requirements of culture media •Nutrients - Energy source - Carbon source - Nitrogen source •Mineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. •A suitable pH – 7.2 – 7.4 •Accessory growth factors - Tryptophan for Salmonella typhi - X & V factors for H. influenzae Dr. Nabil El Aila General Microbiology
- 4. Agar - Agar •Agar is obtained from Sea weeds •Agar contain long chain polysaccharides. Inorganic salts and protein like substance •Used as solidifying agent for culture media in Petri plates, slants, and deeps •Generally not metabolized by microbes •Liquefies at 98 C and Solidifies ~42 C Dr. Nabil El Aila General Microbiology
- 5. 5 Culture medium –is the mixture of various nutrients that is suitable for the growth of microorganisms. Types of Culture Media based on the function and chemical components based on the physical state Dr. Nabil El Aila General Microbiology
- 6. 6 Based on the physical state •Liquid medium: –Without agar. –for the proliferation of bacteria. •Solid medium: –1.5-2.5% agar. –for the isolation and identification of bacteria –e.g., slant, Petri dishes/plates. •Semisolid medium: –0.3-0.5% agar. –for the observation of bacterial motility and preservation of bacteria. Dr. Nabil El Aila General Microbiology
- 7. ii.Growth of bacteria in culture medium i.Liquid medium or Broth 1)Homogeneous turbidity 2)Surface 3)Bottom ii.Solid agar medium Colony and mossy 1)Smooth colony 2)Rough colony 3)Mucoid colony iii.Semi-solid agar medium Dr. Nabil El Aila General Microbiology
- 8. Blood culture – ‘Liquid Medium’
- 9. Semisolid medium The organism in the tube on the left in the figure is motile and the organism in the tube on the right is nonmotile. Dr. Nabil El Aila General Microbiology
- 10. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media Dr. Nabil El Aila General Microbiology
- 11. Types of culture media •Basic media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basic medium. Dr. Nabil El Aila General Microbiology
- 12. Peptone •Peptone contain partially digested proteins •Proteases •Polypeptides •Aminoacids •Inorganic salts Phosphates Potassium and Magnesium Riboflavin Meat exract called as Lab lemco
- 13. Carbohydrate media •Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% soln(1ml) •Dissolve the desired carbohydrate in peptone water and steam for 30 min or sterilize by filtration. •Use: To test the fermenting ability of an organism Dr. Nabil El Aila General Microbiology
- 14. Nutrient Agar •Contain 2% agar added to Nutrient agar commonly used •Concentration can be increased to 6% to prevent swarming •Can be reduced to 0’5%
- 16. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media Dr. Nabil El Aila General Microbiology
- 17. •Enriched media: Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. - Enriched media are used to grow nutritionally exacting (fastidious) bacteria. - Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media. Dr. Nabil El Aila General Microbiology
- 18. Enriched Medium •To culture medium Blood serum or egg are added to medium • eg Blood agar •Chocolate agar •Egg Dr. Nabil El Aila General Microbiology
- 19. Dr. Nabil El Aila General Microbiology
- 20. Different types of hemolysis on Blood Agar Dr. Nabil El Aila General Microbiology
- 21. Other Enrichments – Chocolate Agar •Several organic materials are added to the basic constituents of the Medium such as Blood, yeast, yeast extract etc Dr. Nabil El Aila General Microbiology
- 22. Chocolate agar
- 23. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media
- 24. •Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. - While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. - Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. - Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. - Examples of selective media like Salmonella shigella agar, XLD agar, Heckton Enteric agar, Mannitol salt agar, Lowenstein Jensen’s medium •Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. - Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens.
- 25. Salmonella Shigella agar - For isolation and differential medium for pathogenic Gram-negative bacilli in particular, Salmonella and Shigella. Inhibitor for Coliforms. Dr. Nabil El Aila General Microbiology
- 26. For isolation of Salmonella and Shigella species from clinical samples XLD agar (Xylose Lysine Deoxycholate)
- 27. For isolation of Salmonella and Shigella species from clinical samples Hecktoen Enteric agar Dr. Nabil El Aila General Microbiology
- 28. Lowenstein Jensen Medium - cultivation of Mycobacterium tuberculosis
- 29. Lowenstein-Jensen’s medium •Mineral salt soln - 600ml Malachite green soln - 20ml (2gm% in D.water) Beaten egg - 1000ml (20-22 eggs) •Mix the above •Distribute in Mc Cartney bottles •Sterilize by Inspissation Use: To cultivate Mycobacteria Dr. Nabil El Aila General Microbiology
- 30. • For selective isolation of pathogenic staphylococci • Mannitol fermentation by pathogenic staphylococci is indicated by a yellow halo surrounding the colonies. •Sodium chloride is the inhibitor agent. •Phenol red is the PH indicator Mannitol Salt Agar ( MSA )
- 31. Enrichment Medium •If the sample contain more than one type of bacteria, undesired bacteria grwoth can be reduced or eliminated. •The desired organism is facilitated to grow •Eg Tetrathionate broth •Selenite F broth
- 32. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media Dr. Nabil El Aila General Microbiology
- 33. •Differential media or indicator media: Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. - Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. - Such media are called differential media or indicator media. - Examples: MacConkey’s agar, CLED agar, TCBS agar etc. Dr. Nabil El Aila General Microbiology
- 34. MacConkey agar •MacConkey agar is useful medium for cultivation of enterobacteria •It contains a bile salt to inhibit non intestinal bacteria •Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting Salmonella and Dysentery group
- 35. Differential Medium Mac Conkey's agar •Bringing out different characters of bacteria their atypical characters •Mac Conkey’s medium Contain peptone, Lactose Agar, Neutral red and taurocholate and show growth of Lactose fermenters as pink colored colonies Dr. Nabil El Aila General Microbiology
- 36. Lactose fermenting and Non lactose fermenting
- 37. TCBS medium (Thiosulphate Citrate bile salts sucrose agar) For cultivation of Vibrio cholera Dr. Nabil El Aila General Microbiology
- 38. Cystine-Lactose-Electrolyte-Deficient Agar (CLED agar) For isolation and differentiation of urinary pathogens on the basis of lactose fermentation Dr. Nabil El Aila General Microbiology
- 39. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media Dr. Nabil El Aila General Microbiology
- 40. •Transport media: Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. - This can be achieved by using transport media. - Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. - Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. - Addition of charcoal serves to neutralize inhibitory factors. - Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. - Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery.
- 41. Transport Medium •Stuart’s medium contain reducing agents to prevent oxidation. •Charcoal to neutralize certain bacterial inhibitors to Gonococci, Dr. Nabil El Aila General Microbiology
- 42. Types of culture media •Basic media •Enriched media •Selective and enrichment media •Enrichment media •Differential media or indicator media •Transport media •Anaerobic media Dr. Nabil El Aila General Microbiology
- 43. •Anaerobic media: Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients. •Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. •Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. •Robertson cooked meat that is commonly used to grow Clostridium spps. •Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the thioglycollate medium. Under reduced condition, methylene blue is colorless.
- 44. Anaerobic Medium •Robertson’s cooked meat medium •Thiglyclolate liquid medium
- 45. Dr. Nabil El Aila General Microbiology
- 46. Anaerobic Jar (GasPak) Clamp with clamp screw Lid with O-ring gasket Catalyst pellets in reaction chamber Flash arrester to prevent explosion GasPak disposable anaerobic indicator strip Culture plates GasPak disposable hydrogen and carbon dioxide generator envelope O O O H H H H Oxygen in jar (O2) Water (H2O) Hydrogen gas (H2) + 2 2 Platinum catalyst Dr. Nabil El Aila General Microbiology
- 47. Other/ungrouped media Dr. Nabil El Aila General Microbiology
- 48. Mueller Hinton Agar •It is a microbiological growth medium that support the growth of most microorganism. •It is commonly used for antibiotic susceptibility testing •typically contains (w/v): - 30.0% beef infusion - 1.75% casein hydrolysate - 0.15% starch - 1.7% agar •pH adjusted to neutral at 25 C. •Five percent sheep blood may also be added when susceptibility testing is done on Streptococcus species or the sensitivity can be done on Blood agar.
- 49. Muller Hinton Agar for Antibiotic Testing Dr. Nabil El Aila General Microbiology
- 50. Antibiotic Testing on Blood Agar Medium Dr. Nabil El Aila General Microbiology
- 51. Triple Sugar Iron Agar (TSI): •It is used for identification of a Gram- negative bacilli of the enteric group. •It contains glucose (0.1% ), Lactose (1%), sucrose(1%). And peptone (2%) as nutritional sources. •Sodium Thiosulfate serves as the electron receptor for reduction of sulfur and production of H2S. •Detects fermentation of sucrose, lactose, glucose, as well as production of hydrogen sulfide and /or gas . •Phenol red is the PH indicator; ferric ammonium citrate is H2S indicator. Dr. Nabil El Aila General Microbiology
- 52. 52 Triple sugar iron slant Dr. Nabil El Aila General Microbiology
- 53. The lysine iron agar slant or LIA slant test is used to distinguish bacteria which are able to decarboxylate lysine and/or produce hydrogen sulfide from those that cannot. This test is particularly useful for distinguishing different Gram-negative bacilli—especially among the Enterobacteriaceae Lysine Iron Agar (LIA):
- 54. Simmons' Citrate Agar is a defined, enrichment medium that tests for an organism's ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source. Simmons' citrate agar Dr. Nabil El Aila General Microbiology
- 55. Urea agar base Urea Agar Base (Christensen Agar Base). for the detection of urease producing organisms.
- 56. Sabouraud's Dextrose agar commonly used Fungal Isolation Medium
- 57. Sabouraud's Dextrose Agar Dextrose - 4 gm% Neopeptone - 1 gm% Agar - 1.5 gm% Distilled water - 100 ml •Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4 •Autoclave and dispense 20 ml amount in test tubes Use: For the cultivation of Fungi Dr. Nabil El Aila General Microbiology
- 58. Sterilization of culture media •Media are sterilized in the autoclave at 1210 c for 15 min under 15 lb of Pressure •Heat-labile substances like serum & sugar solutions must be sterilized by free-steam or filtration •Egg containing media –-- Lowenstein-Jensen’s medium, Loeffler's serum slope by inspissation •Discarded culture plates are to be sterilized by autoclaving prior to washing Dr. Nabil El Aila General Microbiology
- 59. Storage of culture media •Prepared media in individual screw capped bottles can be stored for weeks at room temp •Poured plates deteriorate quickly and often contaminated, hence cold storage is necessary •For smaller labs domestic refrigerators & for larger labs insulated cold room(4-5oc) •Deep freeze refrigerators for preservation of sera, antibiotics & amino acids (-10 to - 400c) Dr. Nabil El Aila General Microbiology