1. Culture Media Mohammad Rhbar (PhD) Dep. Of Microbiology, Iranian Reference Health Laboratory.

2. Introduction In the microbiology laboratory many tests and procedures depend on culture media being consistent and providing reproducible results. Several hundreds of formula of dehydrated culture media are commercially available and many more,designed for specific purpose, are described in literature.,in laboratories charring out the microbiological examination of clinical specimens the main objective are growth and rapid detection of pathogenic organisms.

3. Terminology of Culture Medium Lot or batch) Fully traceable unit of a medium referring to defined amount of bulk,semi-finished product or end product which is consistent in type and quality and which has passed the requirements production (in-process control) and quality assurance testing and which has been produced within one defined production period have been assigned the same lot number.

4. Terminology.. Culture media: Formulation of substance,in liquid, semi-solid or in solid form, which contains natural and/or synthetic constituents intended to support the multiplication, or to preserve viability of microorganisms

5. Culture media classified by composition Chemically defined media: culture medium consisting of chemically defined constituents (i.e. of known molecular structure and degree of purity )only Chemically undefined culture media Culture medium consisting entirely or partly of natural materials or other wise,the chemical compositions of which are not completely defined.

6. culture Media classified by consistency Liquid culture medium: Culture medium consisting of an aqueous solution of one or more constituents ( e.g.., peptone water,nutrient broth) Note1 : In some cases,solid particles are added to the liquid medium Note2 : Liquid media in tubes, flasks or bottles are commonly called “ broth”

7. Solid culture medium and semi-solid culture medium. Liquid culture medium containing solidify martial ( e.g. agar-agar,gelatin,et c.,) in different concentrations Note1: Due to the world-wide use of culture media solidified with agar-agar,the shortened term ”agar is s often used synonymously for solid culture media and therefore is connection with nouns, e.g. ‘plate count agar.

8. Culture Media classified by intent use Transport medium: Cultured medium designed to preserve and maintain the viability of microorganisms for the time period between sample collection and laboratory processing of sample. Note1- Transport media usually contain substance that do not permit multiplication of microorganisms but ensure their preservation (e.g.,Stuart's Carry Blair or Amies Transport medium)

9. Preservation medium Culture medium designed or preserve and maintain the viability of microorganisms over an extended period, to protect tem against the adverse influence with may occur during long-term storage and to allow recovery after this period( e.g. Dorset egg medium, Skimed Milk, TSB broth with 15% glycerol.)

10. Enrichment medium Predominantly liquid culture medium which,due to its composition,provide particularly favorable condition for multiplication of microorganisms.

11. Selective enrichment medium Enrichment medium which supports the multiplication of specific microorganisms while inhibiting other microorganisms (e.g..SF,GN broth…. )

12. Non –selective enrichment medium Enrichment medium is not devised to selectively inhibit microorganisms (e.g. nutrient broth)

13. Isolation medium Solid or semi-solid culture medium which supports growth and/or the formation of colonies of microorganism.

14. Selective isolation medium Isolation medium which supports growth of specific microorganisms,which inhibiting other microorganism (e.g. MacConky agar EMB agar)

15. non-selective isolation medium Isolation medium which is not devised to selectively inhibit microorganisms ( e.g. nutrient agar)

16. Differential medium Culture medium which permits the testing of one or more physiological/ biochemical character of the microorganisms for the their identification ( e.g Urea medium,Kiligler Iron agar. LDC.ODC.ADH.Cimmon,s Citrate) Note differential media which can be used as isolation media are referred to as isolation /differential media( e.g. Xylose lysine desoxycholate (XLD ) Hekton Enteric agar (HEA)

17. Identification medium Culture media designed to produce a specific identification reaction which dose not require a further confirmatory tests. Note 1-idetification which can be used as isolation are referred to as isolation/ identification media.

18. Media with multiple intents of use Certain culture media may be assigned to several categories.e.g. Blood agar is a enrichment medium,an isolation medium. and a differential medium for detection of hemolysis.

19. Culture media classified to the form of product Ready –to-use medium: Culture medium which is supplied container s in ready form( e.g. Petri dishes or tubes or other carriers).

20. Dehydrate commercially culture medium Culture medium in dry form which is not reedy to use (e.g. powder,granules lyophilsed products) rehydration will make one of two kinds of medium. 1- a complete ready –to-use medium 2- an incomplete medium to which labile components are added at time o use

21. Practices for quality of culture dehydrated media Documentation required from manufacturesome them only on request1- the following details should be availed from the manufacture, 1--Name of medium and product code 2- Batch code 3-pH value 4-Storage information and expiry date 6-any performance evaluation and control strain used 7-Technical data sheet 8-Quality –control certificate ( fore ready to use media) 9-Safety and /or hazard data where needed

22. Check list by laboratory Laboratory checks following data at the media's delivery 1--Name of medium and batch code 2--Date of receipt expiry date 3- -condition of packaging and integrity i.e. checking of the seal 4- -Container damage if needed

23. Quality management and control for dehydration media and supplements Media nowadays are usually purchased from commercial manufactures. They are delivered in dehydrated powdered or granulated form in sealed container and supplements of different selective or diagnosis substances are supplied in either the lyophilized or liquid state. However purchases of should be planned to encourage a regular turnover of stock. To maintain an effective inventory (i.e. first in first out) further checks should include: Re-checking Date of first opening Visual assessment of contents of opened container

24. Continue… Especially after opening a new container,the quality of the medium may depend on the storage environment. Loss of quality of dehydrated media is shown by change in flow characteristics of the powder,homogeneity,caking, color changes etc.. Any dehydrated medium that has absorbed moisture or shows obvious changes in physical appearance should be discarded

25. Laboratory preparation of media The accurate preparation of culture media is one of the fundamental steps in microbiological examination and it shall be given special care. Good laboratory practice or the manufacture's instructions regarding the handling of dehydrated media and other components,particularly those containing Hazardous material i.e. bile salts or other selective agents.

26. Continue…. When media are prepared from dehydrated commercial formulation follow the manufactures instructions precisely. Document all relevant data i.e. weights,pH date of preparation,sterilization conditions, operator For media prepared from individual components,follow the recipe precisely and record all details,in addition the full identity ( i.e. code and batch number ) of all the components used.

27. Preparation of culture Media Dehydrated media are hygroscopic and are sensitive to moisture,heat and light.They are adversely affected by drastic changes in temperature e.g. hot/cold cycling temperature may which may occur between day and night laboratory temperature in winter.

28. Continue… 1-write on the label the date of receipt in the laboratory. 2- Store as directed on the label; usually below 25 °C in a dry area,away from direct sunlight,autoclaves, drying ovens or other heat sources, where indicated store at 2-8°C 3- Check expiry date on the label,some media significantly shorter shelf –lives than others.

29. Continue…. 4- Use stock in lot/batch number order.Do not open a new bottle until the previous bottle has been emptied.Note on the label the date the container is first opened. After use,make sure the container is tightly closed and return it to the designed storage area.

30. Continue… - order the medium in an appropriate size of container and in a quantity which accords to normal use requirements.A medium in a large container which has been opened many times will deteriorate on storage. Discard the medium if the powder is not free flowing, if the color has changed or if it appears abnormally in any way.

31. Reconstitution of dehydrated media Weighing out Using a top-pan balance with an accuracy of 0.1 gram the powder should be spooned to a weighing boat or clean beaker. Do not tip the media out of container as this will cause excess dust which may be irritant and will certainly need cleaning up. The components of some formulation can irritant so the wearing of a face mask at this stage is advisable.

32. Continue…, Complete instruction for the preparation of culture media are given on the label of each bottle.As a general rule it is wise to prepare one week's requirement only. 1- use water prepared by distillation, deionization or reveres osmosis. Toxic metals such as copper must be absent. Check the pH of water,if below 5.5,heat the water to drive off CO2 and re-check.The conductivity of the water should be below15 Mir siemens (μS). Rinse glassware before use

33. Continue… 2-Prepare the medium in a vessel about twice the final volume of the medium to allow adequate mixing.Follow the instructions given on the label of each product 3-Open the culture medium container away from draughts and moisture,Avoid inhaling the powder and prolong skin contact.Weigh the powder quickly accurately and without creating,clouds of dust. Reclose the container as soon as possible.

34. Continue… 4- pour the half the required volume of the water in the vessel, then the weighed quantity of medium and agitate briskly for a few minutes.Pour the rest of the water down the side of vessel to wash any adherent medium back into solution. This is an important step because dry culture media powder above the level of the water may not be sterilized in the autoclave and may be source of contamination

35. Continue.. Agar free media will usually dissolve with gentle agitation. Media containing agar should be heated to dissolve the agar before autoclaving. Bring the medium to the boil without scorching or burning. Those media which should not be autoclaved will be ready to pour into dishes or other containers after this amount of heating (e.g XLD, TCBS, SS agar). Most culture media will required final sterilization an autoclave at 121.Do not adjust pH before sterilization.

36. Continue… the pH of the dehydrated medium has been adjusted so that the final pH of the prepared medium confirm with the label specification when the medium has been cooled at 25ºC. Do not adjust pH before sterilization.

37. Sterilization of Culture Media Although sterilization of culture media is best carried out in a steam autoclave at temperature around 121C° it has recognized that damage is caused to the medium by the heating process. Heat treatment of culture media which contain peptide,sugars minerals and metals results in nutrient destruction,either by direct thermal degradation or by reaction between the medium components. Toxic products caused by chemo-oxidation can also be formed during heat –treatment.

38. Continue… It is important,therefore,to optimize the heating process that a medium is sterile after heating but minimal damages caused to the intergradient of the medium. As a general rule it is accepted that shrot duration,high- temperature process are more lethal to organisms and less chemically damage than are longer,lower temperature process.

39. Continue… A general instruction for sterilization culture media in volume up to one liter at 121ºC for 15 minutes is given on each label. Autoclaves vary in performance,however,and thermocouple tests using different volume of media should be carried out to determine the,heat-up and cool-down times,It will be essential to do this when volume of media greater than two liters are prepared. In order to avoid overheating large volume units of media,the heat up, and cool-down periods are normally integrated into 121º C holding time

40. Sterilization Cycle the sterilization cycle can be divided into four cycle 1- Chamber heat-up 2- Heat penetration 3- Holding time at the prescribed temperature 4- Cool-down time for the chamber to reach 80ºC

41. Sterilization cycle Sage 1 20-121ºC Stage 2 <100-121ºC Stage 3 121-121ºC Stage 4 121- 80ºC

42. Stage 1 The chamber heat-up time depends on the efficiency of the autoclave (air discharge /steam input) and the size of the load in the chamber. The time required for this stage is measured with a recording probe located in the air-discharge valve located in the base of the chamber.

43. Stage 2 The heat penetration time depends mainly on the volume of the individual containers, although the shape and the heat –transfer properties of the containers may affect this stage.The time required the medium volume to reach 121 is measured with thermocouples placed in the center of the innermost container

44. Continue… Volume (ml) in glass bottles. Time 100 19 500 18 1000 22 2000 20 5000 37

45. Continue…. These time assume that agar media have been dissolved before autoclaving.It is also assumed that maximum exposure to steam is possible Thus although the single 100ml bottle required 12minutes to reac121C when placed in a crate with other bottles it required 19 minute and when placed in the center of staked crates it require 30 minutes.

46. Continue… Stage 3 The holding time at 121C depends on (i) The number of organism originally present In the medium (ii) The fractional number of an organism presumed present after heating.e.g. N=0.001 equivalent o one bottle in everey1000 bottle heated becoming contaminated. (iii) the thermal death rate constant of the presumed organism present at 12ºC

47. continue…. Stage 4 The cool down time depends on the size of the load in the camber and the heat loss rate from the autoclave. water-spray are used to accelerate cooling in commercial sterilizers but very careful control is required to avoid bottle fracture and the ingress of the cooling spray into the sterilizes medium. The latter problem occurs when the vacuum formed in the heat –space during cooling sucks contaminated cooling fluid up the thread of the cap and into the bottle

48. Continue… Culture media autoclaves should be untagged and of moderate chamber capacity only. Thermal locks on the doors should prevent them opening when the chamber temperature is above 80 º C but even in these circumstances care should be taken to avoid thermal shock when removing glass bottles of hot liquid from the autoclave.

49. Continue... When screw-capped containers are placed in an autoclave the caps should be a half-turn free to allow the escape of heated air. When removed from the autoclave the caps are screwed down tight after the contents have cooled to ambient temperature.

50. Continue… All autoclaves should be calibrated and checked at fixed periods of time to ensure that they are functioning efficiently. Physical measurements should be made on temperature and pressure readings, the quality of the steam should be checked,the efficiency of the steam should be checked,the efficiency of the,near –to-steam,air traps in the base of the autoclave should be determined and the safety valves checked. Mandatory inspection of autoclaves as pressure vessels are normally carried out annually by specialist under instructions from insurers of such apparatus

51. Sterilization Checks Biological indicators of sterilization will demonstrate the ability of the autoclaves to destroy spores.Such tests may be compulsory in certain countries. Chemical indicators will show the temperature reached or exceed and some will indicate the time held at the specified temperature. Under autoclaving is usually self-evident because failure to destroy all the bacterial spores naturally present in dehydrated media( the bioburden) will allow grow to take place in the stored or incubated medium. Failure of sterilization should always be suspected when contamination of prepared media occurs with sporing organisms.

52. Faults and Possible Causes in Media sterilization Fault ;;Wrong pH value. Possible causes. pH test carried out above 25ºC Overheating through prolonged sterilization remeltingor overlong period at 50 ºC Incomplete solution of Medium. Poor quality water or container Dehydrated medium stored incorrectly or beyond the stated shelf lif

53. Turbidity,Precipitation Possible causes: Poor quality water or container Overheating or prolonged storage at 50ºC pH value incorrect Incomplete solution

54. Darkening Possible causes: Overheating. Incomplete solution pH drift

55. Soft gel Possible causes: Agar not in solution, poor mixing, prolonged storage at 50ºC Overheating at low pH value Error in weighing or over dilution with inoculum or media supplement.

56. poor bacteriological growth Prolonged and excessive heating,incomplete solution Inhibitory substance in water or container Darkening and pH drift.

57. Preparation of sterilized Media Liquid media which are sterilized in their final concentration should be cooled down room temperature as rapidly as possible. Screw caps should then tightened. Container of agar media which have been sterilized should be placed at 50ºwater bath and the medium dispensed as soon as possible as it reach this temperature, or within a maximum of 3 hours in the bath. The medium should be mixed thoroughly without bubble formation and aseptically dispensed into sterile containers. Do not expose dishes of agar media to sunlight; it causes excessive condensation on the lids and may cause the formation of inhibitory substance by photo-oxidation

58. Continue.. Heat –labile supplements should be added to the medium after it has cooled to 50ºC Allow the sterile supplement to come to room temperature before adding it to the agar medium. Cold liquids may cause agar to gel or form transparent flakes which can easily be seen in blood-enriched agar.Mix all supplements into the medium gently and thoroughly then distribute into the final containers as quickly as possible.

59. Continue.. Blood used for the preparation of the blood agar should be as fresh as possible and should have been stored at 2-8º C( blood must not be frozen) Warm the blood in an incubator to about 35-37º before adding to sterile molten agar base, which has been cooled to 40-45. A adequate mixing in a large head –space vessel is essential to ensure aeration of the blood. Poorly oxygenated blood plates are purplish in color whereas properly aerated blood agar is cherry-red.

60. Sterilty Check All prepared couture media should be checked for sterility. After preparation incubate %5 of all media for 24 hours.

61. Storage of Prepared Media The recommended shelf-life of prepared culture media varies considerably.Screw-capped bottles of nutrient broth and agar can be stored for 6 month at low ambient temperature (12-16C) it is important to store all media away from light.. Do not freeze the culture media. Loss of moisture from agar plates is a common

62. Quality assurance for commercially prepared culture media Testing required by Manufacture: The media must be tested by manufactures for performance.Use of control strains,the incubation condition are important factors;

63. Source of Control Strains American Type culture Collection (ATCC). Commercial Sources Reference Laboratories Patents isolates

64. Maintance of control Strains. Trypticase Soy agar( for non-fastidious ) Deep freeze Lyophilization.

65. Test procedure for Culture Media (briefly) 1-Prepare a 0.5 Mac Farland Suspension 2-For testing the nutritive capacity of plate media( Blood agar Nutrient agar) dilute the basic cell suspension1:100 3-Inoculate 10-µL,the diluted suspension to provide 1to 2x10 4 CFU

66. Continue… For testing the inhibitory capacity(e.g EMB): 1 -Prepare 1: 10 suspension 2-Inoculate 10-µl by streaking inplate to provide 1 to2 x10 5 CFU For testing the performance of tubed media,inoculate with a 10-µL.

67. Reporting Quality Assurance Data to the user The manufactures should indicate that performance testing has beene established and documented. The manufactures should Insert: Label Package Technical manual

68. transport and storage Media shall be shipped to prevent excessive loss of moisture and to provide mechanical and thermal protection. Whenever possible The number of intermediate handlers should be minimized.

69. User There is not necessary performance testing by user ( campylobacter agar and media for isolation Neisseria spp) User of commercialyy prepared media must inspect all media in each shipments for any the following conditions :

70. User inspection Cracked Petri dishes Unequal filling of plates Cracked medium in plates Hemolysis (blood agar) Freezing Excessive numbers of bubbles Contamination.