ESCMID Online Lecture Library. by author. Mike Cox, CEO Anaerobe Systems Morgan Hill, CA

Transcription

1 Mike Cox, CEO Anaerobe Systems Morgan Hill, CA

2 Oxygen Nitrogen Oxygen Other

3 Ground State Singlet State Oxygen Toxicity

4 Ground State O 2

5 Singlet State O 2 = O 2 - OH H 2 O 2

6 Superoxide Anion O 2 + Energy O 2 Ground State Singlet State O 2 + e- O 2 - Singlet State Superoxide Anion

7 Hydrogen Peroxide O e- + 2H + H 2 O 2 Superoxide Anion Hydrogen Peroxide

8 Hydroperoxy Radical H 2 O e- + H + H 2 O + OH Hydrogen Peroxide Hydroperoxy Radical

9 H 2 0 OH + e- + H + H 2 O Hydroperoxy Radical Water

10 Defenses Against Oxygen Toxicity Hemoglobin Superoxide Dismutase Catalase Peroxidase Vitamin C Vitamin E Uric Acid

11 Damage to Anaerobes by Oxygen Oxidation of lipids in membranes Inactivation of enzymes Direct genetic damage

12 Oxygen Damage to Culture Media Oxidation of organic constituents Formation of hydrogen peroxide Damage is accelerated by reducing agents e.g. Cysteine This damage is not reversed by reducing the media

13 Light Oxygen Principle Causes of Media Deterioration Dehydration or hydration

14

15 Pre Reduced Anaerobically Sterilized (PRAS) Boiled free of molecular oxygen Reducing agent then added Autoclaved anaerobically Dispensed anaerobically Stored anaerobically

16 Oxygen Kills Time Procedures that fail due to excessive exposure to oxygen result in delayed reports. Oxygen Kills Confidence You saw the organism in the Gram stain, however, you failed to isolate it. Oxygen Kills Credibility The physician knows the patient has an infection, however, the report says no growth! Oxygen Kills! Oxygen Kills Budgets You committed time and money to work-up a culture, however, you did not obtain the results you needed. Material and time are wasted. Oxygen Kills Media Oxygen is the #1 cause of media deterioration. When exposed to oxygen during manufacturing and storage, media accumulate toxic products of oxidation that will inhibit the growth of many anaerobic pathogens.

17 Oxygen Kills People! When culture results are compromised by processing the specimen in air and on oxidized media, the loser is the patient.

18

19

20

21

22

23

24

25

26

27

28 Atmosphere Chambers, Jars, Bags

29 PRAS Media: yes The VPI Technique Inoculation: anaerobic Incubation: anaerobic Inspection: anaerobic Subculture: anaerobic

30

31 PRAS Media: yes Anaerobic Jars Inoculation: aerobic Inspection: aerobic Subculture: aerobic Incubation: anaerobic Cost per plate: $0.20

32

33 Use of Anaerobic Jars Limit exposure of plates to air to 15 minutes maximum during set up and examination Be sure to reactivate the catalyst after each use

34 PRAS Media: yes Plastic Pouches Inoculation: aerobic Inspection: aerobic Subculture: aerobic Incubation: anaerobic Cost per plate: $1.05

35 Use of Plastic Pouches Convenient for small labs and for dropin samples You can see through the bag to decide when to open it Limit exposure of plates to air to 15 minutes maximum during set up and examination

36 Anaerobic Chambers PRAS Media: yes Inoculation: anaerobic Inspection: anaerobic Subculture: anaerobic Incubation: anaerobic Cost per plate < $0.10

37

38

39

40

41

42 Use of Anaerobic Chambers No exposure to oxygen Work flow can be organized in a massproduction fashion Reactivate the catalyst each day Do not use desiccants in an anaerobic chamber Use activated charcoal to absorb H 2 S Do not bring plates out of the chamber for inspection

43 Gas Mixture is 5% hydrogen, 5% carbon dioxide, and 90% nitrogen Do not exceed 5% hydrogen for safety reasons You can test for the presence of hydrogen by flowing the gas over good catalyst. The catalyst will heat up if hydrogen is present.

44 Catalyst Reactivate by heating for 2 hours at 160º C Test by flowing the anaerobic gas mixture over the catalyst; if it is active, it will produce heat Clean by heating the catalyst to 200º C and then flowing the anaerobic gas mix through the catalyst while it is hot

45 Positive Pressure The manometer shows if you have a positive pressure

46 Keeping the Chamber Anaerobic The Triangle

47

48

49 Concepts: Susceptibility Testing Healthy organism Standardized number of micro organisms Known antibiotic concentration Proper work environment Proper incubation environment Proper temperature

50 Susceptibility Testing Exposure to oxygen could: A. Compromise optimum growth B. Cause degradation of antibiotics C. Result in mutations

51 B. Fragilis H B.frag Growth Chloram 12 mcg/ml B.frag S Clinda 8 mcg/ml B.frag S Pen 2 u/ml B.frag R Carb 40 mcg/ml B.frag R Metro 16 mcg/ml B.frag S Cefoxitin 30 mcg/ml B.frag S

52 B. Fragilis H 2 O 2 Antibiotic H202 Blood B.frag Results B.frag Growth Chloram: 12 mcg/ml 3% 2 Drops 2 Drops B.frag R Clinda: 8 mcg/ml 3% 2 Drops 2 Drops B.frag R Pen: 2 u/ml 3% 2 Drops 2 Drops B.frag R Carb: 40 mcg/ml 3% 2 Drops 2 Drops B.frag R Metro: 16 mcg/ml 3% 2 Drops 2 Drops B.frag R Cefoxitin: 30 mcg/ml 3% 2 Drops 2 Drops B.frag R 0 3% 2 Drops 2 Drops B.Frag Growth 0 3% 2 Drops B.frag No Growth

53 Quality Control Will the media support growth? Time factor 24 or 48 hours Proper end points for QC strains

54 B. Thetaiotaomicron - Anaerobic

55 B. Thetaiotaomicron - Air

56 C. Difficile - Anaerobic

57 C. Difficile - Air