Culture-based techniques for identification of pathogens in environmental samples

1. Culture-based techniques for identification of pathogens in environmental samples

2. Testing Methods

3. (Total) Heterotrophic Count developed and implemented by R. Koch

4. (Total) Heterotrophic Count developed and implemented by R. Koch any bacteria growing in water indicate poor quality (100-500 cfu/ml is acceptable, depending on the country) not suitable for more complex samples

5. (Total) Heterotrophic Count developed and implemented by R. Koch any bacteria growing in water indicate poor quality (100-500 cfu/ml is acceptable, depending on the country) not suitable for more complex samples the cheapest and easiest technique of all good survey technique

6. (Total) Heterotrophic Count any bacteria growing in water indicate poor quality (100-500 cfu/ml is acceptable, depending on the country) not suitable for more complex samples the cheapest and easiest technique of all good survey technique detects only bacteria which grow fast (48 hrs at 30-37oC) on common rich media won�t detect anaerobes won�t detect bacteria with specialized growth requirements (e.g. Campylobacter, Legionella, Listeria) tells little about the identity of cultured bugs. not a Coliform test

7. Detection Media Hundreds of different formulations available. Pre-enrichment/resuscitation medium Enrichment medium Elective enrichment (unique combination of nutrients or physiological conditions) Selective enrichment (inhitors, antibiotics) Isolation medium (contains nutrients for specific microbes) Differential medium (contains dyes/stains/indicators)

8. Differential Media:examples

9. Differential Media: XLD XLD (g/L) Agar:15.0 Lactose:7.5 Sucrose:7.5 Na2S2O3:6.8 L-Lysine:5.0 NaCl:5.0 Xylose:3.5 Yeast extract:3.0 Sodium desoxycholate:2.5 Ferric ammonium citrate:0.8 Phenol Red:0.08

10. Detection Media Advantages: Cheap. (Molecular techniques$$$ > Detection Media$$ > Total Heterotrophic Count$) Easy. Suitable for Field Testing OK for surveys Fairly informative Antibiotic resistance tests may detect bugs of particular concern

11. Detection Media Disadvantages: A negative results does not necessarily mean that the bacterium is not there (e.g. VBNC state, antibiotic concentrations, etc) Tricky to interpret crowded plates Time consuming (at least 24 hrs, more for Legionella, Campylobacter)

12. Detection Media Disadvantages: A negative results does not necessarily mean that the bacterium is not there (e.g. VBNC state, antibiotic concentrations, etc) Tricky to interpret crowded plates Time consuming (at least 24 hrs, more for Legionella, Campylobacter) A single medium won�t support growth of all potential problem bacteria. Not as good for surveys as THPC Pre-enrichment may be required for dilute samples for complex samples (e.g. sludge, soil, bedding) Can�t rely on a single medium for ID.

13. Detection Media for E. coli Membrane filtration only for clear water. debris clog and/or tear membranes. dilution plate cloudy samples Hard to isolate E.coli from complex samples (e.g. feces) with less than 1,000 cfu/g

14. Detection Media for E. coli Membrane filtration only for clear water. debris clog and/or tear membranes. dilution plate cloudy samples Hard to isolate E.coli from complex samples (e.g. feces) with less than 1,000 cfu/g Media contain lactose and pH indicator (MacConkey, Violet Red Bile Agar) MacConkey is a �classic�, not very sensitive.

15. Detection Media for E. coli Most E.coli ferment sorbitol, and lactose. US O157:H7 does not, some European O157:H7 isolates ferment sorbitol rapidly

16. Example:protocols for isolation of Salmonella from foods http://www.cfsan.fda.gov/~ebam/bam-5.html

17. Detection Media for E. coli Most E.coli ferment sorbitol, and lactose. US O157:H7 does not, some European O157:H7 isolates ferment sorbitol rapidly MUG test. E. coli (except O157:H7) are positive. MUG can be incorporated into media (not EMB!) cheaper non-fluorescent substrate (X-gluc) works fine Colilert-18 detects a broad range of coliforms, fecal coliforms are overestimated by 10-1000x (especially marine isolates)

18. Colilert

19. Colilert

20. Colilert

21. Colilert: Nothing magic

22. Colilert: a caveat

23. Detection Media for E. coli(cont�d) Trays with multiple individual substrates (Biolog products) may allow better identification, some will simultaneously test for antibiotic resistance. relatively pricey complex samples should not be subject to multiple substrate tests. Results will be impossible to interpret

24. How would you pick a detection medium? How would you design own detection medium?

25. Most Probable Number

26. Most Probable Number Advantages: simple. Cheap. suitable for field tests can be combined with detection media to better ID the organisms for enterics:

27. Most Probable Number

28. Most Probable Number Disadvantages: sterile technique is crucial under field conditions MPN estimates can easily range +/- 10 fold.

32. Most Probable Number Disadvantages: sterile technique is crucial under field conditions MPN estimates can easily range +/- 10 fold. drinking water tests require detection at 100-500 cfu/ml level. MPN tests may score these as 10 or a 1,000 cfu/ml

33. Most Probable Number Disadvantages: to avoid +/- 10x differences, replicate! when bacteria aggregate, MPN is wildly inaccurate. Vibrio, Pseudomonas, older or stressed cultures aggregate easily VBNC are not detected