Culture-based techniques for identification of pathogens in water samples

Testing Methods
Total heterotrophic Growth on detection plate count media Most Probable Number Culture-dependent techniques
Sample with Bugs

Culture-independent techniques Optical Nucleic Acid-Based Immunological Methods

Chemical

(Total) Heterotrophic Count

developed and implemented by R. Koch

(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

(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

(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
wont detect anaerobes wont detect bacteria with specialized growth requirements (e.g. Campylobacter, Legionella, Listeria) tells little about the identity of cultured bugs. not a Coliform test

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)

Differential Media: examples

Shigella on 5 media
Textbookbacteriology.net

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

Klebsiella

Salmonella

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

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)

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 wont 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)

Cant rely on a single medium for ID.

Most Probable Number

10x dilution 10x dilution 10x dilution 10x dilution

~ 1,000

~100

~ 10

0 cfu/ml

Most Probable Number

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

Most Probable Number

Can be combined with detection media
10x dilution 10x dilution 10x dilution 10x dilution

detection medium A

Medium B

Medium C

Most Probable Number

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

10x dilution

10x dilution

10x dilution

~100

~10

0 cfu

10x dilution

10x dilution

10x dilution

~ 1,000

~99

~9

0 cfu/ml

10x dilution

10x dilution

10x dilution

~ 1,000

~99

~9

0 cfu/ml

~100

~10

0 cfu

Most Probable Number

Disadvantages:
sterile technique is crucial under field conditions MPN estimates can easily range +/- 10 fold.
drinking water tests require detection at 100500 cfu/ml level. MPN tests may score these as 10 or a 1,000 cfu/ml

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