Culture-based techniques for identification of pathogens in

environmental samples

Testing Methods

Culture-dependent techniques

Culture-independent techniques

Total heterotrophic plate count

Growth on detection media

Samplewith Bugs

Optical

Immunological Methods

Nucleic Acid-Based

Chemical

Most ProbableNumber

(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 • 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

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 mediaTextbookbacteriology.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 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.

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

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.

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

Example:protocols for isolation of Salmonella from foods

http://www.cfsan.fda.gov/~ebam/bam-5.html

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)

ColilertAdd your sample into the bottle with the buffers+substrates

Pour into the plate. Seal ⎯ > distributes the bugs

Incubate

Read: yellow = total coliforms, Yellow/fluorescent = E.coli

http://www.idexx.com/water/colilert18/index.jsp

Colilert

Add your sample into the bottle with the buffers+substrates

Pour into the plate. Seal ⎯ > distributes the bugs

Incubate

Read: yellow = total coliforms, fluorescent = E.coli

http://www.idexx.com/water/colilert18/index.jsp

Colilert

http://www.idexx.com/water/colilert18/index.jsp

Colilert: Nothing magic

Protocol: http://www.promega.com/tbs/tb097/tb097.pdf

Protocol essentially as above, minor differences

Colilert: a caveatAdd your sample into the bottle with the buffers+substrates

Pour into the plate. Seal ⎯ > distributes the bugs

Incubate

Read: yellow = not total coliforms, but all culturable bacteria that can utilize galactoseYellow/fluorescent = not only E.coli, but all culturable bacteria that have b-galactosidase and glucuronidase activities

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

• How would you pick a detection medium?

• How would you design own detection medium?

Most Probable Number

~ 1,000 ~100 ~ 10 1 0 cfu/ml

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

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 Number10x dilution 10x dilution 10x dilution 10x dilution

Can be combined with detection media

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 1 0 cfu

~ 1,000 ~99 ~ 9 0 cfu/ml

10x dilution 10x dilution 10x dilution

~ 1,000 ~99 ~ 9 0 cfu/ml

10x dilution 10x dilution 10x dilution

~100 ~10 1 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 100-

500 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