Overview of indicator organisms tủ tài liệu bách khoa

| Safety z May require cultivation and direct manipulation of pathogenic organisms | Time and cost z Each pathogen must be detected using a different test | Requires processing of large

Overview of

Indicator Organisms

Why not test directly for

waterborne pathogens?

| Safety

z May require cultivation and direct

manipulation of pathogenic organisms

| Time and cost

z Each pathogen must be detected using a different test

| Requires processing of large volumes of sample

z Pathogens usually are present in low

concentrations

An Ideal Indicator Organism

| Has an easy testing procedure

| Is of human or animal origin

| Survives as long as, or longer, than

Weaknesses of Indicator

Organisms

| May be present when there is no fecal

contamination

z Total coliforms and C perfringens are found in soil

so they are poor indicators of fecal contamination

| Absent when pathogens are present

z E coli may die off faster than viral pathogens

| Density may not always relate well to the density

of pathogens

z E coli can reproduce in warm, tropical waters.

E coli cells (from USEPA web site)

BACTERIAL INDICATORS

Common Indicator Bacteria

The Coliform Group of Bacteria

Enterobacteriaceae Total coliforms

Fecal coliforms

E coli

Coliform Characteristics

Coliforms are facultative anaerobic, gram-negative, spore forming, and able to ferment lactose

non-| Total coliforms

z Ubiquitous in the environment; because they include fecal

coliforms and E coli they may indicate fecal contamination

z Fresh surface waters

z Ground water that has tested positive for total coliform

Analytical Techniques for Indicator Bacteria

| Membrane filter and direct

Detection and Enumeration Methods: Coliforms

Total coliforms and E coli:

MI Medium

| Under ambient light, E coli

colonies are blue

| The same plate under UV light; total coliform colonies fluoresce

E coli are magenta

modified mTEC

E coli: modified mTEC

Total coliforms and E coli:

Colilert Quantitray

| Under ambient

light, total coliform

colonies are yellow

| The same tray

under UV light;

total coliform

colonies that are E

coli fluoresce

Fecal Coliforms: mFC

medium

| Fecal coliforms are blue or

grayish blue

Common Bacteria Indicators

Enterococci

Group D (includes fecal streptococci)

Streptococcus Group of Bacteria

Fecal Streptococci Characteristics

| Fecal streptococci

z Ubiquitous in the environment but

may be of fecal origin thus a poor fecal indicator

z Gram positive cocci; grow at 35 °C

Detection and Enumeration Methods: Enterococci

| Enterococci

z mE/EIA

z mEI

z Enterolert™

Enterococci: mEI Medium

| Colonies with blue halos are counted

as enterococci, regardless of colony color

Enterococci: Defined Substrate Technology

Common Bacteria Indicators

Processing samples

Membrane filtration Defined substrate technology

Use Aseptic Technique

| Aseptic means “sterile”

| Minimizes sample

contamination

| Assume you are surrounded

by contaminating organisms;

disinfect your work area

before and after use

| Use aseptic techniques

during sample collection,

sample transport, and sample

processing

Dr Harold Sears, U of S Carolina

bacteria on a pin

Sterile buffer water

Manifold with vacuum

Agar plates Pipet &

bulb

Sample water

Dilution bottles

UV light box

Basics of Membrane Filtration

1. A specific volume of sample is passed

through a membrane filter to separate out the bacteria

2. The filter membrane is placed on a plate of

nutrient agar medium that provides the

growth needs of the target organism

3. The plate is incubated at a specific

temperature

4. Target colonies are counted

Step one: Filter the sample

| Filter 3-5 volumes (e.g 1, 3, 10, 30, 100 mL)

| The volumes selected depend on how “dirty”the sample is expected to be

| The goal is to select at least one volume that produces counts within the “ideal” range

| A very dirty sample may need to be diluted to produce a count in the ideal range

Serial Dilutions—an Example

| If a sample has 30,000 target

bacteria per 100 mL;

z For 1 mL filtered, ~3,000

colonies are present—too

many to count.

z But for 0.01 mL filtered, only 30

colonies are present—a count

in the ideal range but a volume

too small to easily measure.

z 1 mL of a 1:100 sample dilution

= 0.01 mL of original sample.

99-mL dilution buffer Sample

1 mL of sample in

99 mL of buffer

makes 1:100 dilution

1:100 dil’n

Step 2: Nutrient Agar

Medium

2. Place filter on a nutrient medium that

promotes target organism growth

Media Contain Selective and

the target bacteria

that allows for its

identification

Enriched lactose medium, rosolic acid favor fecal coliforms

E coli has enzyme

to cleave MUG and produce fluorogen

Sodium lauryl sulfate, sodium desoxycholate

favor E coli;

enzyme cleaves urea phenol

mFC medium

mTEC medium

MUG medium

Step 4: Results

4. Colonies that grow and meet the

differential criteria for target organisms are counted

mFC plate: Count blue colonies NOT gray or cream-

colored

Defined substrate technology (DST)

sample

pre-measured

media

quantitray mixing bottle sterile DI water

How does DST work?

| Nutrient medium for coliforms

| Total coliform enzyme

metabolizes ONPG to form a

yellow product.

| E coli enzyme metabolizes

MUG to form a fluorescent

product.

How does DST work?

| Nutrient medium for

DST Steps

1 Mix media with

sample; pour into

Quantitray

3 Count the positive cells

2 Seal, incubate

Preparing a sample for incubation

Determining a final count

| Compare color

intensity to the

“comparator” tray

| Count the number

of large and small

wells that are

positive

| Look up the most

probable number

on the IDEXX table

test