LaMotte 5553 User manual

NUT/MAC

CODE 5553

USE

Isolation and differentiation of Gram (-) enteric bacilli. Coliform Testing/Recovering of Stressed Coliforms

APPLICATION

In total coliform testing, the coliform organisms tested for include: total coliform, fecal coliform, and E. coli (Escherichia coli).
Detection of fecal coliforms (a subset of total coliforms) or Escherichia coli (a subset of fecal coliforms) can indicate the
potential presence of waterborne pathogens associated with fecal contamination.

PADDLE AGARS

Note: Side 1 of each paddle is marked with an indented laser line.

Side 1: Nutrient-TTC Agar (NUT) – (Color: Yellow) General purpose (relatively non-selective) medium, which

will support the growth of a wide variety of organisms. Suitable for cultivation of both aerobes and anaerobes.
Aerobic coliform bacteria can be detected by their ability to reduce the Triphenyl tetrazolium chloride (TTC) dye
to a red-colored formazan dye. Bacterial colonies appear as red dots on an otherwise yellow medium.

Note: Paddle color is normally LIGHT YELLOW when the NUT agar is cast (about pH 6.0). Some
microorganism growth (even before colonies are OBSERVABLE) will shift the pH from an acidic level to a more
alkaline level (pH 7.0 or higher) – turning the agar a light green.

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Side 2: MacConkey Agar (MAC) – (Color: Brownish-pink) Both selective AND differential; used to differentiate
between Gram negative bacteria while inhibiting the growth of most Gram positive bacteria. The medium also
differentiates between lactose-fermenting coliforms (Lac (+)) and lactose non-fermenters (Lac (-)), which
include potential pathogens.

STORAGE / EXPIRATION

Store tightly sealed BioPaddles® in a cool, dry location. Shield from direct sunlight. Store BioPaddles® at room temperature
(65 - 77°F/18 - 25°C). Avoid sudden temperature changes. Temperature fluctuations may result in condensation settling at the
bottom of the vial. This will not affect the culture properties but could reduce the shelf-life or cause the agar to separate from
the plastic paddle support. Do not refrigerate or store at temperatures above 80°F/27°C. Refrigeration may result in water
condensation. Avoid freezing.

Refer to Best Before End date (See: BBE stamped on vial). Discard if paddle agar appears oxidized and darker than the
expected color or if contaminants appear. The expiration date is based on medium in an intact container that is stored as
directed.

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United State Pharmacopeial Convention. 2007 The United States Pharmacopeia, 31st ed., Amended Chapters 61, 62, 111. The United States Pharmacoeial

Convention, Rockville, MD.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

01.27.15

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AGAR VERIFICATION

These agars have been verified by EMSL Analytical, Inc. using E. coli and E. faecalis cultures. Documentation available upon
request.

SAMPLING

LIQUID SAMPLING PROTOCOL

DIRECT IMMERSION PROTOCOL for Low Viscosity Liquids

1. Twist to remove paddle from vial. Do not touch agar surfaces.

2. Fill vial to 40 mL fill line with the liquid to be sampled and immerse paddle
or immerse paddle directly in the sample. Both agar surfaces must be
completely contacted. Allow at least 15 second contact time (30 seconds is
optimal).

3. Remove paddle. Allow liquid to drain off both agar surfaces.

4. Replace paddle in vial.

5. Incubate.

SPREAD PROTOCOL for High Viscosity Liquids

1. Twist to remove paddle from vial. Do not touch agar surfaces.

2. Hold the contact agar surface on a horizontal plane. Deposit the liquid sample as a single drop
approximately 1 cm from the handle boundary.

3. Position a sterile glass rod between the handle and the drop of sample. Bring the rod in contact with
the drop to create a meniscus. Drag the rod over the agar surface toward the tip of the paddle.

4. Replace paddle in vial.

5. Incubate.

SURFACE SAMPLING PROTOCOL

Recovery Rate is about 50%

1. Twist paddle to remove from vial. Do not touch agar surfaces.

2. Touch the paddle surface (10 cm2) to two different areas of the test surface to cover a total of 20 cm2. Or touch the
paddle to the surface once and multiply the colony count by 2.

3. Allow 15 second contact time.

4. Replace the paddle in the vial.

5. Incubate

AIR SAMPLING PROTOCOL

1. Twist to remove paddle from vial. Do not touch agar surfaces.

2. Invert paddle and insert the circular cap.

3. Expose for 15 minutes.

4. Replace paddle in vial.

5. Incubate.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

01.27.15

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INCUBATION

Incubation of Paddle Growth Incubation Temperature Examine at:

Total Coliform / Bacteria 35 ± 2°C 24 to 48 hours

Total Coliform / Bacteria Room Temperature Up to 5 days

Yeast / Mold 25 to 30°C 48 hours up to 120 hours (5 days)

Yeast / Mold Room Temperature Up to 7 days

Note: Incubation of bacteria after 48 hours may produce confluent growth making enumeration more difficult.

COLONY MEASURING

Each BioPaddles® paddle has molded media attachment points that are 4 mm in length
(point-to-point). This feature provides a useful guidepost to estimating nearby colony
size.

ENUMERATION

Bacteria

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3

10

4

Light Moderate Heavy

Note: Estimation of lower counts is possible, but statistically difficult to justify. Use Light, Moderate and Heavy for Mold
and Mildew growth. Mold and mildew colony growth is more confluent than bacterial growth and therefore more difficult to
quantify. Use Light, Moderate, and Heavy for surface and air testing.

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5

10

6

10

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For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

01.27.15

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DISPOSAL

Twist to remove paddle from vial. Fill vial to 40 mL fill line with 1:9 dilution of household bleach (5.25% sodium hypochlorite).
Replace paddle in vial. Allow 15 minute contact time. Remove paddle. Discard bleach solution. Replace paddle in vial and
dispose. Alternatively, loosen cap and microwave for 30 seconds, autoclave, or incinerate.

IDENTIFICATION

An organism with Growth +++ will grow very well (non-fastidious) on the indicated media. An organism with Growth + is
less likely to grow (fastidious), especially if crowded out by Growth +++ organisms. The media may not contain all of the
nutrients that a Growth + organism needs in order to thrive.

Organism Nutrient-TTC (NUT) Agar MacConkey (MAC) Agar

Aspergillus niger

Bacillus spp.

INHIBITED

Growth: +++
Colony: Granular, jet black conidia with
yellow/gray hyphae, 3-5++ cm

INHIBITED

Growth: +++
Colony: Irregular, raised, lobate (wrinkled),
opaque, with darker center (bullseye)
2-4+ mm

Candida albicans

Growth: +++
Colony: Cream, convex, entire, glossy,
1-2 mm

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

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INHIBITED

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Escherichia coli

Growth: +++
Colony: Yellow/orange/red, convex, entire,
glossy, 2-4 mm

Growth: +++
Colony: Pink/red, convex, entire, glossy,

0.2-0.5 mm

Enterobacter aerogenes

Growth: +++
Colony: Maroon with transparent margin,
convex, entire, glossy, 0.1 - 0.5 mm

Growth: +++
Colony: Pink, thick, round, raised to low­convex, spreading, 0.1-0.5 mm

Enterococcus spp. INHIBITED PARTIAL TO COMPLETE INHIBITION

Klebsiella spp.

Growth: +++
Colony: Amber/red, spreading, 0.5-1.0 mm

Growth: +++
Colony: Colorless/light pink, spreading,

0.5-1.0 mm

Proteus spp.

Growth: +++
Colony: Maroon/red with dark red center
and transparent margin; irregular, glistening
(swarming - transparent field), raised,

Growth: +
Colony: Colorless to yellow, pink/red, circular,
wrinkled (flower-like), umbonate, erose,
2-3 mm

undualte, 1-4 mm

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

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Pseudomonas aeruginosa

Pseudomonas fluorescens

Salmonella (serotype)
enteriditis

Growth: +++
Colony: Maroon with transparent margin,
circular to irregular, raised, entire, 1-2 mm

Growth: +++
Colony: Clear/colorless with grey/dark
center, translucent edges, irregular/
spreading to confluent, 2-4 mm

Growth: +++
Colony: Transparent, convex, entire, glossy

0.1-0.2 mm (punctiform)

Growth: +++
Colony: Clear/pink with dark pink center,
transluecent edges, irregular edges, 2-4 mm

Growth: +++
Colony: Red, full, entire, dull, 0.5-1.0 mm

Growth: +++
Colony: Gray to white (pearl), circular,
umbonate, entire, 1-2 mm

Serratia spp.

Growth: ++
Colony: Red, full, entire, dull, 0.5-1.0 mm

Growth: +
Colony: Pink, convex, dull, entire, 0.1-0.5 mm
(punctiform)

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

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Shigella spp.

Staphylococcus aureus

Streptococcus spp.

Growth: +
Colony: Maroon/red, convex, entire, glossy,

0.5-1.0 mm

Growth: +
Colony: Red, full, entire, dull, 0.5-1.0 mm

Growth + +
Colony: Maroon (red), convex, entire, glossy,

0.1-0. 5mm

Growth: +++
Colony: Transparent to gray (pearl), circular,
raised, dull, entire, 1-2 mm

PARTIAL TO INHIBITED GROWTH

Growth: +
Transparent, circular, umbonate, glistening,
entire, 1-2 mm

Streptomyces griseus

PARTIAL TO COMPLETE INHIBITION

Growth: +
Colony: Yellow, full, entire, dull, 0.5-1.0 mm

Gram (+) Bacteria PARTIAL TO COMPLETE INHIBITION PARTIAL TO COMPLETE INHIBITION

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

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GLOSSARY

Catalase Test Catalase enzyme will react with hydrogen peroxide to produce oxygen if the bacteria is catalase

positive.

Lactose Test Lactose positive bacteria can ferment available lactose in the agar producing an acid which lowers

the pH. Lactose negative bacteria are non-fermenting.

Indole Test Biochemical test to determine the ability of an organism to split indole from the amino acid

tryptophan. P. vulgaris is indole positive while P. mirabilis is indole negative.

Oxidase Test Oxidase positive bacteria contain cytochrome c oxidase which will turn an indicator dark blue. In

contact with oxidase negative bacteria, the indicator will remain colorless.

Urease Test Bacteria containing urease will hydrolyze urea to ammonia and carbon dioxide causing an alkaline

environment which changes the color of a pH indicator from yellow to fuchsia.

-D-Glucoronidase
Reaction

Gram Staining A method for differentiating bacteria into two groups – gram positive and gram negative – based on

The presence of E. coli is determined when both -D-Glucoronidase and Indole are positive, and the
organism is gram negative.

the chemical and physical properties of their cell walls. Often the first step in identifying bacteria.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel
with knowledge of microbacterial techniques in the laboratory. ©LaMotte Company. All rights reserved.

01.27.15

Find Quality Products Online at: [e-mail]

www.GlobalTestSupply.com

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