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Quanti-Tray* Enumeration Laboratory(Draft in progress)

ENV 113 Oceanography

Dr. Frashure

Background Information:

Quanti-Tray is method used by environmental scientists to determine whether or not harmful levels of enterococcus bacteria are present in marine waters. Quanti-tray enumeration determines whether or not marine beach waters are safe for swimming. With Quanti-Tray, a 100 mL sample is divided into 51 wells. Then the Standard Methods’ Most Probable Number (MPN) approach is used to determine the number of bacteria in the original sample. Automation minimizes hands-on time and maximizes throughput (up to 4 samples per minute). The relatively large number of wells provides a high counting range of 1–200 (without dilutions) and very tight 95% confidence limits.

Materials:

· 2 (250 ml)Niskon sampling bottle

· 500 ml Sample of ocean water

· IDEXX Quanti-Tray Sealer

· 4 Graduated cylinders

· 4 Quanti Tray

· Incubator (41 degrees C)

· 4 Results Interpretation Table

· Distilled water

· Biohazard waste bag

· Autoclave

· Sterile gloves

Methods:

1. Marine water samples must be diluted at least tenfold with sterile fresh water.

2. Add contents of one pack to a 100 mL water sample in a sterile vessel.

3. Cap vessel and shake until dissolved. 3. Pour sample/reagent mixture into a Quanti-Tray* or Quanti-Tray*/2000.

4. Seal in an IDEXX Quanti-Tray* Sealer.

5. Place the sealed tray in a 41±0.5°C incubator for 24 hours.

6. Read results according to the Result Interpretation table below.

a. Look for fluorescence with a 6–watt, 365 nm, UV light within 5 inches of the sample in a dark environment.

b. Face light away from your eyes and towards the sample.

c. Enterolert results are definitive at 24–28 hours.Result Interpretation: If the appearance result has a lack of fluorescence, then it is negative for enterococci. If the appearance has a blue fluorescence, then it is positive for enterococci . In addition, positives for enterococci observed before 24 hours and negatives observed after 28 hours are also valid.

7. Count the number of positive wells and refer to the MPN table provided with the trays to obtain a Most Probable Number.

8. Multiply the MPN value by the dilution factor to obtain the proper quantitative result.

9. Aseptic technique should always be followed when using Enterolert. Dispose of in accordance with Good Laboratory Practices.

Results:

EnterolertQuantitray 2000 – Water

To be filled out at time of collection:

Site Name: _________________________ Date: ____/____/___ Time: ___________

MM / DD /YY (military)

To be filled out by laboratory analyst:

Analyzed by (initials): _____________ Analysis Date: ____________

Enterolert lot number: ________________ Time in 41C: ____________

Quantitray lot number: __________________ Time out of incubator: ____________

Read by: ____________ Date trays read: ____________

(Always use sterile distilled or deionized water when making dilutions.)

Well counts:

[Enterococci positive well: fluorescence under UV light]

NWIS Parameter code 99601, Method code BAC06

Sample dilution (circle one): undiluted 1:10 1:100 1:1,000 1:10,000

Positive result	Large well count	Small well count	MPN/100 mL1
Enterococci (fluorescence)

Sample dilution (circle one): undiluted 1:10 1:100 1:1,000 1:10,000

Positive result	Large well count	Small well count	MPN/100 mL1
Enterococci (fluorescence)

1For undiluted samples, results are pulled from MPN table. If diluted, results are pulled from MPN table and multiplied by the dilution factor.

Average results: Enterococci= ___________MPN/100 mL

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