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Water analysis practical

All experiments we have designed for the water analysis:

1-Colilert test:

  1. Warm up the sealer; this takes about 10 minutes.
  2. Prepare lab forms. Fill out appropriate lab forms for each sample and check off the dilutions to prepare, if any.
  3. Label the bottom of the incubation tray with sample information. Label the tray with site name, date, and dilu­tion (if any).
  4. Prepare sample/reagent mixture.
    1. For undiluted sample:
      1. The system will enumerate between 1 and 2,400 MPN/100 mL for the undiluted sample.
      2. Combine 100 mL of sample with one packet of Colilert reagent.
  • Mix to dissolve reagent by gently inverting the bottle. Do not shake vigorously; this will cause foam.
  1. Pour the reagent/sample mixture into the incubation tray. Tap the small wells to release any air and allow foam to settle.
  2. Run the tray through the Quanti-Tray sealer.
  3. Incubate at 35±0.5°C
    1. Colilert-24 results are definitive at 24–28 hours. In addition, positives observed before 24 hours and negatives observed after 28 hours are also valid
  4. Count large and small positive wells that
    1. Fluoresce under a long-wave ultraviolet light as coli
    2. Appear yellow under ambient light as total coliforms
    3. Dim yellow color and dim or off-color fluorescence are not counted as positive results.
    4. The large overflow well at the top of the tray is counted as a large well.
  5. Refer to the MPN table to obtain results.
  6. Record results on a bench sheet form (an example is attached).

Results:

For the Colilert method, three cultures are analyzed:

  • coli—positive for total coliforms and E. coli. Wells will be yellow under normal light and fluoresce under long wave UV light.

Well counts:

[E. coli positive well: fluorescence under UV light; total coliform positive well: yellow under ambient light]

NWIS parameter code using Colilert Quantitray 2000 for: E. coli in water – 50468; total coliforms – 50569

Method codes: Colilert: BAC14 (EC), BAC47 (TC); Colilert-18: BAC15 (EC), BAC48 (TC)

Positive result

Large

well count

Small
well count

MPN/100 mL1

Total Coliforms (yellow)

49

48

>2419.6

E. coli (fluorescence)

3

0

3.1

2- Membrane filtration method:

1- Collect the sample 1 L.

2- We will use two volume of sample used 100 ml and 200 ml

3- pour 100 ml in the measuring cylinder.

4. two Chromocult agar culture medium for two volume samples chosen.

3- Flame the forceps, and remove the membrane from the sterile package.

4. Place the membrane filter into the funnel assembly.

5. Flame the pouring lip of the sample and pour the sample into the funnel

6. Turn on the vacuum and allow the sample to draw completely through the filter.

7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw completely through the filter.

8. Flame the forceps and remove the membrane filter from the funnel.

9. Place the membrane filter into chromocult agar

10. Incubate at 35 C for 24 h.

11. Count the colonies.

12. Confirm the colonies and report the results.

Results for membrane filtration:

In chromocult agar we observed colonies has been grown in both chromoucult agar. For the 100 ml, we counted the blue colonies and it was 15 with different sizes, while in plate 200 ml counted blue colonies were 19. This dark blue to violet colonies indicate E.coli and red to purple colonies indicates the total coliforms which is too much to count.

3-Isolating colonies from chromocult agar for identification tests

These colonies were isolated (blue and purple from the 100 ml and 200 ml sample) and streaked aseptically on 6 PCA plates, 4 chromocult and 4 MacConkey agar plates and incubated at different temperatures 37 and 44 C for 2-3 days in order to do the identification test and differentiate the type of microorganisms present in the sample water. Results next week

4- This Monday identification tests:( not done yet will provide it later)

Colonies from PCA plates will goes under several identification tests:

-Oxidase – catalase reaction

-Gram staining reaction

- API 20 E test

Water analysis practical manual

During class we will assign students to groups, you may end up working with different people than you have in previous activities. Spend some time with your group discussing what you have learned regarding methods for water quality assessment. We will provide some additional literature on methods to detect particular organisms in the water samples, why these are measured, guideline values, etc. As a group decide what environment you would like to sample and how you plan to undertake the sampling.

The goal for this week is for each group to decide on a research question, an environment of interest to sample and develop a sensible strategy regarding sample collection and analysis. All groups will formulate their research question and strategy, then put together a 1-2 slide presentation explaining their experimental design and plan. In the second half of this session each group will have 3 mins to present their research question and plan, this is informal – aim is to make sure you have formulated safe and sensible plans. Each group will also need to submit a list of the resources they require (this is not completely open ended and you will need to work with what we have available!).

Part 1- Experimental design

As a group put together a 1-2 slide PowerPoint presentation explaining your experimental design and plan (e.g. exactly what you will be doing to test the water samples for faecal pathogens). All groups will have 1.5 hrs to formulate their research question and prepare the slide(s).

Some questions to consider (a starting point!):

  • What environment(s) will be interesting to sample?
  • Are there characteristics of the chosen environment that will make testing easier/more difficult? (salinity, accessibility, etc.)
  • How many samples do you need to collect (replicates)?
  • What volume of water do you need?
  • How will you store sample after collection?
  • What types of tests/procedures will you carry out? (think about time limitations – 3 sessions, each 1 week apart to conduct all experiments)

Remember to keep in mind; What scientific question are you setting out to answer?

Part 2- Experimental design

In the second half of this session each group will have 3 mins to go through their presentation. You will only have the allotted time – make sure you keep to it! The point of this task is to provide you with feedback regarding your planned experiments early and to help make sure you have a manageable and sensible plan.

Part 3 - Required materials

You will need to provide Elsa a list (1 page max) of all materials required by the end of this practical so they can be ready for use in the next practical.

Guidelines for Sample Collection

Unfiltered and environmental water samples can be collected from a variety of sources. For this assignment you will be required to collect your water samples according to your own experimental design. For this purpose, thoroughly cleaned and rinsed 1L commercial mineral/still water plastic bottles can be used. We recommend collecting enough empty bottles well in advance of your field-sampling day.

To prevent contamination and improve sampling quality, please rinse your water sampling bottles before you use or fill it with some surface water from your sampling site. This is called conditioning. Discard the water used to rinse the bottle before taking your final sample. For sampling, fill your sample bottles to approximately 5 cm of the bottle rim to leave an air space in the sample bottle to allow for mixing of the sample in the laboratory. Cap your sample bottle and place it into a cooler (if possible). Samples should be taken in duplicates. Collection of samples will ideally occur within 48 hours of your session 9 (absolutely no more than 72 hours prior). Keep samples as cool as possible after collection (ideally 4ºC) and make sure your samples are transported in for practical session (you may be able to leave samples in lab fridges if you have afternoon practical sessions – please organise with Elsa).

Environmental isolates could potentially include human pathogens, so samples and isolates should be treated as a biohazard (double bag bottles with clean bags if you must store water in a home refrigerator for a period of time). Please follow the general laboratory safety and aseptic techniques. Disposable gloves for sampling can be obtained in advance from the laboratory officer.

Sampling considerations

  1. Ensure that the samples collected will be representative of the system being investigated. As part of the sampling process it is necessary to provide relevant detailed descriptions of the sampling area (environment).
  2. Before selecting a sampling site consider:
  • What is the scientific question/hypothesis or study objective(s)?
  • What sampling method(s), post-sampling analysis methods and equipment available to you for the assignment?
  • Consider physical, chemical and biological characteristics of sampling area, e.g., geology, location, climate, water depth, land/water use, tributary, possible source of contaminations, industry, flora and fauna etc. (better to avoid marine environments).
  • If relevant, obtain historical information of sampling site.

Under no circumstances should you collect samples from wastewater plants, sewage, abattoirs, or any other source of likely high level pathogen contamination or can put you at risk of physical injury.

Standard materials we can provide you with

(Please read and familiarise yourself about these media and tests beforehand)

  • Colilert test, including Colilert Quanti-Trays (IDEXX).
  • Chromocult agar.
  • MacConkey agar.
  • API20E bacterial identification test (one per group)
  • Catalase, Oxidase test materials.
  • Filtration units, membrane filters and pumps.
  1. If you want to perform tests which require a specific incubation time you will need to organise with Elsa to remove samples from incubators, at required times in normal work hours please!), she will place them at 4ºC for observation in your following pracical session. Remember labelling will be particularly important in these situations and it will need to be clear what you want removed and when.

We are very lucky to have obliging, helpful technical support for this course- Elsa has numerous responsibilities outside of this course, so please be considerate and do not abuse this service!

  1. For these experiments your demonstrators will be around to help provide technical expertise, not to tell you what to do. They will be happy to sit down and discuss your plans to some degree, but we are expecting you to use these practical sessions to develop your ability to design and conduct an independent research project.

Don't expect to be automatically given answers to questions you should be able to work out on your own.

PRACTICAL 9: Water Analysis Part 2

PRE-LAB TASK (earn marks!)

Each group must organise collection of their environmental water sample (or samples) for microbiological testing. You must bring to class for your practical session this week (you may be able to organise with Elsa to put samples in University 4ºC fridge ahead of time). You should also have notes planning out what experiments you will conduct this week.

Self-directed research

You assemble all the materials you need to do the testing as requested by you on your list, prepare the required media and proceed with the experiment. Remember all members of the group are expected to have a good record of what has been done in your lab books.

PRACTICAL 10: Water Analysis Part 3

PRE-LAB TASK (earn marks!)

  1. Think ahead and plan out what experiments you need to conduct this week. Remember there are only 2 practical sessions following this in which to complete all your tests.
  1. Write up a flow chart of the planned work.
  2. Have you isolated specific microorganisms?
  3. What tests can you carry out to find out likely identity?

Self-directed research

Follow up from last week according to your experimental protocol. Remember all members of the group are expected to have a good record of what has been done in your lab books.

PRACTICAL 11: Water Analysis Part 4

PRE-LAB TASK

Think ahead about what you need to finalise this week. Do you have any last tests to conduct?

Self-directed research

Complete any last tests/procedures. Remember all members of the group are expected to have a good record of what has been done in your lab books.

PRACTICAL 12: Water Analysis Part 5

PRE-LAB TASK

Think ahead about what you need to finalise this week: Do you have any last tests to conduct or results to collect? Have you isolated specific microorganisms? What can you say about them?

Self-directed research

Complete any last tests/procedures. Last week to conduct experiments – use your time constructively. Use this chance to discuss your results and interpretation with others and demonstrators.

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