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Laboratory 6

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Laboratory 6

Laboratory 6 Enumeration

Introduction

     One of the central questions a researcher asks when studying any bacterial system is

    “how many bacteria are present in this sample?” This applies to environmental samples where

    we want to learn about a native population as well as to lab cultures where we are measuring

    physiological functions. There are many ways to count bacteria and each of them has advantages

    and disadvantages.

    Measuring the optical density (O.D.) of a sample is an indirect method of determining the

    number of cells present. The amount of light of a specific wavelength that is absorbed by a

    culture is related to the number of cells. This is a very fast and easy way to count cells when

    possible. Unfortunately, in order to use this method, you first need to calculate a response factor

    (the ratio of the number of cells to the change in absorbance) for the specific cells and media that

    you are using. This requires use of an independent method for counting the cells.

    Another common method for counting cells is to serially dilute the original sample and

    spread-plate the resulting dilutions. This is known as the plate-count method. The colonies that

    arise after incubation are counted to determine the original cell density. This method requires

    more time and supplies than measuring the optical density but it does result in an absolute

    number of cells rather than a relative value. The one important thing to remember when doing

    plate-counts is that it only counts the viable cells (those cells that are able to grow under the

    specific incubation conditions used).

    If you want to count all of the cells present in a sample then a direct-counting method

    should be chosen. This generally involves using a microscope to visualize the cells. Special

    microscope slides that have surfaces calibrated into precise volumes can be used to determine

    exactly how many cells there are in a milliliter of sample. Sometimes stains are used to make the

    cells more visible. This method counts all cells whether they are alive or not. The decision as to

    whether viable or total cell counts are more relevant will depend on the experimental question

    being asked.

    In this lab you will be counting the number of bacteria in an overnight culture of E. coli

    using three different methods: optical density, direct counts and plate counts. You will then be

    able to calculate a response factor for optical density and compare the direct count and viable

    count methods.

Materials

     Cultures Equipment - Escherichia coli (broth)

    - spectrophotometer - microscopes Supplies - Petroff-Hauser chamber - Tryptic Soy Agar (TSA) plates - bunsen burners - Liquid TSB media - spreaders - Pasteur pipettes and bulbs

     - Crystal violet stain

    - 95% ethanol

    Procedures

    Plate-Counts (Counting by Dilution Series)

     Period 1

    1. Make a dilution series of the E. coli culture in liquid TSB media.

    a. Aseptically transfer 1 ml of the culture into a tube containing 9 ml of TSB

    medium.

    b. Transfer 1 ml of the resulting tube into another tube with 9 ml of medium. -1-9 10). c. Repeat this process until you have produced 9 tubes. (10

    2. Spread 0.1 ml of each dilution tube onto a TSA plate.

    3. Incubate at 25?C for two days.

     Period 2

    4. Count the number of colonies on plates that contain between 20 and 200 well-

    separated colonies.

    5. Calculate the number of cells in the starting culture by multiplying the number of

    colonies times the inverse of the dilution.

    Optical Density (Counting by Spectrophotometer)

    1. Turn on the spectrophotometer and set it to 525 nm.

    2. Make a dilution series of the original E. coli culture in liquid TSB media.

    a. Transfer 2 ml of the culture into a tube containing 2 ml of TSB medium.

    b. Transfer 2 ml of the resulting tube into another tube with 2 ml of medium.

    c. Repeat this process until you have produced 4 new tubes. (1:2 1:16).

    3. Set the transmittance to 0 with no sample in the chamber.

    4. Set the absorbance to 0 using un-inoculated TSB medium as a blank.

    5. Measure the absorbance of each of the dilution tubes used in the dilution series.

    6. Plot the absorbance against the dilution factor.

    Direct Counting (Petroff-Hauser Chamber)

    1. Use a pipette to transfer 0.9 ml of the starting culture to a clean test-tube.

    2. Add 0.1 ml of crystal violet stain to the culture.

    3. Use a Pasteur pipette to transfer a drop of the stained culture into the Petroff-Hauser

    slide.

    4. Carefully place the cover slip onto the calibrated surface of the counting chamber.

    5. Place the chamber on the microscope stage and focus under the 20X objective to

    locate the large counting grids.

    6. Switch to high power and count the number of bacteria in at least 20 of the small

    squares.

    (If there are too many bacteria to count, make a 1:10 dilution of the culture and start

    over)

    7. Average the number of cells and calculate the number per ml. (cubic centimeter).

Dilution Series

Lab #6 20 points

Name: Date:

Plate counts

    Count the number of colonies on plates that contain between 20 and 200 well-separated

    colonies.

     colonies dilution

    Calculate the number of cells in the starting culture by multiplying the number of colonies

    times the inverse of the dilution.

    Concentration of cells in starting culture = cells/ml

Optical density

     dilution tube absorbance

     culture

     1:2

     1:4

     1:8

     1:16

    Plot the absorbance against the dilution factor. (Attach graph)

Direct counts

    Average the number of cells/square and calculate the number per ml. (cubic centimeter). thththEach small square is 1/20 X 1/20 of one millimeter square and 1/50 of one millimeter

    deep.

     Average number of cells per square -

     Cells per milliliter -

     Cells per milliliter - (corrected for dilution by dye)

Response factor

    Use the absorbance graph, the plate-count data and the direct-count data to calculate two

    separate response factors (# of cells / absorbance unit) for absorbance at 620nm for E. coli.

     Plate count response factor cells/absorbance unit

     Direct count response factor cells/absorbance unit

Questions:

1). Which response factor is more accurate for correlating absorbance to cell numbers? Why?

2). Give an example of an experiment in which you would want to use a direct count and an

    example of an experiment in which you would want to use a live count for quantifying bacteria.

     Laboratory 7 Coliform Testing

Introduction

     The determination of coliform bacteria in water is one of the oldest and most commonly

    used techniques in environmental microbiology. Coliform bacteria are used as indicators of

    potential health threats. The presence of bacteria in the coliform group in any sample is

    considered to be evidence of fecal contamination and therefore of the possibility that pathogenic

    organisms are present. The coliform group is functionally defined according to the way they are detected as “all aerobic and facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48hr at 35?C”. This definition was devised to detect E. coli but it also includes several other genera in the family

    Enterobacteriaceae including Klebsiella, Enterobacter and Citrobacter. All of these organisms are coliforms and will give a positive result in these tests but not all of them are necessarily the

    result of fecal contamination. In order to help clarify the results, and understand the implications

    of a coliform test, it is helpful to identify the number of “fecal coliforms” or to identify the

    species of bacteria detected. Fecal coliforms are defined as those coliforms that are capable of

    growth at 44?C. The increased temperature is meant to select only those organisms adapted to

    growth in the human body. Species typing is carried out by conducting several biochemical tests.

     In this lab you will use two different methods two measure the number of coliforms

    present in surface water samples. The first method (membrane-filtration) is a one-step process

    where coliform colonies are counted directly from filters that have been incubated on m-Endo

    media. This method is relatively quick and easy but it is more prone to errors than the alternative

    “multiple tube fermentation” technique. Multiple tube fermentation is carried out in three

    separate steps. The “presumptive” step involves testing for gas production from lactose fermentation in selective media. The number of coliforms is determined by using the MPN

    method during this step. This is followed by the “confirmed” step where positive samples from

    the presumptive test are transferred to a more selective media and re-tested for gas production.

    Finally, the test is “completed” by isolating single colonies with a positive reaction on m-Endo

    agar.

     You will further characterize the isolates from the completed step by using a commercial

    kit to determine the species.

Materials

     Cultures Equipment - environmental water samples - incubator (35?C) - Escherichia coli - bunsen burners - Enterobacter aerogenes - inoculating loops - Pseudomonas sp.

     - pipettes and bulbs

     - sterile graduated cylinders

    Media - 100 ml dilution bottles

    - Durham tubes with lauryl tryptose - sterile filter holders and vacuum

    broth (LTB) (2X and 1X) flasks

    - Durham tubes with brilliant green

    bile (BGB) broth

    - m-Endo plates - Enterotube II tests - TSA plates and TSB media in tubes - sterile water - Tryptone broth - Kovac’s reagent

     - 1% aqueous tetramethyl-p-Supplies and Reagents phenylenediamine HCl - membrane filters

    Procedures (Membrane Filtration Technique)

    Period 1

    1. Assemble a sterile filtration apparatus with a membrane filter

    2. Aseptically transfer 25 ml of the sample to 225 ml of sterile distilled water.

    3. Mixed diluted sample well.

    4. Filter 10 ml of diluted water.

    5. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.

    6. Replace the membrane filter and filter 50 ml of diluted water.

    7. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.

    8. Replace the membrane filter and filter 100 ml of diluted water.

    9. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.

    10. Incubate the plates upright at 35?C for 24 hrs.

    Period 2

    11. Count the number of dark red colonies with a metallic sheen.

    12. Calculate the number of coliforms per 100 mL of the water sample.

    Procedures (Multiple Fermentation Tube Technique)

     Period 1

    Most Probable Number (MPN) Determination (Presumptive Test)

    1. Inoculate 5 tubes of 2X lauryl tryptose broth with 10 ml of your water sample and 5

    tubes of 1X broth with 1 ml.

    2. Dilute your water sample 1:10 by adding 10 ml to 90 ml of sterile water.

    3. Inoculate 5 tubes of 1X broth with 1 ml of the diluted sample and 5 tubes of 1X broth

    with 0.1 ml.

    4. Incubate the tubes at 35?C for two days.

    Period 2

    5. Examine each tube for gas production.

    6. Record the results for each tube and use the MPN Table to calculate the number of

    coliforms per 100 mL of the water samples.

Confirmed Test

    1. Inoculate BGB tubes with a loop full of culture from each of the most dilute positive

    tubes from the presumptive test.

    2. Incubate the tubes at 35?C for two days.

Period 3

    3. Examine each tube for gas production.

Completed Test

1. Streak two m-Endo plates from each positive BGB tube.

    2. Incubate one plate at 35?C and the other at 44?C for 24 hrs.

Period 4

    3. Examine the plates for the presence of green, metallic colonies.

Species Typing

1. Select a positive E. coli colony from the m-Endo plates.

    2. Streak a TSA plate with that colony from the m-Endo plate.

    3. Incubate at 35?C for 24 hrs.

    Procedures (Enterotube II kit)

     Period 5

Oxidase Test

    1. Pick a colony from the TSA plate using a wooden applicator stick and smear it onto a

    filter soaked in tetramethyl-p-phenylenediamine.

    2. Development of a purple color indicates a positive reaction.

    3. Record the result on the Data sheet.

     Inoculating the Tube

1. Remove both caps from the Enterotube without touching the wire inside.

    2. Use the tip of the inoculating wire to pick one large (2-3mm) colony from your TSA

    plate (there should be a visible amount of cells on the wire).

    3. Inoculate the tube by first twisting the wire then withdrawing it through all 12

    compartments while slowly turning it.

    4. Reinsert the wire back through all 12 compartments until the notch in the wire is even

    with the opening of the tube.

    5. Break off the handle end of the wire by bending it.

6. Use the handle of the wire to punch holes through the foil covering the air inlets of

    the last eight compartments (adonitol, lactose, arabinose, sorbitol, Voges-Proskauer,

    dulcitol/PA, urea and citrate).

    7. Replace both caps.

    8. Incubate at 37?C for 24 hrs.

Period 6 Reading the Results

1. First read the color reactions for all of the tests except indole and Voges-Proskauer

    and record the results on the ID form.

    2. Use a syringe and needle to pierce the plastic film and add one or two drops of

    Kovac’s reagent to the HS / indole compartment. 2

    3. Development of a red color in the compartment indicates a positive result.

    4. Record the result on the ID form

    5. Calculate the biotype number and determine the isolate’s identity from the code book.

    6. If the code book indicates that it is necessary, perform the Voges-Proskauer test for

    confirmation.

    a. Add two drops of 20% KOH and three drops of alpha naphthol to the

    chamber.

    b. Development of a red color within 20 min. indicates a positive reaction.

Lab #7 20 points

    Name: Date:

Water sample name:

Membrane filter test

     # of coliform colonies:

     10 ml 50 ml 100 ml

     # of coliform cells per 100 mL

MPN test

     Presumptive test

     # of positive tubes

     10 ml 1.0 ml 0.1 ml 0.01 ml

     Most probable number

     Confirmed test

     How many of the BGB tubes are positive? /

     How does this change the estimate of how many coliforms were in the original sample?

     Completed test

     How many of the EMB plates showed typical coliform colonies at 35?C?

     How many of the EMB plates showed typical coliform colonies at 44?C?

     Species typing (Attach Enterotube ID form)

     Indole test Species identity

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