Laboratory 6Enumeration

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

Equipment

-spectrophotometer

-microscopes

-Petroff-Hauser chamber

-bunsen burners

-spreaders

Cultures

-Escherichia coli (broth)

Supplies

-Tryptic Soy Agar (TSA) plates

-Liquid TSB media

-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.
  1. Aseptically transfer 1 ml of the culture into a tube containing 9 ml of TSB medium.
  2. Transfer 1 ml of the resulting tube into another tube with 9 ml of medium.
  3. Repeat this process until you have produced 9 tubes. (10-1 – 10-9).
  1. Spread 0.1 ml of each dilution tube onto a TSA plate.
  2. Incubate at 25C for two days.

Period 2

  1. Count the number of colonies on plates that contain between 20 and 200 well-separated colonies.
  2. 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.
  1. Transfer 2 ml of the culture into a tube containing 2 ml of TSB medium.
  2. Transfer 2 ml of the resulting tube into another tube with 2 ml of medium.
  3. Repeat this process until you have produced 4 new tubes. (1:2 – 1:16).
  1. Set the transmittance to 0 with no sample in the chamber.
  2. Set the absorbance to 0 using un-inoculated TSB medium as a blank.
  3. Measure the absorbance of each of the dilution tubes used in the dilution series.
  4. 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)

  1. 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). Each small square is 1/20th X 1/20th of one millimeter square and 1/50th 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 7Coliform 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

Equipment

-incubator (35C)

-bunsen burners

-inoculating loops

-pipettes and bulbs

-sterile graduated cylinders

-100 ml dilution bottles

-sterile filter holders and vacuum flasks

Cultures

-environmental water samples

-Escherichia coli

-Enterobacter aerogenes

-Pseudomonas sp.

Media

-Durham tubes with lauryl tryptose broth (LTB) (2X and 1X)

-Durham tubes with brilliant green bile (BGB) broth

-m-Endo plates

-TSA plates and TSB media in tubes

-Tryptone broth

Supplies and Reagents

-membrane filters

-Enterotube II tests

-sterile water

-Kovac’s reagent

-1% aqueous tetramethyl-p-phenylenediamine HCl

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

  1. Count the number of dark red colonies with a metallic sheen.
  2. 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

  1. Examine each tube for gas production.
  2. 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
  1. 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

  1. 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 6Reading 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 H2S / indole compartment.
  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.
  1. Add two drops of 20% KOH and three drops of alpha naphthol to the chamber.
  2. 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 ml100 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 testSpecies identity

Laboratory 8Chlorination

Introduction

The final step in the treatment of sewage and other wastewaters often involves disinfecting the treated water before discharging it into a river or lake. This is done to kill any pathogenic organisms that may have survived through the initial stages in treatment. Chlorination is the most common method used for the disinfecting stage of wastewater treatment. Chlorine gas is bubbled through the water where it hydrolyses to form hypochlorous acid (HOCl) and hypochlorite (OCl-).

HydrolysisCL2  HOCl + H+ + CL-

IonizationHOCl  H+ + OCl-

Hypochlorous acid is a strong oxidizing agent that acts by reacting with cell components to cause irreparable damage. Hypoclorite is less effective as an oxidizer. Together these two compounds are referred to as the free residual chlorine. Hypoclorous acid can also be formed by adding sodium hypochlorite (NaOCl) to the wastewater. Sodium hypochlorite is the active ingredient in Clorox bleach.

NaOCl + H2O  HOCl + NaOH

In addition to killing cells, the free residual chlorine also oxidizes non-living organic matter and several inorganic components of wastewater such as ammonia, iron and sulfides. When hypochlorous acid reacts with ammonia, monochloramine and dichloramine are formed. These compounds are also oxidizers and contribute to the disinfection process. They, along with some chloro-organic compounds, are referred to as combined residual chlorine. When enough chlorine has been added to completely oxidize the combined residual chlorine then free residual chlorine will begin to accumulate. This process is known as breakpoint chlorination.

Although chlorination is performed in order to kill all of the remaining pathogens in treated wastewater it is not always completely effective. Some organisms will only be injured by the chlorine and some will escape treatment altogether. When this happens, there is potential for re-growth of the pathogens after they have been discharged into clean water. This is why it is so important to be sure that the breakpoint has been reached. In this lab we will be carrying out a simple chlorination series with different concentrations of sodium hypochlorite and measuring the free residual chlorine and the total chlorine with a colorimetric assay. Then we will evaluate the survival of coliforms in the treated wastewater.

Materials

Equipment

-spectrophotometer

-incubator

Cultures

-sewage samples

Supplies

-Clorox bleach

-Hach Test Kit

-m-Endo plates

-beakers and flasks

-16 X 150mm test tubes with 9.9ml of phosphate buffer

-pipettes and bulbs

Procedures

Chlorination

  1. Dilute a sample of Clorox bleach by adding 1ml of bleach to 499ml of distilled water.
  2. Dispense 60ml portions of “sewage” into seven 125 ml Erlenmeyer flasks.
  3. Add 0, 0.5, 1, 2, 3, 5 or 8 ml of diluted bleach (1:500) to each of the flasks.
  4. Mix each sample well and allow it to sit for 20min.
  5. Follow the directions to measure both TOTAL and FREE Chlorine for each flask.
  6. Select three flasks: one that has no free or total chlorine; one that has no free chlorine but does have measurable total chlorine; and one with a measurablefree chlorine concentration.
  7. Test each of these flasks for coliforms as described below.

Chlorine, Free [Method 10069 (0 – 5.0 mg/L)]

  1. Press [PRGM] key. The display will show “PRGM?”.
  2. Press [8] [ENTER]. The display will show “mg/L, CL2” and the ZERO icon.
  3. Fill two sample cells with 10 ml of the sample.
  4. Add the contents of one “DPD Free Chlorine” packet to one sample cell. Cap the cell and swirl vigorously to dissolve the powder.
  5. Within one minute of reagent addition, add deionized water to each cell in order to fill to the 25 ml mark. Cap and mix both cells.
  6. Place the diluted sample without reagent into the cell holder and tightly cover with the instrument cap.
  7. Press [ZERO]. The cursor will move to the right and the display will read “0.00 mg/L CL2”. Remove the blank.
  8. Place the sample with reagent into the cell holder and tightly cover with the instrument cap.
  9. Press [READ]. The cursor will move to the right and the result will be displayed. Record this value.

Chlorine, Total [Method 10070 (0 – 5.0 mg/L)]

  1. Press [PRGM] key. The display will show “PRGM?”.
  2. Press [8] [ENTER]. The display will show “mg/L, CL2” and the ZERO icon.
  3. Fill two sample cells with 10 ml of the sample.
  4. Add the contents of one “DPD Total Chlorine” packet to one sample cell. Cap the cell and swirl vigorously to dissolve the powder.
  5. Press [TIMER][ENTER]. A two-minute reaction period will begin.
  6. After the timer beeps, add deionized water to each cell in order to fill to the 25 ml mark. Cap and mix both cells.
  7. Place the diluted sample without reagent into the cell holder and tightly cover with the instrument cap.
  8. Press [ZERO]. The cursor will move to the right and the display will read “0.00 mg/L CL2”. Remove the blank.
  9. Place the sample with reagent into the cell holder and tightly cover with the instrument cap.
  10. Press [READ]. The cursor will move to the right and the result will be displayed. Record this value.

Counting Coliforms

Period 1